Quantification of differential gene expression by multiplexed targeted resequencing of cDNA

PubMed Central

Arts, Peer; van der Raadt, Jori; van Gestel, Sebastianus H.C.; Steehouwer, Marloes; Shendure, Jay; Hoischen, Alexander; Albers, Cornelis A.

2017-01-01

Whole-transcriptome or RNA sequencing (RNA-Seq) is a powerful and versatile tool for functional analysis of different types of RNA molecules, but sample reagent and sequencing cost can be prohibitive for hypothesis-driven studies where the aim is to quantify differential expression of a limited number of genes. Here we present an approach for quantification of differential mRNA expression by targeted resequencing of complementary DNA using single-molecule molecular inversion probes (cDNA-smMIPs) that enable highly multiplexed resequencing of cDNA target regions of ∼100 nucleotides and counting of individual molecules. We show that accurate estimates of differential expression can be obtained from molecule counts for hundreds of smMIPs per reaction and that smMIPs are also suitable for quantification of relative gene expression and allele-specific expression. Compared with low-coverage RNA-Seq and a hybridization-based targeted RNA-Seq method, cDNA-smMIPs are a cost-effective high-throughput tool for hypothesis-driven expression analysis in large numbers of genes (10 to 500) and samples (hundreds to thousands). PMID:28474677

Missing data and technical variability in single-cell RNA-sequencing experiments.

PubMed

Hicks, Stephanie C; Townes, F William; Teng, Mingxiang; Irizarry, Rafael A

2017-11-06

Until recently, high-throughput gene expression technology, such as RNA-Sequencing (RNA-seq) required hundreds of thousands of cells to produce reliable measurements. Recent technical advances permit genome-wide gene expression measurement at the single-cell level. Single-cell RNA-Seq (scRNA-seq) is the most widely used and numerous publications are based on data produced with this technology. However, RNA-seq and scRNA-seq data are markedly different. In particular, unlike RNA-seq, the majority of reported expression levels in scRNA-seq are zeros, which could be either biologically-driven, genes not expressing RNA at the time of measurement, or technically-driven, genes expressing RNA, but not at a sufficient level to be detected by sequencing technology. Another difference is that the proportion of genes reporting the expression level to be zero varies substantially across single cells compared to RNA-seq samples. However, it remains unclear to what extent this cell-to-cell variation is being driven by technical rather than biological variation. Furthermore, while systematic errors, including batch effects, have been widely reported as a major challenge in high-throughput technologies, these issues have received minimal attention in published studies based on scRNA-seq technology. Here, we use an assessment experiment to examine data from published studies and demonstrate that systematic errors can explain a substantial percentage of observed cell-to-cell expression variability. Specifically, we present evidence that some of these reported zeros are driven by technical variation by demonstrating that scRNA-seq produces more zeros than expected and that this bias is greater for lower expressed genes. In addition, this missing data problem is exacerbated by the fact that this technical variation varies cell-to-cell. Then, we show how this technical cell-to-cell variability can be confused with novel biological results. Finally, we demonstrate and discuss how batch-effects and confounded experiments can intensify the problem. © The Author 2017. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Heterologous expression of the Pleurotus ostreatus MnP3 gene by the laccase gene promoter in Lentinula edodes.

PubMed

Sato, Toshitsugu; Irie, Toshikazu; Yoshino, Fumihiko

2017-08-01

Lentinula edodes (shiitake), which have a powerful ligninolytic system, is one of the most important edible mushrooms in Asia. In this study, we introduced the manganese peroxidase (MnP, EC 1.11.1.13) gene from Pleurotus ostreatus driven by L. edodes laccase 1 gene promoter into L. edodes for expression. The resulting transformant expressed the recombinant gene and showed a higher level of MnP activity than that of the wild-type strain.

Allen Brain Atlas-Driven Visualizations: A Web-Based Gene Expression Energy Visualization Tool

DTIC Science & Technology

2014-05-21

purposes notwithstanding any copyright anno - tation thereon. The views and conclusions contained herein are those of the authors and should not be...Brain Res. Brain Res. Rev. 28, 309–369. doi: 10.1016/S0165-0173(98)00019-8 Bostock, M., Ogievetsky, V., and Heer, J . (2011). D³ data-driven documents...omnibus: NCBI gene expression and hybridization array data repository. Nucleic Acids Res. 30, 207–210. doi: 10.1093/nar/30.1.207 Eppig, J . T., Blake

A self-initiating eukaryotic transient gene expression system based on contransfection of bacteriophage T7 RNA polymerase and DNA vectors containing a T7 autogene.

PubMed Central

Chen, X; Li, Y; Xiong, K; Wagner, T E

1994-01-01

A novel cytoplasmic gene expression system has been developed. This system differs from other expression systems in that it relies on the co-delivery of plasmid DNA and T7 RNA polymerase (RNAP) during transfection. The plasmid contains a T7 RNAP gene driven by the T7 promoter (T7 autogene) and a functional/reporter gene driven by another T7 promoter (T7T7/T7-gene construct). Once this DNA-enzyme complex is introduced into eukaryotic cells, the transcription of the T7 RNAP and the functional/reporter genes is initiated by the co-delivered T7 RNAP. The T7 RNAP, which is responsible for the initiation and maintenance of expression of both T7 and functional/reporter genes, is replenished by translation of newly synthesized T7 mRNA. This T7 system was designed in such a manner that the expression of the functional/reporter genes can occur in the cytoplasm and does not require any nuclear involvement. When transfected by either a pT7T7/T7Luc or a pT7T7/T7hGH plasmids with the cointroduced T7 RNAP, mouse L cells were found to express high levels of luciferase immediately after transfection, apparently due to the cytoplasmic gene expression; the expression of human growth hormone (hGH) could be sustained for at least 6 days. Both T7 and hGH mRNA were expressed by the cells transfected with pT7T7/T7hGH. These results suggest that this cytoplasmic expression system may be used for certain targets of somatic gene therapy. Images PMID:8029020

GUS expression in sweet oranges (Citrus sinensis L. Osbeck) driven by three different phloem-specific promoters.

PubMed

Miyata, Luzia Yuriko; Harakava, Ricardo; Stipp, Liliane Cristina Libório; Mendes, Beatriz Madalena Januzzi; Appezzato-da-Glória, Beatriz; de Assis Alves Mourão Filho, Francisco

2012-11-01

Huanglongbing (HLB) is associated with Candidatus Liberibacter spp., endogenous, sieve tube-restricted bacteria that are transmitted by citrus psyllid insect vectors. Transgenic expression in the phloem of specific genes that might affect Ca. Liberibacter spp. growth and development may be an adequate strategy to improve citrus resistance to HLB. To study specific phloem gene expression in citrus, we developed three different binary vector constructs with expression cassettes bearing the β-glucuronidase (GUS) reporter gene (uidA) under the control of one of the three different promoters: Citrus phloem protein 2 (CsPP2), Arabidopsis thaliana phloem protein 2 (AtPP2), and Arabidopsis thaliana sucrose transporter 2 (AtSUC2). Transgenic lines of 'Hamlin', 'Pera', and 'Valencia' sweet oranges [Citrus sinensis (L.) Osbeck] were produced via Agrobacterium tumefaciens transformation. The epicotyl segments collected from in vitro germinated seedlings were used as explants. The gene nptII, which confers resistance to the antibiotic kanamycin, was used for selection. The transformation efficiency was expressed as the number of GUS-positive shoots over the total number of explants and varied from 1.54 to 6.08 % among the three cultivars and three constructs studied. Several lines of the three sweet orange cultivars analyzed using PCR and Southern blot analysis were genetically transformed with the three constructs evaluated. The histological GUS activity in the leaves indicates that the uidA gene was preferentially expressed in the phloem, which suggests that the use of the three promoters might be adequate for producing HLB-resistant transgenic sweet oranges. The results reported here conclusively demonstrate the preferential expression of GUS in the phloem driven by two heterologous and one homologous gene promoters. Key message The results reported here conclusively demonstrate the preferential expression of GUS in the phloem driven by two heterologous and one homologous gene promoters.

Misregulation of spermatogenesis genes in Drosophila hybrids is lineage-specific and driven by the combined effects of sterility and fast male regulatory divergence.

PubMed

Gomes, S; Civetta, A

2014-09-01

Hybrid male sterility is a common outcome of crosses between different species. Gene expression studies have found that a number of spermatogenesis genes are differentially expressed in sterile hybrid males, compared with parental species. Late-stage sperm development genes are particularly likely to be misexpressed, with fewer early-stage genes affected. Thus, a link has been posited between misexpression and sterility. A more recent alternative explanation for hybrid gene misexpression has been that it is independent of sterility and driven by divergent evolution of male-specific regulatory elements between species (faster male hypothesis). The faster male hypothesis predicts that misregulation of spermatogenesis genes should be independent of sterility and approximately the same in both hybrids, whereas sterility should only affect gene expression in sterile hybrids. To test the faster male hypothesis vs. the effect of sterility on gene misexpression, we analyse spermatogenesis gene expression in different species pairs of the Drosophila phylogeny, where hybrid male sterility occurs in only one direction of the interspecies cross (i.e. unidirectional sterility). We find significant differences among genes in misexpression with effects that are lineage-specific and caused by sterility or fast male regulatory divergence. © 2014 The Authors. Journal of Evolutionary Biology © 2014 European Society For Evolutionary Biology.

Isolation of the endosperm-specific LPAAT gene promoter from coconut (Cocos nucifera L.) and its functional analysis in transgenic rice plants.

PubMed

Xu, Li; Ye, Rongjian; Zheng, Yusheng; Wang, Zhekui; Zhou, Peng; Lin, Yongjun; Li, Dongdong

2010-09-01

As one of the key tropical crops, coconut (Cocos nucifera L.) is a member of the monocotyledonous family Aracaceae (Palmaceae). In this study, we amplified the upstream region of an endosperm-specific expression gene, Lysophosphatidyl acyltransferase (LPAAT), from the coconut genomic DNA by chromosome walking. In this sequence, we found several types of promoter-related elements including TATA-box, CAAT-box and Skn1-motif. In order to further examine its function, three different 5'-deletion fragments were inserted into pBI101.3, a plant expression vector harboring the LPAAT upstream sequence, leading to pBI101.3-L1, pBI101.3-L2 and pBI101.3-L3, respectively. We obtained transgenic plants of rice by Agrobacterium-mediated callus transformation and plant regeneration and detected the expression of gus gene by histochemical staining and fluorometric determination. We found that gus gene driven by the three deletion fragments was specifically expressed in the endosperm of rice seeds, but not in the empty vector of pBI101.3 and other tissues. The highest expression level of GUS was at 15 DAF in pBI101.3-L3 and pBI101.3-L2 transgenic lines, while the same level was detected at 10 DAF in pBI101.3-L1. The expression driven by the whole fragment was up to 1.76- and 2.8-fold higher than those driven by the -817 bp and -453 bp upstream fragments, and 10.7-fold higher than that driven by the vector without the promoter. Taken together, our results strongly suggest that these promoter fragments from coconut have a significant potential in genetically improving endosperm in main crops.

Association between PD-L1 expression and driven gene status in NSCLC: A meta-analysis.

PubMed

Li, D; Zhu, X; Wang, H; Li, N

2017-07-01

We explored the potential clinical association between programmed death-ligand 1 (PD-L1) expression and driven gene status in non-small cell lung cancer (NSCLC). We systemically searched through October 2015. Odd ratios (ORs) with 95% CIs were calculated to examine the association of PD-L1 expression with driven gene status. A random- or fixed-effects model was used. Nine studies were identified. KRAS-mutant tumors were more likely to be PD-L1 positive than KRAS-wild type tumors (51% vs 36%; OR 1.69; 95% CI 1.01-2.84; p = 0.045). In contrast, PD-L1 expression did not differ by EGFR (OR 0.86; 95% CI 0.43-1.73; p = 0.675) or ALK (OR 1.02; 95% CI 0.44-2.37; p = 0.954) status. In subgroup analysis, there was also no significant association between PD-L1 expression and EGFR status in term of the cut-offs or ethnicity. In conclusion, NSCLC with KRAS mutations showed a trend for higher frequency of positive PD-L1 expression. Copyright © 2017 Elsevier Ltd, BASO ~ The Association for Cancer Surgery, and the European Society of Surgical Oncology. All rights reserved.

Allen Brain Atlas-Driven Visualizations: a web-based gene expression energy visualization tool.

PubMed

Zaldivar, Andrew; Krichmar, Jeffrey L

2014-01-01

The Allen Brain Atlas-Driven Visualizations (ABADV) is a publicly accessible web-based tool created to retrieve and visualize expression energy data from the Allen Brain Atlas (ABA) across multiple genes and brain structures. Though the ABA offers their own search engine and software for researchers to view their growing collection of online public data sets, including extensive gene expression and neuroanatomical data from human and mouse brain, many of their tools limit the amount of genes and brain structures researchers can view at once. To complement their work, ABADV generates multiple pie charts, bar charts and heat maps of expression energy values for any given set of genes and brain structures. Such a suite of free and easy-to-understand visualizations allows for easy comparison of gene expression across multiple brain areas. In addition, each visualization links back to the ABA so researchers may view a summary of the experimental detail. ABADV is currently supported on modern web browsers and is compatible with expression energy data from the Allen Mouse Brain Atlas in situ hybridization data. By creating this web application, researchers can immediately obtain and survey numerous amounts of expression energy data from the ABA, which they can then use to supplement their work or perform meta-analysis. In the future, we hope to enable ABADV across multiple data resources.

[Comparison of TTR and CMV promoters in vivo and in vitro via a secreted luciferase reporter system].

PubMed

Luo, Shun-Tao; Tian, Wen-Hong; Wang, Gang; Dong, Xiao-Yan; Yang, Li; Wu, Xiao-Bing

2009-11-01

GLuc (Gaussia luciferase) is a secreted luciferase with high sensitivity. In this study, we primarily compared expression character of PTTR with that of PCMV, relied on easy secretion, high sensitivity and simple and fast detection of GLuc. We firstly constructed two plasmids pAAV2-neo-TTR-GLuc and pAAV2-neo-CMV-GLuc. Then, 4 cell lines were transfected with the two plasmids in aid of Lipofectamine 2000, including Huh7 and HepG2, which are derived from liver cells, as well as HEK293 and HeLaS3 cells, which are non-liver cell lines. We monitored the expression of GLuc in the supernatant of these cell cultures at different time points post-transfection. Furthermore, we injected the two plasmids with different doses into BALB/c mice by the means of hydrodynamic delivery and monitored the GLuc expression in vivo with 2.5 microl tail tip blood since 2 h post-injection. The cell assay results suggested that the expression of GLuc driven by CMV promoter was significantly higher than that of GLuc driven by TTR promoter. And, the luciferase activity of GLuc driven by CMV promoter was 50-300 times higher than that of GLuc driven by TTR promoter in HEK293 and HeLaS3 cell lines, but less than 10 times higher than that of GLuc driven by TTR promoter in the HepG2 and Huh7 cell lines, indicating the relative liver-specificity of TTR promoter. In the animal assay, the higher luciferase activity was determined in CMV promoter group than in TTR promoter group at different doses of the two plasmids. But the expression patterns for the two promoters differed obviously. The expression of GLuc driven by CMV promoter reached the maximum 10 hours post-injection and declined rapidly; while the expression of GLuc driven by TTR promoter reached the maximum 48 hours after delivery, and declined very slowly. These results implied that PTTR could keep expression of driven gene in a long time although its expression intensity is lower than PCMV's. Thus, it is more suitable for maintaining longer expression of target genes in liver.

Enhancement of expression of survivin promoter-driven CD/TK double suicide genes by the nuclear matrix attachment region in transgenic gastric cancer cells.

PubMed

Niu, Ying; Li, Jian-Sheng; Luo, Xian-Run

2014-01-25

This work aimed to study a novel transgenic expression system of the CD/TK double suicide genes enhanced by the nuclear matrix attachment region (MAR) for gene therapy. The recombinant vector pMS-CD/TK containing the MAR-survivin promoter-CD/TK cassette was developed and transfected into human gastric cancer SGC-7901 cells. Expression of the CD/TK genes was detected by quantitative real-time PCR (qPCR) and Western blot. Cell viability and apoptosis were measured using the methyl thiazolyl tetrazolium (MTT) assay and flow cytometry. When the MAR fragment was inserted into the upstream of the survivin promoter, the qPCR result showed that the expression of the CD/TK genes significantly increased 7.7-fold in the transgenic SGC-7901 cells with plasmid pMS-CD/TK compared with that without MAR. MTT and flow cytometry analyses indicated that treatment with the prodrugs (5-FC+GCV) significantly decreased the cellular survival rate and enhanced the cellular apoptosis in the SGC-7901 cells. The expression of the CD/TK double suicide genes driven by the survivin promoter can be enhanced by the MAR fragment in human gastric cancer cells. Copyright © 2013 Elsevier B.V. All rights reserved.

The cytomegalovirus promoter-driven short hairpin RNA constructs mediate effective RNA interference in zebrafish in vivo.

PubMed

Su, Jianguo; Zhu, Zuoyan; Wang, Yaping; Xiong, Feng; Zou, Jun

2008-01-01

The ability to utilize the RNA interference (RNAi) machinery for silencing target-gene expression has created a lot of excitement in the research community. In the present study, we used a cytomegalovirus (CMV) promoter-driven DNA template approach to induce short hairpin RNA (shRNA) triggered RNAi to block exogenous Enhanced Green Fluorescent Protein (EGFP) and endogenous No Tail (NTL) gene expressions. We constructed three plasmids, pCMV-EGFP-CMV-shGFP-SV40, pCMV-EGFP-CMV-shNTL-SV40, and pCMV-EGFP-CMV-shScrambled-SV40, each containing a CMV promoter driving an EGFP reporter cDNA and DNA coding for one shRNA under the control of another CMV promoter. The three shRNA-generating plasmids and pCMV-EGFP control plasmid were introduced into zebrafish embryos by microinjection. Samples were collected at 48 h after injection. Results were evaluated by phenotype observation and real-time fluorescent quantitative reverse-transcription polymerase chain reaction (Q-PCR). The shGFP-generating plasmid significantly inhibited the EGFP expression viewed under fluorescent microscope and reduced by 70.05 +/- 1.26% of exogenous EGFP gene mRNA levels compared with controls by Q-PCR. The shRNA targeting endogenous NTL gene resulted in obvious NTL phenotype of 30 +/- 4% and decreased the level of their corresponding mRNAs up to 54.52 +/- 2.05% compared with nontargeting control shRNA. These data proved the feasibility of the CMV promoter-driven shRNA expression technique to be used to inhibit exogenous and endogenous gene expressions in zebrafish in vivo.

Effects of Gene Orientation and Use of Multiple Promoters on the Expression of XYL1 and XYL2 in Saccharomyces cerevisiae

NASA Astrophysics Data System (ADS)

Bae, Ju Yun; Laplaza, José; Jeffries, Thomas W.

Orientation of adjacent genes has been reported to affect their expression in eukaryotic systems, and metabolic engineering also often makes repeated use of a few promoters to obtain high expression. To improve transcriptional control in heterologous expression, we examined how these factors affect gene expression and enzymatic activity in Saccharomyces cerevisiae. We assembled d-xylose reductase (XYL1) and d-xylitol dehydrogenase (XYL2) in four ways. Each pair of genes was placed in two different tandem (l→2→ or √1√2), convergent (1→√2), and divergent (√1 2→) orientations in autonomous plasmids. The TEF1 promoter was used to drive XYL1 and the TDH3 promoter to drive XYL2 in each of the constructs. The effects of gene orientation on growth, transcription, and enzyme activity were analyzed. The transcription level as measured by quantitative PCR (q-PCR) correlated with enzyme activities, but our data did not show a significant effect of gene orientation. To test the possible dilution of promoter strength due to multiple use of the same promoter, we examined the level of expression of XYL1 driven by either the TEF1 or TDH3 promoter when carried on a single copy plasmid. We then coexpressed XYL2 from either a single or multicopy plasmid, which was also driven by the same promoter. XYL2 transcript and enzyme expression increased with plasmid copy number, while the expression of XYLl was constant regardless of the number of other TEF1 or TDH3 promoters present in the cell. According to our data, there is no significant effect of gene orientation or multiple promoter use on gene transcription and translation when genes are expressed from plasmids; however, other factors could affect expression of adjacent genes in chromosomes.

Compensation for intracellular environment in expression levels of mammalian circadian clock genes

PubMed Central

Matsumura, Ritsuko; Okamoto, Akihiko; Node, Koichi; Akashi, Makoto

2014-01-01

The circadian clock is driven by transcriptional oscillation of clock genes in almost all body cells. To investigate the effect of cell type-specific intracellular environment on the circadian machinery, we examined gene expression profiles in five peripheral tissues. As expected, the phase relationship between expression rhythms of nine clock genes was similar in all tissues examined. We also compared relative expression levels of clock genes among tissues, and unexpectedly found that quantitative variation remained within an approximately three-fold range, which was substantially smaller than that of metabolic housekeeping genes. Interestingly, circadian gene expression was little affected even when fibroblasts were cultured with different concentrations of serum. Together, these findings support a hypothesis that expression levels of clock genes are quantitatively compensated for the intracellular environment, such as redox potential and metabolite composition. However, more comprehensive studies are required to reach definitive conclusions. PMID:24504324

Co-expression of interleukin 12 enhances antitumor effects of a novel chimeric promoter-mediated suicide gene therapy in an immunocompetent mouse model

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xu, Yu, E-mail: xuyu1001@gmail.com; Hubei Key Laboratory of Tumor Biological Behaviors and Hubei Cancer Clinical Study Center, 169 Donghu Road, Wuhan 430071; Liu, Zhengchun, E-mail: l135027@126.com

Highlights: {yields} A novel chimeric promoter consisting of CArG element and hTERT promoter was developed. {yields} The promoter was characterized with radiation-inducibility and tumor-specificity. {yields} Suicide gene system driven by the promoter showed remarkable cytotoxicity in vitro. {yields} Co-expression of IL12 enhanced the promoter mediated suicide gene therapy in vivo. -- Abstract: The human telomerase reverse transcriptase (hTERT) promoter has been widely used in target gene therapy of cancer. However, low transcriptional activity limited its clinical application. Here, we designed a novel dual radiation-inducible and tumor-specific promoter system consisting of CArG elements and the hTERT promoter, resulting in increased expressionmore » of reporter genes after gamma-irradiation. Therapeutic and side effects of adenovirus-mediated horseradish peroxidase (HRP)/indole-3-acetic (IAA) system downstream of the chimeric promoter were evaluated in mice bearing Lewis lung carcinoma, combining with or without adenovirus-mediated interleukin 12 (IL12) gene driven by the cytomegalovirus promoter. The combination treatment showed more effective suppression of tumor growth than those with single agent alone, being associated with pronounced intratumoral T-lymphocyte infiltration and minor side effects. Our results suggest that the combination treatment with HRP/IAA system driven by the novel chimeric promoter and the co-expression of IL12 might be an effective and safe target gene therapy strategy of cancer.« less

miR-1298 inhibits mutant KRAS-driven tumor growth by repressing FAK and LAMB3

PubMed Central

Zhou, Ying; Dang, Jason; Chang, Kung-Yen; Yau, Edwin; Aza-Blanc, Pedro; Moscat, Jorge; Rana, Tariq M.

2016-01-01

Global microRNA functional screens can offer a strategy to identify synthetic lethal interactions in cancer cells that might be exploited therapeutically. In this study, we applied this strategy to identify novel gene interactions in KRAS mutant cancer cells. In this manner, we discovered miR-1298, a novel miRNA that inhibited the growth of KRAS-driven cells both in vitro and in vivo. Using miR-TRAP affinity purification technology, we identified the tyrosine kinase FAK and the laminin subunit LAMB3 as functional targets of miR-1298. Silencing of FAK or LAMB3 recapitulated the synthetic lethal effects of miR-1298 expression in KRAS-driven cancer cells, whereas co-expression of both proteins was critical to rescue miR-1298-induced cell death. Expression of LAMB3 but not FAK was upregulated by mutant KRAS. In clinical specimens, elevated LAMB3 expression correlated with poorer survival in lung cancer patients with an oncogenic KRAS gene signature, suggesting a novel candidate biomarker in this disease setting. Our results define a novel regulatory pathway in KRAS-driven cancers which offers a potential therapeutic target for their eradication PMID:27698189

Knowledge-driven genomic interactions: an application in ovarian cancer.

PubMed

Kim, Dokyoon; Li, Ruowang; Dudek, Scott M; Frase, Alex T; Pendergrass, Sarah A; Ritchie, Marylyn D

2014-01-01

Effective cancer clinical outcome prediction for understanding of the mechanism of various types of cancer has been pursued using molecular-based data such as gene expression profiles, an approach that has promise for providing better diagnostics and supporting further therapies. However, clinical outcome prediction based on gene expression profiles varies between independent data sets. Further, single-gene expression outcome prediction is limited for cancer evaluation since genes do not act in isolation, but rather interact with other genes in complex signaling or regulatory networks. In addition, since pathways are more likely to co-operate together, it would be desirable to incorporate expert knowledge to combine pathways in a useful and informative manner. Thus, we propose a novel approach for identifying knowledge-driven genomic interactions and applying it to discover models associated with cancer clinical phenotypes using grammatical evolution neural networks (GENN). In order to demonstrate the utility of the proposed approach, an ovarian cancer data from the Cancer Genome Atlas (TCGA) was used for predicting clinical stage as a pilot project. We identified knowledge-driven genomic interactions associated with cancer stage from single knowledge bases such as sources of pathway-pathway interaction, but also knowledge-driven genomic interactions across different sets of knowledge bases such as pathway-protein family interactions by integrating different types of information. Notably, an integration model from different sources of biological knowledge achieved 78.82% balanced accuracy and outperformed the top models with gene expression or single knowledge-based data types alone. Furthermore, the results from the models are more interpretable because they are framed in the context of specific biological pathways or other expert knowledge. The success of the pilot study we have presented herein will allow us to pursue further identification of models predictive of clinical cancer survival and recurrence. Understanding the underlying tumorigenesis and progression in ovarian cancer through the global view of interactions within/between different biological knowledge sources has the potential for providing more effective screening strategies and therapeutic targets for many types of cancer.

Dissecting Daily and Circadian Expression Rhythms of Clock-Controlled Genes in Human Blood.

PubMed

Lech, Karolina; Ackermann, Katrin; Revell, Victoria L; Lao, Oscar; Skene, Debra J; Kayser, Manfred

2016-02-01

The identification and investigation of novel clock-controlled genes (CCGs) has been conducted thus far mainly in model organisms such as nocturnal rodents, with limited information in humans. Here, we aimed to characterize daily and circadian expression rhythms of CCGs in human peripheral blood during a sleep/sleep deprivation (S/SD) study and a constant routine (CR) study. Blood expression levels of 9 candidate CCGs (SREBF1, TRIB1, USF1, THRA1, SIRT1, STAT3, CAPRIN1, MKNK2, and ROCK2), were measured across 48 h in 12 participants in the S/SD study and across 33 h in 12 participants in the CR study. Statistically significant rhythms in expression were observed for STAT3, SREBF1, TRIB1, and THRA1 in samples from both the S/SD and the CR studies, indicating that their rhythmicity is driven by the endogenous clock. The MKNK2 gene was significantly rhythmic in the S/SD but not the CR study, which implies its exogenously driven rhythmic expression. In addition, we confirmed the circadian expression of PER1, PER3, and REV-ERBα in the CR study samples, while BMAL1 and HSPA1B were not significantly rhythmic in the CR samples; all 5 genes previously showed significant expression in the S/SD study samples. Overall, our results demonstrate that rhythmic expression patterns of clock and selected clock-controlled genes in human blood cells are in part determined by exogenous factors (sleep and fasting state) and in part by the endogenous circadian timing system. Knowledge of the exogenous and endogenous regulation of gene expression rhythms is needed prior to the selection of potential candidate marker genes for future applications in medical and forensic settings. © 2015 The Author(s).

Adapted Resistance to the Knockdown Effect of shRNA-Derived Srsf3 siRNAs in Mouse Littermates | Center for Cancer Research

Cancer.gov

Gene silencing techniques are widely used to control gene expression and have potential for RNAi-based therapeutics. In this report, transgenic mouse lines were created for conditional knockdown of Srsf3 (SRp20) expression in liver and mammary gland tissues by expressing Srsf3-specific shRNAs driven by a U6 promoter.

Sustainability of keratinocyte gene transfer and cell survival in vivo.

PubMed

Choate, K A; Khavari, P A

1997-05-20

The epidermis is an attractive site for therapeutic gene delivery because it is accessible and capable of delivering polypeptides to the systemic circulation. A number of difficulties, however, have emerged in attempts at cutaneous gene delivery, and central among these is an inability to sustain therapeutic gene production. We have examined two major potential contributing factors, viral vector stamina and involvement of long-lived epidermal progenitor cells. Human keratinocytes were either untreated or transduced with a retroviral vector for beta-galactosidase (beta-Gal) at > 99% efficiency and then grafted onto immunodeficient mice to regenerate human epidermis. Human epidermis was monitored in vivo after grafting for clinical and histologic appearance as well as for gene expression. Although integrated vector sequences persisted unchanged in engineered epidermis at 10 weeks post-grafting, retroviral long terminal repeat (LTR)-driven beta-Gal expression ceased in vivo after approximately 4 weeks. Endogenous cellular promoters, however, maintained consistently normal gene expression levels without evidence of time-dependent decline, as determined by immunostaining with species-specific antibodies for human involucrin, filaggrin, keratinocyte transglutaminase, keratin 10, type VII collagen, and Laminin 5 proteins out to week 14 post-grafting. Transduced human keratinocytes generated multilayer epidermis sustained through multiple epidermal turnover cycles; this epidermis demonstrated retention of a spatially appropriate pattern of basal and suprabasal epidermal marker gene expression. These results confirm previous findings suggesting that viral promoter-driven gene expression is not durable and demonstrate that keratinocytes passaged in vitro can regenerate and sustain normal epidermis for prolonged periods.

A 310-bp minimal promoter mediates smooth muscle cell-specific expression of telokin.

PubMed

Smith, A F; Bigsby, R M; Word, R A; Herring, B P

1998-05-01

A cell-specific promoter located in an intron of the smooth muscle myosin light chain kinase gene directs transcription of telokin exclusively in smooth muscle cells. Transgenic mice were generated in which a 310-bp rabbit telokin promoter fragment, extending from -163 to +147, was used to drive expression of simian virus 40 large T antigen. Smooth muscle-specific expression of the T-antigen transgene paralleled that of the endogenous telokin gene in all smooth muscle tissues except uterus. The 310-bp promoter fragment resulted in very low levels of transgene expression in uterus; in contrast, a transgene driven by a 2.4-kb fragment (-2250 to +147) resulted in high levels of transgene expression in uterine smooth muscle. Telokin expression levels correlate with the estrogen status of human myometrial tissues, suggesting that deletion of an estrogen response element (ERE) may account for the low levels of transgene expression driven by the 310-bp rabbit telokin promoter in uterine smooth muscle. Experiments in A10 smooth muscle cells directly showed that reporter gene expression driven by the 2.4-kb, but not 310-bp, promoter fragment could be stimulated two- to threefold by estrogen. This stimulation was mediated through an ERE located between -1447 and -1474. Addition of the ERE to the 310-bp fragment restored estrogen responsiveness in A10 cells. These data demonstrate that in addition to a minimal 310-bp proximal promoter at least one distal cis-acting regulatory element is required for telokin expression in uterine smooth muscle. The distal element may include an ERE between -1447 and -1474.

Discovering the Deregulated Molecular Functions Involved in Malignant Transformation of Endometriosis to Endometriosis-Associated Ovarian Carcinoma Using a Data-Driven, Function-Based Analysis

PubMed Central

Chang, Chia-Ming; Yang, Yi-Ping; Chuang, Jen-Hua; Chuang, Chi-Mu; Lin, Tzu-Wei; Wang, Peng-Hui; Yu, Mu-Hsien

2017-01-01

The clinical characteristics of clear cell carcinoma (CCC) and endometrioid carcinoma EC) are concomitant with endometriosis (ES), which leads to the postulation of malignant transformation of ES to endometriosis-associated ovarian carcinoma (EAOC). Different deregulated functional areas were proposed accounting for the pathogenesis of EAOC transformation, and there is still a lack of a data-driven analysis with the accumulated experimental data in publicly-available databases to incorporate the deregulated functions involved in the malignant transformation of EOAC. We used the microarray gene expression datasets of ES, CCC and EC downloaded from the National Center for Biotechnology Information Gene Expression Omnibus (NCBI GEO) database. Then, we investigated the pathogenesis of EAOC by a data-driven, function-based analytic model with the quantified molecular functions defined by 1454 Gene Ontology (GO) term gene sets. This model converts the gene expression profiles to the functionome consisting of 1454 quantified GO functions, and then, the key functions involving the malignant transformation of EOAC can be extracted by a series of filters. Our results demonstrate that the deregulated oxidoreductase activity, metabolism, hormone activity, inflammatory response, innate immune response and cell-cell signaling play the key roles in the malignant transformation of EAOC. These results provide the evidence supporting the specific molecular pathways involved in the malignant transformation of EAOC. PMID:29113136

Insulators to improve expression of a 3(')IgH LCR-driven reporter gene in transgenic mouse models.

PubMed

Guglielmi, Laurence; Le Bert, Marc; Truffinet, Véronique; Cogné, Michel; Denizot, Yves

2003-08-01

A locus control region (LCR) containing four transcriptional enhancers lies downstream of the IgH chain locus. We studied transgenes carrying a 3(')IgH LCR-driven GFP reporter gene for expression and B cell differentiation stage specificity. We also compared transgenes that were or were not flanked by two copies of the beta-globin HS4 insulator, an element defined by its ability to protect transgenes from the influences of surrounding genes at the insertion site. Results indicate that insulators are instrumental in sustaining GFP expression in GFP-3(')LCR transgenic mice when they were included. Flow cytometry experiments reported a strictly B cell specific GFP expression from pre-B cells in bone marrow to mature B cells in spleen. Despite addition of 5(')HS4 insulators to the GFP-3(')LCR construct, complete transgene silencing occurred in some transgenic lines and was systematically observed in ageing animals from all lines.

NRIP enhances HPV gene expression via interaction with either GR or E2

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chang, Szu-Wei; Lu, Pei-Yu; Guo, Jih-Huong

We previously identified a gene, nuclear receptor-interaction protein (NRIP), which functions as a transcription cofactor in glucocorticoid receptor (GR) and human papillomavirus E2 (HPV E2)-driven gene expression. Here, we comprehensively evaluated the role of NRIP in HPV-16 gene expression. NRIP acts as a transcription cofactor to enhance GR-regulated HPV-16 gene expression in the presence of hormone. NRIP also can form complex with E2 that caused NRIP-induced HPV gene expression via E2-binding sites in a hormone-independent manner. Furthermore, NRIP can associate with GR and E2 to form tri-protein complex to activate HPV gene expression via GRE, not the E2-binding site, inmore » a hormone-dependent manner. These results indicate that NRIP and GR are viral E2-binding proteins and that NRIP regulates HPV gene expression via GRE and/or E2 binding site in the HPV promoter in a hormone-dependent or independent manner, respectively.« less

8-Oxoguanine DNA glycosylase1-driven DNA repair-A paradoxical role in lung aging.

PubMed

German, Peter; Saenz, David; Szaniszlo, Peter; Aguilera-Aguirre, Leopoldo; Pan, Lang; Hegde, Muralidhar L; Bacsi, Attila; Hajas, Gyorgy; Radak, Zsolt; Ba, Xueqing; Mitra, Sankar; Papaconstantinou, John; Boldogh, Istvan

2017-01-01

Age-associated changes in lung structure and function are some of the most important predictors of overall health, cognitive activities and longevity. Common to all aging cells is an increase in oxidatively modified DNA bases, primarily 8-oxo-7,8-dihydroguanine (8-oxoG). It is repaired via DNA base excision repair pathway driven by 8-oxoguanine DNA glycosylase-1 (OGG1-BER), whose role in aging has been the focus of many studies. This study hypothesizes that signaling and consequent gene expression during cellular response to OGG1-BER "wires" senescence/aging processes. To test OGG1-BER was mimicked by repeatedly exposing diploid lung fibroblasts cells and airways of mice to 8-oxoG base. Results showed that repeated exposures led to G1 cell cycle arrest and pre-matured senescence of cultured cells in which over 1000 genes were differentially expressed -86% of them been identical to those in naturally senesced cells. Gene ontology analysis of gene expression displayed biological processes driven by small GTPases, phosphoinositide 3-kinase and mitogen activated kinase cascades both in cultured cells and lungs. These results together, points to a new paradigm about the role of DNA damage and repair by OGG1 in aging and age-associated disease processes. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Development of siRNA expression vector utilizing rock bream beta-actin promoter: a potential therapeutic tool against viral infection in fish.

PubMed

Zenke, Kosuke; Nam, Yoon Kwon; Kim, Ki Hong

2010-01-01

In the present study, we have developed short interfering RNA (siRNA) expression vector utilizing rock bream beta-actin promoter and examined the possible use for the inhibition of highly pathogenic fish virus, rock bream iridovirus (RBIV), replication in vitro. Initially, in order to express siRNA effectively, we added several modifications to wild-type rock bream beta-actin promoter. Next, we succeeded in knocking down the expression of enhanced green fluorescent protein reporter gene expression in fish cells using newly developed vector more effectively than the fugu U6 promoter-driven vector we described previously. Finally, we could observe that cells transfected with modified rock bream beta-actin promoter-driven siRNA expression vector targeting major capsid protein (MCP) gene of RBIV exhibited more resistance to RBIV challenge than other control cells. Our results indicate that this novel siRNA expression vector can be used as a new tool for therapeutics in virus infection in fish species.

ICG: a wiki-driven knowledgebase of internal control genes for RT-qPCR normalization.

PubMed

Sang, Jian; Wang, Zhennan; Li, Man; Cao, Jiabao; Niu, Guangyi; Xia, Lin; Zou, Dong; Wang, Fan; Xu, Xingjian; Han, Xiaojiao; Fan, Jinqi; Yang, Ye; Zuo, Wanzhu; Zhang, Yang; Zhao, Wenming; Bao, Yiming; Xiao, Jingfa; Hu, Songnian; Hao, Lili; Zhang, Zhang

2018-01-04

Real-time quantitative PCR (RT-qPCR) has become a widely used method for accurate expression profiling of targeted mRNA and ncRNA. Selection of appropriate internal control genes for RT-qPCR normalization is an elementary prerequisite for reliable expression measurement. Here, we present ICG (http://icg.big.ac.cn), a wiki-driven knowledgebase for community curation of experimentally validated internal control genes as well as their associated experimental conditions. Unlike extant related databases that focus on qPCR primers in model organisms (mainly human and mouse), ICG features harnessing collective intelligence in community integration of internal control genes for a variety of species. Specifically, it integrates a comprehensive collection of more than 750 internal control genes for 73 animals, 115 plants, 12 fungi and 9 bacteria, and incorporates detailed information on recommended application scenarios corresponding to specific experimental conditions, which, collectively, are of great help for researchers to adopt appropriate internal control genes for their own experiments. Taken together, ICG serves as a publicly editable and open-content encyclopaedia of internal control genes and accordingly bears broad utility for reliable RT-qPCR normalization and gene expression characterization in both model and non-model organisms. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Cell-Specific Actions of a Human LHX3 Gene Enhancer During Pituitary and Spinal Cord Development

PubMed Central

Park, Soyoung; Mullen, Rachel D.

2013-01-01

The LIM class of homeodomain protein 3 (LHX3) transcription factor is essential for pituitary gland and nervous system development in mammals. In humans, mutations in the LHX3 gene underlie complex pediatric syndromes featuring deficits in anterior pituitary hormones and defects in the nervous system. The mechanisms that control temporal and spatial expression of the LHX3 gene are poorly understood. The proximal promoters of the human LHX3 gene are insufficient to guide expression in vivo and downstream elements including a conserved enhancer region appear to play a role in tissue-specific expression in the pituitary and nervous system. Here we characterized the activity of this downstream enhancer region in regulating gene expression at the cellular level during development. Human LHX3 enhancer-driven Cre reporter transgenic mice were generated to facilitate studies of enhancer actions. The downstream LHX3 enhancer primarily guides gene transcription in α-glycoprotein subunit -expressing cells secreting the TSHβ, LHβ, or FSHβ hormones and expressing the GATA2 and steroidogenic factor 1 transcription factors. In the developing nervous system, the enhancer serves as a targeting module active in V2a interneurons. These results demonstrate that the downstream LHX3 enhancer is important in specific endocrine and neural cell types but also indicate that additional regulatory elements are likely involved in LHX3 gene expression. Furthermore, these studies revealed significant gonadotrope cell heterogeneity during pituitary development, providing insights into the cellular physiology of this key reproductive regulatory cell. The human LHX3 enhancer-driven Cre reporter transgenic mice also provide a valuable tool for further developmental studies of cell determination and differentiation in the pituitary and nervous system. PMID:24100213

Simvastatin and Dipentyl Phthalate Lower Ex vivo Testicular Testosterone Production and Exhibit Additive Effects on Testicular Testosterone and Gene Expression Via Distinct Mechanistic Pathways in the Fetal Rat

EPA Science Inventory

Sex differentiation of the male reproductive tract in mammals is driven, in part, by fetal androgen production. In utero, some phthalate esters (PEs) alter fetal Leydig cell differentiation, reducing the expression of several genes associated with steroid synthesis/transport, and...

The bZIP transcription factor HY5 interacts with the promoter of the monoterpene synthase gene QH6 in modulating its rhythmic expression.

PubMed

Zhou, Fei; Sun, Tian-Hu; Zhao, Lei; Pan, Xi-Wu; Lu, Shan

2015-01-01

The Artemisia annua L. β-pinene synthase QH6 was previously determined to be circadian-regulated at the transcriptional level, showing a rhythmic fluctuation of steady-state transcript abundances. Here we isolated both the genomic sequence and upstream promoter region of QH6. Different regulatory elements, such as G-box (TGACACGTGGCA, -421 bp from the translation initiation site) which might have effects on rhythmic gene expression, were found. Using the yeast one-hybrid and electrophoretic mobility shift assay (EMSA), we confirmed that the bZIP transcription factor HY5 binds to this motif of QH6. Studies with promoter truncations before and after this motif suggested that this G-box was important for the diurnal fluctuation of the transgenic β-glucuronidase gene (GUS) transcript abundance in Arabidopsis thaliana. GUS gene driven by the promoter region immediately after G-box showed an arrhythmic expression in both light/dark (LD) and constant dark (DD) conditions, whereas the control with G-box retained its fluctuation in both LD and DD. We further transformed A. thaliana with the luciferase gene (LUC) driven by an 1400 bp fragment upstream QH6 with its G-box intact or mutated, respectively. The luciferase activity assay showed that a peak in the early morning disappeared in the mutant. Gene expression analysis also demonstrated that the rhythmic expression of LUC was abolished in the hy5-1 mutant.

SoxB1-driven transcriptional network underlies neural-specific interpretation of morphogen signals.

PubMed

Oosterveen, Tony; Kurdija, Sanja; Ensterö, Mats; Uhde, Christopher W; Bergsland, Maria; Sandberg, Magnus; Sandberg, Rickard; Muhr, Jonas; Ericson, Johan

2013-04-30

The reiterative deployment of a small cadre of morphogen signals underlies patterning and growth of most tissues during embyogenesis, but how such inductive events result in tissue-specific responses remains poorly understood. By characterizing cis-regulatory modules (CRMs) associated with genes regulated by Sonic hedgehog (Shh), retinoids, or bone morphogenetic proteins in the CNS, we provide evidence that the neural-specific interpretation of morphogen signaling reflects a direct integration of these pathways with SoxB1 proteins at the CRM level. Moreover, expression of SoxB1 proteins in the limb bud confers on mesodermal cells the potential to activate neural-specific target genes upon Shh, retinoid, or bone morphogenetic protein signaling, and the collocation of binding sites for SoxB1 and morphogen-mediatory transcription factors in CRMs faithfully predicts neural-specific gene activity. Thus, an unexpectedly simple transcriptional paradigm appears to conceptually explain the neural-specific interpretation of pleiotropic signaling during vertebrate development. Importantly, genes induced in a SoxB1-dependent manner appear to constitute repressive gene regulatory networks that are directly interlinked at the CRM level to constrain the regional expression of patterning genes. Accordingly, not only does the topology of SoxB1-driven gene regulatory networks provide a tissue-specific mode of gene activation, but it also determines the spatial expression pattern of target genes within the developing neural tube.

Whole transcriptome analysis of the coral Acropora millepora reveals complex responses to CO₂-driven acidification during the initiation of calcification.

PubMed

Moya, A; Huisman, L; Ball, E E; Hayward, D C; Grasso, L C; Chua, C M; Woo, H N; Gattuso, J-P; Forêt, S; Miller, D J

2012-05-01

The impact of ocean acidification (OA) on coral calcification, a subject of intense current interest, is poorly understood in part because of the presence of symbionts in adult corals. Early life history stages of Acropora spp. provide an opportunity to study the effects of elevated CO(2) on coral calcification without the complication of symbiont metabolism. Therefore, we used the Illumina RNAseq approach to study the effects of acute exposure to elevated CO(2) on gene expression in primary polyps of Acropora millepora, using as reference a novel comprehensive transcriptome assembly developed for this study. Gene ontology analysis of this whole transcriptome data set indicated that CO(2) -driven acidification strongly suppressed metabolism but enhanced extracellular organic matrix synthesis, whereas targeted analyses revealed complex effects on genes implicated in calcification. Unexpectedly, expression of most ion transport proteins was unaffected, while many membrane-associated or secreted carbonic anhydrases were expressed at lower levels. The most dramatic effect of CO(2) -driven acidification, however, was on genes encoding candidate and known components of the skeletal organic matrix that controls CaCO(3) deposition. The skeletal organic matrix effects included elevated expression of adult-type galaxins and some secreted acidic proteins, but down-regulation of other galaxins, secreted acidic proteins, SCRiPs and other coral-specific genes, suggesting specialized roles for the members of these protein families and complex impacts of OA on mineral deposition. This study is the first exhaustive exploration of the transcriptomic response of a scleractinian coral to acidification and provides an unbiased perspective on its effects during the early stages of calcification. © 2012 Blackwell Publishing Ltd.

Modulation of Estrogen Response Element-Driven Gene Expressions and Cellular Proliferation with Polar Directions by Designer Transcription Regulators

PubMed Central

Muyan, Mesut; Güpür, Gizem; Yaşar, Pelin; Ayaz, Gamze; User, Sırma Damla; Kazan, Hasan Hüseyin; Huang, Yanfang

2015-01-01

Estrogen receptor α (ERα), as a ligand-dependent transcription factor, mediates 17β-estradiol (E2) effects. ERα is a modular protein containing a DNA binding domain (DBD) and transcription activation domains (AD) located at the amino- and carboxyl-termini. The interaction of the E2-activated ERα dimer with estrogen response elements (EREs) of genes constitutes the initial step in the ERE-dependent signaling pathway necessary for alterations of cellular features. We previously constructed monomeric transcription activators, or monotransactivators, assembled from an engineered ERE-binding module (EBM) using the ERα-DBD and constitutively active ADs from other transcription factors. Monotransactivators modulated cell proliferation by activating and repressing ERE-driven gene expressions that simulate responses observed with E2-ERα. We reasoned here that integration of potent heterologous repression domains (RDs) into EBM could generate monotransrepressors that alter ERE-bearing gene expressions and cellular proliferation in directions opposite to those observed with E2-ERα or monotransactivators. Consistent with this, monotransrepressors suppressed reporter gene expressions that emulate the ERE-dependent signaling pathway. Moreover, a model monotransrepressor regulated DNA synthesis, cell cycle progression and proliferation of recombinant adenovirus infected ER-negative cells through decreasing as well as increasing gene expressions with polar directions compared with E2-ERα or monotransactivator. Our results indicate that an ‘activator’ or a ‘repressor’ possesses both transcription activating/enhancing and repressing/decreasing abilities within a chromatin context. Offering a protein engineering platform to alter signal pathway-specific gene expressions and cell growth, our approach could also be used for the development of tools for epigenetic modifications and for clinical interventions wherein multigenic de-regulations are an issue. PMID:26295471

RNAi-Mediated Knockdown of IKK1 in Transgenic Mice Using a Transgenic Construct Containing the Human H1 Promoter

PubMed Central

Moreno-Maldonado, Rodolfo; Murillas, Rodolfo; Page, Angustias; Suarez-Cabrera, Cristian; Alameda, Josefa P.; Bravo, Ana; Casanova, M. Llanos

2014-01-01

Inhibition of gene expression through siRNAs is a tool increasingly used for the study of gene function in model systems, including transgenic mice. To achieve perdurable effects, the stable expression of siRNAs by an integrated transgenic construct is necessary. For transgenic siRNA expression, promoters transcribed by either RNApol II or III (such as U6 or H1 promoters) can be used. Relatively large amounts of small RNAs synthesis are achieved when using RNApol III promoters, which can be advantageous in knockdown experiments. To study the feasibility of H1 promoter-driven RNAi-expressing constructs for protein knockdown in transgenic mice, we chose IKK1 as the target gene. Our results indicate that constructs containing the H1 promoter are sensitive to the presence of prokaryotic sequences and to transgene position effects, similar to RNApol II promoters-driven constructs. We observed variable expression levels of transgenic siRNA among different tissues and animals and a reduction of up to 80% in IKK1 expression. Furthermore, IKK1 knockdown led to hair follicle alterations. In summary, we show that constructs directed by the H1 promoter can be used for knockdown of genes of interest in different organs and for the generation of animal models complementary to knockout and overexpression models. PMID:24523631

A Systems' Biology Approach to Study MicroRNA-Mediated Gene Regulatory Networks

PubMed Central

Kunz, Manfred; Vera, Julio; Wolkenhauer, Olaf

2013-01-01

MicroRNAs (miRNAs) are potent effectors in gene regulatory networks where aberrant miRNA expression can contribute to human diseases such as cancer. For a better understanding of the regulatory role of miRNAs in coordinating gene expression, we here present a systems biology approach combining data-driven modeling and model-driven experiments. Such an approach is characterized by an iterative process, including biological data acquisition and integration, network construction, mathematical modeling and experimental validation. To demonstrate the application of this approach, we adopt it to investigate mechanisms of collective repression on p21 by multiple miRNAs. We first construct a p21 regulatory network based on data from the literature and further expand it using algorithms that predict molecular interactions. Based on the network structure, a detailed mechanistic model is established and its parameter values are determined using data. Finally, the calibrated model is used to study the effect of different miRNA expression profiles and cooperative target regulation on p21 expression levels in different biological contexts. PMID:24350286

[Construction of a plant effective expression vector containing the gene of hepatitis B virus surface antigen].

PubMed

Lin, Bing-Ying; Jin, Zhi-Qiang; Li, Mei

2006-11-01

To construct a plant effective expression vector driven by a fruit specific promoter for the expression of hepatitis B virus surface antigen (HBsAg), to further improve the expression of exogenous gene in plant, and to prepare for the development of an effective anti-hepatitis vaccine. Tomato fruit-specific promoters' gene 2A12 and E8 were respectively introduced to pBPFOmega7 to form pB2A12 and pBE8. The DNA fragment containing HBsAg-s gene from plasmid YEP-HBs was inserted respectively into pB2A12 and pBE8 to form pB2A12-HBs and pBE8-HBs. The fragment containing "p35S+2A12+Omega+HBsAg-s+Tnos" of the pB2A12-HBs was sub-cloned into plasmid pCAMBIA1301 to yield the reconstructed plant binary expression plasmid pCAM2A12-HBs, and the fragment containing "p35S+E8+Omega+HBsAg-s+Tnos" of the pBE8-HBs was sub-cloned into plasmid pCAMBIA1301 to yield the plasmid pCAME8-HBs. The inserted gene HBsAg and fruit-specific promoters in the reconstructed plant binary expression vectors were confirmed by sequencing. Then, pCAM2A12-HBs and pCAME8-HBs were directly introduced into Agrobacterium tumefaciens strain EHA105. Digestion with restriction enzymes proved that all recombinant vectors had the inserts with expected length of the target fragments, and the sequencing results were confirmed correct. In this study, plant expression vector containing HBsAg gene driven by fruit specific promoter and CaMV35s promoter was successfully constructed.

Comprehensive identification of genes driven by ERV9-LTRs reveals TNFRSF10B as a re-activatable mediator of testicular cancer cell death

PubMed Central

Beyer, U; Krönung, S K; Leha, A; Walter, L; Dobbelstein, M

2016-01-01

The long terminal repeat (LTR) of human endogenous retrovirus type 9 (ERV9) acts as a germline-specific promoter that induces the expression of a proapoptotic isoform of the tumor suppressor homologue p63, GTAp63, in male germline cells. Testicular cancer cells silence this promoter, but inhibitors of histone deacetylases (HDACs) restore GTAp63 expression and give rise to apoptosis. We show here that numerous additional transcripts throughout the genome are driven by related ERV9-LTRs. 3' Rapid amplification of cDNA ends (3'RACE) was combined with next-generation sequencing to establish a large set of such mRNAs. HDAC inhibitors induce these ERV9-LTR-driven genes but not the LTRs from other ERVs. In particular, a transcript encoding the death receptor DR5 originates from an ERV9-LTR inserted upstream of the protein coding regions of the TNFRSF10B gene, and it shows an expression pattern similar to GTAp63. When treating testicular cancer cells with HDAC inhibitors as well as the death ligand TNF-related apoptosis-inducing ligand (TRAIL), rapid cell death was observed, which depended on TNFRSF10B expression. HDAC inhibitors also cooperate with cisplatin (cDDP) to promote apoptosis in testicular cancer cells. ERV9-LTRs not only drive a large set of human transcripts, but a subset of them acts in a proapoptotic manner. We propose that this avoids the survival of damaged germ cells. HDAC inhibition represents a strategy of restoring the expression of a class of ERV9-LTR-mediated genes in testicular cancer cells, thereby re-enabling tumor suppression. PMID:26024393

Mesenchymal stem cell-based HSP70 promoter-driven VEGFA induction by resveratrol promotes angiogenesis in a mouse model.

PubMed

Chen, Young-Bin; Lan, Ying-Wei; Hung, Tsai-Hsien; Chen, Lih-Geeng; Choo, Kong-Bung; Cheng, Winston T K; Lee, Hsuan-Shu; Chong, Kowit-Yu

2015-07-01

Several studies of stem cell-based gene therapy have indicated that long-lasting regeneration following vessel ischemia may be stimulated through VEGFA gene therapy and/or MSC transplantation for reduction of ischemic injury in limb ischemia and heart failure. The therapeutic potential of MSC transplantation can be further improved by genetically modifying MSCs with genes which enhance angiogenesis following ischemic injury. In the present study, we aimed to develop an approach in MSC-based therapy for repair and mitigation of ischemic injury and regeneration of damaged tissues in ischemic disease. HSP70 promoter-driven VEGFA expression was induced by resveratrol (RSV) in MSCs, and in combination with known RSV biological functions, the protective effects of our approach were investigated by using ex vivo aortic ring coculture system and a 3D scaffolds in vivo model. Results of this investigation demonstrated that HSP promoter-driven VEGFA expression in MSC increased approximately 2-fold over the background VEGFA levels upon HSP70 promoter induction by RSV. Exposure of HUVEC cells to medium containing MSC in which VEGFA had been induced by cis-RSV enhanced tube formation in the treated HUVEC cells. RSV-treated MSC cells differentiated into endothelial-like phenotypes, exhibiting markedly elevated expression of endothelial cell markers. These MSCs also induced aortic ring sprouting, characteristic of neovascular formation from pre-existing vessels, and additionally promoted neovascularization at the MSC transplantation site in a mouse model. These observations support a hypothesis that VEGFA expression induced by cis-RSV acting on the HSP70 promoter in transplanted MSC augments the angiogenic effects of stem cell gene therapy. The use of an inducible system also vastly reduces possible clinical risks associated with constitutive VEGFA expression.

Overexpression of Transcription Factor Sp1 Leads to Gene Expression Perturbations and Cell Cycle Inhibition

PubMed Central

Deniaud, Emmanuelle; Baguet, Joël; Chalard, Roxane; Blanquier, Bariza; Brinza, Lilia; Meunier, Julien; Michallet, Marie-Cécile; Laugraud, Aurélie; Ah-Soon, Claudette; Wierinckx, Anne; Castellazzi, Marc; Lachuer, Joël; Gautier, Christian

2009-01-01

Background The ubiquitous transcription factor Sp1 regulates the expression of a vast number of genes involved in many cellular functions ranging from differentiation to proliferation and apoptosis. Sp1 expression levels show a dramatic increase during transformation and this could play a critical role for tumour development or maintenance. Although Sp1 deregulation might be beneficial for tumour cells, its overexpression induces apoptosis of untransformed cells. Here we further characterised the functional and transcriptional responses of untransformed cells following Sp1 overexpression. Methodology and Principal Findings We made use of wild-type and DNA-binding-deficient Sp1 to demonstrate that the induction of apoptosis by Sp1 is dependent on its capacity to bind DNA. Genome-wide expression profiling identified genes involved in cancer, cell death and cell cycle as being enriched among differentially expressed genes following Sp1 overexpression. In silico search to determine the presence of Sp1 binding sites in the promoter region of modulated genes was conducted. Genes that contained Sp1 binding sites in their promoters were enriched among down-regulated genes. The endogenous sp1 gene is one of the most down-regulated suggesting a negative feedback loop induced by overexpressed Sp1. In contrast, genes containing Sp1 binding sites in their promoters were not enriched among up-regulated genes. These results suggest that the transcriptional response involves both direct Sp1-driven transcription and indirect mechanisms. Finally, we show that Sp1 overexpression led to a modified expression of G1/S transition regulatory genes such as the down-regulation of cyclin D2 and the up-regulation of cyclin G2 and cdkn2c/p18 expression. The biological significance of these modifications was confirmed by showing that the cells accumulated in the G1 phase of the cell cycle before the onset of apoptosis. Conclusion This study shows that the binding to DNA of overexpressed Sp1 induces an inhibition of cell cycle progression that precedes apoptosis and a transcriptional response targeting genes containing Sp1 binding sites in their promoter or not suggesting both direct Sp1-driven transcription and indirect mechanisms. PMID:19753117

m6A-Driver: Identifying Context-Specific mRNA m6A Methylation-Driven Gene Interaction Networks

PubMed Central

Zhang, Song-Yao; Zhang, Shao-Wu; Liu, Lian; Huang, Yufei

2016-01-01

As the most prevalent mammalian mRNA epigenetic modification, N6-methyladenosine (m6A) has been shown to possess important post-transcriptional regulatory functions. However, the regulatory mechanisms and functional circuits of m6A are still largely elusive. To help unveil the regulatory circuitry mediated by mRNA m6A methylation, we develop here m6A-Driver, an algorithm for predicting m6A-driven genes and associated networks, whose functional interactions are likely to be actively modulated by m6A methylation under a specific condition. Specifically, m6A-Driver integrates the PPI network and the predicted differential m6A methylation sites from methylated RNA immunoprecipitation sequencing (MeRIP-Seq) data using a Random Walk with Restart (RWR) algorithm and then builds a consensus m6A-driven network of m6A-driven genes. To evaluate the performance, we applied m6A-Driver to build the context-specific m6A-driven networks for 4 known m6A (de)methylases, i.e., FTO, METTL3, METTL14 and WTAP. Our results suggest that m6A-Driver can robustly and efficiently identify m6A-driven genes that are functionally more enriched and associated with higher degree of differential expression than differential m6A methylated genes. Pathway analysis of the constructed context-specific m6A-driven gene networks further revealed the regulatory circuitry underlying the dynamic interplays between the methyltransferases and demethylase at the epitranscriptomic layer of gene regulation. PMID:28027310

Dynamic CRM occupancy reflects a temporal map of developmental progression.

PubMed

Wilczyński, Bartek; Furlong, Eileen E M

2010-06-22

Development is driven by tightly coordinated spatio-temporal patterns of gene expression, which are initiated through the action of transcription factors (TFs) binding to cis-regulatory modules (CRMs). Although many studies have investigated how spatial patterns arise, precise temporal control of gene expression is less well understood. Here, we show that dynamic changes in the timing of CRM occupancy is a prevalent feature common to all TFs examined in a developmental ChIP time course to date. CRMs exhibit complex binding patterns that cannot be explained by the sequence motifs or expression of the TFs themselves. The temporal changes in TF binding are highly correlated with dynamic patterns of target gene expression, which in turn reflect transitions in cellular function during different stages of development. Thus, it is not only the timing of a TF's expression, but also its temporal occupancy in refined time windows, which determines temporal gene expression. Systematic measurement of dynamic CRM occupancy may therefore serve as a powerful method to decode dynamic changes in gene expression driving developmental progression.

Fractal Clustering and Knowledge-driven Validation Assessment for Gene Expression Profiling.

PubMed

Wang, Lu-Yong; Balasubramanian, Ammaiappan; Chakraborty, Amit; Comaniciu, Dorin

2005-01-01

DNA microarray experiments generate a substantial amount of information about the global gene expression. Gene expression profiles can be represented as points in multi-dimensional space. It is essential to identify relevant groups of genes in biomedical research. Clustering is helpful in pattern recognition in gene expression profiles. A number of clustering techniques have been introduced. However, these traditional methods mainly utilize shape-based assumption or some distance metric to cluster the points in multi-dimension linear Euclidean space. Their results shows poor consistence with the functional annotation of genes in previous validation study. From a novel different perspective, we propose fractal clustering method to cluster genes using intrinsic (fractal) dimension from modern geometry. This method clusters points in such a way that points in the same clusters are more self-affine among themselves than to the points in other clusters. We assess this method using annotation-based validation assessment for gene clusters. It shows that this method is superior in identifying functional related gene groups than other traditional methods.

Integrated pathway-based transcription regulation network mining and visualization based on gene expression profiles.

PubMed

Kibinge, Nelson; Ono, Naoaki; Horie, Masafumi; Sato, Tetsuo; Sugiura, Tadao; Altaf-Ul-Amin, Md; Saito, Akira; Kanaya, Shigehiko

2016-06-01

Conventionally, workflows examining transcription regulation networks from gene expression data involve distinct analytical steps. There is a need for pipelines that unify data mining and inference deduction into a singular framework to enhance interpretation and hypotheses generation. We propose a workflow that merges network construction with gene expression data mining focusing on regulation processes in the context of transcription factor driven gene regulation. The pipeline implements pathway-based modularization of expression profiles into functional units to improve biological interpretation. The integrated workflow was implemented as a web application software (TransReguloNet) with functions that enable pathway visualization and comparison of transcription factor activity between sample conditions defined in the experimental design. The pipeline merges differential expression, network construction, pathway-based abstraction, clustering and visualization. The framework was applied in analysis of actual expression datasets related to lung, breast and prostrate cancer. Copyright © 2016 Elsevier Inc. All rights reserved.

Novel electric power-driven hydrodynamic injection system for gene delivery: safety and efficacy of human factor IX delivery in rats.

PubMed

Yokoo, T; Kamimura, K; Suda, T; Kanefuji, T; Oda, M; Zhang, G; Liu, D; Aoyagi, Y

2013-08-01

The development of a safe and reproducible gene delivery system is an essential step toward the clinical application of the hydrodynamic gene delivery (HGD) method. For this purpose, we have developed a novel electric power-driven injection system called the HydroJector-EM, which can replicate various time-pressure curves preloaded into the computer program before injection. The assessment of the reproducibility and safety of gene delivery system in vitro and in vivo demonstrated the precise replication of intravascular time-pressure curves and the reproducibility of gene delivery efficiency. The highest level of luciferase expression (272 pg luciferase per mg of proteins) was achieved safely using the time-pressure curve, which reaches 30 mm Hg in 10 s among various curves tested. Using this curve, the sustained expression of a therapeutic level of human factor IX protein (>500 ng ml(-1)) was maintained for 2 months after the HGD of the pBS-HCRHP-FIXIA plasmid. Other than a transient increase in liver enzymes that recovered in a few days, no adverse events were seen in rats. These results confirm the effectiveness of the HydroJector-EM for reproducible gene delivery and demonstrate that long-term therapeutic gene expression can be achieved by automatic computer-controlled hydrodynamic injection that can be performed by anyone.

Respiratory epithelial cells convert inactive vitamin D to its active form: potential effects on host defense.

PubMed

Hansdottir, Sif; Monick, Martha M; Hinde, Sara L; Lovan, Nina; Look, Dwight C; Hunninghake, Gary W

2008-11-15

The role of vitamin D in innate immunity is increasingly recognized. Recent work has identified a number of tissues that express the enzyme 1alpha-hydroxylase and are able to activate vitamin D. This locally produced vitamin D is believed to have important immunomodulatory effects. In this paper, we show that primary lung epithelial cells express high baseline levels of activating 1alpha-hydroxylase and low levels of inactivating 24-hydroxylase. The result of this enzyme expression is that airway epithelial cells constitutively convert inactive 25-dihydroxyvitamin D(3) to the active 1,25-dihydroxyvitamin D(3). Active vitamin D that is generated by lung epithelium leads to increased expression of vitamin D-regulated genes with important innate immune functions. These include the cathelicidin antimicrobial peptide gene and the TLR coreceptor CD14. dsRNA increases the expression of 1alpha-hydroxylase, augments the production of active vitamin D, and synergizes with vitamin D to increase expression of cathelicidin. In contrast to induction of the antimicrobial peptide, vitamin D attenuates dsRNA-induced expression of the NF-kappaB-driven gene IL-8. We conclude that primary epithelial cells generate active vitamin D, which then influences the expression of vitamin D-driven genes that play a major role in host defense. Furthermore, the presence of vitamin D alters induction of antimicrobial peptides and inflammatory cytokines in response to viruses. These observations suggest a novel mechanism by which local conversion of inactive to active vitamin D alters immune function in the lung.

Expression analysis of dihydroflavonol 4-reductase genes in Petunia hybrida.

PubMed

Chu, Y X; Chen, H R; Wu, A Z; Cai, R; Pan, J S

2015-05-12

Dihydroflavonol 4-reductase (DFR) genes from Rosa chinensis (Asn type) and Calibrachoa hybrida (Asp type), driven by a CaMV 35S promoter, were integrated into the petunia (Petunia hybrida) cultivar 9702. Exogenous DFR gene expression characteristics were similar to flower-color changes, and effects on anthocyanin concentration were observed in both types of DFR gene transformants. Expression analysis showed that exogenous DFR genes were expressed in all of the tissues, but the expression levels were significantly different. However, both of them exhibited a high expression level in petals that were starting to open. The introgression of DFR genes may significantly change DFR enzyme activity. Anthocyanin ultra-performance liquid chromatography results showed that anthocyanin concentrations changed according to DFR enzyme activity. Therefore, the change in flower color was probably the result of a DFR enzyme change. Pelargonidin 3-O-glucoside was found in two different transgenic petunias, indicating that both CaDFR and RoDFR could catalyze dihydrokaempferol. Our results also suggest that transgenic petunias with DFR gene of Asp type could biosynthesize pelargonidin 3-O-glucoside.

Converting cancer genes into killer genes.

PubMed Central

Da Costa, L T; Jen, J; He, T C; Chan, T A; Kinzler, K W; Vogelstein, B

1996-01-01

Over the past decade, it has become clear that tumorigenesis is driven by alterations in genes that control cell growth or cell death. Theoretically, the proteins encoded by these genes provide excellent targets for new therapeutic agents. Here, we describe a gene therapy approach to specifically kill tumor cells expressing such oncoproteins. In outline, the target oncoprotein binds to exogenously introduced gene products, resulting in transcriptional activation of a toxic gene. As an example, we show that this approach can be used to specifically kill cells overexpressing a mutant p53 gene in cell culture. The strategy may be generally applicable to neoplastic diseases in which the underlying patterns of genetic alterations or abnormal gene expression are known. Images Fig. 1 Fig. 2 Fig. 4 Fig. 5 PMID:8633039

Epithelial reticulon 4B (Nogo-B) is an endogenous regulator of Th2-driven lung inflammation

PubMed Central

Wright, Paulette L.; Yu, Jun; Di, Y.P. Peter; Homer, Robert J.; Chupp, Geoffrey; Elias, Jack A.; Cohn, Lauren

2010-01-01

Nogo-B is a member of the reticulon family of proteins (RTN-4B) that is highly expressed in lung tissue; however, its function remains unknown. We show that mice with Th2-driven lung inflammation results in a loss of Nogo expression in airway epithelium and smooth muscle compared with nonallergic mice, a finding which is replicated in severe human asthma. Mice lacking Nogo-A/B (Nogo-KO) display an exaggerated asthma-like phenotype, and epithelial reconstitution of Nogo-B in transgenic mice blunts Th2-mediated lung inflammation. Microarray analysis of lungs from Nogo-KO mice reveals a marked reduction in palate lung and nasal clone (PLUNC) gene expression, and the levels of PLUNC are enhanced in epithelial Nogo-B transgenic mice. Finally, transgenic expression of PLUNC into Nogo-KO mice rescues the enhanced asthmatic-like responsiveness in these KO mice. These data identify Nogo-B as a novel protective gene expressed in lung epithelia, and its expression regulates the levels of the antibacterial antiinflammatory protein PLUNC. PMID:20975041

BdCESA7, BdCESA8, and BdPMT utility promoter constructs for targeted expression to secondary cell-wall-forming cells of grasses

DOE PAGES

Petrik, Deborah L.; Cass, Cynthia L.; Padmakshan, Dharshana; ...

2016-02-04

Utility vectors with promoters that confer desired spatial and temporal expression patterns are useful tools for studying gene and cellular function and for industrial applications. To target the expression of DNA sequences of interest to cells forming plant secondary cell walls, which generate most of the vegetative biomass, upstream regulatory sequences of the Brachypodium distachyon lignin biosynthetic gene BdPMT and the cellulose synthase genes BdCESA7 and BdCESA8 were isolated and cloned into binary vectors designed for Agrobacterium-mediated transformation of monocots. Expression patterns were assessed using the β-glucuronidase gene GUSPlus and X-glucuronide staining. All three promoters showed strong expression levels inmore » stem tissue at the base of internodes where cell wall deposition is most active, in both vascular bundle xylem vessels and tracheids, and in interfascicular tissues, with expression less pronounced in developmentally older tissues. In leaves, BdCESA7 and BdCESA8 promoter-driven expression was strongest in leaf veins, leaf margins, and trichomes; relatively weaker and patchy expression was observed in the epidermis. BdPMT promoter-driven expression was similar to the BdCESA promoters expression patterns, including strong expression in trichomes. The intensity and extent of GUS staining varied considerably between transgenic lines, suggesting that positional effects influenced promoter activity. Introducing the BdPMT and BdCESA8 Open Reading Frames into BdPMT and BdCESA8 utility promoter binary vectors, respectively, and transforming those constructs into Brachypodium pmt and cesa8 loss-of-function mutants resulted in rescue of the corresponding mutant phenotypes. This work therefore validates the functionality of these utility promoter binary vectors for use in Brachypodium and likely other grass species. Lastly, the identification, in Bdcesa8-1 T-DNA mutant stems, of an 80% reduction in crystalline cellulose levels confirms that the BdCESA8 gene is a secondary-cell-wall-forming cellulose synthase.« less

BdCESA7, BdCESA8, and BdPMT utility promoter constructs for targeted expression to secondary cell-wall-forming cells of grasses

DOE Office of Scientific and Technical Information (OSTI.GOV)

Petrik, Deborah L.; Cass, Cynthia L.; Padmakshan, Dharshana

Utility vectors with promoters that confer desired spatial and temporal expression patterns are useful tools for studying gene and cellular function and for industrial applications. To target the expression of DNA sequences of interest to cells forming plant secondary cell walls, which generate most of the vegetative biomass, upstream regulatory sequences of the Brachypodium distachyon lignin biosynthetic gene BdPMT and the cellulose synthase genes BdCESA7 and BdCESA8 were isolated and cloned into binary vectors designed for Agrobacterium-mediated transformation of monocots. Expression patterns were assessed using the β-glucuronidase gene GUSPlus and X-glucuronide staining. All three promoters showed strong expression levels inmore » stem tissue at the base of internodes where cell wall deposition is most active, in both vascular bundle xylem vessels and tracheids, and in interfascicular tissues, with expression less pronounced in developmentally older tissues. In leaves, BdCESA7 and BdCESA8 promoter-driven expression was strongest in leaf veins, leaf margins, and trichomes; relatively weaker and patchy expression was observed in the epidermis. BdPMT promoter-driven expression was similar to the BdCESA promoters expression patterns, including strong expression in trichomes. The intensity and extent of GUS staining varied considerably between transgenic lines, suggesting that positional effects influenced promoter activity. Introducing the BdPMT and BdCESA8 Open Reading Frames into BdPMT and BdCESA8 utility promoter binary vectors, respectively, and transforming those constructs into Brachypodium pmt and cesa8 loss-of-function mutants resulted in rescue of the corresponding mutant phenotypes. This work therefore validates the functionality of these utility promoter binary vectors for use in Brachypodium and likely other grass species. Lastly, the identification, in Bdcesa8-1 T-DNA mutant stems, of an 80% reduction in crystalline cellulose levels confirms that the BdCESA8 gene is a secondary-cell-wall-forming cellulose synthase.« less

Frontotemporal dementia: insights into the biological underpinnings of disease through gene co-expression network analysis.

PubMed

Ferrari, Raffaele; Forabosco, Paola; Vandrovcova, Jana; Botía, Juan A; Guelfi, Sebastian; Warren, Jason D; Momeni, Parastoo; Weale, Michael E; Ryten, Mina; Hardy, John

2016-02-24

In frontotemporal dementia (FTD) there is a critical lack in the understanding of biological and molecular mechanisms involved in disease pathogenesis. The heterogeneous genetic features associated with FTD suggest that multiple disease-mechanisms are likely to contribute to the development of this neurodegenerative condition. We here present a systems biology approach with the scope of i) shedding light on the biological processes potentially implicated in the pathogenesis of FTD and ii) identifying novel potential risk factors for FTD. We performed a gene co-expression network analysis of microarray expression data from 101 individuals without neurodegenerative diseases to explore regional-specific co-expression patterns in the frontal and temporal cortices for 12 genes (MAPT, GRN, CHMP2B, CTSC, HLA-DRA, TMEM106B, C9orf72, VCP, UBQLN2, OPTN, TARDBP and FUS) associated with FTD and we then carried out gene set enrichment and pathway analyses, and investigated known protein-protein interactors (PPIs) of FTD-genes products. Gene co-expression networks revealed that several FTD-genes (such as MAPT and GRN, CTSC and HLA-DRA, TMEM106B, and C9orf72, VCP, UBQLN2 and OPTN) were clustering in modules of relevance in the frontal and temporal cortices. Functional annotation and pathway analyses of such modules indicated enrichment for: i) DNA metabolism, i.e. transcription regulation, DNA protection and chromatin remodelling (MAPT and GRN modules); ii) immune and lysosomal processes (CTSC and HLA-DRA modules), and; iii) protein meta/catabolism (C9orf72, VCP, UBQLN2 and OPTN, and TMEM106B modules). PPI analysis supported the results of the functional annotation and pathway analyses. This work further characterizes known FTD-genes and elaborates on their biological relevance to disease: not only do we indicate likely impacted regional-specific biological processes driven by FTD-genes containing modules, but also do we suggest novel potential risk factors among the FTD-genes interactors as targets for further mechanistic characterization in hypothesis driven cell biology work.

miRNA-embedded shRNAs for Lineage-specific BCL11A Knockdown and Hemoglobin F Induction

PubMed Central

Guda, Swaroopa; Brendel, Christian; Renella, Raffaele; Du, Peng; Bauer, Daniel E; Canver, Matthew C; Grenier, Jennifer K; Grimson, Andrew W; Kamran, Sophia C; Thornton, James; de Boer, Helen; Root, David E; Milsom, Michael D; Orkin, Stuart H; Gregory, Richard I; Williams, David A

2015-01-01

RNA interference (RNAi) technology using short hairpin RNAs (shRNAs) expressed via RNA polymerase (pol) III promoters has been widely exploited to modulate gene expression in a variety of mammalian cell types. For certain applications, such as lineage-specific knockdown, embedding targeting sequences into pol II-driven microRNA (miRNA) architecture is required. Here, using the potential therapeutic target BCL11A, we demonstrate that pol III-driven shRNAs lead to significantly increased knockdown but also increased cytotoxcity in comparison to pol II-driven miRNA adapted shRNAs (shRNAmiR) in multiple hematopoietic cell lines. We show that the two expression systems yield mature guide strand sequences that differ by a 4 bp shift. This results in alternate seed sequences and consequently influences the efficacy of target gene knockdown. Incorporating a corresponding 4 bp shift into the guide strand of shRNAmiRs resulted in improved knockdown efficiency of BCL11A. This was associated with a significant de-repression of the hemoglobin target of BCL11A, human γ-globin or the murine homolog Hbb-y. Our results suggest the requirement for optimization of shRNA sequences upon incorporation into a miRNA backbone. These findings have important implications in future design of shRNAmiRs for RNAi-based therapy in hemoglobinopathies and other diseases requiring lineage-specific expression of gene silencing sequences. PMID:26080908

Transcriptional regulation of ABI3- and ABA-responsive genes including RD29B and RD29A in seeds, germinating embryos, and seedlings of Arabidopsis.

PubMed

Nakashima, Kazuo; Fujita, Yasunari; Katsura, Koji; Maruyama, Kyonoshin; Narusaka, Yoshihiro; Seki, Motoaki; Shinozaki, Kazuo; Yamaguchi-Shinozaki, Kazuko

2006-01-01

ABA-responsive elements (ABREs) are cis-acting elements and basic leucine zipper (bZIP)-type ABRE-binding proteins (AREBs) are transcriptional activators that function in the expression of RD29B in vegetative tissue of Arabidopsis in response to abscisic acid (ABA) treatment. Dehydration-responsive elements (DREs) function as coupling elements of ABRE in the expression of RD29A in response to ABA. Expression analysis using abi3 and abi5 mutants showed that ABI3 and ABI5 play important roles in the expression of RD29B in seeds. Base-substitution analysis showed that two ABREs function strongly and one ABRE coupled with DRE functions weakly in the expression of RD29A in embryos. In a transient transactivation experiment, ABI3, ABI5 and AREB1 activated transcription of a GUS reporter gene driven by the RD29B promoter strongly but these proteins activated the transcription driven by the RD29A promoter weakly. In 35S::ABI3 Arabidopsis plants, the expression of RD29B was up-regulated strongly, but that of RD29A was up-regulated weakly. These results indicate that the expression of RD29B having ABREs in the promoter is up-regulated strongly by ABI3, whereas that of RD29A having one ABRE coupled with DREs in the promoter is up-regulated weakly by ABI3. We compared the expression of 7000 Arabidopsis genes in response to ABA treatment during germination and in the vegetative growth stage, and that in 35S::ABI3 plants using a full-length cDNA microarray. The expression of ABI3- and/or ABA-responsive genes and cis-elements in the promoters are discussed.

Development of an Expression Vector to Overexpress or Downregulate Genes in Curvularia protuberata.

PubMed

Liu, Chengke; Cleckler, Blake; Morsy, Mustafa

2018-05-05

Curvularia protuberata , an endophytic fungus in the Ascomycota, provides plants with thermotolerance only when it carries a mycovirus known as Curvularia thermotolerance virus (CThTV), and forms a three-way symbiotic relationship among these organisms. Under heat stress, several genes are expressed differently between virus-free C. protuberata (VF) and C. protuberata carrying CThTV (AN). We developed an expression vector, pM2Z-fun, carrying a zeocin resistance gene driven by the ToxA promoter, to study gene functions in C. protuberata to better understand this three-way symbiosis. Using this new 3.7-kb vector, five genes that are differentially expressed in C. protuberata —including genes involved in the trehalose, melanin, and catalase biosynthesis pathways—were successfully overexpressed or downregulated in VF or AN C. protuberata strains, respectively. The VF overexpression lines showed higher metabolite and enzyme activity than in the control VF strain. Furthermore, downregulation of expression of the same genes in the AN strain resulted in lower metabolite and enzyme activity than in the control AN strain. The newly generated expression vector, pM2Z-fun, has been successfully used to express target genes in C. protuberata and will be useful in further functional expression studies in other Ascomycota fungi.

Circadian processes in the RNA life cycle.

PubMed

Torres, Manon; Becquet, Denis; Franc, Jean-Louis; François-Bellan, Anne-Marie

2018-05-01

The circadian clock drives daily rhythms of multiple physiological processes, allowing organisms to anticipate and adjust to periodic changes in environmental conditions. These physiological rhythms are associated with robust oscillations in the expression of at least 30% of expressed genes. While the ability for the endogenous timekeeping system to generate a 24-hr cycle is a cell-autonomous mechanism based on negative autoregulatory feedback loops of transcription and translation involving core-clock genes and their protein products, it is now increasingly evident that additional mechanisms also govern the circadian oscillations of clock-controlled genes. Such mechanisms can take place post-transcriptionally during the course of the RNA life cycle. It has been shown that many steps during RNA processing are regulated in a circadian manner, thus contributing to circadian gene expression. These steps include mRNA capping, alternative splicing, changes in splicing efficiency, and changes in RNA stability controlled by the tail length of polyadenylation or the use of alternative polyadenylation sites. RNA transport can also follow a circadian pattern, with a circadian nuclear retention driven by rhythmic expression within the nucleus of particular bodies (the paraspeckles) and circadian export to the cytoplasm driven by rhythmic proteins acting like cargo. Finally, RNA degradation may also follow a circadian pattern through the rhythmic involvement of miRNAs. In this review, we summarize the current knowledge of the post-transcriptional circadian mechanisms known to play a prominent role in shaping circadian gene expression in mammals. This article is categorized under: RNA Processing > Splicing Regulation/Alternative Splicing RNA Processing > RNA Editing and Modification RNA Export and Localization > Nuclear Export/Import. © 2018 Wiley Periodicals, Inc.

Environmental regulation of plant gene expression: an RT-qPCR laboratory project for an upper-level undergraduate biochemistry or molecular biology course.

PubMed

Eickelberg, Garrett J; Fisher, Alison J

2013-01-01

We present a novel laboratory project employing "real-time" RT-qPCR to measure the effect of environment on the expression of the FLOWERING LOCUS C gene, a key regulator of floral timing in Arabidopsis thaliana plants. The project requires four 3-hr laboratory sessions and is aimed at upper-level undergraduate students in biochemistry or molecular biology courses. The project provides students with hands-on experience with RT-qPCR, the current "gold standard" for gene expression analysis, including detailed data analysis using the common 2-ΔΔCT method. Moreover, it provides a convenient starting point for many inquiry-driven projects addressing diverse questions concerning ecological biochemistry, naturally occurring genetic variation, developmental biology, and the regulation of gene expression in nature. Copyright © 2013 Wiley Periodicals, Inc.

Tau pathology does not affect experience-driven single-neuron and network-wide Arc/Arg3.1 responses.

PubMed

Rudinskiy, Nikita; Hawkes, Jonathan M; Wegmann, Susanne; Kuchibhotla, Kishore V; Muzikansky, Alona; Betensky, Rebecca A; Spires-Jones, Tara L; Hyman, Bradley T

2014-06-10

Intraneuronal neurofibrillary tangles (NFTs) - a characteristic pathological feature of Alzheimer's and several other neurodegenerative diseases - are considered a major target for drug development. Tangle load correlates well with the severity of cognitive symptoms and mouse models of tauopathy are behaviorally impaired. However, there is little evidence that NFTs directly impact physiological properties of host neurons. Here we used a transgenic mouse model of tauopathy to study how advanced tau pathology in different brain regions affects activity-driven expression of immediate-early gene Arc required for experience-dependent consolidation of long-term memories. We demonstrate in vivo that visual cortex neurons with tangles are as likely to express comparable amounts of Arc in response to structured visual stimulation as their neighbors without tangles. Probability of experience-dependent Arc response was not affected by tau tangles in both visual cortex and hippocampal pyramidal neurons as determined postmortem. Moreover, whole brain analysis showed that network-wide activity-driven Arc expression was not affected by tau pathology in any of the brain regions, including brain areas with the highest tangle load. Our findings suggest that intraneuronal NFTs do not affect signaling cascades leading to experience-dependent gene expression required for long-term synaptic plasticity.

Engrailed negatively regulates the expression of cell adhesion molecules connectin and neuroglian in embryonic Drosophila nervous system.

PubMed

Siegler, M V; Jia, X X

1999-02-01

Engrailed is expressed in subsets of interneurons that do not express Connectin or appreciable Neuroglian, whereas other neurons that are Engrailed negative strongly express these adhesion molecules. Connectin and Neuroglian expression are virtually eliminated in interneurons when engrailed expression is driven ubiquitously in neurons, and greatly increased when engrailed genes are lacking in mutant embryos. The data suggest that Engrailed is normally a negative regulator of Connectin and neuroglian. These are the first two "effector" genes identified in the nervous system of Drosophila as regulatory targets for Engrailed. We argue that differential Engrailed expression is crucial in determining the pattern of expression of cell adhesion molecules and thus constitutes an important determinant of neuronal shape and perhaps connectivity.

Gene expression profiling of MYC-driven tumor signatures in porcine liver stem cells by transcriptome sequencing

USDA-ARS?s Scientific Manuscript database

It is now well-established that cancer stem cells (CSCs) drive tumor growth and that the cancer gene, c-Myc, plays a critical role in converting cells to CSCs. However, little is known about the genes that are induced and regulated by c-Myc to generate tumors, and, in particular, tumors of the live...

Transient, Inducible, Placenta-Specific Gene Expression in Mice

PubMed Central

Fan, Xiujun; Petitt, Matthew; Gamboa, Matthew; Huang, Mei; Dhal, Sabita; Druzin, Maurice L.; Wu, Joseph C.

2012-01-01

Molecular understanding of placental functions and pregnancy disorders is limited by the absence of methods for placenta-specific gene manipulation. Although persistent placenta-specific gene expression has been achieved by lentivirus-based gene delivery methods, developmentally and physiologically important placental genes have highly stage-specific functions, requiring controllable, transient expression systems for functional analysis. Here, we describe an inducible, placenta-specific gene expression system that enables high-level, transient transgene expression and monitoring of gene expression by live bioluminescence imaging in mouse placenta at different stages of pregnancy. We used the third generation tetracycline-responsive tranactivator protein Tet-On 3G, with 10- to 100-fold increased sensitivity to doxycycline (Dox) compared with previous versions, enabling unusually sensitive on-off control of gene expression in vivo. Transgenic mice expressing Tet-On 3G were created using a new integrase-based, site-specific approach, yielding high-level transgene expression driven by a ubiquitous promoter. Blastocysts from these mice were transduced with the Tet-On 3G-response element promoter-driving firefly luciferase using lentivirus-mediated placenta-specific gene delivery and transferred into wild-type pseudopregnant recipients for placenta-specific, Dox-inducible gene expression. Systemic Dox administration at various time points during pregnancy led to transient, placenta-specific firefly luciferase expression as early as d 5 of pregnancy in a Dox dose-dependent manner. This system enables, for the first time, reliable pregnancy stage-specific induction of gene expression in the placenta and live monitoring of gene expression during pregnancy. It will be widely applicable to studies of both placental development and pregnancy, and the site-specific Tet-On G3 mouse will be valuable for studies in a broad range of tissues. PMID:23011919

Effect of external and internal factors on the expression of reporter genes driven by the N resistance gene promoter.

PubMed

Kathiria, Palak; Sidler, Corinne; Woycicki, Rafal; Yao, Youli; Kovalchuk, Igor

2013-07-01

The role of resistance (R) genes in plant pathogen interaction has been studied extensively due to its economical impact on agriculture. Interaction between tobacco mosaic virus (TMV) and the N protein from tobacco is one of the most widely used models to understand various aspects of pathogen resistance. The transcription activity governed by N gene promoter is one of the least understood elements of the model. In this study, the N gene promoter was cloned and fused with two different reporter genes, one encoding β-glucuronidase (N::GUS) and another, luciferase (N::LUC). Tobacco plants transformed with the N::GUS or N::LUC reporter constructs were screened for homozygosity and stable expression. Histochemical analysis of N::GUS tobacco plants revealed that the expression is organ specific and developmentally regulated. Whereas two week old plants expressed GUS in midveins only, 6-wk-old plants also expressed GUS in leaf lamella. Roots did not show GUS expression at any time during development. Experiments to address effects of external stress were performed using N::LUC tobacco plants. These experiments showed that N gene promoter expression was suppressed when plants were exposed to high but not low temperatures. Expression was also upregulated in response to TMV, but no changes were observed in plants treated with SA.

Expression of a suicidal gene under control of the human secreted protein acidic and rich in cysteine (SPARC) promoter in tumor or stromal cells led to the inhibition of tumor cell growth

PubMed Central

Lopez, María V.; Blanco, Patricia; Viale, Diego L.; Cafferata, Eduardo G.; Carbone, Cecilia; Gould, David; Chernajovsky, Yuti; Podhajcer, Osvaldo L.

2009-01-01

The successful use of transcriptional targeting for cancer therapy depends on the activity of a given promoter inside the malignant cell. Because solid human tumors evolve as a “cross-talk” between the different cell types within the tumor, we hypothesized that targeting the entire tumor mass might have better therapeutic effect. Secreted protein acidic and rich in cysteine (SPARC) is a matricellular protein overexpressed in different human cancers malignant melanomas both in the malignant cells compartment as in the stromal one (fibroblasts and endothelial cells). We have shown that expression of the herpes simplex virus-thymidine kinase (TK) gene driven by the SPARC promoter in combination with ganciclovir inhibited human melanoma cell growth in monolayer as well as in multicellular spheroids. This inhibitory effect was observed both in homotypic spheroids composed of melanoma cells alone as well as in spheroids made of melanoma cells and stromal cells. Expression of the TK gene was also efficient to inhibit the in vivo tumor growth of established melanomas when TK was expressed either by the malignant cells themselves or by coadministered endothelial cells. Our data suggest that the use of therapeutic genes driven by SPARC promoter could be a valuable strategy for cancer therapy aiming to target all the cellular components of the tumor mass. PMID:17041094

Casein kinase 1α–dependent feedback loop controls autophagy in RAS-driven cancers

PubMed Central

Cheong, Jit Kong; Zhang, Fuquan; Chua, Pei Jou; Bay, Boon Huat; Thorburn, Andrew; Virshup, David M.

2015-01-01

Activating mutations in the RAS oncogene are common in cancer but are difficult to therapeutically target. RAS activation promotes autophagy, a highly regulated catabolic process that metabolically buffers cells in response to diverse stresses. Here we report that casein kinase 1α (CK1α), a ubiquitously expressed serine/threonine kinase, is a key negative regulator of oncogenic RAS–induced autophagy. Depletion or pharmacologic inhibition of CK1α enhanced autophagic flux in oncogenic RAS–driven human fibroblasts and multiple cancer cell lines. FOXO3A, a master longevity mediator that transcriptionally regulates diverse autophagy genes, was a critical target of CK1α, as depletion of CK1α reduced levels of phosphorylated FOXO3A and increased expression of FOXO3A-responsive genes. Oncogenic RAS increased CK1α protein abundance via activation of the PI3K/AKT/mTOR pathway. In turn, elevated levels of CK1α increased phosphorylation of nuclear FOXO3A, thereby inhibiting transactivation of genes critical for RAS-induced autophagy. In both RAS-driven cancer cells and murine xenograft models, pharmacologic CK1α inactivation synergized with lysosomotropic agents to inhibit growth and promote tumor cell death. Together, our results identify a kinase feedback loop that influences RAS-dependent autophagy and suggest that targeting CK1α-regulated autophagy offers a potential therapeutic opportunity to treat oncogenic RAS–driven cancers. PMID:25798617

Functional Characterization of a Strong Bi-directional Constitutive Plant Promoter Isolated from Cotton Leaf Curl Burewala Virus

PubMed Central

Khan, Zainul A.; Abdin, Malik Z.; Khan, Jawaid A.

2015-01-01

Cotton leaf curl Burewala virus (CLCuBuV), belonging to the genus Begomovirus, possesses single-stranded monopartite DNA genome. The bidirectional promoters representing Rep and coat protein (CP) genes of CLCuBuV were characterized and their efficacy was assayed. Rep and CP promoters of CLCuBuV and 35S promoter of Cauliflower mosaic virus (CaMV) were fused with β-glucuronidase (GUS) and green fluorescent protein (GFP) reporter genes. GUS activity in individual plant cells driven by Rep, CP and 35S promoters was estimated using real-time PCR and fluorometric GUS assay. Histochemical staining of GUS in transformed tobacco (Nicotiana tabacum cv. Xanthi) leaves showed highest expression driven by Rep promoter followed by 35S promoter and CP promoter. The expression level of GUS driven by Rep promoter in transformed tobacco plants was shown to be two to four-fold higher than that of 35S promoter, while the expression by CP promoter was slightly lower. Further, the expression of GFP was monitored in agroinfiltrated leaves of N. benthamiana, N. tabacum and cotton (Gossypium hirsutum) plants using confocal laser scanning microscopy. Rep promoter showed strong consistent transient expression in tobacco and cotton leaves as compared to 35S promoter. The strong constitutive CLCuBuV Rep promoter developed in this study could be very useful for high level expression of transgenes in a wide variety of plant cells. PMID:25799504

Functional characterization of a strong bi-directional constitutive plant promoter isolated from cotton leaf curl Burewala virus.

PubMed

Khan, Zainul A; Abdin, Malik Z; Khan, Jawaid A

2015-01-01

Cotton leaf curl Burewala virus (CLCuBuV), belonging to the genus Begomovirus, possesses single-stranded monopartite DNA genome. The bidirectional promoters representing Rep and coat protein (CP) genes of CLCuBuV were characterized and their efficacy was assayed. Rep and CP promoters of CLCuBuV and 35S promoter of Cauliflower mosaic virus (CaMV) were fused with β-glucuronidase (GUS) and green fluorescent protein (GFP) reporter genes. GUS activity in individual plant cells driven by Rep, CP and 35S promoters was estimated using real-time PCR and fluorometric GUS assay. Histochemical staining of GUS in transformed tobacco (Nicotiana tabacum cv. Xanthi) leaves showed highest expression driven by Rep promoter followed by 35S promoter and CP promoter. The expression level of GUS driven by Rep promoter in transformed tobacco plants was shown to be two to four-fold higher than that of 35S promoter, while the expression by CP promoter was slightly lower. Further, the expression of GFP was monitored in agroinfiltrated leaves of N. benthamiana, N. tabacum and cotton (Gossypium hirsutum) plants using confocal laser scanning microscopy. Rep promoter showed strong consistent transient expression in tobacco and cotton leaves as compared to 35S promoter. The strong constitutive CLCuBuV Rep promoter developed in this study could be very useful for high level expression of transgenes in a wide variety of plant cells.

Identification of airway mucosal type 2 inflammation by using clinical biomarkers in asthmatic patients.

PubMed

Silkoff, Philip E; Laviolette, Michel; Singh, Dave; FitzGerald, J Mark; Kelsen, Steven; Backer, Vibeke; Porsbjerg, Celeste M; Girodet, Pierre-Olivier; Berger, Patrick; Kline, Joel N; Chupp, Geoffrey; Susulic, Vedrana S; Barnathan, Elliot S; Baribaud, Frédéric; Loza, Matthew J

2017-09-01

The Airways Disease Endotyping for Personalized Therapeutics (ADEPT) study profiled patients with mild, moderate, and severe asthma and nonatopic healthy control subjects. We explored this data set to define type 2 inflammation based on airway mucosal IL-13-driven gene expression and how this related to clinically accessible biomarkers. IL-13-driven gene expression was evaluated in several human cell lines. We then defined type 2 status in 25 healthy subjects, 28 patients with mild asthma, 29 patients with moderate asthma, and 26 patients with severe asthma based on airway mucosal expression of (1) CCL26 (the most differentially expressed gene), (2) periostin, or (3) a multigene IL-13 in vitro signature (IVS). Clinically accessible biomarkers included fraction of exhaled nitric oxide (Feno) values, blood eosinophil (bEOS) counts, serum CCL26 expression, and serum CCL17 expression. Expression of airway mucosal CCL26, periostin, and IL-13-IVS all facilitated segregation of subjects into type 2-high and type 2-low asthmatic groups, but in the ADEPT study population CCL26 expression was optimal. All subjects with high airway mucosal CCL26 expression and moderate-to-severe asthma had Feno values (≥35 ppb) and/or high bEOS counts (≥300 cells/mm 3 ) compared with a minority (36%) of subjects with low airway mucosal CCL26 expression. A combination of Feno values, bEOS counts, and serum CCL17 and CCL26 expression had 100% positive predictive value and 87% negative predictive value for airway mucosal CCL26-high status. Clinical variables did not differ between subjects with type 2-high and type 2-low status. Eosinophilic inflammation was associated with but not limited to airway mucosal type 2 gene expression. A panel of clinical biomarkers accurately classified type 2 status based on airway mucosal CCL26, periostin, or IL-13-IVS gene expression. Use of Feno values, bEOS counts, and serum marker levels (eg, CCL26 and CCL17) in combination might allow patient selection for novel type 2 therapeutics. Copyright © 2017 American Academy of Allergy, Asthma & Immunology. All rights reserved.

Robust variable selection method for nonparametric differential equation models with application to nonlinear dynamic gene regulatory network analysis.

PubMed

Lu, Tao

2016-01-01

The gene regulation network (GRN) evaluates the interactions between genes and look for models to describe the gene expression behavior. These models have many applications; for instance, by characterizing the gene expression mechanisms that cause certain disorders, it would be possible to target those genes to block the progress of the disease. Many biological processes are driven by nonlinear dynamic GRN. In this article, we propose a nonparametric differential equation (ODE) to model the nonlinear dynamic GRN. Specially, we address following questions simultaneously: (i) extract information from noisy time course gene expression data; (ii) model the nonlinear ODE through a nonparametric smoothing function; (iii) identify the important regulatory gene(s) through a group smoothly clipped absolute deviation (SCAD) approach; (iv) test the robustness of the model against possible shortening of experimental duration. We illustrate the usefulness of the model and associated statistical methods through a simulation and a real application examples.

Regulatory Architecture of Gene Expression Variation in the Threespine Stickleback Gasterosteus aculeatus.

PubMed

Pritchard, Victoria L; Viitaniemi, Heidi M; McCairns, R J Scott; Merilä, Juha; Nikinmaa, Mikko; Primmer, Craig R; Leder, Erica H

2017-01-05

Much adaptive evolutionary change is underlain by mutational variation in regions of the genome that regulate gene expression rather than in the coding regions of the genes themselves. An understanding of the role of gene expression variation in facilitating local adaptation will be aided by an understanding of underlying regulatory networks. Here, we characterize the genetic architecture of gene expression variation in the threespine stickleback (Gasterosteus aculeatus), an important model in the study of adaptive evolution. We collected transcriptomic and genomic data from 60 half-sib families using an expression microarray and genotyping-by-sequencing, and located expression quantitative trait loci (eQTL) underlying the variation in gene expression in liver tissue using an interval mapping approach. We identified eQTL for several thousand expression traits. Expression was influenced by polymorphism in both cis- and trans-regulatory regions. Trans-eQTL clustered into hotspots. We did not identify master transcriptional regulators in hotspot locations: rather, the presence of hotspots may be driven by complex interactions between multiple transcription factors. One observed hotspot colocated with a QTL recently found to underlie salinity tolerance in the threespine stickleback. However, most other observed hotspots did not colocate with regions of the genome known to be involved in adaptive divergence between marine and freshwater habitats. Copyright © 2017 Pritchard et al.

Regulatory Architecture of Gene Expression Variation in the Threespine Stickleback Gasterosteus aculeatus

PubMed Central

Pritchard, Victoria L.; Viitaniemi, Heidi M.; McCairns, R. J. Scott; Merilä, Juha; Nikinmaa, Mikko; Primmer, Craig R.; Leder, Erica H.

2016-01-01

Much adaptive evolutionary change is underlain by mutational variation in regions of the genome that regulate gene expression rather than in the coding regions of the genes themselves. An understanding of the role of gene expression variation in facilitating local adaptation will be aided by an understanding of underlying regulatory networks. Here, we characterize the genetic architecture of gene expression variation in the threespine stickleback (Gasterosteus aculeatus), an important model in the study of adaptive evolution. We collected transcriptomic and genomic data from 60 half-sib families using an expression microarray and genotyping-by-sequencing, and located expression quantitative trait loci (eQTL) underlying the variation in gene expression in liver tissue using an interval mapping approach. We identified eQTL for several thousand expression traits. Expression was influenced by polymorphism in both cis- and trans-regulatory regions. Trans-eQTL clustered into hotspots. We did not identify master transcriptional regulators in hotspot locations: rather, the presence of hotspots may be driven by complex interactions between multiple transcription factors. One observed hotspot colocated with a QTL recently found to underlie salinity tolerance in the threespine stickleback. However, most other observed hotspots did not colocate with regions of the genome known to be involved in adaptive divergence between marine and freshwater habitats. PMID:27836907

An essential role for UTX in resolution and activation of bivalent promoters

PubMed Central

Dhar, Shilpa S.; Lee, Sung-Hun; Chen, Kaifu; Zhu, Guangjing; Oh, WonKyung; Allton, Kendra; Gafni, Ohad; Kim, Young Zoon; Tomoiga, Alin S.; Barton, Michelle Craig; Hanna, Jacob H.; Wang, Zhibin; Li, Wei; Lee, Min Gyu

2016-01-01

Trimethylated histone H3 lysine 27 (H3K27me3) is linked to gene silencing, whereas H3K4me3 is associated with gene activation. These two marks frequently co-occupy gene promoters, forming bivalent domains. Bivalency signifies repressed but activatable states of gene expression and can be resolved to active, H3K4me3-prevalent states during multiple cellular processes, including differentiation, development and epithelial mesenchymal transition. However, the molecular mechanism underlying bivalency resolution remains largely unknown. Here, we show that the H3K27 demethylase UTX (also called KDM6A) is required for the resolution and activation of numerous retinoic acid (RA)-inducible bivalent genes during the RA-driven differentiation of mouse embryonic stem cells (ESCs). Notably, UTX loss in mouse ESCs inhibited the RA-driven bivalency resolution and activation of most developmentally critical homeobox (Hox) a–d genes. The UTX-mediated resolution and activation of many bivalent Hox genes during mouse ESC differentiation were recapitulated during RA-driven differentiation of human NT2/D1 embryonal carcinoma cells. In support of the importance of UTX in bivalency resolution, Utx-null mouse ESCs and UTX-depleted NT2/D1 cells displayed defects in RA-driven cellular differentiation. Our results define UTX as a bivalency-resolving histone modifier necessary for stem cell differentiation. PMID:26762983

Tumor-specific expression of shVEGF and suicide gene as a novel strategy for esophageal cancer therapy.

PubMed

Liu, Ting; Wu, Hai-Jun; Liang, Yu; Liang, Xu-Jun; Huang, Hui-Chao; Zhao, Yan-Zhong; Liao, Qing-Chuan; Chen, Ya-Qi; Leng, Ai-Min; Yuan, Wei-Jian; Zhang, Gui-Ying; Peng, Jie; Chen, Yong-Heng

2016-06-21

To develop a potent and safe gene therapy for esophageal cancer. An expression vector carrying fusion suicide gene (yCDglyTK) and shRNA against vascular endothelial growth factor (VEGF) was constructed and delivered into EC9706 esophageal cancer cells by calcium phosphate nanoparticles (CPNP). To achieve tumor selectivity, expression of the fusion suicide gene was driven by a tumor-specific human telomerase reverse transcriptase (hTERT) promoter. The biologic properties and therapeutic efficiency of the vector, in the presence of prodrug 5-fluorocytosine (5-FC), were evaluated in vitro and in vivo. Both in vitro and in vivo testing showed that the expression vector was efficiently introduced by CPNP into tumor cells, leading to cellular expression of yCDglyTK and decreased VEGF level. With exposure to 5-FC, it exhibited strong anti-tumor effects against esophageal cancer. Combination of VEGF shRNA with the fusion suicide gene demonstrated strong anti-tumor activity. The shVEGF-hTERT-yCDglyTK/5-FC system provided a novel approach for esophageal cancer-targeted gene therapy.

UMG Lenti: novel lentiviral vectors for efficient transgene- and reporter gene expression in human early hematopoietic progenitors.

PubMed

Chiarella, Emanuela; Carrà, Giovanna; Scicchitano, Stefania; Codispoti, Bruna; Mega, Tiziana; Lupia, Michela; Pelaggi, Daniela; Marafioti, Maria G; Aloisio, Annamaria; Giordano, Marco; Nappo, Giovanna; Spoleti, Cristina B; Grillone, Teresa; Giovannone, Emilia D; Spina, Raffaella; Bernaudo, Francesca; Moore, Malcolm A S; Bond, Heather M; Mesuraca, Maria; Morrone, Giovanni

2014-01-01

Lentiviral vectors are widely used to investigate the biological properties of regulatory proteins and/or of leukaemia-associated oncogenes by stably enforcing their expression in hematopoietic stem and progenitor cells. In these studies it is critical to be able to monitor and/or sort the infected cells, typically via fluorescent proteins encoded by the modified viral genome. The most popular strategy to ensure co-expression of transgene and reporter gene is to insert between these cDNAs an IRES element, thus generating bi-cistronic mRNAs whose transcription is driven by a single promoter. However, while the product of the gene located upstream of the IRES is generally abundantly expressed, the translation of the downstream cDNA (typically encoding the reporter protein) is often inconsistent, which hinders the detection and the isolation of transduced cells. To overcome these limitations, we developed novel lentiviral dual-promoter vectors (named UMG-LV5 and -LV6) where transgene expression is driven by the potent UBC promoter and that of the reporter protein, EGFP, by the minimal regulatory element of the WASP gene. These vectors, harboring two distinct transgenes, were tested in a variety of human haematopoietic cell lines as well as in primary human CD34+ cells in comparison with the FUIGW vector that contains the expression cassette UBC-transgene-IRES-EGFP. In these experiments both UMG-LV5 and UMG-LV6 yielded moderately lower transgene expression than FUIGW, but dramatically higher levels of EGFP, thereby allowing the easy distinction between transduced and non-transduced cells. An additional construct was produced, in which the cDNA encoding the reporter protein is upstream, and the transgene downstream of the IRES sequence. This vector, named UMG-LV11, proved able to promote abundant expression of both transgene product and EGFP in all cells tested. The UMG-LVs represent therefore useful vectors for gene transfer-based studies in hematopoietic stem and progenitor cells, as well as in non-hematopoietic cells.

Promoter polymorphisms in genes involved in porcine myogenesis influence their transcriptional activity.

PubMed

Bongiorni, Silvia; Tilesi, Francesca; Bicorgna, Silvia; Iacoponi, Francesca; Willems, Daniela; Gargani, Maria; D'Andrea, MariaSilvia; Pilla, Fabio; Valentini, Alessio

2014-11-07

Success of meat production and selection for improvement of meat quality is among the primary aims in animal production. Meat quality traits are economically important in swine; however, the underlying genetic nature is very complex. Therefore, an improved pork production strongly depends on identifying and studying how genetic variations contribute to modulate gene expression. Promoters are key regions in gene modulation as they harbour several binding motifs to transcription regulatory factors. Therefore, polymorphisms in these regions are likely to deeply affect RNA levels and consequently protein synthesis. In this study, we report the identification of single nucleotide polymorphisms (SNPs) in promoter regions of candidate genes involved in development, cellular differentiation and muscle growth in Sus scrofa. We identified SNPs in the promoter regions of genes belonging to the Myogenic Regulatory Factors (MRF) gene family (the Myogenic Differentiation gene, MYOD1) and to Growth and Differentiation Factors (GDF) gene family (Myostatin gene, MSTN, GDF8), in Casertana and Large White breeds. The purpose of this study was to investigate if polymorphisms in the promoters could affect the transcriptional activity of these genes. With this aim, we evaluated in vitro the functional activity of the luciferase reporter gene luc2 activity, driven by two constructs carrying different promoter haplotypes. We tested the effects of the G302A (U12574) transition on the promoter efficiency in MYOD1 gene. We ascertained a difference in transcription efficiency for the two variants. A stronger activity of the A-carrying construct is more evident in C2C12. The luciferase expression driven by the MYOD1-A allelic variant displayed a 3.8-fold increased transcriptional activity. We investigated the activity of two haplotype variants (AY527152) in the promoter of GDF8 gene. The haploptype-1 (A435-A447-A879) up-regulated the expression of the reporter gene by a two-fold increase, and hence presumably of the GDF8 gene, in both CHO and C2C12 cultured cells. In vitro the MYOD1-A allelic variant could up-regulate the expression of MYOD1 gene. Additionally, we could assess a different response of in vitro gene expression according to cell type used to transfect constructs, suggesting that MyoD activation is regulated by mechanisms that are specific of myoblasts.

Characterization of Pseudomonas putida Genes Responsive to Nutrient Limitation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Syn, Chris K.; Magnuson, Jon K.; Kingsley, Mark T.

2004-06-01

The low bioavailability of nutrients and oxygen in the soil environment has hampered successful expression of biodegradation/biocontrol genes that are driven by promoters highly active during routine laboratory conditions of high nutrient- and oxygen-availability. Hence, in the present study, expression of the gus-tagged genes in 12 Tn5-gus mutants of the soil microbe Pseudomonas putida PNL-MK25 was examined under various conditions chosen to mimic the soil environment: low carbon, phosphate, nitrate, or oxygen, and in the rhizosphere. Based on their expression profiles, three nutrient-responsive mutant (NRM) strains, NRM5, NRM7, and NRM17, were selected for identification of the tagged genes. In themore » mutant strain NRM5, expression of the glutamate dehydrogenase (gdhA) gene was increased between 4.9- to 26.4-fold under various low nutrient conditions. In NRM7, expression of the novel NADPH:quinone oxidoreductase-like (nql) gene was consistently amongst the highest and was synergistically upregulated by low nutrient and anoxic conditions. The cyoD gene in NRM17, which encodes the fourth subunit of the cytochrome o ubiquinol oxidase complex, had decreased expression in low nutrient conditions but its absolute expression levels was still amongst the highest. Additionally, it was independent of oxygen availability, in contrast to that in E. coli.« less

Artificial selection on brain-expressed genes during the domestication of dog.

PubMed

Li, Yan; Vonholdt, Bridgett M; Reynolds, Andy; Boyko, Adam R; Wayne, Robert K; Wu, Dong-Dong; Zhang, Ya-Ping

2013-08-01

Domesticated dogs have many unique behaviors not found in gray wolves that have augmented their interaction and communication with humans. The genetic basis of such unique behaviors in dogs remains poorly understood. We found that genes within regions highly differentiated between outbred Chinese native dogs (CNs) and wolves show high bias for expression localized to brain tissues, particularly the prefrontal cortex, a specific region responsible for complex cognitive behaviors. In contrast, candidate genes showing high population differentiation between CNs and German Shepherd dogs (GSs) did not demonstrate significant expression bias. These observations indicate that these candidate genes highly expressed in the brain have rapidly evolved. This rapid evolution was probably driven by artificial selection during the primary transition from wolves to ancient dogs and was consistent with the evolution of dog-specific characteristics, such as behavior transformation, for thousands of years.

Identifying stochastic oscillations in single-cell live imaging time series using Gaussian processes

PubMed Central

Manning, Cerys; Rattray, Magnus

2017-01-01

Multiple biological processes are driven by oscillatory gene expression at different time scales. Pulsatile dynamics are thought to be widespread, and single-cell live imaging of gene expression has lead to a surge of dynamic, possibly oscillatory, data for different gene networks. However, the regulation of gene expression at the level of an individual cell involves reactions between finite numbers of molecules, and this can result in inherent randomness in expression dynamics, which blurs the boundaries between aperiodic fluctuations and noisy oscillators. This underlies a new challenge to the experimentalist because neither intuition nor pre-existing methods work well for identifying oscillatory activity in noisy biological time series. Thus, there is an acute need for an objective statistical method for classifying whether an experimentally derived noisy time series is periodic. Here, we present a new data analysis method that combines mechanistic stochastic modelling with the powerful methods of non-parametric regression with Gaussian processes. Our method can distinguish oscillatory gene expression from random fluctuations of non-oscillatory expression in single-cell time series, despite peak-to-peak variability in period and amplitude of single-cell oscillations. We show that our method outperforms the Lomb-Scargle periodogram in successfully classifying cells as oscillatory or non-oscillatory in data simulated from a simple genetic oscillator model and in experimental data. Analysis of bioluminescent live-cell imaging shows a significantly greater number of oscillatory cells when luciferase is driven by a Hes1 promoter (10/19), which has previously been reported to oscillate, than the constitutive MoMuLV 5’ LTR (MMLV) promoter (0/25). The method can be applied to data from any gene network to both quantify the proportion of oscillating cells within a population and to measure the period and quality of oscillations. It is publicly available as a MATLAB package. PMID:28493880

TESTING HIGH-DIMENSIONAL COVARIANCE MATRICES, WITH APPLICATION TO DETECTING SCHIZOPHRENIA RISK GENES

PubMed Central

Zhu, Lingxue; Lei, Jing; Devlin, Bernie; Roeder, Kathryn

2017-01-01

Scientists routinely compare gene expression levels in cases versus controls in part to determine genes associated with a disease. Similarly, detecting case-control differences in co-expression among genes can be critical to understanding complex human diseases; however statistical methods have been limited by the high dimensional nature of this problem. In this paper, we construct a sparse-Leading-Eigenvalue-Driven (sLED) test for comparing two high-dimensional covariance matrices. By focusing on the spectrum of the differential matrix, sLED provides a novel perspective that accommodates what we assume to be common, namely sparse and weak signals in gene expression data, and it is closely related with Sparse Principal Component Analysis. We prove that sLED achieves full power asymptotically under mild assumptions, and simulation studies verify that it outperforms other existing procedures under many biologically plausible scenarios. Applying sLED to the largest gene-expression dataset obtained from post-mortem brain tissue from Schizophrenia patients and controls, we provide a novel list of genes implicated in Schizophrenia and reveal intriguing patterns in gene co-expression change for Schizophrenia subjects. We also illustrate that sLED can be generalized to compare other gene-gene “relationship” matrices that are of practical interest, such as the weighted adjacency matrices. PMID:29081874

TESTING HIGH-DIMENSIONAL COVARIANCE MATRICES, WITH APPLICATION TO DETECTING SCHIZOPHRENIA RISK GENES.

PubMed

Zhu, Lingxue; Lei, Jing; Devlin, Bernie; Roeder, Kathryn

2017-09-01

Scientists routinely compare gene expression levels in cases versus controls in part to determine genes associated with a disease. Similarly, detecting case-control differences in co-expression among genes can be critical to understanding complex human diseases; however statistical methods have been limited by the high dimensional nature of this problem. In this paper, we construct a sparse-Leading-Eigenvalue-Driven (sLED) test for comparing two high-dimensional covariance matrices. By focusing on the spectrum of the differential matrix, sLED provides a novel perspective that accommodates what we assume to be common, namely sparse and weak signals in gene expression data, and it is closely related with Sparse Principal Component Analysis. We prove that sLED achieves full power asymptotically under mild assumptions, and simulation studies verify that it outperforms other existing procedures under many biologically plausible scenarios. Applying sLED to the largest gene-expression dataset obtained from post-mortem brain tissue from Schizophrenia patients and controls, we provide a novel list of genes implicated in Schizophrenia and reveal intriguing patterns in gene co-expression change for Schizophrenia subjects. We also illustrate that sLED can be generalized to compare other gene-gene "relationship" matrices that are of practical interest, such as the weighted adjacency matrices.

A microRNA embedded AAV alpha-synuclein gene silencing vector for dopaminergic neurons

PubMed Central

Han, Ye; Khodr, Christina E.; Sapru, Mohan K.; Pedapati, Jyothi; Bohn, Martha C.

2011-01-01

Alpha-synuclein (SNCA), an abundantly expressed presynaptic protein, is implicated in Parkinson disease (PD). Since over-expression of human SNCA (hSNCA) leads to death of dopaminergic (DA) neurons in human, rodent and fly brain, hSNCA gene silencing may reduce levels of toxic forms of SNCA and ameliorate degeneration of DA neurons in PD. To begin to develop a gene therapy for PD based on hSNCA gene silencing, two AAV gene silencing vectors were designed, and tested for efficiency and specificity of silencing, as well as toxicity in vitro. The same hSNCA silencing sequence (shRNA) was used in both vectors, but in one vector, the shRNA was embedded in a microRNA backbone and driven by a pol II promoter, and in the other the shRNA was not embedded in a microRNA and was driven by a pol III promoter. Both vectors silenced hSNCA to the same extent in 293T cells transfected with hSNCA. In DA PC12 cells, neither vector decreased expression of rat SNCA, tyrosine hydroxylase (TH), dopamine transporter (DAT) or the vesicular monoamine transporter (VMAT). However, the mir30 embedded vector was significantly less toxic to both PC12 and SH-SY5Y cells. Our in vitro data suggest that this miRNA-embedded silencing vector may be ideal for chronic in vivo SNCA gene silencing in DA neurons. PMID:21338582

MOPED 2.5—An Integrated Multi-Omics Resource: Multi-Omics Profiling Expression Database Now Includes Transcriptomics Data

PubMed Central

Montague, Elizabeth; Stanberry, Larissa; Higdon, Roger; Janko, Imre; Lee, Elaine; Anderson, Nathaniel; Choiniere, John; Stewart, Elizabeth; Yandl, Gregory; Broomall, William; Kolker, Natali

2014-01-01

Abstract Multi-omics data-driven scientific discovery crucially rests on high-throughput technologies and data sharing. Currently, data are scattered across single omics repositories, stored in varying raw and processed formats, and are often accompanied by limited or no metadata. The Multi-Omics Profiling Expression Database (MOPED, http://moped.proteinspire.org) version 2.5 is a freely accessible multi-omics expression database. Continual improvement and expansion of MOPED is driven by feedback from the Life Sciences Community. In order to meet the emergent need for an integrated multi-omics data resource, MOPED 2.5 now includes gene relative expression data in addition to protein absolute and relative expression data from over 250 large-scale experiments. To facilitate accurate integration of experiments and increase reproducibility, MOPED provides extensive metadata through the Data-Enabled Life Sciences Alliance (DELSA Global, http://delsaglobal.org) metadata checklist. MOPED 2.5 has greatly increased the number of proteomics absolute and relative expression records to over 500,000, in addition to adding more than four million transcriptomics relative expression records. MOPED has an intuitive user interface with tabs for querying different types of omics expression data and new tools for data visualization. Summary information including expression data, pathway mappings, and direct connection between proteins and genes can be viewed on Protein and Gene Details pages. These connections in MOPED provide a context for multi-omics expression data exploration. Researchers are encouraged to submit omics data which will be consistently processed into expression summaries. MOPED as a multi-omics data resource is a pivotal public database, interdisciplinary knowledge resource, and platform for multi-omics understanding. PMID:24910945

Computational genetic neuroanatomy of the developing mouse brain: dimensionality reduction, visualization, and clustering.

PubMed

Ji, Shuiwang

2013-07-11

The structured organization of cells in the brain plays a key role in its functional efficiency. This delicate organization is the consequence of unique molecular identity of each cell gradually established by precise spatiotemporal gene expression control during development. Currently, studies on the molecular-structural association are beginning to reveal how the spatiotemporal gene expression patterns are related to cellular differentiation and structural development. In this article, we aim at a global, data-driven study of the relationship between gene expressions and neuroanatomy in the developing mouse brain. To enable visual explorations of the high-dimensional data, we map the in situ hybridization gene expression data to a two-dimensional space by preserving both the global and the local structures. Our results show that the developing brain anatomy is largely preserved in the reduced gene expression space. To provide a quantitative analysis, we cluster the reduced data into groups and measure the consistency with neuroanatomy at multiple levels. Our results show that the clusters in the low-dimensional space are more consistent with neuroanatomy than those in the original space. Gene expression patterns and developing brain anatomy are closely related. Dimensionality reduction and visual exploration facilitate the study of this relationship.

The reverse evolution from multicellularity to unicellularity during carcinogenesis.

PubMed

Chen, Han; Lin, Fangqin; Xing, Ke; He, Xionglei

2015-03-09

Theoretical reasoning suggests that cancer may result from a knockdown of the genetic constraints that evolved for the maintenance of metazoan multicellularity. By characterizing the whole-life history of a xenograft tumour, here we show that metastasis is driven by positive selection for general loss-of-function mutations on multicellularity-related genes. Expression analyses reveal mainly downregulation of multicellularity-related genes and an evolving expression profile towards that of embryonic stem cells, the cell type resembling unicellular life in its capacity of unlimited clonal proliferation. Also, the emergence of metazoan multicellularity ~600 Myr ago is accompanied by an elevated birth rate of cancer genes, and there are more loss-of-function tumour suppressors than activated oncogenes in a typical tumour. These data collectively suggest that cancer represents a loss-of-function-driven reverse evolution back to the unicellular 'ground state'. This cancer evolution model may account for inter-/intratumoural genetic heterogeneity, could explain distant-organ metastases and hold implications for cancer therapy.

Biasogram: Visualization of Confounding Technical Bias in Gene Expression Data

PubMed Central

Krzystanek, Marcin; Szallasi, Zoltan; Eklund, Aron C.

2013-01-01

Gene expression profiles of clinical cohorts can be used to identify genes that are correlated with a clinical variable of interest such as patient outcome or response to a particular drug. However, expression measurements are susceptible to technical bias caused by variation in extraneous factors such as RNA quality and array hybridization conditions. If such technical bias is correlated with the clinical variable of interest, the likelihood of identifying false positive genes is increased. Here we describe a method to visualize an expression matrix as a projection of all genes onto a plane defined by a clinical variable and a technical nuisance variable. The resulting plot indicates the extent to which each gene is correlated with the clinical variable or the technical variable. We demonstrate this method by applying it to three clinical trial microarray data sets, one of which identified genes that may have been driven by a confounding technical variable. This approach can be used as a quality control step to identify data sets that are likely to yield false positive results. PMID:23613961

Modulation of light-driven arousal by LIM-homeodomain transcription factor Apterous in large PDF-positive lateral neurons of the Drosophila brain

PubMed Central

Shimada, Naoto; Inami, Show; Sato, Shoma; Kitamoto, Toshihiro; Sakai, Takaomi

2016-01-01

Apterous (Ap), the best studied LIM-homeodomain transcription factor in Drosophila, cooperates with the cofactor Chip (Chi) to regulate transcription of specific target genes. Although Ap regulates various developmental processes, its function in the adult brain remains unclear. Here, we report that Ap and Chi in the neurons expressing PDF, a neuropeptide, play important roles in proper sleep/wake regulation in adult flies. PDF-expressing neurons consist of two neuronal clusters: small ventral-lateral neurons (s-LNvs) acting as the circadian pacemaker and large ventral-lateral neurons (l-LNvs) regulating light-driven arousal. We identified that Ap localizes to the nuclei of s-LNvs and l-LNvs. In light-dark (LD) cycles, RNAi knockdown or the targeted expression of dominant-negative forms of Ap or Chi in PDF-expressing neurons or l-LNvs promoted arousal. In contrast, in constant darkness, knockdown of Ap in PDF-expressing neurons did not promote arousal, indicating that a reduced Ap function in PDF-expressing neurons promotes light-driven arousal. Furthermore, Ap expression in l-LNvs showed daily rhythms (peaking at midnight), which are generated by a direct light-dependent mechanism rather than by the endogenous clock. These results raise the possibility that the daily oscillation of Ap expression in l-LNvs may contribute to the buffering of light-driven arousal in wild-type flies. PMID:27853240

Modulation of light-driven arousal by LIM-homeodomain transcription factor Apterous in large PDF-positive lateral neurons of the Drosophila brain.

PubMed

Shimada, Naoto; Inami, Show; Sato, Shoma; Kitamoto, Toshihiro; Sakai, Takaomi

2016-11-17

Apterous (Ap), the best studied LIM-homeodomain transcription factor in Drosophila, cooperates with the cofactor Chip (Chi) to regulate transcription of specific target genes. Although Ap regulates various developmental processes, its function in the adult brain remains unclear. Here, we report that Ap and Chi in the neurons expressing PDF, a neuropeptide, play important roles in proper sleep/wake regulation in adult flies. PDF-expressing neurons consist of two neuronal clusters: small ventral-lateral neurons (s-LNvs) acting as the circadian pacemaker and large ventral-lateral neurons (l-LNvs) regulating light-driven arousal. We identified that Ap localizes to the nuclei of s-LNvs and l-LNvs. In light-dark (LD) cycles, RNAi knockdown or the targeted expression of dominant-negative forms of Ap or Chi in PDF-expressing neurons or l-LNvs promoted arousal. In contrast, in constant darkness, knockdown of Ap in PDF-expressing neurons did not promote arousal, indicating that a reduced Ap function in PDF-expressing neurons promotes light-driven arousal. Furthermore, Ap expression in l-LNvs showed daily rhythms (peaking at midnight), which are generated by a direct light-dependent mechanism rather than by the endogenous clock. These results raise the possibility that the daily oscillation of Ap expression in l-LNvs may contribute to the buffering of light-driven arousal in wild-type flies.

Functional Analysis of Maize Silk-Specific ZmbZIP25 Promoter.

PubMed

Li, Wanying; Yu, Dan; Yu, Jingjuan; Zhu, Dengyun; Zhao, Qian

2018-03-12

ZmbZIP25 ( Zea mays bZIP (basic leucine zipper) transcription factor 25) is a function-unknown protein that belongs to the D group of the bZIP transcription factor family. RNA-seq data showed that the expression of ZmbZIP25 was tissue-specific in maize silks, and this specificity was confirmed by RT-PCR (reverse transcription-polymerase chain reaction). In situ RNA hybridization showed that ZmbZIP25 was expressed exclusively in the xylem of maize silks. A 5' RACE (rapid amplification of cDNA ends) assay identified an adenine residue as the transcription start site of the ZmbZIP25 gene. To characterize this silk-specific promoter, we isolated and analyzed a 2450 bp (from -2083 to +367) and a 2600 bp sequence of ZmbZIP25 (from -2083 to +517, the transcription start site was denoted +1). Stable expression assays in Arabidopsis showed that the expression of the reporter gene GUS driven by the 2450 bp ZmbZIP25 5'-flanking fragment occurred exclusively in the papillae of Arabidopsis stigmas. Furthermore, transient expression assays in maize indicated that GUS and GFP expression driven by the 2450 bp ZmbZIP25 5'-flanking sequences occurred only in maize silks and not in other tissues. However, no GUS or GFP expression was driven by the 2600 bp ZmbZIP25 5'-flanking sequences in either stable or transient expression assays. A series of deletion analyses of the 2450 bp ZmbZIP25 5'-flanking sequence was performed in transgenic Arabidopsis plants, and probable elements prediction analysis revealed the possible presence of negative regulatory elements within the 161 bp region from -1117 to -957 that were responsible for the specificity of the ZmbZIP25 5'-flanking sequence.

Functional Analysis of Maize Silk-Specific ZmbZIP25 Promoter

PubMed Central

Li, Wanying; Yu, Dan; Yu, Jingjuan; Zhu, Dengyun; Zhao, Qian

2018-01-01

ZmbZIP25 (Zea mays bZIP (basic leucine zipper) transcription factor 25) is a function-unknown protein that belongs to the D group of the bZIP transcription factor family. RNA-seq data showed that the expression of ZmbZIP25 was tissue-specific in maize silks, and this specificity was confirmed by RT-PCR (reverse transcription-polymerase chain reaction). In situ RNA hybridization showed that ZmbZIP25 was expressed exclusively in the xylem of maize silks. A 5′ RACE (rapid amplification of cDNA ends) assay identified an adenine residue as the transcription start site of the ZmbZIP25 gene. To characterize this silk-specific promoter, we isolated and analyzed a 2450 bp (from −2083 to +367) and a 2600 bp sequence of ZmbZIP25 (from −2083 to +517, the transcription start site was denoted +1). Stable expression assays in Arabidopsis showed that the expression of the reporter gene GUS driven by the 2450 bp ZmbZIP25 5′-flanking fragment occurred exclusively in the papillae of Arabidopsis stigmas. Furthermore, transient expression assays in maize indicated that GUS and GFP expression driven by the 2450 bp ZmbZIP25 5′-flanking sequences occurred only in maize silks and not in other tissues. However, no GUS or GFP expression was driven by the 2600 bp ZmbZIP25 5′-flanking sequences in either stable or transient expression assays. A series of deletion analyses of the 2450 bp ZmbZIP25 5′-flanking sequence was performed in transgenic Arabidopsis plants, and probable elements prediction analysis revealed the possible presence of negative regulatory elements within the 161 bp region from −1117 to −957 that were responsible for the specificity of the ZmbZIP25 5′-flanking sequence. PMID:29534529

Primate Lentiviruses Modulate NF-κB Activity by Multiple Mechanisms to Fine-Tune Viral and Cellular Gene Expression

PubMed Central

Heusinger, Elena; Kirchhoff, Frank

2017-01-01

The transcription factor nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) plays a complex role during the replication of primate lentiviruses. On the one hand, NF-κB is essential for induction of efficient proviral gene expression. On the other hand, this transcription factor contributes to the innate immune response and induces expression of numerous cellular antiviral genes. Recent data suggest that primate lentiviruses cope with this challenge by boosting NF-κB activity early during the replication cycle to initiate Tat-driven viral transcription and suppressing it at later stages to minimize antiviral gene expression. Human and simian immunodeficiency viruses (HIV and SIV, respectively) initially exploit their accessory Nef protein to increase the responsiveness of infected CD4+ T cells to stimulation. Increased NF-κB activity initiates Tat expression and productive replication. These events happen quickly after infection since Nef is rapidly expressed at high levels. Later during infection, Nef proteins of HIV-2 and most SIVs exert a very different effect: by down-modulating the CD3 receptor, an essential factor for T cell receptor (TCR) signaling, they prevent stimulation of CD4+ T cells via antigen-presenting cells and hence suppress further induction of NF-κB and an effective antiviral immune response. Efficient LTR-driven viral transcription is maintained because it is largely independent of NF-κB in the presence of Tat. In contrast, human immunodeficiency virus type 1 (HIV-1) and its simian precursors have lost the CD3 down-modulation function of Nef and use the late viral protein U (Vpu) to inhibit NF-κB activity by suppressing its nuclear translocation. In this review, we discuss how HIV-1 and other primate lentiviruses might balance viral and antiviral gene expression through a tight temporal regulation of NF-κB activity throughout their replication cycle. PMID:28261165

Epidermal Growth Factor and Interleukin-1β Utilize Divergent Signaling Pathways to Synergistically Upregulate Cyclooxygenase-2 Gene Expression in Human Amnion-Derived WISH Cells1

PubMed Central

Ackerman, William E.; Rovin, Brad H.; Kniss, Douglas A.

2006-01-01

In human parturition, uterotonic prostaglandins (PGs) arise predominantly via increased expression of cyclooxygenase-2 (COX-2 [also known as prostaglandin synthase 2]) within intra-uterine tissues. Interleukin-1 (IL-1) and epidermal growth factor (EGF), both inducers of COX-2 transcription, are among numerous factors that accumulate within amniotic fluid with advancing gestation. It was previously demonstrated that EGF could potentiate IL-1β-driven PGE2 production in amnion and amnion-derived (WISH) cells. To define the mechanism for this observation, we hypothesized that EGF and IL-1β might exhibit synergism in regulating COX-2 gene expression. In WISH cells, combined treatment with EGF and IL-1β resulted in a greater-than-additive increase in COX-2 mRNA relative to challenge with either agent independently. Augmentation of IL-1β-induced transactivation by EGF was not observed in cells harboring reporter plasmids bearing nuclear factor-kappa B (NFκB) regulatory elements alone, but was evident when a fragment (−891/+9) of the COX-2 gene 5′-promoter was present. Both agents transiently activated intermediates of multiple signaling pathways potentially involved in the regulation of COX-2 gene expression. The 26 S proteasome inhibitor, MG-132, selectively abrogated IL-1β-driven NFκB activation and COX-2 mRNA expression. Only pharmacologic blockade of the p38 mitogen-activated protein kinase eliminated COX-2 expression following EGF stimulation. We conclude that EGF and IL-1β appear to signal through different signaling cascades leading to COX-2 gene expression. IL-1β employs the NFκB pathway predominantly, while the spectrum of EGF signaling is broader and includes p38 kinase. The synergism observed between IL-1β and EGF does not rely on augmented NFκB function, but rather, occurs through differential use of independent response elements within the COX-2 promoter. PMID:15329330

Analysis of a cis-Acting Element Involved in Regulation by Estrogen of Human Angiotensinogen Gene Expression.

PubMed

Zhao, Yan-Yan; Sun, Kai-Lai; Ashok, Kumar

1998-01-01

The work was aimed to identify the estrogen responsive element in the human angiotensinogen gene. The nucleotide sequence between the transcription initiation site and TATA box in angiotensinogen gene promoter was found to be strongly homologous with the consensus estrogen responsive element. This sequence was confirmed as the estrogen responsive element (HAG ERE) by electrophoretic mobility shift assay. The recombinant expression vectors were constructed in which chloramphenicol acetyltransferase (CAT) reporter gene was driven by angiotensinogen core promoter with HAG ERE of by TK core promoter with multiplied HAG ERE, and were used in cotransfection with the human estrogen receptor expression vector into HepG(2) cells; CAT assays showed an increase of the CAT activity on 17beta-estradiol treatment in those transfectants. These results suggest that the human angiotensinogen gene is transcriptionally up-regulated by estrogen through the estrogen responsive element near TATA box of the promoter.

Modeling genome-wide dynamic regulatory network in mouse lungs with influenza infection using high-dimensional ordinary differential equations.

PubMed

Wu, Shuang; Liu, Zhi-Ping; Qiu, Xing; Wu, Hulin

2014-01-01

The immune response to viral infection is regulated by an intricate network of many genes and their products. The reverse engineering of gene regulatory networks (GRNs) using mathematical models from time course gene expression data collected after influenza infection is key to our understanding of the mechanisms involved in controlling influenza infection within a host. A five-step pipeline: detection of temporally differentially expressed genes, clustering genes into co-expressed modules, identification of network structure, parameter estimate refinement, and functional enrichment analysis, is developed for reconstructing high-dimensional dynamic GRNs from genome-wide time course gene expression data. Applying the pipeline to the time course gene expression data from influenza-infected mouse lungs, we have identified 20 distinct temporal expression patterns in the differentially expressed genes and constructed a module-based dynamic network using a linear ODE model. Both intra-module and inter-module annotations and regulatory relationships of our inferred network show some interesting findings and are highly consistent with existing knowledge about the immune response in mice after influenza infection. The proposed method is a computationally efficient, data-driven pipeline bridging experimental data, mathematical modeling, and statistical analysis. The application to the influenza infection data elucidates the potentials of our pipeline in providing valuable insights into systematic modeling of complicated biological processes.

The Prx1 limb enhancers: targeted gene expression in developing zebrafish pectoral fins.

PubMed

Hernández-Vega, Amayra; Minguillón, Carolina

2011-08-01

Limbs represent an excellent model to study the induction, growth, and patterning of several organs. A breakthrough to study gene function in various tissues has been the characterization of regulatory elements that allow tissue-specific interference of gene function. The mouse Prx1 promoter has been used to generate limb-specific mutants and overexpress genes in tetrapod limbs. Although zebrafish possess advantages that favor their use to study limb morphogenesis, there is no driver described suitable for specifically interfering with gene function in developing fins. We report the generation of zebrafish lines that express enhanced green fluorescent protein (EGFP) driven by the mouse Prx1 enhancer in developing pectoral fins. We also describe the expression pattern of the zebrafish prrx1 genes and identify three conserved non-coding elements (CNEs) that we use to generate fin-specific EGFP reporter lines. Finally, we show that the mouse and zebrafish regulatory elements may be used to modify gene function in pectoral fins. Copyright © 2011 Wiley-Liss, Inc.

Selection and validation of reliable housekeeping genes to evaluate Piscirickettsia salmonis gene expression.

PubMed

Flores-Herrera, Patricio; Arredondo-Zelada, Oscar; Marshall, Sergio H; Gómez, Fernando A

2018-06-01

Piscirickettsia salmonis is a highly aggressive facultative intracellular bacterium that challenges the sustainability of Chilean salmon production. Due to the limited knowledge of its biology, there is a need to identify key molecular markers that could help define the pathogenic potential of this bacterium. We think a model system should be implemented that efficiently evaluates the expression of putative bacterial markers by using validated, stable, and highly specific housekeeping genes to properly select target genes, which could lead to identifying those responsible for infection and disease induction in naturally infected fish. Here, we selected a set of validated reference or housekeeping genes for RT-qPCR expression analyses of P. salmonis under different growth and stress conditions, including an in vitro infection kinetic. After a thorough screening, we selected sdhA as the most reliable housekeeping gene able to represent stable and highly specific host reference genes for RT-qPCR-driven P. salmonis analysis. Copyright © 2018. Published by Elsevier B.V.

Gene Therapy for Human Lung Adenocarcinoma Using a Suicide Gene Driven by a Lung-Specific Promoter Delivered by JC Virus-Like Particles.

PubMed

Chao, Chun-Nun; Lin, Mien-Chun; Fang, Chiung-Yao; Chen, Pei-Lain; Chang, Deching; Shen, Cheng-Huang; Wang, Meilin

2016-01-01

Lung adenocarcinoma, the most commonly diagnosed type of lung cancer, has a poor prognosis even with combined surgery, chemotherapy, or molecular targeted therapies. Most patients are diagnosed with an in-operable advanced or metastatic disease, both pointing to the necessity of developing effective therapies for lung adenocarcinoma. Surfactant protein B (SP-B) has been found to be overexpressed in lung adenocarcinoma. In addition, it has also been demonstrated that human lung adenocarcinoma cells are susceptible to the JC polyomavirus (JCPyV) infection. Therefore, we designed that the JCPyV virus-like particle (VLP) packaged with an SP-B promoter-driven thymidine kinase suicide gene (pSPB-tk) for possible gene therapy of human lung adenocarcinoma. Plasmids expressing the GFP (pSPB-gfp) or thymidine kinase gene (pSPB-tk) under the control of the human SP-B promoter were constructed. The promoter's tissue specificity was tested by transfection of pSPB-gfp into A549, CH27, and H460 human lung carcinoma cells and non-lung cells. The JCPyV VLP's gene transfer efficiency and the selective cytotoxicity of pSPB-tk combined with ganciclovir (GCV) were tested in vitro and in a xenograft mouse model. In the current study, we found that SP-B promoter-driven GFP was specifically expressed in human lung adenocarcinoma (A549) and large cell carcinoma (H460) cells. JCPyV VLPs were able to deliver a GFP reporter gene into A549 cells for expression. Selective cytotoxicity was observed in A549 but not non-lung cells that were transfected with pSPB-tk or infected with pSPB-tk-carrying JCPyV VLPs. In mice injected with pSPB-tk-carrying JCPyV VLPs through the tail vein and treated with ganciclovir (GCV), a potent 80% inhibition of growth of human lung adenocarcinoma nodules resulted. The JCPyV VLPs combined with the use of SP-B promoter demonstrates effectiveness as a potential gene therapy against human lung adenocarcinoma.

Gene expression in gastrointestinal stromal tumors is distinguished by KIT genotype and anatomic site.

PubMed

Antonescu, Cristina R; Viale, Agnes; Sarran, Lisa; Tschernyavsky, Sylvia J; Gonen, Mithat; Segal, Neil H; Maki, Robert G; Socci, Nicholas D; DeMatteo, Ronald P; Besmer, Peter

2004-05-15

Gastrointestinal stromal tumors (GISTs) are specific KIT expressing and KIT-signaling driven mesenchymal tumors of the human digestive tract, many of which have KIT-activating mutations. Previous studies have found a relatively homogeneous gene expression profile in GIST, as compared with other histological types of sarcomas. Transcriptional heterogeneity within clinically or molecularly defined subsets of GISTs has not been previously reported. We tested the hypothesis that the gene expression profile in GISTs might be related to KIT genotype and possibly to other clinicopathological factors. An HG-U133A Affymetrix chip (22,000 genes) platform was used to determine the variability of gene expression in 28 KIT-expressing GIST samples from 24 patients. A control group of six intra-abdominal leiomyosarcomas was also included for comparison. Statistical analyses (t tests) were performed to identify discriminatory gene lists among various GIST subgroups. The levels of expression of various GIST subsets were also linked to a modified version of the growth factor/KIT signaling pathway to analyze differences at various steps in signal transduction. Genes involved in KIT signaling were differentially expressed among wild-type and mutant GISTs. High gene expression of potential drug targets, such as VEGF, MCSF, and BCL2 in the wild-type group, and Mesothelin in exon 9 GISTs were found. There was a striking difference in gene expression between stomach and small bowel GISTs. This finding was validated in four separate tumors, two gastric and two intestinal, from a patient with familial GIST with a germ-line KIT W557R substitution. GISTs have heterogeneous gene expression depending on KIT genotype and tumor location, which is seen at both the genomic level and the KIT signaling pathway in particular. These findings may explain their variable clinical behavior and response to therapy.

MicroRNAs shape circadian hepatic gene expression on a transcriptome-wide scale

PubMed Central

Du, Ngoc-Hien; Arpat, Alaaddin Bulak; De Matos, Mara; Gatfield, David

2014-01-01

A considerable proportion of mammalian gene expression undergoes circadian oscillations. Post-transcriptional mechanisms likely make important contributions to mRNA abundance rhythms. We have investigated how microRNAs (miRNAs) contribute to core clock and clock-controlled gene expression using mice in which miRNA biogenesis can be inactivated in the liver. While the hepatic core clock was surprisingly resilient to miRNA loss, whole transcriptome sequencing uncovered widespread effects on clock output gene expression. Cyclic transcription paired with miRNA-mediated regulation was thus identified as a frequent phenomenon that affected up to 30% of the rhythmic transcriptome and served to post-transcriptionally adjust the phases and amplitudes of rhythmic mRNA accumulation. However, only few mRNA rhythms were actually generated by miRNAs. Overall, our study suggests that miRNAs function to adapt clock-driven gene expression to tissue-specific requirements. Finally, we pinpoint several miRNAs predicted to act as modulators of rhythmic transcripts, and identify rhythmic pathways particularly prone to miRNA regulation. DOI: http://dx.doi.org/10.7554/eLife.02510.001 PMID:24867642

An interspecific fungal hybrid reveals cross-kingdom rules for allopolyploid gene expression patterns.

PubMed

Cox, Murray P; Dong, Ting; Shen, Genggeng; Dalvi, Yogesh; Scott, D Barry; Ganley, Austen R D

2014-03-01

Polyploidy, a state in which the chromosome complement has undergone an increase, is a major force in evolution. Understanding the consequences of polyploidy has received much attention, and allopolyploids, which result from the union of two different parental genomes, are of particular interest because they must overcome a suite of biological responses to this merger, known as "genome shock." A key question is what happens to gene expression of the two gene copies following allopolyploidization, but until recently the tools to answer this question on a genome-wide basis were lacking. Here we utilize high throughput transcriptome sequencing to produce the first genome-wide picture of gene expression response to allopolyploidy in fungi. A novel pipeline for assigning sequence reads to the gene copies was used to quantify their expression in a fungal allopolyploid. We find that the transcriptional response to allopolyploidy is predominantly conservative: both copies of most genes are retained; over half the genes inherit parental gene expression patterns; and parental differential expression is often lost in the allopolyploid. Strikingly, the patterns of gene expression change are highly concordant with the genome-wide expression results of a cotton allopolyploid. The very different nature of these two allopolyploids implies a conserved, eukaryote-wide transcriptional response to genome merger. We provide evidence that the transcriptional responses we observe are mostly driven by intrinsic differences between the regulatory systems in the parent species, and from this propose a mechanistic model in which the cross-kingdom conservation in transcriptional response reflects conservation of the mutational processes underlying eukaryotic gene regulatory evolution. This work provides a platform to develop a universal understanding of gene expression response to allopolyploidy and suggests that allopolyploids are an exceptional system to investigate gene regulatory changes that have evolved in the parental species prior to allopolyploidization.

CRISPR/Cas9 mediates efficient conditional mutagenesis in Drosophila.

PubMed

Xue, Zhaoyu; Wu, Menghua; Wen, Kejia; Ren, Menda; Long, Li; Zhang, Xuedi; Gao, Guanjun

2014-09-05

Existing transgenic RNA interference (RNAi) methods greatly facilitate functional genome studies via controlled silencing of targeted mRNA in Drosophila. Although the RNAi approach is extremely powerful, concerns still linger about its low efficiency. Here, we developed a CRISPR/Cas9-mediated conditional mutagenesis system by combining tissue-specific expression of Cas9 driven by the Gal4/upstream activating site system with various ubiquitously expressed guide RNA transgenes to effectively inactivate gene expression in a temporally and spatially controlled manner. Furthermore, by including multiple guide RNAs in a transgenic vector to target a single gene, we achieved a high degree of gene mutagenesis in specific tissues. The CRISPR/Cas9-mediated conditional mutagenesis system provides a simple and effective tool for gene function analysis, and complements the existing RNAi approach. Copyright © 2014 Xue et al.

An miRNA-mediated therapy for SCA6 blocks IRES-driven translation of the CACNA1A second cistron.

PubMed

Miyazaki, Yu; Du, Xiaofei; Muramatsu, Shin-Ichi; Gomez, Christopher M

2016-07-13

Spinocerebellar ataxia type 6 (SCA6) is a dominantly inherited neurodegenerative disease characterized by slowly progressive ataxia and Purkinje cell degeneration. SCA6 is caused by a polyglutamine repeat expansion within a second CACNA1A gene product, α1ACT. α1ACT expression is under the control of an internal ribosomal entry site (IRES) present within the CACNA1A coding region. Whereas SCA6 allele knock-in mice show indistinguishable phenotypes from wild-type littermates, expression of SCA6-associated α1ACT (α1ACTSCA6) driven by a Purkinje cell-specific promoter in mice produces slowly progressive ataxia and cerebellar atrophy. We developed an early-onset SCA6 mouse model using an adeno-associated virus (AAV)-based gene delivery system to ectopically express CACNA1A IRES-driven α1ACTSCA6 to test the potential of CACNA1A IRES-targeting therapies. Mice expressing AAV9-mediated CACNA1A IRES-driven α1ACTSCA6 exhibited early-onset ataxia, motor deficits, and Purkinje cell degeneration. We identified miR-3191-5p as a microRNA (miRNA) that targeted CACNA1A IRES and preferentially inhibited the CACNA1A IRES-driven translation of α1ACT in an Argonaute 4 (Ago4)-dependent manner. We found that eukaryotic initiation factors (eIFs), eIF4AII and eIF4GII, interacted with the CACNA1A IRES to enhance α1ACT translation. Ago4-bound miR-3191-5p blocked the interaction of eIF4AII and eIF4GII with the CACNA1A IRES, attenuating IRES-driven α1ACT translation. Furthermore, AAV9-mediated delivery of miR-3191-5p protected mice from the ataxia, motor deficits, and Purkinje cell degeneration caused by CACNA1A IRES-driven α1ACTSCA6 We have established proof of principle that viral delivery of an miRNA can rescue a disease phenotype through modulation of cellular IRES activity in a mouse model. Copyright © 2016, American Association for the Advancement of Science.

An miRNA-mediated therapy for SCA6 blocks IRES-driven translation of the CACNA1A second cistron

PubMed Central

Miyazaki, Yu; Du, Xiaofei; Muramatsu, Shin-ichi; Gomez, Christopher M.

2017-01-01

Spinocerebellar ataxia type 6 (SCA6) is a dominantly inherited neurodegenerative disease characterized by slowly progressive ataxia and Purkinje cell degeneration. SCA6 is caused by a polyglutamine repeat expansion within a second CACNA1A gene product, α1ACT. α1ACT expression is under the control of an internal ribosomal entry site (IRES) present within the CACNA1A coding region. Whereas SCA6 allele knock-in mice show indistinguishable phenotypes from wild-type littermates, expression of SCA6-associated α1ACT (α1ACTSCA6) driven by a Purkinje cell–specific promoter in mice produces slowly progressive ataxia and cerebellar atrophy. We developed an early-onset SCA6 mouse model using an adeno-associated virus (AAV)–based gene delivery system to ectopically express CACNA1A IRES–driven α1ACTSCA6 to test the potential of CACNA1A IRES–targeting therapies. Mice expressing AAV9-mediated CACNA1A IRES–driven α1ACTSCA6 exhibited early-onset ataxia, motor deficits, and Purkinje cell degeneration. We identified miR-3191-5p as a microRNA (miRNA) that targeted CACNA1A IRES and preferentially inhibited the CACNA1A IRES–driven translation of α1ACT in an Argonaute 4 (Ago4)–dependent manner. We found that eukaryotic initiation factors (eIFs), eIF4AII and eIF4GII, interacted with the CACNA1A IRES to enhance α1ACT translation. Ago4-bound miR-3191-5p blocked the interaction of eIF4AII and eIF4GII with the CACNA1A IRES, attenuating IRES-driven α1ACT translation. Furthermore, AAV9-mediated delivery of miR-3191-5p protected mice from the ataxia, motor deficits, and Purkinje cell degeneration caused by CACNA1A IRES–driven α1ACTSCA6. We have established proof of principle that viral delivery of an miRNA can rescue a disease phenotype through modulation of cellular IRES activity in a mouse model. PMID:27412786

Sodium-driven energy conversion for flagellar rotation of the earliest divergent hyperthermophilic bacterium.

PubMed

Takekawa, Norihiro; Nishiyama, Masayoshi; Kaneseki, Tsuyoshi; Kanai, Tamotsu; Atomi, Haruyuki; Kojima, Seiji; Homma, Michio

2015-08-05

Aquifex aeolicus is a hyperthermophilic, hydrogen-oxidizing and carbon-fixing bacterium that can grow at temperatures up to 95 °C. A. aeolicus has an almost complete set of flagellar genes that are conserved in bacteria. Here we observed that A. aeolicus has polar flagellum and can swim with a speed of 90 μm s(-1) at 85 °C. We expressed the A. aeolicus mot genes (motA and motB), which encode the torque generating stator proteins of the flagellar motor, in a corresponding mot nonmotile mutant of Escherichia coli. Its motility was slightly recovered by expression of A. aeolicus MotA and chimeric MotB whose periplasmic region was replaced with that of E. coli. A point mutation in the A. aeolicus MotA cytoplasmic region remarkably enhanced the motility. Using this system in E. coli, we demonstrate that the A. aeolicus motor is driven by Na(+). As motor proteins from hyperthermophilic bacteria represent the earliest motor proteins in evolution, this study strongly suggests that ancient bacteria used Na(+) for energy coupling of the flagellar motor. The Na(+)-driven flagellar genes might have been laterally transferred from early-branched bacteria into late-branched bacteria and the interaction surfaces of the stator and rotor seem not to change in evolution.

GeneMesh: a web-based microarray analysis tool for relating differentially expressed genes to MeSH terms.

PubMed

Jani, Saurin D; Argraves, Gary L; Barth, Jeremy L; Argraves, W Scott

2010-04-01

An important objective of DNA microarray-based gene expression experimentation is determining inter-relationships that exist between differentially expressed genes and biological processes, molecular functions, cellular components, signaling pathways, physiologic processes and diseases. Here we describe GeneMesh, a web-based program that facilitates analysis of DNA microarray gene expression data. GeneMesh relates genes in a query set to categories available in the Medical Subject Headings (MeSH) hierarchical index. The interface enables hypothesis driven relational analysis to a specific MeSH subcategory (e.g., Cardiovascular System, Genetic Processes, Immune System Diseases etc.) or unbiased relational analysis to broader MeSH categories (e.g., Anatomy, Biological Sciences, Disease etc.). Genes found associated with a given MeSH category are dynamically linked to facilitate tabular and graphical depiction of Entrez Gene information, Gene Ontology information, KEGG metabolic pathway diagrams and intermolecular interaction information. Expression intensity values of groups of genes that cluster in relation to a given MeSH category, gene ontology or pathway can be displayed as heat maps of Z score-normalized values. GeneMesh operates on gene expression data derived from a number of commercial microarray platforms including Affymetrix, Agilent and Illumina. GeneMesh is a versatile web-based tool for testing and developing new hypotheses through relating genes in a query set (e.g., differentially expressed genes from a DNA microarray experiment) to descriptors making up the hierarchical structure of the National Library of Medicine controlled vocabulary thesaurus, MeSH. The system further enhances the discovery process by providing links between sets of genes associated with a given MeSH category to a rich set of html linked tabular and graphic information including Entrez Gene summaries, gene ontologies, intermolecular interactions, overlays of genes onto KEGG pathway diagrams and heatmaps of expression intensity values. GeneMesh is freely available online at http://proteogenomics.musc.edu/genemesh/.

Turning the gene tap off; implications of regulating gene expression for cancer therapeutics

PubMed Central

Curtin, James F.; Candolfi, Marianela; Xiong, Weidong; Lowenstein, Pedro R.; Castro, Maria G.

2008-01-01

Cancer poses a tremendous therapeutic challenge worldwide, highlighting the critical need for developing novel therapeutics. A promising cancer treatment modality is gene therapy, which is a form of molecular medicine designed to introduce into target cells genetic material with therapeutic intent. Anticancer gene therapy strategies currently used in preclinical models, and in some cases in the clinic, include proapoptotic genes, oncolytic/replicative vectors, conditional cytotoxic approaches, inhibition of angiogenesis, inhibition of growth factor signaling, inactivation of oncogenes, inhibition of tumor invasion and stimulation of the immune system. The translation of these novel therapeutic modalities from the preclinical setting to the clinic has been driven by encouraging preclinical efficacy data and advances in gene delivery technologies. One area of intense research involves the ability to accurately regulate the levels of therapeutic gene expression to achieve enhanced efficacy and provide the capability to switch gene expression off completely if adverse side effects should arise. This feature could also be implemented to switch gene expression off when a successful therapeutic outcome ensues. Here, we will review recent developments related to the engineering of transcriptional switches within gene delivery systems, which could be implemented in clinical gene therapy applications directed at the treatment of cancer. PMID:18347132

Comparative ecological transcriptomics and the contribution of gene expression to the evolutionary potential of a threatened fish.

PubMed

Brauer, Chris J; Unmack, Peter J; Beheregaray, Luciano B

2017-12-01

Understanding whether small populations with low genetic diversity can respond to rapid environmental change via phenotypic plasticity is an outstanding research question in biology. RNA sequencing (RNA-seq) has recently provided the opportunity to examine variation in gene expression, a surrogate for phenotypic variation, in nonmodel species. We used a comparative RNA-seq approach to assess expression variation within and among adaptively divergent populations of a threatened freshwater fish, Nannoperca australis, found across a steep hydroclimatic gradient in the Murray-Darling Basin, Australia. These populations evolved under contrasting selective environments (e.g., dry/hot lowland; wet/cold upland) and represent opposite ends of the species' spectrum of genetic diversity and population size. We tested the hypothesis that environmental variation among isolated populations has driven the evolution of divergent expression at ecologically important genes using differential expression (DE) analysis and an anova-based comparative phylogenetic expression variance and evolution model framework based on 27,425 de novo assembled transcripts. Additionally, we tested whether gene expression variance within populations was correlated with levels of standing genetic diversity. We identified 290 DE candidate transcripts, 33 transcripts with evidence for high expression plasticity, and 50 candidates for divergent selection on gene expression after accounting for phylogenetic structure. Variance in gene expression appeared unrelated to levels of genetic diversity. Functional annotation of the candidate transcripts revealed that variation in water quality is an important factor influencing expression variation for N. australis. Our findings suggest that gene expression variation can contribute to the evolutionary potential of small populations. © 2017 John Wiley & Sons Ltd.

Global Profiling of Rice and Poplar Transcriptomes Highlights Key Conserved Circadian-Controlled Pathways and cis-Regulatory Modules

PubMed Central

Filichkin, Sergei A.; Breton, Ghislain; Priest, Henry D.; Dharmawardhana, Palitha; Jaiswal, Pankaj; Fox, Samuel E.; Michael, Todd P.; Chory, Joanne; Kay, Steve A.; Mockler, Todd C.

2011-01-01

Background Circadian clocks provide an adaptive advantage through anticipation of daily and seasonal environmental changes. In plants, the central clock oscillator is regulated by several interlocking feedback loops. It was shown that a substantial proportion of the Arabidopsis genome cycles with phases of peak expression covering the entire day. Synchronized transcriptome cycling is driven through an extensive network of diurnal and clock-regulated transcription factors and their target cis-regulatory elements. Study of the cycling transcriptome in other plant species could thus help elucidate the similarities and differences and identify hubs of regulation common to monocot and dicot plants. Methodology/Principal Findings Using a combination of oligonucleotide microarrays and data mining pipelines, we examined daily rhythms in gene expression in one monocotyledonous and one dicotyledonous plant, rice and poplar, respectively. Cycling transcriptomes were interrogated under different diurnal (driven) and circadian (free running) light and temperature conditions. Collectively, photocycles and thermocycles regulated about 60% of the expressed nuclear genes in rice and poplar. Depending on the condition tested, up to one third of oscillating Arabidopsis-poplar-rice orthologs were phased within three hours of each other suggesting a high degree of conservation in terms of rhythmic gene expression. We identified clusters of rhythmically co-expressed genes and searched their promoter sequences to identify phase-specific cis-elements, including elements that were conserved in the promoters of Arabidopsis, poplar, and rice. Conclusions/Significance Our results show that the cycling patterns of many circadian clock genes are highly conserved across poplar, rice, and Arabidopsis. The expression of many orthologous genes in key metabolic and regulatory pathways is diurnal and/or circadian regulated and phased to similar times of day. Our results confirm previous findings in Arabidopsis of three major classes of cis-regulatory modules within the plant circadian network: the morning (ME, GBOX), evening (EE, GATA), and midnight (PBX/TBX/SBX) modules. Identification of identical overrepresented motifs in the promoters of cycling genes from different species suggests that the core diurnal/circadian cis-regulatory network is deeply conserved between mono- and dicotyledonous species. PMID:21694767

Use of rhenium-188 for in vivo imaging and treatment of human cervical cancer cells transfected with lentivirus expressing sodium iodide symporter.

PubMed

Zhang, Min; Shi, Shuo; Guo, Rui; Miao, Yin; Li, Biao

2016-10-01

Although survival rates for cervical cancer have improved, they need further improvement in patients with distant metastases. The sodium iodine symporter (NIS) gene has often been used in cancer therapy and imaging. We examined the therapeutic effects of rhenium-188 (188Re) in a cervical cancer xenograft model expressing the NIS gene under the control of the tumor-specific human telomerase reverse transcriptase (hTERT) promoter. We constructed two recombinant lentiviral vectors expressing enhanced green fluorescent protein (eGFP) or the NIS gene driven by the hTERT promoter. To determine the tumor-specific transcriptional activity of the hTERT promoter, the eGFP-expressing vector was stably transfected into tumor cells and normal cells. A cervical cancer HeLa cell line stably expressing NIS (HeLa-TERTNIS) was created and examined in a similar way. HeLa and HeLa-TERTNIS tumor xenografts were transplanted in nude mice, and in vivo 188Re distribution was measured using micro-SPECT/CT imaging. The therapeutic effects of 188Re were assessed over 21 days on the basis of tumor volume and the immunohistochemical findings of excised tumors. eGFP expression controlled by the hTERT promoter was substantially higher in the tumor cells than normal cells. Quantitative PCR and western blotting confirmed that HeLa-TERTNIS cells expressed high levels of NIS mRNA and protein, respectively. Further, 188Re uptake and accumulation were significantly higher in HeLa-TERTNIS cells and xenografts than HeLa cells and xenografts. In vitro and in vivo, 188Re significantly reduced the survival of HeLa-TERTNIS cells and inhibited the growth of HeLa-TERTNIS xenografts, respectively. Immunohistochemical staining showed that HeLa-TERTNIS xenograft tumors expressed higher levels of NIS and caspase-3 and lower levels of Ki-67 than HeLa xenograft tumors. Our findings indicated that hTERT promoter-driven expression of the NIS gene in HeLa cells led to 188Re uptake and therapeutic effects. Thus, NIS-based gene therapy and imaging using the hTERT promoter and 188Re may be possible.

Involvement of the PRKCB1 gene in autistic disorder: significant genetic association and reduced neocortical gene expression.

PubMed

Lintas, C; Sacco, R; Garbett, K; Mirnics, K; Militerni, R; Bravaccio, C; Curatolo, P; Manzi, B; Schneider, C; Melmed, R; Elia, M; Pascucci, T; Puglisi-Allegra, S; Reichelt, K-L; Persico, A M

2009-07-01

Protein kinase C enzymes play an important role in signal transduction, regulation of gene expression and control of cell division and differentiation. The fsI and betaII isoenzymes result from the alternative splicing of the PKCbeta gene (PRKCB1), previously found to be associated with autism. We performed a family-based association study in 229 simplex and 5 multiplex families, and a postmortem study of PRKCB1 gene expression in temporocortical gray matter (BA41/42) of 11 autistic patients and controls. PRKCB1 gene haplotypes are significantly associated with autism (P<0.05) and have the autistic endophenotype of enhanced oligopeptiduria (P<0.05). Temporocortical PRKCB1 gene expression was reduced on average by 35 and 31% for the PRKCB1-1 and PRKCB1-2 isoforms (P<0.01 and <0.05, respectively) according to qPCR. Protein amounts measured for the PKCbetaII isoform were similarly decreased by 35% (P=0.05). Decreased gene expression characterized patients carrying the 'normal' PRKCB1 alleles, whereas patients homozygous for the autism-associated alleles displayed mRNA levels comparable to those of controls. Whole genome expression analysis unveiled a partial disruption in the coordinated expression of PKCbeta-driven genes, including several cytokines. These results confirm the association between autism and PRKCB1 gene variants, point toward PKCbeta roles in altered epithelial permeability, demonstrate a significant downregulation of brain PRKCB1 gene expression in autism and suggest that it could represent a compensatory adjustment aimed at limiting an ongoing dysreactive immune process. Altogether, these data underscore potential PKCbeta roles in autism pathogenesis and spur interest in the identification and functional characterization of PRKCB1 gene variants conferring autism vulnerability.

Modeling Bi-modality Improves Characterization of Cell Cycle on Gene Expression in Single Cells

PubMed Central

Danaher, Patrick; Finak, Greg; Krouse, Michael; Wang, Alice; Webster, Philippa; Beechem, Joseph; Gottardo, Raphael

2014-01-01

Advances in high-throughput, single cell gene expression are allowing interrogation of cell heterogeneity. However, there is concern that the cell cycle phase of a cell might bias characterizations of gene expression at the single-cell level. We assess the effect of cell cycle phase on gene expression in single cells by measuring 333 genes in 930 cells across three phases and three cell lines. We determine each cell's phase non-invasively without chemical arrest and use it as a covariate in tests of differential expression. We observe bi-modal gene expression, a previously-described phenomenon, wherein the expression of otherwise abundant genes is either strongly positive, or undetectable within individual cells. This bi-modality is likely both biologically and technically driven. Irrespective of its source, we show that it should be modeled to draw accurate inferences from single cell expression experiments. To this end, we propose a semi-continuous modeling framework based on the generalized linear model, and use it to characterize genes with consistent cell cycle effects across three cell lines. Our new computational framework improves the detection of previously characterized cell-cycle genes compared to approaches that do not account for the bi-modality of single-cell data. We use our semi-continuous modelling framework to estimate single cell gene co-expression networks. These networks suggest that in addition to having phase-dependent shifts in expression (when averaged over many cells), some, but not all, canonical cell cycle genes tend to be co-expressed in groups in single cells. We estimate the amount of single cell expression variability attributable to the cell cycle. We find that the cell cycle explains only 5%–17% of expression variability, suggesting that the cell cycle will not tend to be a large nuisance factor in analysis of the single cell transcriptome. PMID:25032992

Id expression in amphioxus and lamprey highlights the role of gene cooption during neural crest evolution

NASA Technical Reports Server (NTRS)

Meulemans, Daniel; McCauley, David; Bronner-Fraser, Marianne

2003-01-01

Neural crest cells are unique to vertebrates and generate many of the adult structures that differentiate them from their closest invertebrate relatives, the cephalochordates. Id genes are robust markers of neural crest cells at all stages of development. We compared Id gene expression in amphioxus and lamprey to ask if cephalochordates deploy Id genes at the neural plate border and dorsal neural tube in a manner similar to vertebrates. Furthermore, we examined whether Id expression in these cells is a basal vertebrate trait or a derived feature of gnathostomes. We found that while expression of Id genes in the mesoderm and endoderm is conserved between amphioxus and vertebrates, expression in the lateral neural plate border and dorsal neural tube is a vertebrate novelty. Furthermore, expression of lamprey Id implies that recruitment of Id genes to these cells occurred very early in the vertebrate lineage. Based on expression in amphioxus we postulate that Id cooption conferred sensory cell progenitor-like properties upon the lateral neurectoderm, and pharyngeal mesoderm-like properties upon cranial neural crest. Amphioxus Id expression is also consistent with homology between the anterior neurectoderm of amphioxus and the presumptive placodal ectoderm of vertebrates. These observations support the idea that neural crest evolution was driven in large part by cooption of multipurpose transcriptional regulators from other tissues and cell types.

Synaptic Plasticity and NO-cGMP-PKG Signaling Coordinately Regulate ERK-Driven Gene Expression in the Lateral Amygdala and in the Auditory Thalamus Following Pavlovian Fear Conditioning

ERIC Educational Resources Information Center

Ota, Kristie T.; Monsey, Melissa S.; Wu, Melissa S.; Young, Grace J.; Schafe, Glenn E.

2010-01-01

We have recently hypothesized that NO-cGMP-PKG signaling in the lateral nucleus of the amygdala (LA) during auditory fear conditioning coordinately regulates ERK-driven transcriptional changes in both auditory thalamic (MGm/PIN) and LA neurons that serve to promote pre- and postsynaptic alterations at thalamo-LA synapses, respectively. In the…

Advancing biopharmaceutical process development by system-level data analysis and integration of omics data.

PubMed

Schaub, Jochen; Clemens, Christoph; Kaufmann, Hitto; Schulz, Torsten W

2012-01-01

Development of efficient bioprocesses is essential for cost-effective manufacturing of recombinant therapeutic proteins. To achieve further process improvement and process rationalization comprehensive data analysis of both process data and phenotypic cell-level data is essential. Here, we present a framework for advanced bioprocess data analysis consisting of multivariate data analysis (MVDA), metabolic flux analysis (MFA), and pathway analysis for mapping of large-scale gene expression data sets. This data analysis platform was applied in a process development project with an IgG-producing Chinese hamster ovary (CHO) cell line in which the maximal product titer could be increased from about 5 to 8 g/L.Principal component analysis (PCA), k-means clustering, and partial least-squares (PLS) models were applied to analyze the macroscopic bioprocess data. MFA and gene expression analysis revealed intracellular information on the characteristics of high-performance cell cultivations. By MVDA, for example, correlations between several essential amino acids and the product concentration were observed. Also, a grouping into rather cell specific productivity-driven and process control-driven processes could be unraveled. By MFA, phenotypic characteristics in glycolysis, glutaminolysis, pentose phosphate pathway, citrate cycle, coupling of amino acid metabolism to citrate cycle, and in the energy yield could be identified. By gene expression analysis 247 deregulated metabolic genes were identified which are involved, inter alia, in amino acid metabolism, transport, and protein synthesis.

Deletion of Nrf2 reduces skeletal mechanical properties and decreases load-driven bone formation.

PubMed

Sun, Yong-Xin; Li, Lei; Corry, Kylie A; Zhang, Pei; Yang, Yang; Himes, Evan; Mihuti, Cristina Layla; Nelson, Cecilia; Dai, Guoli; Li, Jiliang

2015-05-01

Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor expressed in many cell types, including osteoblasts, osteocytes, and osteoclasts. Nrf2 has been considered a master regulator of cytoprotective genes against oxidative and chemical insults. The lack of Nrf2 can induce pathologies in multiple organs. The aim of this study was to investigate the role of Nrf2 in load-driven bone metabolism using Nrf2 knockout (KO) mice. Compared to age-matched littermate wild-type controls, Nrf2 KO mice have significantly lowered femoral bone mineral density (-7%, p<0.05), bone formation rate (-40%, p<0.05), as well as ultimate force (-11%, p<0.01). The ulna loading experiment showed that Nrf2 KO mice were less responsive than littermate controls, as indicated by reduction in relative mineralizing surface (rMS/BS, -69%, p<0.01) and relative bone formation rate (rBFR/BS, -84%, p<0.01). Furthermore, deletion of Nrf2 suppressed the load-driven gene expression of antioxidant enzymes and Wnt5a in cultured primary osteoblasts. Taken together, the results suggest that the loss-of-function mutation of Nrf2 in bone impairs bone metabolism and diminishes load-driven bone formation. Copyright © 2015 Elsevier Inc. All rights reserved.

Metatranscriptomics reveals temperature-driven functional changes in microbiome impacting cheese maturation rate

PubMed Central

De Filippis, Francesca; Genovese, Alessandro; Ferranti, Pasquale; Gilbert, Jack A.; Ercolini, Danilo

2016-01-01

Traditional cheeses harbour complex microbial consortia that play an important role in shaping typical sensorial properties. However, the microbial metabolism is considered difficult to control. Microbial community succession and the related gene expression were analysed during ripening of a traditional Italian cheese, identifying parameters that could be modified to accelerate ripening. Afterwards, we modulated ripening conditions and observed consistent changes in microbial community structure and function. We provide concrete evidence of the essential contribution of non-starter lactic acid bacteria in ripening-related activities. An increase in the ripening temperature promoted the expression of genes related to proteolysis, lipolysis and amino acid/lipid catabolism and significantly increases the cheese maturation rate. Moreover, temperature-promoted microbial metabolisms were consistent with the metabolomic profiles of proteins and volatile organic compounds in the cheese. The results clearly indicate how processing-driven microbiome responses can be modulated in order to optimize production efficiency and product quality. PMID:26911915

Querying Co-regulated Genes on Diverse Gene Expression Datasets Via Biclustering.

PubMed

Deveci, Mehmet; Küçüktunç, Onur; Eren, Kemal; Bozdağ, Doruk; Kaya, Kamer; Çatalyürek, Ümit V

2016-01-01

Rapid development and increasing popularity of gene expression microarrays have resulted in a number of studies on the discovery of co-regulated genes. One important way of discovering such co-regulations is the query-based search since gene co-expressions may indicate a shared role in a biological process. Although there exist promising query-driven search methods adapting clustering, they fail to capture many genes that function in the same biological pathway because microarray datasets are fraught with spurious samples or samples of diverse origin, or the pathways might be regulated under only a subset of samples. On the other hand, a class of clustering algorithms known as biclustering algorithms which simultaneously cluster both the items and their features are useful while analyzing gene expression data, or any data in which items are related in only a subset of their samples. This means that genes need not be related in all samples to be clustered together. Because many genes only interact under specific circumstances, biclustering may recover the relationships that traditional clustering algorithms can easily miss. In this chapter, we briefly summarize the literature using biclustering for querying co-regulated genes. Then we present a novel biclustering approach and evaluate its performance by a thorough experimental analysis.

The Arabidopsis polyamine transporter LHR1/PUT3 modulates heat responsive gene expression by enhancing mRNA stability.

PubMed

Shen, Yun; Ruan, Qingxia; Chai, Haoxi; Yuan, Yongze; Yang, Wannian; Chen, Junping; Xin, Zhanguo; Shi, Huazhong

2016-12-01

Polyamines involve in gene regulation by interacting with and modulating the functions of various anionic macromolecules such as DNA, RNA and proteins. In this study, we identified an important function of the polyamine transporter LHR1 (LOWER EXPRESSION OF HEAT RESPONSIVE GENE1) in heat-inducible gene expression in Arabidopsis thaliana. The lhr1 mutant was isolated through a forward genetic screening for altered expression of the luciferase reporter gene driven by the promoter from the heat-inducible gene AtHSP18.2. The lhr1 mutant showed reduced induction of the luciferase gene in response to heat stress and was more sensitive to high temperature than the wild type. Map-based cloning identified that the LHR1 gene encodes the polyamine transporter PUT3 (POLYAMINE UPTAKE TRANSPORTER 3) localized in the plasma membrane. The LHR1/PUT3 is required for the uptake of extracellular polyamines and plays an important role in stabilizing the mRNAs of several crucial heat stress responsive genes under high temperature. Genome-wide gene expression analysis using RNA-seq identified an array of differentially expressed genes, among which the transcript levels of some of the heat shock protein genes significantly reduced in response to prolonged heat stress in the lhr1 mutant. Our findings revealed an important heat stress response and tolerance mechanism involving polyamine influx which modulates mRNA stability of heat-inducible genes under heat stress conditions. © 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.

Computational genetic neuroanatomy of the developing mouse brain: dimensionality reduction, visualization, and clustering

PubMed Central

2013-01-01

Background The structured organization of cells in the brain plays a key role in its functional efficiency. This delicate organization is the consequence of unique molecular identity of each cell gradually established by precise spatiotemporal gene expression control during development. Currently, studies on the molecular-structural association are beginning to reveal how the spatiotemporal gene expression patterns are related to cellular differentiation and structural development. Results In this article, we aim at a global, data-driven study of the relationship between gene expressions and neuroanatomy in the developing mouse brain. To enable visual explorations of the high-dimensional data, we map the in situ hybridization gene expression data to a two-dimensional space by preserving both the global and the local structures. Our results show that the developing brain anatomy is largely preserved in the reduced gene expression space. To provide a quantitative analysis, we cluster the reduced data into groups and measure the consistency with neuroanatomy at multiple levels. Our results show that the clusters in the low-dimensional space are more consistent with neuroanatomy than those in the original space. Conclusions Gene expression patterns and developing brain anatomy are closely related. Dimensionality reduction and visual exploration facilitate the study of this relationship. PMID:23845024

Stability of transgene integration and expression in subsequent generations of doubled haploid oilseed rape transformed with chitinase and beta-1,3-glucanase genes in a double-gene construct.

PubMed

Melander, Margareta; Kamnert, Iréne; Happstadius, Ingrid; Liljeroth, Erland; Bryngelsson, Tomas

2006-09-01

A double-gene construct with one chitinase and one beta-1,3-glucanase gene from barley, both driven by enhanced 35S promoters, was transformed into oilseed rape. From six primary transformants expressing both transgenes 10 doubled haploid lines were produced and studied for five generations. The number of inserted copies for both the genes was determined by Southern blotting and real-time PCR with full agreement between the two methods. When copy numbers were analysed in different generations, discrepancies were found, indicating that at least part of the inserted sequences were lost in one of the alleles of some doubled haploids. Chitinase and beta-1,3-glucanase expression was analysed by Western blotting in all five doubled haploid generations. Despite that both the genes were present on the same T-DNA and directed by the same promoter their expression pattern between generations was different. The beta-1,3-glucanase was expressed at high and stable levels in all generations, while the chitinase displayed lower expression that varied between generations. The transgenic plants did not show any major impact on fungal resistance when assayed in greenhouse, although purified beta-1,3-glucanase and chitinase caused retardment of fungal growth in vitro.

Sexual selection drives evolution and rapid turnover of male gene expression.

PubMed

Harrison, Peter W; Wright, Alison E; Zimmer, Fabian; Dean, Rebecca; Montgomery, Stephen H; Pointer, Marie A; Mank, Judith E

2015-04-07

The profound and pervasive differences in gene expression observed between males and females, and the unique evolutionary properties of these genes in many species, have led to the widespread assumption that they are the product of sexual selection and sexual conflict. However, we still lack a clear understanding of the connection between sexual selection and transcriptional dimorphism, often termed sex-biased gene expression. Moreover, the relative contribution of sexual selection vs. drift in shaping broad patterns of expression, divergence, and polymorphism remains unknown. To assess the role of sexual selection in shaping these patterns, we assembled transcriptomes from an avian clade representing the full range of sexual dimorphism and sexual selection. We use these species to test the links between sexual selection and sex-biased gene expression evolution in a comparative framework. Through ancestral reconstruction of sex bias, we demonstrate a rapid turnover of sex bias across this clade driven by sexual selection and show it to be primarily the result of expression changes in males. We use phylogenetically controlled comparative methods to demonstrate that phenotypic measures of sexual selection predict the proportion of male-biased but not female-biased gene expression. Although male-biased genes show elevated rates of coding sequence evolution, consistent with previous reports in a range of taxa, there is no association between sexual selection and rates of coding sequence evolution, suggesting that expression changes may be more important than coding sequence in sexual selection. Taken together, our results highlight the power of sexual selection to act on gene expression differences and shape genome evolution.

Regulating unfolded protein response activator HAC1p for production of thermostable raw-starch hydrolyzing α-amylase in Pichia pastoris.

PubMed

Huang, Mengmeng; Gao, Yanyun; Zhou, Xiangshan; Zhang, Yuanxing; Cai, Menghao

2017-03-01

Unfolded protein response (UPR) usually happens when expressing heterologous proteins in high level, which may help cells to facilitate protein processing. Here, we evaluated the effects of the UPR activator HAC1p on a raw-starch hydrolyzing α-amylase (Gs4j-amyA), so as to improve heterologous production of the enzyme in Pichia pastoris. The gene (amyA) encoding Gs4j-amyA was first codon-optimized and expressed in P. pastoris under the control of the AOX1 promoter. A high gene dosage (12 copies) of amyA facilitated amylase expression which produced an enzyme activity of 305 U/ml. A spliced HAC1 encoding an UPR activator HAC1p was then co-expressed and the dosage effects of HAC1 on amylase expression was investigated. Six copies of HAC1 driven by AOX1 promoter produced a high amylase activity of 2200 U/ml, further increasing by 621%. However, excessive gene dosages driven by the same promoter led to a titration effect of its transcription factors and decreased the amount of amyA transcripts. Thus, constitutive expression of HAC1 by GAP promotor was further involved and Gs4j-amyA activity reached 3700 U/ml finally, which was further increased by 68.2%. Moreover, Gs4j-amyA was glycosylated in P. pastoris which generated higher enzyme activity than that in E. coli. Generally, regulating HAC1p expression by different strategies enhanced amylase production by 11.1 folds, indicating a reference for expression of other proteins in P. pastoris.

Knockdown of the bovine prion gene PRNP by RNA interference (RNAi) technology.

PubMed

Sutou, Shizuyo; Kunishi, Miho; Kudo, Toshiyuki; Wongsrikeao, Pimprapar; Miyagishi, Makoto; Otoi, Takeshige

2007-07-26

Since prion gene-knockout mice do not contract prion diseases and animals in which production of prion protein (PrP) is reduced by half are resistant to the disease, we hypothesized that bovine animals with reduced PrP would be tolerant to BSE. Hence, attempts were made to produce bovine PRNP (bPRNP) that could be knocked down by RNA interference (RNAi) technology. Before an in vivo study, optimal conditions for knocking down bPRNP were determined in cultured mammalian cell systems. Factors examined included siRNA (short interfering RNA) expression plasmid vectors, target sites of PRNP, and lengths of siRNAs. Four siRNA expression plasmid vectors were used: three harboring different cloning sites were driven by the human U6 promoter (hU6), and one by the human tRNAVal promoter. Six target sites of bovine PRNP were designed using an algorithm. From 1 (22 mer) to 9 (19, 20, 21, 22, 23, 24, 25, 27, and 29 mer) siRNA expression vectors were constructed for each target site. As targets of siRNA, the entire bPRNP coding sequence was connected to the reporter gene of the fluorescent EGFP, or of firefly luciferase or Renilla luciferase. Target plasmid DNA was co-transfected with siRNA expression vector DNA into HeLaS3 cells, and fluorescence or luminescence was measured. The activities of siRNAs varied widely depending on the target sites, length of the siRNAs, and vectors used. Longer siRNAs were less effective, and 19 mer or 21 mer was generally optimal. Although 21 mer GGGGAGAACTTCACCGAAACT expressed by a hU6-driven plasmid with a Bsp MI cloning site was best under the present experimental conditions, the corresponding tRNA promoter-driven plasmid was almost equally useful. The effectiveness of this siRNA was confirmed by immunostaining and Western blotting. Four siRNA expression plasmid vectors, six target sites of bPRNP, and various lengths of siRNAs from 19 mer to 29 mer were examined to establish optimal conditions for knocking down of bPRNP in vitro. The most effective siRNA so far tested was 21 mer GGGGAGAACTTCACCGAAACT driven either by a hU6 or tRNA promoter, a finding that provides a basis for further studies in vivo.

A negative regulator encoded by a rice WRKY gene represses both abscisic acid and gibberellins signaling in aleurone cells.

PubMed

Zhang, Zhong-Lin; Shin, Margaret; Zou, Xiaolu; Huang, Jianzhi; Ho, Tun-hua David; Shen, Qingxi J

2009-05-01

Abscisic acid (ABA) and gibberellins (GAs) control several developmental processes including seed maturation, dormancy, and germination. The antagonism of these two hormones is well-documented. However, recent data from transcription profiling studies indicate that they can function as agonists in regulating the expression of many genes although the underlying mechanism is unclear. Here we report a rice WRKY gene, OsWRKY24, which encodes a protein that functions as a negative regulator of both GA and ABA signaling. Overexpression of OsWRKY24 via particle bombardment-mediated transient expression in aleurone cells represses the expression of two reporter constructs: the beta-glucuronidase gene driven by the GA-inducible Amy32b alpha-amylase promoter (Amy32b-GUS) and the ABA-inducible HVA22 promoter (HVA22-GUS). OsWRKY24 is unlikely a general repressor because it has little effect on the expression of the luciferase reporter gene driven by a constitutive ubiquitin promoter (UBI-Luciferase). As to the GA signaling, OsWRKY24 differs from OsWRKY51 and -71, two negative regulators specifically function in the GA signaling pathway, in several ways. First, OsWRKY24 contains two WRKY domains while OsWRKY51 and -71 have only one; both WRKY domains are essential for the full repressing activity of OsWRKY24. Second, binding of OsWRKY24 to the Amy32b promoter appears to involve sequences in addition to the TGAC cores of the W-boxes. Third, unlike OsWRKY71, OsWRKY24 is stable upon GA treatment. Together, these data demonstrate that OsWRKY24 is a novel type of transcriptional repressor that inhibits both GA and ABA signaling.

Adeno-associated virus type 2 rep gene-mediated inhibition of basal gene expression of human immunodeficiency virus type 1 involves its negative regulatory functions.

PubMed Central

Oelze, I; Rittner, K; Sczakiel, G

1994-01-01

Adeno-associated virus type 2 (AAV-2), a human parvovirus which is apathogenic in adults, inhibits replication and gene expression of human immunodeficiency virus type 1 (HIV-1) in human cells. The rep gene of AAV-2, which was shown earlier to be sufficient for this negative interference, also down-regulated the expression of heterologous sequences driven by the long terminal repeat (LTR) of HIV-1. This effect was observed in the absence of the HIV-1 transactivator Tat, i.e., at basal levels of LTR-driven transcription. In this work, we studied the involvement of functional subsequences of the HIV-1 LTR in rep-mediated inhibition in the absence of Tat. Mutated LTRs driving an indicator gene (cat) were cointroduced into human SW480 cells together with rep alone or with double-stranded DNA fragments or RNA containing sequences of the HIV-1 LTR. The results indicate that rep strongly enhances the function of negative regulatory elements of the LTR. In addition, the experiments revealed a transcribed sequence element located within the TAR-coding sequence termed AHHH (AAV-HIV homology element derived from HIV-1) which is involved in rep-mediated inhibition. The AHHH element is also involved in down-regulation of basal expression levels in the absence of rep, suggesting that AHHH also contributes to negative regulatory functions of the LTR of HIV-1. In contrast, positive regulatory elements of the HIV-1 LTR such as the NF kappa B and SP1 binding sites have no significant influence on the rep-mediated inhibition. Images PMID:8289357

UMG Lenti: Novel Lentiviral Vectors for Efficient Transgene- and Reporter Gene Expression in Human Early Hematopoietic Progenitors

PubMed Central

Chiarella, Emanuela; Carrà, Giovanna; Scicchitano, Stefania; Codispoti, Bruna; Mega, Tiziana; Lupia, Michela; Pelaggi, Daniela; Marafioti, Maria G.; Aloisio, Annamaria; Giordano, Marco; Nappo, Giovanna; Spoleti, Cristina B.; Grillone, Teresa; Giovannone, Emilia D.; Spina, Raffaella; Bernaudo, Francesca; Moore, Malcolm A. S.; Bond, Heather M.; Mesuraca, Maria; Morrone, Giovanni

2014-01-01

Lentiviral vectors are widely used to investigate the biological properties of regulatory proteins and/or of leukaemia-associated oncogenes by stably enforcing their expression in hematopoietic stem and progenitor cells. In these studies it is critical to be able to monitor and/or sort the infected cells, typically via fluorescent proteins encoded by the modified viral genome. The most popular strategy to ensure co-expression of transgene and reporter gene is to insert between these cDNAs an IRES element, thus generating bi-cistronic mRNAs whose transcription is driven by a single promoter. However, while the product of the gene located upstream of the IRES is generally abundantly expressed, the translation of the downstream cDNA (typically encoding the reporter protein) is often inconsistent, which hinders the detection and the isolation of transduced cells. To overcome these limitations, we developed novel lentiviral dual-promoter vectors (named UMG-LV5 and –LV6) where transgene expression is driven by the potent UBC promoter and that of the reporter protein, EGFP, by the minimal regulatory element of the WASP gene. These vectors, harboring two distinct transgenes, were tested in a variety of human haematopoietic cell lines as well as in primary human CD34+ cells in comparison with the FUIGW vector that contains the expression cassette UBC-transgene-IRES-EGFP. In these experiments both UMG-LV5 and UMG–LV6 yielded moderately lower transgene expression than FUIGW, but dramatically higher levels of EGFP, thereby allowing the easy distinction between transduced and non-transduced cells. An additional construct was produced, in which the cDNA encoding the reporter protein is upstream, and the transgene downstream of the IRES sequence. This vector, named UMG-LV11, proved able to promote abundant expression of both transgene product and EGFP in all cells tested. The UMG-LVs represent therefore useful vectors for gene transfer-based studies in hematopoietic stem and progenitor cells, as well as in non-hematopoietic cells. PMID:25502183

Transcriptomic response of sea urchin larvae Strongylocentrotus purpuratus to CO2-driven seawater acidification.

PubMed

Todgham, Anne E; Hofmann, Gretchen E

2009-08-01

Ocean acidification from the uptake of anthropogenic CO(2) is expected to have deleterious consequences for many calcifying marine animals. Forecasting the vulnerability of these marine organisms to climate change is linked to an understanding of whether species possess the physiological capacity to compensate for the potentially adverse effects of ocean acidification. We carried out a microarray-based transcriptomic analysis of the physiological response of larvae of a calcifying marine invertebrate, the purple sea urchin, Strongylocentrotus purpuratus, to CO(2)-driven seawater acidification. In lab-based cultures, larvae were raised under conditions approximating current ocean pH conditions (pH 8.01) and at projected, more acidic pH conditions (pH 7.96 and 7.88) in seawater aerated with CO(2) gas. Targeting expression of approximately 1000 genes involved in several biological processes, this study captured changes in gene expression patterns that characterize the transcriptomic response to CO(2)-driven seawater acidification of developing sea urchin larvae. In response to both elevated CO(2) scenarios, larvae underwent broad scale decreases in gene expression in four major cellular processes: biomineralization, cellular stress response, metabolism and apoptosis. This study underscores that physiological processes beyond calcification are impacted greatly, suggesting that overall physiological capacity and not just a singular focus on biomineralization processes is essential for forecasting the impact of future CO(2) conditions on marine organisms. Conducted on targeted and vulnerable species, genomics-based studies, such as the one highlighted here, have the potential to identify potential ;weak links' in physiological function that may ultimately determine an organism's capacity to tolerate future ocean conditions.

Chromatin programming by developmentally regulated transcription factors: lessons from the study of haematopoietic stem cell specification and differentiation.

PubMed

Obier, Nadine; Bonifer, Constanze

2016-11-01

Although the body plan of individuals is encoded in their genomes, each cell type expresses a different gene expression programme and therefore has access to only a subset of this information. Alterations to gene expression programmes are the underlying basis for the differentiation of multiple cell types and are driven by tissue-specific transcription factors (TFs) that interact with the epigenetic regulatory machinery to programme the chromatin landscape into transcriptionally active and inactive states. The haematopoietic system has long served as a paradigm for studying the molecular principles that regulate gene expression in development. In this review article, we summarize the current knowledge on the mechanism of action of TFs regulating haematopoietic stem cell specification and differentiation, and place this information into the context of general principles governing development. © 2016 Federation of European Biochemical Societies.

Novel transcriptional networks regulated by CLOCK in human neurons.

PubMed

Fontenot, Miles R; Berto, Stefano; Liu, Yuxiang; Werthmann, Gordon; Douglas, Connor; Usui, Noriyoshi; Gleason, Kelly; Tamminga, Carol A; Takahashi, Joseph S; Konopka, Genevieve

2017-11-01

The molecular mechanisms underlying human brain evolution are not fully understood; however, previous work suggested that expression of the transcription factor CLOCK in the human cortex might be relevant to human cognition and disease. In this study, we investigated this novel transcriptional role for CLOCK in human neurons by performing chromatin immunoprecipitation sequencing for endogenous CLOCK in adult neocortices and RNA sequencing following CLOCK knockdown in differentiated human neurons in vitro. These data suggested that CLOCK regulates the expression of genes involved in neuronal migration, and a functional assay showed that CLOCK knockdown increased neuronal migratory distance. Furthermore, dysregulation of CLOCK disrupts coexpressed networks of genes implicated in neuropsychiatric disorders, and the expression of these networks is driven by hub genes with human-specific patterns of expression. These data support a role for CLOCK-regulated transcriptional cascades involved in human brain evolution and function. © 2017 Fontenot et al.; Published by Cold Spring Harbor Laboratory Press.

Dynamics of lineage commitment revealed by single-cell transcriptomics of differentiating embryonic stem cells.

PubMed

Semrau, Stefan; Goldmann, Johanna E; Soumillon, Magali; Mikkelsen, Tarjei S; Jaenisch, Rudolf; van Oudenaarden, Alexander

2017-10-23

Gene expression heterogeneity in the pluripotent state of mouse embryonic stem cells (mESCs) has been increasingly well-characterized. In contrast, exit from pluripotency and lineage commitment have not been studied systematically at the single-cell level. Here we measure the gene expression dynamics of retinoic acid driven mESC differentiation from pluripotency to lineage commitment, using an unbiased single-cell transcriptomics approach. We find that the exit from pluripotency marks the start of a lineage transition as well as a transient phase of increased susceptibility to lineage specifying signals. Our study reveals several transcriptional signatures of this phase, including a sharp increase of gene expression variability and sequential expression of two classes of transcriptional regulators. In summary, we provide a comprehensive analysis of the exit from pluripotency and lineage commitment at the single cell level, a potential stepping stone to improved lineage manipulation through timing of differentiation cues.

A Dynamic Bronchial Airway Gene Expression Signature of Chronic Obstructive Pulmonary Disease and Lung Function Impairment

PubMed Central

Steiling, Katrina; van den Berge, Maarten; Hijazi, Kahkeshan; Florido, Roberta; Campbell, Joshua; Liu, Gang; Xiao, Ji; Zhang, Xiaohui; Duclos, Grant; Drizik, Eduard; Si, Huiqing; Perdomo, Catalina; Dumont, Charles; Coxson, Harvey O.; Alekseyev, Yuriy O.; Sin, Don; Pare, Peter; Hogg, James C.; McWilliams, Annette; Hiemstra, Pieter S.; Sterk, Peter J.; Timens, Wim; Chang, Jeffrey T.; Sebastiani, Paola; O’Connor, George T.; Bild, Andrea H.; Postma, Dirkje S.; Lam, Stephen

2013-01-01

Rationale: Molecular phenotyping of chronic obstructive pulmonary disease (COPD) has been impeded in part by the difficulty in obtaining lung tissue samples from individuals with impaired lung function. Objectives: We sought to determine whether COPD-associated processes are reflected in gene expression profiles of bronchial airway epithelial cells obtained by bronchoscopy. Methods: Gene expression profiling of bronchial brushings obtained from 238 current and former smokers with and without COPD was performed using Affymetrix Human Gene 1.0 ST Arrays. Measurements and Main Results: We identified 98 genes whose expression levels were associated with COPD status, FEV1% predicted, and FEV1/FVC. In silico analysis identified activating transcription factor 4 (ATF4) as a potential transcriptional regulator of genes with COPD-associated airway expression, and ATF4 overexpression in airway epithelial cells in vitro recapitulates COPD-associated gene expression changes. Genes with COPD-associated expression in the bronchial airway epithelium had similarly altered expression profiles in prior studies performed on small-airway epithelium and lung parenchyma, suggesting that transcriptomic alterations in the bronchial airway epithelium reflect molecular events found at more distal sites of disease activity. Many of the airway COPD-associated gene expression changes revert toward baseline after therapy with the inhaled corticosteroid fluticasone in independent cohorts. Conclusions: Our findings demonstrate a molecular field of injury throughout the bronchial airway of active and former smokers with COPD that may be driven in part by ATF4 and is modifiable with therapy. Bronchial airway epithelium may ultimately serve as a relatively accessible tissue in which to measure biomarkers of disease activity for guiding clinical management of COPD. PMID:23471465

A Search for Parent-of-Origin Effects on Honey Bee Gene Expression.

PubMed

Kocher, Sarah D; Tsuruda, Jennifer M; Gibson, Joshua D; Emore, Christine M; Arechavaleta-Velasco, Miguel E; Queller, David C; Strassmann, Joan E; Grozinger, Christina M; Gribskov, Michael R; San Miguel, Phillip; Westerman, Rick; Hunt, Greg J

2015-06-05

Parent-specific gene expression (PSGE) is little known outside of mammals and plants. PSGE occurs when the expression level of a gene depends on whether an allele was inherited from the mother or the father. Kin selection theory predicts that there should be extensive PSGE in social insects because social insect parents can gain inclusive fitness benefits by silencing parental alleles in female offspring. We searched for evidence of PSGE in honey bees using transcriptomes from reciprocal crosses between European and Africanized strains. We found 46 transcripts with significant parent-of-origin effects on gene expression, many of which overexpressed the maternal allele. Interestingly, we also found a large proportion of genes showing a bias toward maternal alleles in only one of the reciprocal crosses. These results indicate that PSGE may occur in social insects. The nonreciprocal effects could be largely driven by hybrid incompatibility between these strains. Future work will help to determine if these are indeed parent-of-origin effects that can modulate inclusive fitness benefits. Copyright © 2015 Kocher et al.

nm23-H1 gene driven by hTERT promoter induces inhibition of invasive phenotype and metastasis of lung cancer xenograft in mice.

PubMed

Fan, Yu; Yao, Yibing; Li, Lu; Wu, Zhihao; Xu, Feng; Hou, Mei; Wu, Heng; Shen, Yali; Wan, Haisu; Zhou, Qinghua

2013-02-01

Lung cancer is the leading cause of cancer death in both men and women worldwide. Tumor metastasis is an essential aspect of lung cancer progression and patient death. The nm23-H1 gene has been extensively investigated as a metastasis suppressor gene. Our previous studies have revealed: that a significant relationship exists between the low-level expression nm23-H1 in primary non-small cell lung cancer (NSCLC) with increased metastasis and a poor prognosis; that L9981-nm23-H1 cells (a nm23-H1 transfactant cell) exhibited lower cell proliferation rates, more G0/G1 phase growth, and an increase in apoptosis with a dramatic decrease in the tumor cells' ability to invade than L9981 cells did; and that L9981- nm23-H1 cells also demonstrated a significantly reduced lymph node and distant metastatic capacity in vivo than L9981 cells did in nude mice. In this study, we construct a plasmid containing the nm23-H1 gene, which was driven by the human telomerase reverse transcriptase (hTERT) promoter. We evaluated the anti-invasion and anti-metastatic effects of pGL3-hTP-nm23 on L9981, a human large cell lung cancer cell line with nm23-H1 negative expression, by transwell assay in vitro and bioluminescence in nude mice models. The toxicity of pGL3-hTP-nm23 and its effects on tumor growth were evaluated in nude mice models after gene therapy. The cell cycles, apoptosis, and proliferation of the nm23-H1 transfactant were also detected by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT assay) and flow cytometry (FCM). The results showed that the hTERT-promoter dramatically drives nm23-H1 gene expression, and induces inhibition of cell growth and migration in L9981-luc cells and MRC-5 cells in vitro. nm23-H1 also significantly inhibited the tumorigenesis and distant metastasis of L9981-luc cell in vivo. Moreover, no obvious side effect was detected in normal mouse tissues after intratumoral injection of the vector. The treatment of the nm23-H1 gene driven by hTERT promoter appears to be a promising approach for the gene therapy of nm23-H1 low-expressed tumors. © 2012 Tianjin Lung Cancer Institute and Wiley Publishing Asia Pty Ltd.

Gene-based association study of genes linked to hippocampal sclerosis of aging neuropathology: GRN, TMEM106B, ABCC9, and KCNMB2

PubMed Central

Katsumata, Yuriko; Nelson, Peter T.; Ellingson, Sally R.; Fardo, David W.

2017-01-01

Hippocampal sclerosis of aging (HS-Aging) is a common neurodegenerative condition associated with dementia. To learn more about genetic risk of HS-Aging pathology, we tested gene-based associations of the GRN, TMEM106B, ABCC9, and KCNMB2 genes, which were reported to be associated with HS-Aging pathology in previous studies. Genetic data were obtained from the Alzheimer’s Disease Genetics Consortium (ADGC), linked to autopsy-derived neuropathological outcomes from the National Alzheimer’s Coordinating Center (NACC). Of the 3,251 subjects included in the study, 271 (8.3%) were identified as an HS-Aging case. The significant gene-based association between the ABCC9 gene and HS-Aging appeared to be driven by a region in which a significant haplotype-based association was found. We tested this haplotype as an expression Quantitative Trait Locus (eQTL) using two different public-access brain gene expression databases. The HS-Aging pathology protective ABCC9 haplotype was associated with decreased ABCC9 expression, indicating a possible toxic gain of function. PMID:28131462

Human Papillomavirus Drives Tumor Development Throughout the Head and Neck: Improved Prognosis Is Associated With an Immune Response Largely Restricted to the Oropharynx

PubMed Central

Chakravarthy, Ankur; Henderson, Stephen; Thirdborough, Stephen M.; Ottensmeier, Christian H.; Su, Xiaoping; Lechner, Matt; Feber, Andrew; Thomas, Gareth J.

2016-01-01

Purpose In squamous cell carcinomas of the head and neck (HNSCC), the increasing incidence of oropharyngeal squamous cell carcinomas (OPSCCs) is attributable to human papillomavirus (HPV) infection. Despite commonly presenting at late stage, HPV-driven OPSCCs are associated with improved prognosis compared with HPV-negative disease. HPV DNA is also detectable in nonoropharyngeal (non-OPSCC), but its pathogenic role and clinical significance are unclear. The objectives of this study were to determine whether HPV plays a causal role in non-OPSCC and to investigate whether HPV confers a survival benefit in these tumors. Methods Meta-analysis was used to build a cross-tissue gene-expression signature for HPV-driven cancer. Classifiers trained by machine-learning approaches were used to predict the HPV status of 520 HNSCCs profiled by The Cancer Genome Atlas project. DNA methylation data were similarly used to classify 464 HNSCCs and these analyses were integrated with genomic, histopathology, and survival data to permit a comprehensive comparison of HPV transcript-positive OPSCC and non-OPSCC. Results HPV-driven tumors accounted for 4.1% of non-OPSCCs. Regardless of anatomic site, HPV+ HNSCCs shared highly similar gene expression and DNA methylation profiles; nonkeratinizing, basaloid histopathological features; and lack of TP53 or CDKN2A alterations. Improved overall survival, however, was largely restricted to HPV-driven OPSCCs, which were associated with increased levels of tumor-infiltrating lymphocytes compared with HPV-driven non-OPSCCs. Conclusion Our analysis identified a causal role for HPV in transcript-positive non-OPSCCs throughout the head and neck. Notably, however, HPV-driven non-OPSCCs display a distinct immune microenvironment and clinical behavior compared with HPV-driven OPSCCs. PMID:27863190

Vascular smooth muscle-specific knockdown of the noncardiac form of the L-type calcium channel by microRNA-based short hairpin RNA as a potential antihypertensive therapy.

PubMed

Rhee, Sung W; Stimers, Joseph R; Wang, Wenze; Pang, Li

2009-05-01

In different rodent models of hypertension, vascular voltage-gated L-type calcium channel (Ca(L)) current and vascular tone is increased because of increased expression of the noncardiac form of the Ca(L) (Ca(v)1.2). The objective of this study was to develop a small interfering RNA (siRNA) expression system against the noncardiac form of Ca(v)1.2 to reduce its expression in vascular smooth muscle cells (VSMCs). siRNAs expressing plasmids and appropriate controls were constructed and first screened in human embryonic kidney (HEK) 293 cells cotransfected with a rat Ca(v)1.2 expression vector. The most effective gene silencing was achieved with a modified mir-30a-based short hairpin RNA (shRNAmir) driven by the cytomegalovirus promoter. In A7r5 cells, a vascular smooth muscle cell line, two copies of shRNAmir driven by a chimeric VSMC-specific enhancer/promoter reduced endogenous Ca(v)1.2 expression by 61% and decreased the Ca(L) current carried by barium by 47%. Moreover, the chimeric vascular smooth muscle-specific enhancer/promoter displayed almost no activity in non-VSMCs (PC-12 and HEK 293). Because the proposed siRNA was designed to only target the noncardiac form of Ca(v)1.2, it did not affect the Ca(L) expression and function in cultured cardiomyocytes, even when driven by a stronger cytomegalovirus promoter. In conclusion, vascular Ca(v)1.2 expression and function were effectively reduced by VSMC-specific delivery of the noncardiac form of Ca(v)1.2 siRNA without similarly affecting cardiac Ca(L) expression and function. When coupled with a viral vector, this molecular intervention in vivo may provide a novel long-term vascular-specific gene therapy for hypertension.

Vascular Smooth Muscle-Specific Knockdown of the Noncardiac Form of the L-Type Calcium Channel by MicroRNA-Based Short Hairpin RNA as a Potential Antihypertensive Therapy

PubMed Central

Rhee, Sung W.; Stimers, Joseph R.; Wang, Wenze; Pang, Li

2009-01-01

In different rodent models of hypertension, vascular voltage-gated L-type calcium channel (CaL) current and vascular tone is increased because of increased expression of the noncardiac form of the CaL (Cav1.2). The objective of this study was to develop a small interfering RNA (siRNA) expression system against the noncardiac form of Cav1.2 to reduce its expression in vascular smooth muscle cells (VSMCs). siRNAs expressing plasmids and appropriate controls were constructed and first screened in human embryonic kidney (HEK) 293 cells cotransfected with a rat Cav1.2 expression vector. The most effective gene silencing was achieved with a modified mir-30a-based short hairpin RNA (shRNAmir) driven by the cytomegalovirus promoter. In A7r5 cells, a vascular smooth muscle cell line, two copies of shRNAmir driven by a chimeric VSMC-specific enhancer/promoter reduced endogenous Cav1.2 expression by 61% and decreased the CaL current carried by barium by 47%. Moreover, the chimeric vascular smooth muscle-specific enhancer/promoter displayed almost no activity in non-VSMCs (PC-12 and HEK 293). Because the proposed siRNA was designed to only target the noncardiac form of Cav1.2, it did not affect the CaL expression and function in cultured cardiomyocytes, even when driven by a stronger cytomegalovirus promoter. In conclusion, vascular Cav1.2 expression and function were effectively reduced by VSMC-specific delivery of the noncardiac form of Cav1.2 siRNA without similarly affecting cardiac CaL expression and function. When coupled with a viral vector, this molecular intervention in vivo may provide a novel long-term vascular-specific gene therapy for hypertension. PMID:19244098

KRAS-driven lung adenocarcinoma: combined DDR1/Notch inhibition as an effective therapy

PubMed Central

Ambrogio, Chiara; Nadal, Ernest; Villanueva, Alberto; Gómez-López, Gonzalo; Cash, Timothy P; Barbacid, Mariano; Santamaría, David

2016-01-01

Understanding the early evolution of cancer heterogeneity during the initial steps of tumorigenesis can uncover vulnerabilities of cancer cells that may be masked at later stages. We describe a comprehensive approach employing gene expression analysis in early lesions to identify novel therapeutic targets and the use of mouse models to test synthetic lethal drug combinations to treat human Kirsten rat sarcoma viral oncogene homologue (KRAS)-driven lung adenocarcinoma. PMID:27843638

Seed-Specific Expression of OsDWF4, a Rate-Limiting Gene Involved in Brassinosteroids Biosynthesis, Improves Both Grain Yield and Quality in Rice.

PubMed

Li, Qian-Feng; Yu, Jia-Wen; Lu, Jun; Fei, Hong-Yuan; Luo, Ming; Cao, Bu-Wei; Huang, Li-Chun; Zhang, Chang-Quan; Liu, Qiao-Quan

2018-04-18

Brassinosteroids (BRs) are essential plant-specific steroidal hormones that regulate diverse growth and developmental processes in plants. We evaluated the effects of OsDWF4, a gene that encodes a rate-limiting enzyme in BR biosynthesis, on both rice yield and quality when driven by the Gt1 or Ubi promoter, which correspond to seed-specific or constitutive expression, respectively. Generally, transgenic plants expressing OsDWF4 showed increased grain yield with more tillers and longer and heavier seeds. Moreover, the starch physicochemical properties of the transgenic rice were also improved. Interestingly, OsDWF4 was found to exert different effects on either rice yield or quality when driven by the different promoters. The overall performance of the pGt1::OsDWF4 lines was better than that of the pUbi::OsDWF4 lines. Our data not only demonstrate the effects of OsDWF4 overexpression on both rice yield and quality but also suggest that a seed-specific promoter is a good choice in BR-mediated rice breeding programs.

The Epstein–Barr virus nuclear protein SM is both a post-transcriptional inhibitor and activator of gene expression

PubMed Central

Ruvolo, Vivian; Wang, Eryu; Boyle, Sarah; Swaminathan, Sankar

1998-01-01

The Epstein–Barr virus (EBV) nuclear protein BS-MLF1 (SM) is expressed early after entry of EBV into the lytic cycle. SM transactivates reporter gene constructs driven by a wide variety of promoters, but the mechanism of SM action is poorly understood. In this study, we demonstrate that the SM protein inhibits expression of intron-containing genes and activates expression of intron-less genes. We demonstrate that SM has the predicted inhibitory effect on expression of a spliced EBV gene but activates an unspliced early EBV gene. SM inhibited gene expression at the post-transcriptional level by preventing the accumulation of nuclear and cytoplasmic RNA transcripts. Conversely, SM led to increased accumulation of nuclear mRNA from intron-less genes without affecting the rate of transcription, indicating that SM enhances nuclear RNA stability. The ratio of cytoplasmic to nuclear polyadenylated mRNA was increased in the presence of SM, suggesting that SM also enhances nucleo-cytoplasmic mRNA transport. The degree of transactivation by SM was dependent on the sequence of the 3′-untranslated region of the target mRNA. Finally, we demonstrate that the amino-terminal portion of SM fused to glutathione-S-transferase binds radioactively labeled RNA in vitro, indicating that SM is a single-stranded RNA binding protein. Importantly, the latent and immediate-early genes of EBV contain introns whereas many early and late genes do not. Thus, SM may down-regulate synthesis of host cell proteins and latent EBV proteins while simultaneously enhancing expression of specific lytic EBV genes by binding to mRNA and modulating its stability and transport. PMID:9671768

The Epstein-Barr virus nuclear protein SM is both a post-transcriptional inhibitor and activator of gene expression.

PubMed

Ruvolo, V; Wang, E; Boyle, S; Swaminathan, S

1998-07-21

The Epstein-Barr virus (EBV) nuclear protein BS-MLF1 (SM) is expressed early after entry of EBV into the lytic cycle. SM transactivates reporter gene constructs driven by a wide variety of promoters, but the mechanism of SM action is poorly understood. In this study, we demonstrate that the SM protein inhibits expression of intron-containing genes and activates expression of intron-less genes. We demonstrate that SM has the predicted inhibitory effect on expression of a spliced EBV gene but activates an unspliced early EBV gene. SM inhibited gene expression at the post-transcriptional level by preventing the accumulation of nuclear and cytoplasmic RNA transcripts. Conversely, SM led to increased accumulation of nuclear mRNA from intron-less genes without affecting the rate of transcription, indicating that SM enhances nuclear RNA stability. The ratio of cytoplasmic to nuclear polyadenylated mRNA was increased in the presence of SM, suggesting that SM also enhances nucleo-cytoplasmic mRNA transport. The degree of transactivation by SM was dependent on the sequence of the 3'-untranslated region of the target mRNA. Finally, we demonstrate that the amino-terminal portion of SM fused to glutathione-S-transferase binds radioactively labeled RNA in vitro, indicating that SM is a single-stranded RNA binding protein. Importantly, the latent and immediate-early genes of EBV contain introns whereas many early and late genes do not. Thus, SM may down-regulate synthesis of host cell proteins and latent EBV proteins while simultaneously enhancing expression of specific lytic EBV genes by binding to mRNA and modulating its stability and transport.

Preferential epigenetic programming of estrogen response after in utero xenoestrogen (bisphenol-A) exposure

PubMed Central

Jorgensen, Elisa M.; Alderman, Myles H.; Taylor, Hugh S.

2016-01-01

Bisphenol-A (BPA) is an environmentally ubiquitous estrogen-like endocrine-disrupting compound. Exposure to BPA in utero has been linked to female reproductive disorders, including endometrial hyperplasia and breast cancer. Estrogens are an etiological factor in many of these conditions. We sought to determine whether in utero exposure to BPA altered the global CpG methylation pattern of the uterine genome, subsequent gene expression, and estrogen response. Pregnant mice were exposed to an environmentally relevant dose of BPA or DMSO control. Uterine DNA and RNA were examined by using methylated DNA immunoprecipitation methylation microarray, expression microarray, and quantitative PCR. In utero BPA exposure altered the global CpG methylation profile of the uterine genome and subsequent gene expression. The effect on gene expression was not apparent until sexual maturation, which suggested that estrogen response was the primary alteration. Indeed, prenatal BPA exposure preferentially altered adult estrogen-responsive gene expression. Changes in estrogen response were accompanied by altered methylation that preferentially affected estrogen receptor-α (ERα)–binding genes. The majority of genes that demonstrated both altered expression and ERα binding had decreased methylation. BPA selectively altered the normal developmental programming of estrogen-responsive genes via modification of the genes that bind ERα. Gene–environment interactions driven by early life xenoestrogen exposure likely contributes to increased risk of estrogen-related disease in adults.—Jorgensen, E. M., Alderman, M. H., III, Taylor, H. S. Preferential epigenetic programming of estrogen response after in utero xenoestrogen (bisphenol-A) exposure. PMID:27312807

Classification of early-stage non-small cell lung cancer by weighing gene expression profiles with connectivity information.

PubMed

Zhang, Ao; Tian, Suyan

2018-05-01

Pathway-based feature selection algorithms, which utilize biological information contained in pathways to guide which features/genes should be selected, have evolved quickly and become widespread in the field of bioinformatics. Based on how the pathway information is incorporated, we classify pathway-based feature selection algorithms into three major categories-penalty, stepwise forward, and weighting. Compared to the first two categories, the weighting methods have been underutilized even though they are usually the simplest ones. In this article, we constructed three different genes' connectivity information-based weights for each gene and then conducted feature selection upon the resulting weighted gene expression profiles. Using both simulations and a real-world application, we have demonstrated that when the data-driven connectivity information constructed from the data of specific disease under study is considered, the resulting weighted gene expression profiles slightly outperform the original expression profiles. In summary, a big challenge faced by the weighting method is how to estimate pathway knowledge-based weights more accurately and precisely. Only until the issue is conquered successfully will wide utilization of the weighting methods be impossible. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Price, Morgan N.; Arkin, Adam P.; Alm, Eric J.

Operons are a major feature of all prokaryotic genomes, but how and why operon structures vary is not well understood. To elucidate the life-cycle of operons, we compared gene order between Escherichia coli K12 and its relatives and identified the recently formed and destroyed operons in E. coli. This allowed us to determine how operons form, how they become closely spaced, and how they die. Our findings suggest that operon evolution is driven by selection on gene expression patterns. First, both operon creation and operon destruction lead to large changes in gene expression patterns. For example, the removal of lysAmore » and ruvA from ancestral operons that contained essential genes allowed their expression to respond to lysine levels and DNA damage, respectively. Second, some operons have undergone accelerated evolution, with multiple new genes being added during a brief period. Third, although most operons are closely spaced because of a neutral bias towards deletion and because of selection against large overlaps, highly expressed operons tend to be widely spaced because of regulatory fine-tuning by intervening sequences. Although operon evolution seems to be adaptive, it need not be optimal: new operons often comprise functionally unrelated genes that were already in proximity before the operon formed.« less

Inhibition of PRMT3 activity selectively impairs LXR-driven transcription of hepatic lipogenic genes in vivo.

PubMed

Nahon, Joya E; Groeneveldt, Christianne; Geerling, Janine J; Van Eck, Miranda; Hoekstra, Menno

2018-05-18

Agonists for the liver X receptor (LXR) are considered promising therapeutic moieties in cholesterol-driven diseases by promoting cellular cholesterol efflux pathways. However, current clinical application of these agents is hampered by the concomitant LXR-induced activation of a lipogenic transcriptional network, leading to hepatic steatosis. Recent studies have suggested that protein arginine methyltransferase 3 (PRMT3) may act as a selective co-activator of LXR activity. Here we verified the hypothesis that PRMT3 inhibition selectively disrupts the ability of LXR to stimulate lipogenesis, while maintaining the capacity of LXR to modulate macrophage cholesterol homeostasis. A combination of the LXR agonist T0901317 and palm oil was administered to C57BL/6 mice to maximally stimulate LXR and PRMT3 activity. PRMT3 activity was inhibited using the allosteric inhibitor SGC707. Treatment with the PRMT3 inhibitor SGC707 did not negatively influence the T0901317/palm oil induced upregulation of the cholesterol efflux genes ABCA1 and ABCG1 in peritoneal cells. In contrast, SGC707 treatment was associated with a significant decrease in the hepatic expression of the lipogenic gene FAS (-64%). A similar trend was observed for SCD1 and ACC expression (-43%; -56%) This obstruction of lipogenic gene transcription coincided with a significant 2.3-fold decrease in liver triglyceride content as compared to the T0901317 and palm oil treated control group. We have shown that inhibition of PRMT3 activity by SGC707 treatment selectively impairs LXR-driven transcription of hepatic lipogenic genes, while the positive effect of LXR stimulation on macrophage cholesterol efflux pathways is maintained. This article is protected by copyright. All rights reserved.

Differential maturation of rhythmic clock gene expression during early development in medaka (Oryzias latipes).

PubMed

Cuesta, Ines H; Lahiri, Kajori; Lopez-Olmeda, Jose Fernando; Loosli, Felix; Foulkes, Nicholas S; Vallone, Daniela

2014-05-01

One key challenge for the field of chronobiology is to identify how circadian clock function emerges during early embryonic development. Teleosts such as the zebrafish are ideal models for studying circadian clock ontogeny since the entire process of development occurs ex utero in an optically transparent chorion. Medaka (Oryzias latipes) represents another powerful fish model for exploring early clock function with, like the zebrafish, many tools available for detailed genetic analysis. However, to date there have been no reports documenting circadian clock gene expression during medaka development. Here we have characterized the expression of key clock genes in various developmental stages and in adult tissues of medaka. As previously reported for other fish, light dark cycles are required for the emergence of clock gene expression rhythms in this species. While rhythmic expression of per and cry genes is detected very early during development and seems to be light driven, rhythmic clock and bmal expression appears much later around hatching time. Furthermore, the maturation of clock function seems to correlate with the appearance of rhythmic expression of these positive elements of the clock feedback loop. By accelerating development through elevated temperatures or by artificially removing the chorion, we show an earlier onset of rhythmicity in clock and bmal expression. Thus, differential maturation of key elements of the medaka clock mechanism depends on the developmental stage and the presence of the chorion.

Advanced Design of Dumbbell-shaped Genetic Minimal Vectors Improves Non-coding and Coding RNA Expression.

PubMed

Jiang, Xiaoou; Yu, Han; Teo, Cui Rong; Tan, Genim Siu Xian; Goh, Sok Chin; Patel, Parasvi; Chua, Yiqiang Kevin; Hameed, Nasirah Banu Sahul; Bertoletti, Antonio; Patzel, Volker

2016-09-01

Dumbbell-shaped DNA minimal vectors lacking nontherapeutic genes and bacterial sequences are considered a stable, safe alternative to viral, nonviral, and naked plasmid-based gene-transfer systems. We investigated novel molecular features of dumbbell vectors aiming to reduce vector size and to improve the expression of noncoding or coding RNA. We minimized small hairpin RNA (shRNA) or microRNA (miRNA) expressing dumbbell vectors in size down to 130 bp generating the smallest genetic expression vectors reported. This was achieved by using a minimal H1 promoter with integrated transcriptional terminator transcribing the RNA hairpin structure around the dumbbell loop. Such vectors were generated with high conversion yields using a novel protocol. Minimized shRNA-expressing dumbbells showed accelerated kinetics of delivery and transcription leading to enhanced gene silencing in human tissue culture cells. In primary human T cells, minimized miRNA-expressing dumbbells revealed higher stability and triggered stronger target gene suppression as compared with plasmids and miRNA mimics. Dumbbell-driven gene expression was enhanced up to 56- or 160-fold by implementation of an intron and the SV40 enhancer compared with control dumbbells or plasmids. Advanced dumbbell vectors may represent one option to close the gap between durable expression that is achievable with integrating viral vectors and short-term effects triggered by naked RNA.

Age-Related Gene Expression Differences in Monocytes from Human Neonates, Young Adults, and Older Adults

PubMed Central

Tong, Ann-Jay; Kollmann, Tobias R.; Smale, Stephen T.

2015-01-01

A variety of age-related differences in the innate and adaptive immune systems have been proposed to contribute to the increased susceptibility to infection of human neonates and older adults. The emergence of RNA sequencing (RNA-seq) provides an opportunity to obtain an unbiased, comprehensive, and quantitative view of gene expression differences in defined cell types from different age groups. An examination of ex vivo human monocyte responses to lipopolysaccharide stimulation or Listeria monocytogenes infection by RNA-seq revealed extensive similarities between neonates, young adults, and older adults, with an unexpectedly small number of genes exhibiting statistically significant age-dependent differences. By examining the differentially induced genes in the context of transcription factor binding motifs and RNA-seq data sets from mutant mouse strains, a previously described deficiency in interferon response factor-3 activity could be implicated in most of the differences between newborns and young adults. Contrary to these observations, older adults exhibited elevated expression of inflammatory genes at baseline, yet the responses following stimulation correlated more closely with those observed in younger adults. Notably, major differences in the expression of constitutively expressed genes were not observed, suggesting that the age-related differences are driven by environmental influences rather than cell-autonomous differences in monocyte development. PMID:26147648

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Jing; Ma, Zihao; Carr, Steven A.

Coexpression of mRNAs under multiple conditions is commonly used to infer cofunctionality of their gene products despite well-known limitations of this “guilt-by-association” (GBA) approach. Recent advancements in mass spectrometry-based proteomic technologies have enabled global expression profiling at the protein level; however, whether proteome profiling data can outperform transcriptome profiling data for coexpression based gene function prediction has not been systematically investigated. Here, we address this question by constructing and analyzing mRNA and protein coexpression networks for three cancer types with matched mRNA and protein profiling data from The Cancer Genome Atlas (TCGA) and the Clinical Proteomic Tumor Analysis Consortium (CPTAC).more » Our analyses revealed a marked difference in wiring between the mRNA and protein coexpression networks. Whereas protein coexpression was driven primarily by functional similarity between coexpressed genes, mRNA coexpression was driven by both cofunction and chromosomal colocalization of the genes. Functionally coherent mRNA modules were more likely to have their edges preserved in corresponding protein networks than functionally incoherent mRNA modules. Proteomic data strengthened the link between gene expression and function for at least 75% of Gene Ontology (GO) biological processes and 90% of KEGG pathways. A web application Gene2Net (http://cptac.gene2net.org) developed based on the three protein coexpression networks revealed novel gene-function relationships, such as linking ERBB2 (HER2) to lipid biosynthetic process in breast cancer, identifying PLG as a new gene involved in complement activation, and identifying AEBP1 as a new epithelial-mesenchymal transition (EMT) marker. Our results demonstrate that proteome profiling outperforms transcriptome profiling for coexpression based gene function prediction. Proteomics should be integrated if not preferred in gene function and human disease studies. Molecular & Cellular Proteomics 16: 10.1074/mcp.M116.060301, 121–134, 2017.« less

Synthetic dual-input mammalian genetic circuits enable tunable and stringent transcription control by chemical and light.

PubMed

Chen, Xianjun; Li, Ting; Wang, Xue; Du, Zengmin; Liu, Renmei; Yang, Yi

2016-04-07

Programmable transcription factors can enable precise control of gene expression triggered by a chemical inducer or light. To obtain versatile transgene system with combined benefits of a chemical inducer and light inducer, we created various chimeric promoters through the assembly of different copies of the tet operator and Gal4 operator module, which simultaneously responded to a tetracycline-responsive transcription factor and a light-switchable transactivator. The activities of these chimeric promoters can be regulated by tetracycline and blue light synergistically or antagonistically. Further studies of the antagonistic genetic circuit exhibited high spatiotemporal resolution and extremely low leaky expression, which therefore could be used to spatially and stringently control the expression of highly toxic protein Diphtheria toxin A for light regulated gene therapy. When transferring plasmids engineered for the gene switch-driven expression of a firefly luciferase (Fluc) into mice, the Fluc expression levels of the treated animals directly correlated with the tetracycline and light input program. We suggest that dual-input genetic circuits using TET and light that serve as triggers to achieve expression profiles may enable the design of robust therapeutic gene circuits for gene- and cell-based therapies. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Circadian Enhancers Coordinate Multiple Phases of Rhythmic Gene Transcription In Vivo

PubMed Central

Fang, Bin; Everett, Logan J.; Jager, Jennifer; Briggs, Erika; Armour, Sean M.; Feng, Dan; Roy, Ankur; Gerhart-Hines, Zachary; Sun, Zheng; Lazar, Mitchell A.

2014-01-01

SUMMARY Mammalian transcriptomes display complex circadian rhythms with multiple phases of gene expression that cannot be accounted for by current models of the molecular clock. We have determined the underlying mechanisms by measuring nascent RNA transcription around the clock in mouse liver. Unbiased examination of eRNAs that cluster in specific circadian phases identified functional enhancers driven by distinct transcription factors (TFs). We further identify on a global scale the components of the TF cistromes that function to orchestrate circadian gene expression. Integrated genomic analyses also revealed novel mechanisms by which a single circadian factor controls opposing transcriptional phases. These findings shed new light on the diversity and specificity of TF function in the generation of multiple phases of circadian gene transcription in a mammalian organ. PMID:25416951

Cloning, expression, and sequence analysis of the Bacillus methanolicus C1 methanol dehydrogenase gene.

PubMed Central

de Vries, G E; Arfman, N; Terpstra, P; Dijkhuizen, L

1992-01-01

The gene (mdh) coding for methanol dehydrogenase (MDH) of thermotolerant, methylotroph Bacillus methanolicus C1 has been cloned and sequenced. The deduced amino acid sequence of the mdh gene exhibited similarity to those of five other alcohol dehydrogenase (type III) enzymes, which are distinct from the long-chain zinc-containing (type I) or short-chain zinc-lacking (type II) enzymes. Highly efficient expression of the mdh gene in Escherichia coli was probably driven from its own promoter sequence. After purification of MDH from E. coli, the kinetic and biochemical properties of the enzyme were investigated. The physiological effect of MDH synthesis in E. coli and the role of conserved sequence patterns in type III alcohol dehydrogenases have been analyzed and are discussed. Images PMID:1644761

Circadian enhancers coordinate multiple phases of rhythmic gene transcription in vivo.

PubMed

Fang, Bin; Everett, Logan J; Jager, Jennifer; Briggs, Erika; Armour, Sean M; Feng, Dan; Roy, Ankur; Gerhart-Hines, Zachary; Sun, Zheng; Lazar, Mitchell A

2014-11-20

Mammalian transcriptomes display complex circadian rhythms with multiple phases of gene expression that cannot be accounted for by current models of the molecular clock. We have determined the underlying mechanisms by measuring nascent RNA transcription around the clock in mouse liver. Unbiased examination of enhancer RNAs (eRNAs) that cluster in specific circadian phases identified functional enhancers driven by distinct transcription factors (TFs). We further identify on a global scale the components of the TF cistromes that function to orchestrate circadian gene expression. Integrated genomic analyses also revealed mechanisms by which a single circadian factor controls opposing transcriptional phases. These findings shed light on the diversity and specificity of TF function in the generation of multiple phases of circadian gene transcription in a mammalian organ.

Diversity and Gene Expression of Phosphatase Genes Provide Insight into Soil Phosphorus Dynamics in a New Zealand Managed Grassland

NASA Astrophysics Data System (ADS)

Dunfield, K. E.; Gaiero, J. R.; Condron, L.

2017-12-01

Healthy and diverse communities of soil organisms influence key soil ecosystem services such as carbon sequestration, water quality protection, climate regulation and nutrient cycling. Microbially driven mineralization of organic phosphorus is an important contributor to plant available inorganic orthophosphates. In acidic soils, microbes produce non-specific acid phosphatases (NSAPs) which act on common forms of organic phosphorus (P). Our current understanding of P turnover in soils has been limited by lack of research tools capable of targeting these genes. Thus, we developed a set of oligonucleotide PCR primers that targeted bacteria with the genetic potential for acid phosphatase production. A long term randomized-block pasture trial was sampled following 22 years of continued aerial biomass removal and retention. Primers were used to target genes encoding alkaline phosphatase (phoD) and the three classes (CAAP, CBAP, CCAP) of non-specific acid phosphatases. PCR amplicons targeting total genes and gene transcripts were sequenced using Illumina MiSeq to understand the diversity of the bacterial phosphatase producing communities. In general, the majority of operational taxonomic units (OTUs) were shared across both treatments and across metagenomes and transcriptomes. However, analysis of DNA OTUs revealed significantly different communities driven by treatment differences (P < 0.05). Transcript expression was highest in the removed biomass treatment which corresponded the reduced Olsen P levels (15 vs. 36 mg kg-1 in retained treatment). Acid phosphatase activity was measured in all samples, and found to be highest in the biomass retained treatment (16.8 vs. 11.4 µmol g-1 dry soil h-1), likely elevated due to plant-derived enzymes; however, was still correlated to bacterial gene abundances. Overall, the phosphatase producing microbial communities responded to the effect of consistent P limitation as expected, through alteration in the composition of the community structure and through increased levels of gene expression of the phosphatase genes.

Co-transfection of decorin and interleukin-10 modulates pro-fibrotic extracellular matrix gene expression in human tenocyte culture

NASA Astrophysics Data System (ADS)

Abbah, Sunny A.; Thomas, Dilip; Browne, Shane; O'Brien, Timothy; Pandit, Abhay; Zeugolis, Dimitrios I.

2016-02-01

Extracellular matrix synthesis and remodelling are driven by increased activity of transforming growth factor beta 1 (TGF-β1). In tendon tissue repair, increased activity of TGF-β1 leads to progressive fibrosis. Decorin (DCN) and interleukin 10 (IL-10) antagonise pathological collagen synthesis by exerting a neutralising effect via downregulation of TGF-β1. Herein, we report that the delivery of DCN and IL-10 transgenes from a collagen hydrogel system supresses the constitutive expression of TGF-β1 and a range of pro-fibrotic extracellular matrix genes.

Genome-wide RNAi screening identifies protein damage as a regulator of osmoprotective gene expression.

PubMed

Lamitina, Todd; Huang, Chunyi George; Strange, Kevin

2006-08-08

The detection, stabilization, and repair of stress-induced damage are essential requirements for cellular life. All cells respond to osmotic stress-induced water loss with increased expression of genes that mediate accumulation of organic osmolytes, solutes that function as chemical chaperones and restore osmotic homeostasis. The signals and signaling mechanisms that regulate osmoprotective gene expression in animal cells are poorly understood. Here, we show that gpdh-1 and gpdh-2, genes that mediate the accumulation of the organic osmolyte glycerol, are essential for survival of the nematode Caenorhabditis elegans during osmotic stress. Expression of GFP driven by the gpdh-1 promoter (P(gpdh-1)::GFP) is detected only during hypertonic stress but is not induced by other stressors. Using P(gpdh-1)::GFP expression as a phenotype, we screened approximately 16,000 genes by RNAi feeding and identified 122 that cause constitutive activation of gpdh-1 expression and glycerol accumulation. Many of these genes function to regulate protein translation and cotranslational protein folding and to target and degrade denatured proteins, suggesting that the accumulation of misfolded proteins functions as a signal to activate osmoprotective gene expression and organic osmolyte accumulation in animal cells. Consistent with this hypothesis, 73% of these protein-homeostasis genes have been shown to slow age-dependent protein aggregation in C. elegans. Because diverse environmental stressors and numerous disease states result in protein misfolding, mechanisms must exist that discriminate between osmotically induced and other forms of stress-induced protein damage. Our findings provide a foundation for understanding how these damage-selectivity mechanisms function.

Genome-wide RNAi screening identifies protein damage as a regulator of osmoprotective gene expression

PubMed Central

Lamitina, Todd; Huang, Chunyi George; Strange, Kevin

2006-01-01

The detection, stabilization, and repair of stress-induced damage are essential requirements for cellular life. All cells respond to osmotic stress-induced water loss with increased expression of genes that mediate accumulation of organic osmolytes, solutes that function as chemical chaperones and restore osmotic homeostasis. The signals and signaling mechanisms that regulate osmoprotective gene expression in animal cells are poorly understood. Here, we show that gpdh-1 and gpdh-2, genes that mediate the accumulation of the organic osmolyte glycerol, are essential for survival of the nematode Caenorhabditis elegans during osmotic stress. Expression of GFP driven by the gpdh-1 promoter (Pgpdh-1::GFP) is detected only during hypertonic stress but is not induced by other stressors. Using Pgpdh-1::GFP expression as a phenotype, we screened ≈16,000 genes by RNAi feeding and identified 122 that cause constitutive activation of gpdh-1 expression and glycerol accumulation. Many of these genes function to regulate protein translation and cotranslational protein folding and to target and degrade denatured proteins, suggesting that the accumulation of misfolded proteins functions as a signal to activate osmoprotective gene expression and organic osmolyte accumulation in animal cells. Consistent with this hypothesis, 73% of these protein-homeostasis genes have been shown to slow age-dependent protein aggregation in C. elegans. Because diverse environmental stressors and numerous disease states result in protein misfolding, mechanisms must exist that discriminate between osmotically induced and other forms of stress-induced protein damage. Our findings provide a foundation for understanding how these damage-selectivity mechanisms function. PMID:16880390

CoGAPS matrix factorization algorithm identifies transcriptional changes in AP-2alpha target genes in feedback from therapeutic inhibition of the EGFR network

PubMed Central

Thakar, Manjusha; Howard, Jason D.; Kagohara, Luciane T.; Krigsfeld, Gabriel; Ranaweera, Ruchira S.; Hughes, Robert M.; Perez, Jimena; Jones, Siân; Favorov, Alexander V.; Carey, Jacob; Stein-O'Brien, Genevieve; Gaykalova, Daria A.; Ochs, Michael F.; Chung, Christine H.

2016-01-01

Patients with oncogene driven tumors are treated with targeted therapeutics including EGFR inhibitors. Genomic data from The Cancer Genome Atlas (TCGA) demonstrates molecular alterations to EGFR, MAPK, and PI3K pathways in previously untreated tumors. Therefore, this study uses bioinformatics algorithms to delineate interactions resulting from EGFR inhibitor use in cancer cells with these genetic alterations. We modify the HaCaT keratinocyte cell line model to simulate cancer cells with constitutive activation of EGFR, HRAS, and PI3K in a controlled genetic background. We then measure gene expression after treating modified HaCaT cells with gefitinib, afatinib, and cetuximab. The CoGAPS algorithm distinguishes a gene expression signature associated with the anticipated silencing of the EGFR network. It also infers a feedback signature with EGFR gene expression itself increasing in cells that are responsive to EGFR inhibitors. This feedback signature has increased expression of several growth factor receptors regulated by the AP-2 family of transcription factors. The gene expression signatures for AP-2alpha are further correlated with sensitivity to cetuximab treatment in HNSCC cell lines and changes in EGFR expression in HNSCC tumors with low CDKN2A gene expression. In addition, the AP-2alpha gene expression signatures are also associated with inhibition of MEK, PI3K, and mTOR pathways in the Library of Integrated Network-Based Cellular Signatures (LINCS) data. These results suggest that AP-2 transcription factors are activated as feedback from EGFR network inhibition and may mediate EGFR inhibitor resistance. PMID:27650546

PKCθ promotes c-Rel–driven mammary tumorigenesis in mice and humans by repressing estrogen receptor α synthesis

PubMed Central

Belguise, Karine; Sonenshein, Gail E.

2007-01-01

The vast majority of primary human breast cancer tissues display aberrant nuclear NF-κB c-Rel expression. A causal role for c-Rel in mammary tumorigenesis has been demonstrated using a c-Rel transgenic mouse model; however, tumors developed with a long latency, suggesting a second event is needed to trigger tumorigenesis. Here we show that c-Rel activity in the mammary gland is repressed by estrogen receptor α (ERα) signaling, and we identify an epigenetic mechanism in breast cancer mediated by activation of what we believe is a novel PKCθ-Akt pathway that leads to downregulation of ERα synthesis and derepression of c-Rel. ERα levels were lower in c-Rel–induced mammary tumors compared with normal mammary gland tissue. PKCθ induced c-Rel activity and target gene expression and promoted growth of c-Rel- and c-RelxCK2α–driven mouse mammary tumor–derived cell lines. RNA expression levels of PKCθ and c-Rel target genes were inversely correlated with ERα levels in human breast cancer specimens. PKCθ activated Akt, thereby inactivating forkhead box O protein 3a (FOXO3a) and leading to decreased synthesis of its target genes, ERα and p27Kip1. Thus we have shown that activation of PKCθ inhibits the FOXO3a/ERα/p27Kip1 axis that normally maintains an epithelial cell phenotype and induces c-Rel target genes, thereby promoting proliferation, survival, and more invasive breast cancer. PMID:18037997

Integration of High-Risk Human Papillomavirus into Cellular Cancer-Related Genes in Head and Neck Cancer Cell Lines

PubMed Central

Walline, Heather M; Komarck, Christine M; McHugh, Jonathan B; Tang, Alice L; Owen, John H; Teh, Bin T; McKean, Erin; Glover, Thomas; Graham, Martin P; Prince, Mark E; Chepeha, Douglas B; Chinn, Steven B; Ferris, Robert L; Gollin, Susanne M; Hoffmann, Thomas K; Bier, Henning; Brakenhoff, Ruud; Bradford, Carol R; Carey, Thomas E

2017-01-01

Background HPV-positive oropharyngeal cancer is generally associated with excellent response to therapy, but some HPV-positive tumors progress despite aggressive therapy. This study evaluates viral oncogene expression and viral integration sites in HPV16 and HPV18-positive squamous carcinoma cell lines. Methods E6-E7 alternate transcripts were assessed by RT-PCR. Detection of integrated papillomavirus sequences (DIPS-PCR) and sequencing identified viral insertion sites and affected host genes. Cellular gene expression was assessed across viral integration sites. Results All HPV-positive cell lines expressed alternate HPVE6/E7 splicing indicative of active viral oncogenesis. HPV integration occurred within cancer-related genes TP63, DCC, JAK1, TERT, ATR, ETV6, PGR, PTPRN2, and TMEM237 in 8 HNSCC lines but UM-SCC-105 and UM-GCC-1 had only intergenic integration. Conclusions HPV integration into cancer-related genes occurred in 7/9 HPV-positive cell lines and of these six were from tumors that progressed. HPV integration into cancer-related genes may be a secondary carcinogenic driver in HPV-driven tumors. PMID:28236344

Amniotic fluid RNA gene expression profiling provides insights into the phenotype of Turner syndrome.

PubMed

Massingham, Lauren J; Johnson, Kirby L; Scholl, Thomas M; Slonim, Donna K; Wick, Heather C; Bianchi, Diana W

2014-09-01

Turner syndrome is a sex chromosome aneuploidy with characteristic malformations. Amniotic fluid, a complex biological material, could contribute to the understanding of Turner syndrome pathogenesis. In this pilot study, global gene expression analysis of cell-free RNA in amniotic fluid supernatant was utilized to identify specific genes/organ systems that may play a role in Turner syndrome pathophysiology. Cell-free RNA from amniotic fluid of five mid-trimester Turner syndrome fetuses and five euploid female fetuses matched for gestational age was extracted, amplified, and hybridized onto Affymetrix(®) U133 Plus 2.0 arrays. Significantly differentially regulated genes were identified using paired t tests. Biological interpretation was performed using Ingenuity Pathway Analysis and BioGPS gene expression atlas. There were 470 statistically significantly differentially expressed genes identified. They were widely distributed across the genome. XIST was significantly down-regulated (p < 0.0001); SHOX was not differentially expressed. One of the most highly represented organ systems was the hematologic/immune system, distinguishing the Turner syndrome transcriptome from other aneuploidies we previously studied. Manual curation of the differentially expressed gene list identified genes of possible pathologic significance, including NFATC3, IGFBP5, and LDLR. Transcriptomic differences in the amniotic fluid of Turner syndrome fetuses are due to genome-wide dysregulation. The hematologic/immune system differences may play a role in early-onset autoimmune dysfunction. Other genes identified with possible pathologic significance are associated with cardiac and skeletal systems, which are known to be affected in females with Turner syndrome. The discovery-driven approach described here may be useful in elucidating novel mechanisms of disease in Turner syndrome.

Gene transfer with a vector expressing Maxi-K from a smooth muscle-specific promoter restores erectile function in the aging rat.

PubMed

Melman, A; Biggs, G; Davies, K; Zhao, W; Tar, M T; Christ, G J

2008-03-01

Previous reports have demonstrated that gene transfer with the alpha, or pore-forming, subunit of the human Maxi-K channel (hSlo) restores the decline in erectile capacity observed in established rat models of diabetes and aging. Preliminary data from a human clinical trial also showed safety and potential efficacy in 11 men treated with the same plasmid construct expressing the Maxi-K channel. In all instances, the original plasmid was driven by the heterologous cytomegalovirus promoter which is broadly active in a wide variety of cell and tissue types. To more precisely determine the contribution of the corporal myocyte to the observed physiological effects in vivo, we report here our initial work using a distinct vector (pSMAA-hSlo) in which hSlo gene expression was driven off the mouse smooth muscle alpha-actin (SMAA) promoter. Specifically, older rats, with diminished erectile capacity, were given a single intracorporal injection with either 100 mug pVAX-hSlo or 10, 100 or 1000 mug pSMAA-hSlo, or vector or vehicle alone. Significantly increased intracavernous pressure (ICP) responses to cavernous nerve stimulation were observed for all doses of both plasmids encoding hSlo, relative to control injections. These data confirm and extend previous observations to document that smooth muscle cell-specific expression of hSlo in corporal tissue is both necessary and sufficient to restore erectile function in aging rats.

Deciphering the role of the signal- and Sty1 kinase-dependent phosphorylation of the stress-responsive transcription factor Atf1 on gene activation.

PubMed

Salat-Canela, Clàudia; Paulo, Esther; Sánchez-Mir, Laura; Carmona, Mercè; Ayté, José; Oliva, Baldo; Hidalgo, Elena

2017-08-18

Adaptation to stress triggers the most dramatic shift in gene expression in fission yeast ( Schizosaccharomyces pombe ), and this response is driven by signaling via the MAPK Sty1. Upon activation, Sty1 accumulates in the nucleus and stimulates expression of hundreds of genes via the nuclear transcription factor Atf1, including expression of atf1 itself. However, the role of stress-induced, Sty1-mediated Atf1 phosphorylation in transcriptional activation is unclear. To this end, we expressed Atf1 phosphorylation mutants from a constitutive promoter to uncouple Atf1 activity from endogenous, stress-activated Atf1 expression. We found that cells expressing a nonphosphorylatable Atf1 variant are sensitive to oxidative stress because of impaired transcription of a subset of stress genes whose expression is also controlled by another transcription factor, Pap1. Furthermore, cells expressing a phospho-mimicking Atf1 mutant display enhanced stress resistance, and although expression of the Pap1-dependent genes still relied on stress induction, another subset of stress-responsive genes was constitutively expressed in these cells. We also observed that, in cells expressing the phospho-mimicking Atf1 mutant, the presence of Sty1 was completely dispensable, with all stress defects of Sty1-deficient cells being suppressed by expression of the Atf1 mutant. We further demonstrated that Sty1-mediated Atf1 phosphorylation does not stimulate binding of Atf1 to DNA but, rather, establishes a platform of interactions with the basal transcriptional machinery to facilitate transcription initiation. In summary, our results provide evidence that Atf1 phosphorylation by the MAPK Sty1 is required for oxidative stress responses in fission yeast cells by promoting transcription initiation. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Transcriptional regulation of arylalkylamine-N-acetyltransferase-2 gene in the pineal gland of the gilthead seabream.

PubMed

Zilberman-Peled, B; Appelbaum, L; Vallone, D; Foulkes, N S; Anava, S; Anzulovich, A; Coon, S L; Klein, D C; Falcón, J; Ron, B; Gothilf, Y

2007-01-01

Pineal serotonin-N-acetyltransferase (arylalkylamine-N-acetyltransferase; AANAT) is considered the key enzyme in the generation of circulating melatonin rhythms; the rate of melatonin production is determined by AANAT activity. In all the examined species, AANAT activity is regulated at the post-translational level and, to a variable degree, also at the transcriptional level. Here, the transcriptional regulation of pineal aanat (aanat2) of the gilthead seabream (Sparus aurata) was investigated. Real-time polymerase chain reaction quantification of aanat2 mRNA levels in the pineal gland collected throughout the 24-h cycle revealed a rhythmic expression pattern. In cultured pineal glands, the amplitude was reduced, but the daily rhythmic expression pattern was maintained under constant illumination, indicating a circadian clock-controlled regulation of seabream aanat2. DNA constructs were prepared in which green fluorescent protein was driven by the aanat2 promoters of seabream and Northern pike. In vivo transient expression analyses in zebrafish embryos indicated that these promoters contain the necessary elements to drive enhanced expression in the pineal gland. In the light-entrainable clock-containing PAC-2 zebrafish cell line, a stably transfected seabream aanat2 promoter-luciferase DNA construct exhibited a clock-controlled circadian rhythm of luciferase activity, characteristic for an E-box-driven expression. In NIH-3T3 cells, the seabream aanat2 promoter was activated by a synergistic action of BMAL/CLOCK and orthodenticle homeobox 5 (OTX5). Promoter sequence analyses revealed the presence of the photoreceptor conserved element and an extended E-box (i.e. the binding sites for BMAL/CLOCK and OTX5 that have been previously associated with pineal-specific and rhythmic gene expression). These results suggest that seabream aanat2 is a clock-controlled gene that is regulated by conserved mechanisms.

PAX6 MiniPromoters drive restricted expression from rAAV in the adult mouse retina

PubMed Central

Hickmott, Jack W; Chen, Chih-yu; Arenillas, David J; Korecki, Andrea J; Lam, Siu Ling; Molday, Laurie L; Bonaguro, Russell J; Zhou, Michelle; Chou, Alice Y; Mathelier, Anthony; Boye, Sanford L; Hauswirth, William W; Molday, Robert S; Wasserman, Wyeth W; Simpson, Elizabeth M

2016-01-01

Current gene therapies predominantly use small, strong, and readily available ubiquitous promoters. However, as the field matures, the availability of small, cell-specific promoters would be greatly beneficial. Here we design seven small promoters from the human paired box 6 (PAX6) gene and test them in the adult mouse retina using recombinant adeno-associated virus. We chose the retina due to previous successes in gene therapy for blindness, and the PAX6 gene since it is: well studied; known to be driven by discrete regulatory regions; expressed in therapeutically interesting retinal cell types; and mutated in the vision-loss disorder aniridia, which is in need of improved therapy. At the PAX6 locus, 31 regulatory regions were bioinformatically predicted, and nine regulatory regions were constructed into seven MiniPromoters. Driving Emerald GFP, these MiniPromoters were packaged into recombinant adeno-associated virus, and injected intravitreally into postnatal day 14 mice. Four MiniPromoters drove consistent retinal expression in the adult mouse, driving expression in combinations of cell-types that endogenously express Pax6: ganglion, amacrine, horizontal, and Müller glia. Two PAX6-MiniPromoters drive expression in three of the four cell types that express PAX6 in the adult mouse retina. Combined, they capture all four cell types, making them potential tools for research, and PAX6-gene therapy for aniridia. PMID:27556059

PAX6 MiniPromoters drive restricted expression from rAAV in the adult mouse retina.

PubMed

Hickmott, Jack W; Chen, Chih-Yu; Arenillas, David J; Korecki, Andrea J; Lam, Siu Ling; Molday, Laurie L; Bonaguro, Russell J; Zhou, Michelle; Chou, Alice Y; Mathelier, Anthony; Boye, Sanford L; Hauswirth, William W; Molday, Robert S; Wasserman, Wyeth W; Simpson, Elizabeth M

2016-01-01

Current gene therapies predominantly use small, strong, and readily available ubiquitous promoters. However, as the field matures, the availability of small, cell-specific promoters would be greatly beneficial. Here we design seven small promoters from the human paired box 6 (PAX6) gene and test them in the adult mouse retina using recombinant adeno-associated virus. We chose the retina due to previous successes in gene therapy for blindness, and the PAX6 gene since it is: well studied; known to be driven by discrete regulatory regions; expressed in therapeutically interesting retinal cell types; and mutated in the vision-loss disorder aniridia, which is in need of improved therapy. At the PAX6 locus, 31 regulatory regions were bioinformatically predicted, and nine regulatory regions were constructed into seven MiniPromoters. Driving Emerald GFP, these MiniPromoters were packaged into recombinant adeno-associated virus, and injected intravitreally into postnatal day 14 mice. Four MiniPromoters drove consistent retinal expression in the adult mouse, driving expression in combinations of cell-types that endogenously express Pax6: ganglion, amacrine, horizontal, and Müller glia. Two PAX6-MiniPromoters drive expression in three of the four cell types that express PAX6 in the adult mouse retina. Combined, they capture all four cell types, making them potential tools for research, and PAX6-gene therapy for aniridia.

Adaptive induction of NF-E2-related factor-2-driven antioxidant genes in endothelial cells in response to hyperglycemia.

PubMed

Ungvari, Zoltan; Bailey-Downs, Lora; Gautam, Tripti; Jimenez, Rosario; Losonczy, Gyorgy; Zhang, Cuihua; Ballabh, Praveen; Recchia, Fabio A; Wilkerson, Donald C; Sonntag, William E; Pearson, Kevin; de Cabo, Rafael; Csiszar, Anna

2011-04-01

Hyperglycemia in diabetes mellitus promotes oxidative stress in endothelial cells, which contributes to development of cardiovascular diseases. Nuclear factor erythroid 2-related factor-2 (Nrf2) is a transcription factor activated by oxidative stress that regulates expression of numerous reactive oxygen species (ROS) detoxifying and antioxidant genes. This study was designed to elucidate the homeostatic role of adaptive induction of Nrf2-driven free radical detoxification mechanisms in endothelial protection under diabetic conditions. Using a Nrf2/antioxidant response element (ARE)-driven luciferase reporter gene assay we found that in a cultured coronary arterial endothelial cell model hyperglycemia (10-30 mmol/l glucose) significantly increases transcriptional activity of Nrf2 and upregulates the expression of the Nrf2 target genes NQO1, GCLC, and HMOX1. These effects of high glucose were significantly attenuated by small interfering RNA (siRNA) downregulation of Nrf2 or overexpression of Keap-1, which inactivates Nrf2. High-glucose-induced upregulation of NQO1, GCLC, and HMOX1 was also prevented by pretreatment with polyethylene glycol (PEG)-catalase or N-acetylcysteine, whereas administration of H(2)O(2) mimicked the effect of high glucose. To test the effects of metabolic stress in vivo, Nrf2(+/+) and Nrf2(-/-) mice were fed a high-fat diet (HFD). HFD elicited significant increases in mRNA expression of Gclc and Hmox1 in aortas of Nrf2(+/+) mice, but not Nrf2(-/-) mice, compared with respective standard diet-fed control mice. Additionally, HFD-induced increases in vascular ROS levels were significantly greater in Nrf2(-/-) than Nrf2(+/+) mice. HFD-induced endothelial dysfunction was more severe in Nrf2(-/-) mice, as shown by the significantly diminished acetylcholine-induced relaxation of aorta of these animals compared with HFD-fed Nrf2(+/+) mice. Our results suggest that adaptive activation of the Nrf2/ARE pathway confers endothelial protection under diabetic conditions.

Pineal-specific expression of green fluorescent protein under the control of the serotonin-N-acetyltransferase gene regulatory regions in transgenic zebrafish.

PubMed

Gothilf, Yoav; Toyama, Reiko; Coon, Steven L; Du, Shao-Jun; Dawid, Igor B; Klein, David C

2002-11-01

Zebrafish serotonin-N-acetyltransferase-2 (zfAANAT-2) mRNA is exclusively expressed in the pineal gland (epiphysis) at the embryonic stage. Here, we have initiated an effort to study the mechanisms underlying tissue-specific expression of this gene. DNA constructs were prepared in which green fluorescent protein (GFP) is driven by regulatory regions of the zfAANAT-2 gene. In vivo transient expression analysis in zebrafish embryos indicated that in addition to the 5'-flanking region, a regulatory sequence in the 3'-flanking region is required for pineal-specific expression. This finding led to an effort to produce transgenic lines expressing GFP under the control of the 5' and 3' regulatory regions of the zfAANAT-2 gene. Embryos transiently expressing GFP were raised to maturity and tested for germ cell transmission of the transgene. Three transgenic lines were produced in which GFP fluorescence in the pineal was detected starting 1 to 2 days after fertilization. One line was crossed with mindbomb and floating head mutants that cause abnormal development of the pineal and an elevation or reduction of zfAANAT-2 mRNA levels, respectively. Homozygous mutant transgenic embryos exhibited similar effects on GFP expression in the pineal gland. These observations indicate that the transgenic lines described here will be useful in studying the development of the pineal gland and the mechanisms that determine pineal-specific gene expression in the zebrafish. Published 2002 Wiley-Liss, Inc.

PW1 gene/paternally expressed gene 3 (PW1/Peg3) identifies multiple adult stem and progenitor cell populations

PubMed Central

Besson, Vanessa; Smeriglio, Piera; Wegener, Amélie; Relaix, Frédéric; Nait Oumesmar, Brahim; Sassoon, David A.; Marazzi, Giovanna

2011-01-01

A variety of markers are invaluable for identifying and purifying stem/progenitor cells. Here we report the generation of a murine reporter line driven by Pw1 that reveals cycling and quiescent progenitor/stem cells in all adult tissues thus far examined, including the intestine, blood, testis, central nervous system, bone, skeletal muscle, and skin. Neurospheres generated from the adult PW1-reporter mouse show near 100% reporter-gene expression following a single passage. Furthermore, epidermal stem cells can be purified solely on the basis of reporter-gene expression. These cells are clonogenic, repopulate the epidermal stem-cell niches, and give rise to new hair follicles. Finally, we demonstrate that only PW1 reporter-expressing epidermal cells give rise to follicles that are capable of self-renewal following injury. Our data demonstrate that PW1 serves as an invaluable marker for competent self-renewing stem cells in a wide array of adult tissues, and the PW1-reporter mouse serves as a tool for rapid stem cell isolation and characterization. PMID:21709251

Unraveling the role of hypoxia-inducible factor (HIF)-1α and HIF-2α in the adaption process of human microvascular endothelial cells (HMEC-1) to hypoxia: Redundant HIF-dependent regulation of macrophage migration inhibitory factor.

PubMed

Hahne, Martin; Schumann, Peggy; Mursell, Mathias; Strehl, Cindy; Hoff, Paula; Buttgereit, Frank; Gaber, Timo

2018-03-01

Hypoxia driven angiogenesis is a prominent feature of tissue regeneration, inflammation and tumor growth and is regulated by hypoxia-inducible factor (HIF)-1 and -2. The distinct functions of HIFs in the hypoxia-induced angiogenesis and metabolic switch of endothelial cells are still unknown and therefore aim of this study. We investigated the role of HIF-1 and -2 in the adaptation of immortalized human microvascular endothelial cells (HMEC-1) to hypoxic conditions (1% O 2 ) in terms of angiogenesis, cytokine secretion, gene expression and ATP/ADP-ratio using shRNA-mediated reduction of the oxygen sensitive α-subunits of either HIF-1 or HIF-2 or the combination of both. Reduction of HIF-1α diminished cellular energy, hypoxia-induced glycolytic gene expression, and angiogenesis not altering pro-angiogenic factors. Reduction of HIF-2α diminished hypoxia-induced pro-angiogenic factors, enhanced anti-angiogenic factors and attenuated angiogenesis not altering glycolytic gene expression. Reduction of both HIFs reduced cell survival, gene expression of glycolytic enzymes and pro-angiogenic factors as compared to the corresponding control. Finally, we identified the macrophage migration inhibitory factor (MIF) to be redundantly regulated by HIF-1 and HIF-2 and to be essential in the process of hypoxia-driven angiogenesis. Our results demonstrate a major impact of HIF-1 and HIF-2 on hypoxia-induced angiogenesis indicating distinct but also overlapping functions of HIF-1 and HIF-2. These findings open new possibilities for therapeutic approaches by specifically targeting the HIF-1 and HIF-2 or their target MIF. Copyright © 2017 Elsevier Inc. All rights reserved.

RNAi-dependent and -independent antiviral phenotypes of chromosomally integrated shRNA clones: role of VASP in respiratory syncytial virus growth.

PubMed

Musiyenko, Alla; Bitko, Vira; Barik, Sailen

2007-07-01

Stable RNA interference (RNAi) is commonly achieved by recombinant expression of short hairpin RNA (shRNA). To generate virus-resistant cell lines, we cloned a shRNA cassette against the phosphoprotein gene of respiratory syncytial virus (RSV) into a polIII-driven plasmid vector. Analysis of individual stable transfectants showed a spectrum of RSV resistance correlating with the levels of shRNA expressed from different chromosomal locations. Interestingly, resistance in a minority of clones was due to mono-allelic disruption of the cellular gene for vasodilator-stimulated phosphoprotein (VASP). Thus, pure clones of chromosomally integrated DNA-directed RNAi can exhibit gene disruption phenotypes resembling but unrelated to RNAi.

The effects of simvastatin and dipentyl phthalate on fetal cholesterol and testosterone production

EPA Science Inventory

Sex differentiation of the male reproductive tract in mammals is driven, in part, by fetal androgen production. In utero, some phthalate esters (PEs) alter fetal Leydig cell differentiation, reducing the expression of genes associated with steroid synthesis/transport, and conseq...

Metatranscriptomics reveals temperature-driven functional changes in microbiome impacting cheese maturation rate

DOE Office of Scientific and Technical Information (OSTI.GOV)

De Filippis, Francesca; Genovese, Alessandro; Ferranti, Pasquale

Traditional cheeses harbour complex microbial consortia that play an important role in shaping typical sensorial properties. However, the microbial metabolism is considered difficult to control. Microbial community succession and the related gene expression were analysed during ripening of a traditional Italian cheese, identifying parameters that could be modified to accelerate ripening. Afterwards, we modulated ripening conditions and observed consistent changes in microbial community structure and function. We provide concrete evidence of the essential contribution of non-starter lactic acid bacteria in ripening-related activities. An increase in the ripening temperature promoted the expression of genes related to proteolysis, lipolysis and amino acid/lipidmore » catabolism and significantly increases the cheese maturation rate. Moreover, temperature-promoted microbial metabolisms were consistent with the metabolomic profiles of proteins and volatile organic compounds in the cheese. Finally, the results clearly indicate how processing-driven microbiome responses can be modulated in order to optimize production efficiency and product quality.« less

Metatranscriptomics reveals temperature-driven functional changes in microbiome impacting cheese maturation rate

DOE PAGES

De Filippis, Francesca; Genovese, Alessandro; Ferranti, Pasquale; ...

2016-02-25

Traditional cheeses harbour complex microbial consortia that play an important role in shaping typical sensorial properties. However, the microbial metabolism is considered difficult to control. Microbial community succession and the related gene expression were analysed during ripening of a traditional Italian cheese, identifying parameters that could be modified to accelerate ripening. Afterwards, we modulated ripening conditions and observed consistent changes in microbial community structure and function. We provide concrete evidence of the essential contribution of non-starter lactic acid bacteria in ripening-related activities. An increase in the ripening temperature promoted the expression of genes related to proteolysis, lipolysis and amino acid/lipidmore » catabolism and significantly increases the cheese maturation rate. Moreover, temperature-promoted microbial metabolisms were consistent with the metabolomic profiles of proteins and volatile organic compounds in the cheese. Finally, the results clearly indicate how processing-driven microbiome responses can be modulated in order to optimize production efficiency and product quality.« less

DNA-Binding Kinetics Determines the Mechanism of Noise-Induced Switching in Gene Networks

PubMed Central

Tse, Margaret J.; Chu, Brian K.; Roy, Mahua; Read, Elizabeth L.

2015-01-01

Gene regulatory networks are multistable dynamical systems in which attractor states represent cell phenotypes. Spontaneous, noise-induced transitions between these states are thought to underlie critical cellular processes, including cell developmental fate decisions, phenotypic plasticity in fluctuating environments, and carcinogenesis. As such, there is increasing interest in the development of theoretical and computational approaches that can shed light on the dynamics of these stochastic state transitions in multistable gene networks. We applied a numerical rare-event sampling algorithm to study transition paths of spontaneous noise-induced switching for a ubiquitous gene regulatory network motif, the bistable toggle switch, in which two mutually repressive genes compete for dominant expression. We find that the method can efficiently uncover detailed switching mechanisms that involve fluctuations both in occupancies of DNA regulatory sites and copy numbers of protein products. In addition, we show that the rate parameters governing binding and unbinding of regulatory proteins to DNA strongly influence the switching mechanism. In a regime of slow DNA-binding/unbinding kinetics, spontaneous switching occurs relatively frequently and is driven primarily by fluctuations in DNA-site occupancies. In contrast, in a regime of fast DNA-binding/unbinding kinetics, switching occurs rarely and is driven by fluctuations in levels of expressed protein. Our results demonstrate how spontaneous cell phenotype transitions involve collective behavior of both regulatory proteins and DNA. Computational approaches capable of simulating dynamics over many system variables are thus well suited to exploring dynamic mechanisms in gene networks. PMID:26488666

The Agrobacterium-mediated transformation of common wheat (Triticum aestivum L.) and triticale (x Triticosecale Wittmack): role of the binary vector system and selection cassettes.

PubMed

Bińka, Agnieszka; Orczyk, Wacław; Nadolska-Orczyk, Anna

2012-02-01

The influence of two binary vector systems, pGreen and pCAMBIA, on the Agrobacterium-mediated transformation ability of wheat and triticale was studied. Both vectors carried selection cassettes with bar or nptII driven by different promoters. Two cultivars of wheat, Kontesa and Torka, and one cultivar of triticale, Wanad, were tested. The transformation rates for the wheat cultivars ranged from 0.00 to 3.58% and from 0.00 to 6.79% for triticale. The best values for wheat were 3.58% for Kontesa and 3.14% for Torka, and these were obtained after transformation with the pGreen vector carrying the nptII selection gene under the control of 35S promoter. In the case of the bar selection system, the best transformation rates were, respectively, 1.46 and 1.79%. Such rates were obtained when the 35S::bar cassette was carried by the pCAMBIA vector; they were significantly lower with the pGreen vector. The triticale cultivar Wanad had its highest transformation rate after transformation with nptII driven by 35S in pCAMBIA. The bar selection system for the same triticale cultivar was better when the gene was driven by nos and the selection cassette was carried by pGreen. The integration of the transgenes was confirmed with at least three pairs of specific starters amplifying the fragments of nptII, bar, or gus. The expression of selection genes, measured by reverse transcriptase polymerase chain reaction (RT-PCR) in relation to the actin gene, was low, ranging from 0.00 to 0.63 for nptII and from 0.00 to 0.33 for bar. The highest relative transcript accumulation was observed for nptII driven by 35S and expressed in Kontesa that had been transformed with pGreen.

Rationally designed, heterologous S. cerevisiae transcripts expose novel expression determinants

PubMed Central

Ben-Yehezkel, Tuval; Atar, Shimshi; Zur, Hadas; Diament, Alon; Goz, Eli; Marx, Tzipy; Cohen, Rafael; Dana, Alexandra; Feldman, Anna; Shapiro, Ehud; Tuller, Tamir

2015-01-01

Deducing generic causal relations between RNA transcript features and protein expression profiles from endogenous gene expression data remains a major unsolved problem in biology. The analysis of gene expression from heterologous genes contributes significantly to solving this problem, but has been heavily biased toward the study of the effect of 5′ transcript regions and to prokaryotes. Here, we employ a synthetic biology driven approach that systematically differentiates the effect of different regions of the transcript on gene expression up to 240 nucleotides into the ORF. This enabled us to discover new causal effects between features in previously unexplored regions of transcripts, and gene expression in natural regimes. We rationally designed, constructed, and analyzed 383 gene variants of the viral HRSVgp04 gene ORF, with multiple synonymous mutations at key positions along the transcript in the eukaryote S. cerevisiae. Our results show that a few silent mutations at the 5′UTR can have a dramatic effect of up to 15 fold change on protein levels, and that even synonymous mutations in positions more than 120 nucleotides downstream from the ORF 5′end can modulate protein levels up to 160%–300%. We demonstrate that the correlation between protein levels and folding energy increases with the significance of the level of selection of the latter in endogenous genes, reinforcing the notion that selection for folding strength in different parts of the ORF is related to translation regulation. Our measured protein abundance correlates notably(correlation up to r = 0.62 (p=0.0013)) with mean relative codon decoding times, based on ribosomal densities (Ribo-Seq) in endogenous genes, supporting the conjecture that translation elongation and adaptation to the tRNA pool can modify protein levels in a causal/direct manner. This report provides an improved understanding of transcript evolution, design principles of gene expression regulation, and suggests simple rules for engineering synthetic gene expression in eukaryotes. PMID:26176266

Rationally designed, heterologous S. cerevisiae transcripts expose novel expression determinants.

PubMed

Ben-Yehezkel, Tuval; Atar, Shimshi; Zur, Hadas; Diament, Alon; Goz, Eli; Marx, Tzipy; Cohen, Rafael; Dana, Alexandra; Feldman, Anna; Shapiro, Ehud; Tuller, Tamir

2015-01-01

Deducing generic causal relations between RNA transcript features and protein expression profiles from endogenous gene expression data remains a major unsolved problem in biology. The analysis of gene expression from heterologous genes contributes significantly to solving this problem, but has been heavily biased toward the study of the effect of 5' transcript regions and to prokaryotes. Here, we employ a synthetic biology driven approach that systematically differentiates the effect of different regions of the transcript on gene expression up to 240 nucleotides into the ORF. This enabled us to discover new causal effects between features in previously unexplored regions of transcripts, and gene expression in natural regimes. We rationally designed, constructed, and analyzed 383 gene variants of the viral HRSVgp04 gene ORF, with multiple synonymous mutations at key positions along the transcript in the eukaryote S. cerevisiae. Our results show that a few silent mutations at the 5'UTR can have a dramatic effect of up to 15 fold change on protein levels, and that even synonymous mutations in positions more than 120 nucleotides downstream from the ORF 5'end can modulate protein levels up to 160%-300%. We demonstrate that the correlation between protein levels and folding energy increases with the significance of the level of selection of the latter in endogenous genes, reinforcing the notion that selection for folding strength in different parts of the ORF is related to translation regulation. Our measured protein abundance correlates notably(correlation up to r = 0.62 (p=0.0013)) with mean relative codon decoding times, based on ribosomal densities (Ribo-Seq) in endogenous genes, supporting the conjecture that translation elongation and adaptation to the tRNA pool can modify protein levels in a causal/direct manner. This report provides an improved understanding of transcript evolution, design principles of gene expression regulation, and suggests simple rules for engineering synthetic gene expression in eukaryotes.

Genome-Wide Analysis of SREBP1 Activity around the Clock Reveals Its Combined Dependency on Nutrient and Circadian Signals

PubMed Central

Naldi, Aurélien; Baruchet, Michaël; Canella, Donatella; Le Martelot, Gwendal; Guex, Nicolas; Desvergne, Béatrice; Delorenzi, Mauro; Deplancke, Bart; Desvergne, Béatrice; Guex, Nicolas; Herr, Winship; Naef, Felix; Rougemont, Jacques; Schibler, Ueli; Deplancke, Bart; Guex, Nicolas; Herr, Winship; Guex, Nicolas; Andersin, Teemu; Cousin, Pascal; Gilardi, Federica; Gos, Pascal; Martelot, Gwendal Le; Lammers, Fabienne; Canella, Donatella; Gilardi, Federica; Raghav, Sunil; Fabbretti, Roberto; Fortier, Arnaud; Long, Li; Vlegel, Volker; Xenarios, Ioannis; Migliavacca, Eugenia; Praz, Viviane; Guex, Nicolas; Naef, Felix; Rougemont, Jacques; David, Fabrice; Jarosz, Yohan; Kuznetsov, Dmitry; Liechti, Robin; Martin, Olivier; Delafontaine, Julien; Sinclair, Lucas; Cajan, Julia; Krier, Irina; Leleu, Marion; Migliavacca, Eugenia; Molina, Nacho; Naldi, Aurélien; Rey, Guillaume; Symul, Laura; Guex, Nicolas; Naef, Felix; Rougemont, Jacques; Bernasconi, David; Delorenzi, Mauro; Andersin, Teemu; Canella, Donatella; Gilardi, Federica; Martelot, Gwendal Le; Lammers, Fabienne; Baruchet, Michaël; Raghav, Sunil

2014-01-01

In mammals, the circadian clock allows them to anticipate and adapt physiology around the 24 hours. Conversely, metabolism and food consumption regulate the internal clock, pointing the existence of an intricate relationship between nutrient state and circadian homeostasis that is far from being understood. The Sterol Regulatory Element Binding Protein 1 (SREBP1) is a key regulator of lipid homeostasis. Hepatic SREBP1 function is influenced by the nutrient-response cycle, but also by the circadian machinery. To systematically understand how the interplay of circadian clock and nutrient-driven rhythm regulates SREBP1 activity, we evaluated the genome-wide binding of SREBP1 to its targets throughout the day in C57BL/6 mice. The recruitment of SREBP1 to the DNA showed a highly circadian behaviour, with a maximum during the fed status. However, the temporal expression of SREBP1 targets was not always synchronized with its binding pattern. In particular, different expression phases were observed for SREBP1 target genes depending on their function, suggesting the involvement of other transcription factors in their regulation. Binding sites for Hepatocyte Nuclear Factor 4 (HNF4) were specifically enriched in the close proximity of SREBP1 peaks of genes, whose expression was shifted by about 8 hours with respect to SREBP1 binding. Thus, the cross-talk between hepatic HNF4 and SREBP1 may underlie the expression timing of this subgroup of SREBP1 targets. Interestingly, the proper temporal expression profile of these genes was dramatically changed in Bmal1 −/− mice upon time-restricted feeding, for which a rhythmic, but slightly delayed, binding of SREBP1 was maintained. Collectively, our results show that besides the nutrient-driven regulation of SREBP1 nuclear translocation, a second layer of modulation of SREBP1 transcriptional activity, strongly dependent from the circadian clock, exists. This system allows us to fine tune the expression timing of SREBP1 target genes, thus helping to temporally separate the different physiological processes in which these genes are involved. PMID:24603613

OmoMYC blunts promoter invasion by oncogenic MYC to inhibit gene expression characteristic of MYC-dependent tumors.

PubMed

Jung, L A; Gebhardt, A; Koelmel, W; Ade, C P; Walz, S; Kuper, J; von Eyss, B; Letschert, S; Redel, C; d'Artista, L; Biankin, A; Zender, L; Sauer, M; Wolf, E; Evan, G; Kisker, C; Eilers, M

2017-04-06

MYC genes have both essential roles during normal development and exert oncogenic functions during tumorigenesis. Expression of a dominant-negative allele of MYC, termed OmoMYC, can induce rapid tumor regression in mouse models with little toxicity for normal tissues. How OmoMYC discriminates between physiological and oncogenic functions of MYC is unclear. We have solved the crystal structure of OmoMYC and show that it forms a stable homodimer and as such recognizes DNA in the same manner as the MYC/MAX heterodimer. OmoMYC attenuates both MYC-dependent activation and repression by competing with MYC/MAX for binding to chromatin, effectively lowering MYC/MAX occupancy at its cognate binding sites. OmoMYC causes the largest decreases in promoter occupancy and changes in expression on genes that are invaded by oncogenic MYC levels. A signature of OmoMYC-regulated genes defines subgroups with high MYC levels in multiple tumor entities and identifies novel targets for the eradication of MYC-driven tumors.

P19-dependent and P19-independent reversion of F1-V gene silencing in tomato.

PubMed

Alvarez, M Lucrecia; Pinyerd, Heidi L; Topal, Emel; Cardineau, Guy A

2008-09-01

As a part of a project to develop a plant-made plague vaccine, we expressed the Yersinia pestis F1-V antigen fusion protein in tomato. We discovered that in some of these plants the expression of the f1-v gene was undetectable in leaves and fruit by ELISA, even though they had multiple copies of f1-v according to Southern-blot analysis. A likely explanation of these results is the phenomenon of RNA silencing, a group of RNA-based processes that produces sequence-specific inhibition of gene expression and may result in transgene silencing in plants. Here we report the reversion of the f1-v gene silencing in transgenic tomato plants through two different mechanisms. In the P19-dependent Reversion or Type I, the viral suppressor of gene silencing, P19, induces the reversion of gene silencing. In the P19-independent Reversion or Type II, the f1-v gene expression is restored after the substantial loss of gene copies as a consequence of transgene segregation in the progeny. The transient and stable expression of the p19 gene driven by a constitutive promoter as well as an ethanol inducible promoter induced a P19-dependent reversion of f1-v gene silencing. In particular, the second generation plant 3D1.6 had the highest P19 protein levels and correlated with the highest F1-V protein accumulation, almost a three-fold increase of F1-V protein levels in fruit than that previously reported for the non-silenced F1-V elite tomato lines. These results confirm the potential exploitation of P19 to substantially increase the expression of value-added proteins in plants.

Whole-transcriptome brain expression and exon-usage profiling in major depression and suicide: evidence for altered glial, endothelial and ATPase activity

PubMed Central

Pantazatos, Spiro P.; Huang, Yung-yu; Rosoklija, Gorazd B.; Dwork, Andrew J.; Arango, Victoria; Mann, J. John

2016-01-01

Brain gene expression profiling studies of suicide and depression using oligonucleotide microarrays have often failed to distinguish these two phenotypes. Moreover, next generation sequencing (NGS) approaches are more accurate in quantifying gene expression and can detect alternative splicing. Using RNA-seq, we examined whole-exome gene and exon expression in non-psychiatric controls (CON, N=29), DSM-IV major depressive disorder suicides (MDD-S, N=21) and MDD non-suicides (MDD, N=9) in dorsal lateral prefrontal cortex (Brodmann Area 9) of sudden-death medication-free individuals postmortem. Using small RNA-seq, we also examined miRNA expression (9 samples per group). DeSeq2 identified thirty-five genes differentially expressed between groups and surviving adjustment for false discovery rate (adjusted p<0.1). In depression, altered genes include humanin like-8 (MTRNRL8), interleukin-8 (IL8), and serpin peptidase inhibitor, clade H (SERPINH1) and chemokine ligand 4 (CCL4), while exploratory gene ontology (GO) analyses revealed lower expression of immune-related pathways such as chemokine receptor activity, chemotaxis and cytokine biosynthesis, and angiogenesis and vascular development in (adjusted p<0.1). Hypothesis-driven GO analysis suggests lower expression of genes involved in oligodendrocyte differentiation, regulation of glutamatergic neurotransmission, and oxytocin receptor expression in both suicide and depression, and provisional evidence for altered DNA-dependent ATPase expression in suicide only. DEXSEq analysis identified differential exon usage in ATPase, class II, type 9B (adjusted p<0.1) in depression. Differences in miRNA expression or structural gene variants were not detected. Results lend further support for models in which deficits in microglial, endothelial (blood-brain barrier), ATPase activity and astrocytic cell functions contribute to MDD and suicide, and identify putative pathways and mechanisms for further study in these disorders. PMID:27528462

Whole-transcriptome brain expression and exon-usage profiling in major depression and suicide: evidence for altered glial, endothelial and ATPase activity.

PubMed

Pantazatos, S P; Huang, Y-Y; Rosoklija, G B; Dwork, A J; Arango, V; Mann, J J

2017-05-01

Brain gene expression profiling studies of suicide and depression using oligonucleotide microarrays have often failed to distinguish these two phenotypes. Moreover, next generation sequencing approaches are more accurate in quantifying gene expression and can detect alternative splicing. Using RNA-seq, we examined whole-exome gene and exon expression in non-psychiatric controls (CON, N=29), DSM-IV major depressive disorder suicides (MDD-S, N=21) and MDD non-suicides (MDD, N=9) in the dorsal lateral prefrontal cortex (Brodmann Area 9) of sudden death medication-free individuals post mortem. Using small RNA-seq, we also examined miRNA expression (nine samples per group). DeSeq2 identified 35 genes differentially expressed between groups and surviving adjustment for false discovery rate (adjusted P<0.1). In depression, altered genes include humanin-like-8 (MTRNRL8), interleukin-8 (IL8), and serpin peptidase inhibitor, clade H (SERPINH1) and chemokine ligand 4 (CCL4), while exploratory gene ontology (GO) analyses revealed lower expression of immune-related pathways such as chemokine receptor activity, chemotaxis and cytokine biosynthesis, and angiogenesis and vascular development in (adjusted P<0.1). Hypothesis-driven GO analysis suggests lower expression of genes involved in oligodendrocyte differentiation, regulation of glutamatergic neurotransmission, and oxytocin receptor expression in both suicide and depression, and provisional evidence for altered DNA-dependent ATPase expression in suicide only. DEXSEq analysis identified differential exon usage in ATPase, class II, type 9B (adjusted P<0.1) in depression. Differences in miRNA expression or structural gene variants were not detected. Results lend further support for models in which deficits in microglial, endothelial (blood-brain barrier), ATPase activity and astrocytic cell functions contribute to MDD and suicide, and identify putative pathways and mechanisms for further study in these disorders.

Manipulation of NF-KappaBetta Activity in the Macrophage Lineage as a Novel Therapeutic Approach

DTIC Science & Technology

2007-05-01

Sadikot, J. W. Christman, and T. S. Blackwell. 2003. Bioluminescent detection of endotoxin effects on HIV-1 LTR-driven transcription in vivo. J...differences in proliferation rates, expression of downstream gene expression and effects mediated by altered macrophages on associated epithelial...kappaB activity within macrophages has significant effects on mammary ductal development. 15. SUBJECT TERMS NF-kappaB, macrophages, mammary ductal

Time-Course Gene Set Analysis for Longitudinal Gene Expression Data

PubMed Central

Hejblum, Boris P.; Skinner, Jason; Thiébaut, Rodolphe

2015-01-01

Gene set analysis methods, which consider predefined groups of genes in the analysis of genomic data, have been successfully applied for analyzing gene expression data in cross-sectional studies. The time-course gene set analysis (TcGSA) introduced here is an extension of gene set analysis to longitudinal data. The proposed method relies on random effects modeling with maximum likelihood estimates. It allows to use all available repeated measurements while dealing with unbalanced data due to missing at random (MAR) measurements. TcGSA is a hypothesis driven method that identifies a priori defined gene sets with significant expression variations over time, taking into account the potential heterogeneity of expression within gene sets. When biological conditions are compared, the method indicates if the time patterns of gene sets significantly differ according to these conditions. The interest of the method is illustrated by its application to two real life datasets: an HIV therapeutic vaccine trial (DALIA-1 trial), and data from a recent study on influenza and pneumococcal vaccines. In the DALIA-1 trial TcGSA revealed a significant change in gene expression over time within 69 gene sets during vaccination, while a standard univariate individual gene analysis corrected for multiple testing as well as a standard a Gene Set Enrichment Analysis (GSEA) for time series both failed to detect any significant pattern change over time. When applied to the second illustrative data set, TcGSA allowed the identification of 4 gene sets finally found to be linked with the influenza vaccine too although they were found to be associated to the pneumococcal vaccine only in previous analyses. In our simulation study TcGSA exhibits good statistical properties, and an increased power compared to other approaches for analyzing time-course expression patterns of gene sets. The method is made available for the community through an R package. PMID:26111374

Nuclear envelope and genome interactions in cell fate

PubMed Central

Talamas, Jessica A.; Capelson, Maya

2015-01-01

The eukaryotic cell nucleus houses an organism’s genome and is the location within the cell where all signaling induced and development-driven gene expression programs are ultimately specified. The genome is enclosed and separated from the cytoplasm by the nuclear envelope (NE), a double-lipid membrane bilayer, which contains a large variety of trans-membrane and associated protein complexes. In recent years, research regarding multiple aspects of the cell nucleus points to a highly dynamic and coordinated concert of efforts between chromatin and the NE in regulation of gene expression. Details of how this concert is orchestrated and how it directs cell differentiation and disease are coming to light at a rapid pace. Here we review existing and emerging concepts of how interactions between the genome and the NE may contribute to tissue specific gene expression programs to determine cell fate. PMID:25852741

Engineered promoters enable constant gene expression at any copy number in bacteria.

PubMed

Segall-Shapiro, Thomas H; Sontag, Eduardo D; Voigt, Christopher A

2018-04-01

The internal environment of growing cells is variable and dynamic, making it difficult to introduce reliable parts, such as promoters, for genetic engineering. Here, we applied control-theoretic ideas to design promoters that maintained constant levels of expression at any copy number. Theory predicts that independence to copy number can be achieved by using an incoherent feedforward loop (iFFL) if the negative regulation is perfectly non-cooperative. We engineered iFFLs into Escherichia coli promoters using transcription-activator-like effectors (TALEs). These promoters had near-identical expression in different genome locations and plasmids, even when their copy number was perturbed by genomic mutations or changes in growth medium composition. We applied the stabilized promoters to show that a three-gene metabolic pathway to produce deoxychromoviridans could retain function without re-tuning when the stabilized-promoter-driven genes were moved from a plasmid into the genome.

Expression and regulation of Icer mRNA in the Syrian hamster pineal gland.

PubMed

Diaz, Elena; Garidou, Marie-Laure; Dardente, Hugues; Salingre, Anthony; Pévet, Paul; Simonneaux, Valérie

2003-04-10

Inducible-cAMP early repressor (ICER) is a potent inhibitor of CRE (cAMP-related element)-driven gene transcription. In the rat pineal gland, it has been proposed to be part of the mechanisms involved in the shutting down of the transcription of the gene coding for arylalkylamine N-acetyltransferase (AA-NAT, the melatonin rhythm-generating enzyme). In this study, we report that ICER is expressed in the pineal gland of the photoperiodic rodent Syrian hamster although with some difference compared to the rat. In the Syrian hamster pineal, Icer mRNA levels, low at daytime, displayed a 20-fold increase during the night. Nighttime administration of a beta-adrenergic antagonist, propranolol, significantly reduced Icer mRNA levels although daytime administration of a beta-adrenergic agonist, isoproterenol, was unable to raise the low amount of Icer mRNA. These observations indicate that Icer mRNA expression is induced by the clock-driven norepinephrine release and further suggest that this stimulation is restricted to nighttime, as already observed for Aa-nat gene transcription. Furthermore, we found that the daily profile of Icer mRNA displayed photoperiodic variation with a lengthening of the nocturnal peak in short versus long photoperiod. These data indicate that ICER may be involved in both daily and seasonal regulation of melatonin synthesis in the Syrian hamster.

Transcriptional mechanisms of resistance to anti-PD-1 therapy

PubMed Central

Ascierto, Maria L.; Makohon-Moore, Alvin; Lipson, Evan J.; Taube, Janis M.; McMiller, Tracee L.; Berger, Alan E.; Fan, Jinshui; Kaunitz, Genevieve J.; Cottrell, Tricia R.; Kohutek, Zachary A.; Favorov, Alexander; Makarov, Vladimir; Riaz, Nadeem; Chan, Timothy A.; Cope, Leslie; Hruban, Ralph H.; Pardoll, Drew M.; Taylor, Barry S.; Solit, David B.; Iacobuzio-Donahue, Christine A; Topalian, Suzanne L.

2017-01-01

Purpose To explore factors associated with response and resistance to anti-PD-1 therapy, we analyzed multiple disease sites at autopsy in a patient with widely metastatic melanoma who had a heterogeneous response. Materials and Methods Twenty-six melanoma specimens (four pre-mortem, 22 post-mortem) were subjected to whole-exome sequencing. Candidate immunologic markers and gene expression were assessed in ten cutaneous metastases showing response or progression during therapy. Results The melanoma was driven by biallelic inactivation of NF1. All lesions had highly concordant mutational profiles and copy number alterations, indicating linear clonal evolution. Expression of candidate immunologic markers was similar in responding and progressing lesions. However, progressing cutaneous metastases were associated with over-expression of genes associated with extracellular matrix and neutrophil function. Conclusions Although mutational and immunologic differences have been proposed as the primary determinants of heterogeneous response/resistance to targeted therapies and immunotherapies, respectively, differential lesional gene expression profiles may also dictate anti-PD-1 outcomes. PMID:28193624

Expression of short hairpin RNAs using the compact architecture of retroviral microRNA genes.

PubMed

Burke, James M; Kincaid, Rodney P; Aloisio, Francesca; Welch, Nicole; Sullivan, Christopher S

2017-09-29

Short hairpin RNAs (shRNAs) are effective in generating stable repression of gene expression. RNA polymerase III (RNAP III) type III promoters (U6 or H1) are typically used to drive shRNA expression. While useful for some knockdown applications, the robust expression of U6/H1-driven shRNAs can induce toxicity and generate heterogeneous small RNAs with undesirable off-target effects. Additionally, typical U6/H1 promoters encompass the majority of the ∼270 base pairs (bp) of vector space required for shRNA expression. This can limit the efficacy and/or number of delivery vector options, particularly when delivery of multiple gene/shRNA combinations is required. Here, we develop a compact shRNA (cshRNA) expression system based on retroviral microRNA (miRNA) gene architecture that uses RNAP III type II promoters. We demonstrate that cshRNAs coded from as little as 100 bps of total coding space can precisely generate small interfering RNAs (siRNAs) that are active in the RNA-induced silencing complex (RISC). We provide an algorithm with a user-friendly interface to design cshRNAs for desired target genes. This cshRNA expression system reduces the coding space required for shRNA expression by >2-fold as compared to the typical U6/H1 promoters, which may facilitate therapeutic RNAi applications where delivery vector space is limiting. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Natural selection in avian protein-coding genes expressed in brain.

PubMed

Axelsson, Erik; Hultin-Rosenberg, Lina; Brandström, Mikael; Zwahlén, Martin; Clayton, David F; Ellegren, Hans

2008-06-01

The evolution of birds from theropod dinosaurs took place approximately 150 million years ago, and was associated with a number of specific adaptations that are still evident among extant birds, including feathers, song and extravagant secondary sexual characteristics. Knowledge about the molecular evolutionary background to such adaptations is lacking. Here, we analyse the evolution of > 5000 protein-coding gene sequences expressed in zebra finch brain by comparison to orthologous sequences in chicken. Mean d(N)/d(S) is 0.085 and genes with their maximal expression in the eye and central nervous system have the lowest mean d(N)/d(S) value, while those expressed in digestive and reproductive tissues exhibit the highest. We find that fast-evolving genes (those which have higher than expected rate of nonsynonymous substitution, indicative of adaptive evolution) are enriched for biological functions such as fertilization, muscle contraction, defence response, response to stress, wounding and endogenous stimulus, and cell death. After alignment to mammalian orthologues, we identify a catalogue of 228 genes that show a significantly higher rate of protein evolution in the two bird lineages than in mammals. These accelerated bird genes, representing candidates for avian-specific adaptations, include genes implicated in vocal learning and other cognitive processes. Moreover, colouration genes evolve faster in birds than in mammals, which may have been driven by sexual selection for extravagant plumage characteristics.

Stochastic switching in biology: from genotype to phenotype

NASA Astrophysics Data System (ADS)

Bressloff, Paul C.

2017-03-01

There has been a resurgence of interest in non-equilibrium stochastic processes in recent years, driven in part by the observation that the number of molecules (genes, mRNA, proteins) involved in gene expression are often of order 1-1000. This means that deterministic mass-action kinetics tends to break down, and one needs to take into account the discrete, stochastic nature of biochemical reactions. One of the major consequences of molecular noise is the occurrence of stochastic biological switching at both the genotypic and phenotypic levels. For example, individual gene regulatory networks can switch between graded and binary responses, exhibit translational/transcriptional bursting, and support metastability (noise-induced switching between states that are stable in the deterministic limit). If random switching persists at the phenotypic level then this can confer certain advantages to cell populations growing in a changing environment, as exemplified by bacterial persistence in response to antibiotics. Gene expression at the single-cell level can also be regulated by changes in cell density at the population level, a process known as quorum sensing. In contrast to noise-driven phenotypic switching, the switching mechanism in quorum sensing is stimulus-driven and thus noise tends to have a detrimental effect. A common approach to modeling stochastic gene expression is to assume a large but finite system and to approximate the discrete processes by continuous processes using a system-size expansion. However, there is a growing need to have some familiarity with the theory of stochastic processes that goes beyond the standard topics of chemical master equations, the system-size expansion, Langevin equations and the Fokker-Planck equation. Examples include stochastic hybrid systems (piecewise deterministic Markov processes), large deviations and the Wentzel-Kramers-Brillouin (WKB) method, adiabatic reductions, and queuing/renewal theory. The major aim of this review is to provide a self-contained survey of these mathematical methods, mainly within the context of biological switching processes at both the genotypic and phenotypic levels. However, applications to other examples of biological switching are also discussed, including stochastic ion channels, diffusion in randomly switching environments, bacterial chemotaxis, and stochastic neural networks.

An ontology-driven semantic mash-up of gene and biological pathway information: Application to the domain of nicotine dependence

PubMed Central

Sahoo, Satya S.; Bodenreider, Olivier; Rutter, Joni L.; Skinner, Karen J.; Sheth, Amit P.

2008-01-01

Objectives This paper illustrates how Semantic Web technologies (especially RDF, OWL, and SPARQL) can support information integration and make it easy to create semantic mashups (semantically integrated resources). In the context of understanding the genetic basis of nicotine dependence, we integrate gene and pathway information and show how three complex biological queries can be answered by the integrated knowledge base. Methods We use an ontology-driven approach to integrate two gene resources (Entrez Gene and HomoloGene) and three pathway resources (KEGG, Reactome and BioCyc), for five organisms, including humans. We created the Entrez Knowledge Model (EKoM), an information model in OWL for the gene resources, and integrated it with the extant BioPAX ontology designed for pathway resources. The integrated schema is populated with data from the pathway resources, publicly available in BioPAX-compatible format, and gene resources for which a population procedure was created. The SPARQL query language is used to formulate queries over the integrated knowledge base to answer the three biological queries. Results Simple SPARQL queries could easily identify hub genes, i.e., those genes whose gene products participate in many pathways or interact with many other gene products. The identification of the genes expressed in the brain turned out to be more difficult, due to the lack of a common identification scheme for proteins. Conclusion Semantic Web technologies provide a valid framework for information integration in the life sciences. Ontology-driven integration represents a flexible, sustainable and extensible solution to the integration of large volumes of information. Additional resources, which enable the creation of mappings between information sources, are required to compensate for heterogeneity across namespaces. Resource page http://knoesis.wright.edu/research/lifesci/integration/structured_data/JBI-2008/ PMID:18395495

An ontology-driven semantic mashup of gene and biological pathway information: application to the domain of nicotine dependence.

PubMed

Sahoo, Satya S; Bodenreider, Olivier; Rutter, Joni L; Skinner, Karen J; Sheth, Amit P

2008-10-01

This paper illustrates how Semantic Web technologies (especially RDF, OWL, and SPARQL) can support information integration and make it easy to create semantic mashups (semantically integrated resources). In the context of understanding the genetic basis of nicotine dependence, we integrate gene and pathway information and show how three complex biological queries can be answered by the integrated knowledge base. We use an ontology-driven approach to integrate two gene resources (Entrez Gene and HomoloGene) and three pathway resources (KEGG, Reactome and BioCyc), for five organisms, including humans. We created the Entrez Knowledge Model (EKoM), an information model in OWL for the gene resources, and integrated it with the extant BioPAX ontology designed for pathway resources. The integrated schema is populated with data from the pathway resources, publicly available in BioPAX-compatible format, and gene resources for which a population procedure was created. The SPARQL query language is used to formulate queries over the integrated knowledge base to answer the three biological queries. Simple SPARQL queries could easily identify hub genes, i.e., those genes whose gene products participate in many pathways or interact with many other gene products. The identification of the genes expressed in the brain turned out to be more difficult, due to the lack of a common identification scheme for proteins. Semantic Web technologies provide a valid framework for information integration in the life sciences. Ontology-driven integration represents a flexible, sustainable and extensible solution to the integration of large volumes of information. Additional resources, which enable the creation of mappings between information sources, are required to compensate for heterogeneity across namespaces. RESOURCE PAGE: http://knoesis.wright.edu/research/lifesci/integration/structured_data/JBI-2008/

Photoperiod regulates multiple gene expression in the suprachiasmatic nuclei and pars tuberalis of the Siberian hamster (Phodopus sungorus).

PubMed

Johnston, Jonathan D; Ebling, Francis J P; Hazlerigg, David G

2005-06-01

Photoperiod regulates the seasonal physiology of many mammals living in temperate latitudes. Photoperiodic information is decoded by the master circadian clock in the suprachiasmatic nuclei (SCN) of the hypothalamus and then transduced via pineal melatonin secretion. This neurochemical signal is interpreted by tissues expressing melatonin receptors (e.g. the pituitary pars tuberalis, PT) to drive physiological changes. In this study we analysed the photoperiodic regulation of the circadian clockwork in the SCN and PT of the Siberian hamster. Female hamsters were exposed to either long or short photoperiod for 8 weeks and sampled at 2-h intervals across the 24-h cycle. In the SCN, rhythmic expression of the clock genes Per1, Per2, Cry1, Rev-erbalpha, and the clock-controlled genes arginine vasopressin (AVP) and d-element binding protein (DBP) was modulated by photoperiod. All of these E-box-containing genes tracked dawn, with earlier peak mRNA expression in long, compared to short, photoperiod. This response occurred irrespective of the presence of additional regulatory cis-elements, suggesting photoperiodic regulation of SCN gene expression through a common E-box-related mechanism. In long photoperiod, expression of Cry1 and Per1 in the PT tracked the onset and offset of melatonin secretion, respectively. However, whereas Cry1 tracked melatonin onset in short period, Per1 expression was not detectably rhythmic. We therefore propose that, in the SCN, photoperiodic regulation of clock gene expression primarily occurs via E-boxes, whereas melatonin-driven signal transduction drives the phasing of a subset of clock genes in the PT, independently of the E-box.

Gibberellin 3-oxidase Gene Expression Patterns Influence Gibberellin Biosynthesis, Growth, and Development in Pea1[W][OPEN

PubMed Central

Reinecke, Dennis M.; Wickramarathna, Aruna D.; Ozga, Jocelyn A.; Kurepin, Leonid V.; Jin, Alena L.; Good, Allen G.; Pharis, Richard P.

2013-01-01

Gibberellins (GAs) are key modulators of plant growth and development. PsGA3ox1 (LE) encodes a GA 3β-hydroxylase that catalyzes the conversion of GA20 to biologically active GA1. To further clarify the role of GA3ox expression during pea (Pisum sativum) plant growth and development, we generated transgenic pea lines (in a lele background) with cauliflower mosaic virus-35S-driven expression of PsGA3ox1 (LE). PsGA3ox1 transgene expression led to higher GA1 concentrations in a tissue-specific and development-specific manner, altering GA biosynthesis and catabolism gene expression and plant phenotype. PsGA3ox1 transgenic plants had longer internodes, tendrils, and fruits, larger stipules, and displayed delayed flowering, increased apical meristem life, and altered vascular development relative to the null controls. Transgenic PsGA3ox1 overexpression lines were then compared with lines where endogenous PsGA3ox1 (LE) was introduced, by a series of backcrosses, into the same genetic background (BC LEle). Most notably, the BC LEle plants had substantially longer internodes containing much greater GA1 levels than the transgenic PsGA3ox1 plants. Induction of expression of the GA deactivation gene PsGA2ox1 appears to make an important contribution to limiting the increase of internode GA1 to modest levels for the transgenic lines. In contrast, PsGA3ox1 (LE) expression driven by its endogenous promoter was coordinated within the internode tissue to avoid feed-forward regulation of PsGA2ox1, resulting in much greater GA1 accumulation. These studies further our fundamental understanding of the regulation of GA biosynthesis and catabolism at the tissue and organ level and demonstrate that the timing/localization of GA3ox expression within an organ affects both GA homeostasis and GA1 levels, and thereby growth. PMID:23979969

Gibberellin 3-oxidase gene expression patterns influence gibberellin biosynthesis, growth, and development in pea.

PubMed

Reinecke, Dennis M; Wickramarathna, Aruna D; Ozga, Jocelyn A; Kurepin, Leonid V; Jin, Alena L; Good, Allen G; Pharis, Richard P

2013-10-01

Gibberellins (GAs) are key modulators of plant growth and development. PsGA3ox1 (LE) encodes a GA 3β-hydroxylase that catalyzes the conversion of GA20 to biologically active GA1. To further clarify the role of GA3ox expression during pea (Pisum sativum) plant growth and development, we generated transgenic pea lines (in a lele background) with cauliflower mosaic virus-35S-driven expression of PsGA3ox1 (LE). PsGA3ox1 transgene expression led to higher GA1 concentrations in a tissue-specific and development-specific manner, altering GA biosynthesis and catabolism gene expression and plant phenotype. PsGA3ox1 transgenic plants had longer internodes, tendrils, and fruits, larger stipules, and displayed delayed flowering, increased apical meristem life, and altered vascular development relative to the null controls. Transgenic PsGA3ox1 overexpression lines were then compared with lines where endogenous PsGA3ox1 (LE) was introduced, by a series of backcrosses, into the same genetic background (BC LEle). Most notably, the BC LEle plants had substantially longer internodes containing much greater GA1 levels than the transgenic PsGA3ox1 plants. Induction of expression of the GA deactivation gene PsGA2ox1 appears to make an important contribution to limiting the increase of internode GA1 to modest levels for the transgenic lines. In contrast, PsGA3ox1 (LE) expression driven by its endogenous promoter was coordinated within the internode tissue to avoid feed-forward regulation of PsGA2ox1, resulting in much greater GA1 accumulation. These studies further our fundamental understanding of the regulation of GA biosynthesis and catabolism at the tissue and organ level and demonstrate that the timing/localization of GA3ox expression within an organ affects both GA homeostasis and GA1 levels, and thereby growth.

A synthetic cGMP-sensitive gene switch providing Viagra(®)-controlled gene expression in mammalian cells and mice.

PubMed

Kim, Taeuk; Folcher, Marc; Charpin-El Hamri, Ghislaine; Fussenegger, Martin

2015-05-01

Cyclic guanosine monophosphate (cGMP) is a universal second messenger that is synthesized from guanosine triphosphate (GTP) by guanylyl cyclases (GCs) and hydrolyzed into guanosine monophosphate (GMP) by phosphodiesterases (PDEs). Small-molecule drugs that induce high cGMP levels in specialized tissues by boosting GC activity or inhibiting PDE activity have become the predominant treatment strategy for a wide range of medical conditions, including congestive heart failure, pulmonary hypertension, atherosclerosis-based claudication and erectile dysfunction. By fusing the cGMP receptor protein (CRP) of Rhodospirillum centenum to the Herpes simplex-derived transactivation domain VP16, we created a novel synthetic mammalian cGMP-sensing transcription factor (GTA) that activates synthetic promoters (PGTA) containing newly identified GTA-specific operator sites in a concentration-dependent manner. In cell lines expressing endogenous natriuretic peptide receptor A (NPR-A) (HeLa), GTA/PGTA-driven transgene expression was induced by B-type natriuretic peptide (BNP; Nesiritide(®)) in a concentration-dependent manner, which activated NPR-A׳s intracellular GC domain and triggered a corresponding cGMP surge. Ectopic expression of NPR-A in NPR-A-negative cell lines (HEK-293T) produced high cGMP levels and mediated maximum GTA/PGTA-driven transgene expression, which was suppressed by co-expression of PDEs (PDE-3A, PDE-5A and PDE-9A) and was re-triggered by the corresponding PDE inhibitor drugs (Pletal(®), Perfan(®), Primacor(®) (PDE-3A), Viagra(®), Levitra(®), Cialis(®) (PDE-5A) and BAY73-6691 (PDE-9A)). Mice implanted with microencapsulated designer cells co-expressing the GTA/PGTA device with NPR-A and PDE-5A showed control of blood SEAP levels through administration of sildenafil (Viagra(®)). Designer cells engineered for PDE inhibitor-modulated transgene expression may provide a cell-based PDE-targeting drug discovery platform and enable drug-adjusted gene- and cell-based therapies. Copyright © 2015 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

Changes in gene expression associated with reproductive maturation in wild female baboons.

PubMed

Babbitt, Courtney C; Tung, Jenny; Wray, Gregory A; Alberts, Susan C

2012-01-01

Changes in gene expression during development play an important role in shaping morphological and behavioral differences, including between humans and nonhuman primates. Although many of the most striking developmental changes occur during early development, reproductive maturation represents another critical window in primate life history. However, this process is difficult to study at the molecular level in natural primate populations. Here, we took advantage of ovarian samples made available through an unusual episode of human-wildlife conflict to identify genes that are important in this process. Specifically, we used RNA sequencing (RNA-Seq) to compare genome-wide gene expression patterns in the ovarian tissue of juvenile and adult female baboons from Amboseli National Park, Kenya. We combined this information with prior evidence of selection occurring on two primate lineages (human and chimpanzee). We found that in cases in which genes were both differentially expressed over the course of ovarian maturation and also linked to lineage-specific selection this selective signature was much more likely to occur in regulatory regions than in coding regions. These results suggest that adaptive change in the development of the primate ovary may be largely driven at the mechanistic level by selection on gene regulation, potentially in relationship to the physiology or timing of female reproductive maturation.

Changes in Gene Expression Associated with Reproductive Maturation in Wild Female Baboons

PubMed Central

Babbitt, Courtney C.; Tung, Jenny; Wray, Gregory A.; Alberts, Susan C.

2012-01-01

Changes in gene expression during development play an important role in shaping morphological and behavioral differences, including between humans and nonhuman primates. Although many of the most striking developmental changes occur during early development, reproductive maturation represents another critical window in primate life history. However, this process is difficult to study at the molecular level in natural primate populations. Here, we took advantage of ovarian samples made available through an unusual episode of human–wildlife conflict to identify genes that are important in this process. Specifically, we used RNA sequencing (RNA-Seq) to compare genome-wide gene expression patterns in the ovarian tissue of juvenile and adult female baboons from Amboseli National Park, Kenya. We combined this information with prior evidence of selection occurring on two primate lineages (human and chimpanzee). We found that in cases in which genes were both differentially expressed over the course of ovarian maturation and also linked to lineage-specific selection this selective signature was much more likely to occur in regulatory regions than in coding regions. These results suggest that adaptive change in the development of the primate ovary may be largely driven at the mechanistic level by selection on gene regulation, potentially in relationship to the physiology or timing of female reproductive maturation. PMID:22155733

Inhibition of Hedgehog-Signaling Driven Genes in Prostate Cancer Cells by Sutherlandia frutescens Extract

PubMed Central

Lu, Yuan; Starkey, Nicholas; Lei, Wei; Li, Jilong; Cheng, Jianlin; Folk, William R.; Lubahn, Dennis B.

2015-01-01

Sutherlandia frutescens (L) R. Br. (Sutherlandia) is a South African botanical that is traditionally used to treat a variety of health conditions, infections and diseases, including cancer. We hypothesized Sutherlandia might act through Gli/ Hedgehog (Hh)-signaling in prostate cancer cells and used RNA-Seq transcription profiling to profile gene expression in TRAMPC2 murine prostate cancer cells with or without Sutherlandia extracts. We found 50% of Hh-responsive genes can be repressed by Sutherlandia ethanol extract, including the canonical Hh-responsive genes Gli1 and Ptch1 as well as newly distinguished Hh-responsive genes Hsd11b1 and Penk. PMID:26710108

Site-specific methylation of the rat prolactin and growth hormone promoters correlates with gene expression.

PubMed Central

Ngô, V; Gourdji, D; Laverrière, J N

1996-01-01

The methylation patterns of the rat prolactin (rPRL) (positions -440 to -20) and growth hormone (rGH) (positions -360 to -110) promoters were analyzed by bisulfite genomic sequencing. Two normal tissues, the anterior pituitary and the liver, and three rat pituitary GH3 cell lines that differ considerably in their abilities to express both genes were tested. High levels of rPRL gene expression were correlated with hypomethylation of the CpG dinucleotides located at positions -277 and -97, near or within positive cis-acting regulatory elements. For the nine CpG sites analyzed in the rGH promoter, an overall hypomethylation-expression coupling was also observed for the anterior pituitary, the liver, and two of the cell lines. The effect of DNA methylation was tested by measuring the transient expression of the chloramphenicol acetyltransferase reporter gene driven by a regionally methylated rPRL promoter. CpG methylation resulted in a decrease in the activity of the rPRL promoter which was proportional to the number of modified CpG sites. The extent of the inhibition was also found to be dependent on the position of methylated sites. Taken together, these data suggest that site-specific methylation may modulate the action of transcription factors that dictate the tissue-specific expression of the rPRL and rGH genes in vivo. PMID:8668139

Dual systemic tumor targeting with ligand-directed phage and Grp78 promoter induces tumor regression.

PubMed

Kia, Azadeh; Przystal, Justyna M; Nianiaris, Nastasia; Mazarakis, Nicholas D; Mintz, Paul J; Hajitou, Amin

2012-12-01

The tumor-specific Grp78 promoter is overexpressed in aggressive tumors. Cancer patients would benefit greatly from application of this promoter in gene therapy and molecular imaging; however, clinical benefit is limited by lack of strategies to target the systemic delivery of Grp78-driven transgenes to tumors. This study aims to assess the systemic efficacy of Grp78-guided expression of therapeutic and imaging transgenes relative to the standard cytomegalovirus (CMV) promoter. Combination of ligand and Grp78 transcriptional targeting into a single vector would facilitate systemic applications of the Grp78 promoter. We generated a dual tumor-targeted phage containing the arginine-glycine-aspartic acid tumor homing ligand and Grp78 promoter. Next, we combined flow cytometry, Western blot analysis, bioluminescence imaging of luciferase, and HSVtk/ganciclovir gene therapy and compared efficacy to conventional phage carrying the CMV promoter in vitro and in vivo in subcutaneous models of rat and human glioblastoma. We show that double-targeted phage provides persistent transgene expression in vitro and in tumors in vivo after systemic administration compared with conventional phage. Next, we showed significant tumor killing in vivo using the HSVtk/ganciclovir gene therapy and found a systemic antitumor effect of Grp78-driven HSVtk against therapy-resistant tumors. Finally, we uncovered a novel mechanism of Grp78 promoter activation whereby HSVtk/ganciclovir therapy upregulates Grp78 and transgene expression via the conserved unfolded protein response signaling cascade. These data validate the potential of Grp78 promoter in systemic cancer gene therapy and report the efficacy of a dual tumor targeting phage that may prove useful for translation into gene therapy and molecular imaging applications.

A sustained increase in plasma NEFA upregulates the Toll-like receptor network in human muscle

PubMed Central

Hussey, Sophie E.; Lum, Helen; Alvarez, Andrea; Cipriani, Yolanda; Garduño-Garcia, José de Jesús; Anaya, Luis; Dube, John; Musi, Nicolas

2014-01-01

Aims/hypothesis Insulin-sensitive tissues (muscle, liver) of individuals with obesity and type 2 diabetes mellitus are in a state of low-grade inflammation, characterised by increased Toll-like receptor (TLR) expression and TLR-driven signalling. However, the cause of this mild inflammatory state is unclear. We tested the hypothesis that a prolonged mild increase in plasma NEFA will increase TLR expression and TLR-driven signalling (nuclear factor κB [NFκB] and mitogen-activated kinase [MAPK]) and impair insulin action in muscle of lean healthy individuals. Methods Twelve lean, normal-glucose-tolerant participants were randomised to receive a 48 h infusion (30 ml/h) of saline or Intralipid followed by a euglycaemic–hyperinsulinaemic clamp. Vastus lateralis muscle biopsies were performed before and during the clamp. Results Lipid infusion impaired insulin-stimulated IRS-1 tyrosine phosphorylation and reduced peripheral insulin sensitivity (p < 0.01). The elevation in circulating NEFA increased expression of TLR3, TLR4 and TLR5, and several MAPK (MAPK8, MAP4K4, MAP2K3) and inhibitor of κB kinase-NFκB (CHUK [IKKA], c-REL [REL] and p65 [RELA, NFKB3,p65]) signalling genes (p < 0.05). The lipid infusion also increased extracellular signal-regulated kinase (ERK) phosphorylation (p < 0.05) and tended to reduce the content of nuclear factor of light polypeptide gene enhancer in B cells inhibitor α (p = 0.09). The muscle content of most diacyglycerol, ceramide and acylcarnitine species was unaffected. In summary, insulin resistance induced by prolonged low-dose lipid infusion occurs together with increased TLR-driven inflammatory signalling and impaired insulin-stimulated IRS-1 tyrosine phosphorylation. Conclusions/interpretation A sustained, mild elevation in plasma NEFA is sufficient to increase TLR expression and TLR-driven signalling (NFκB and MAPK) in lean individuals. The activation of this pathway by NEFA may be involved in the pathogenesis of insulin resistance in humans. PMID:24337154

Protocadherin-1 binds to SMAD3 and suppresses TGF-β1-induced gene transcription

PubMed Central

Faura Tellez, Grissel; Vandepoele, Karl; Brouwer, Uilke; Koning, Henk; Elderman, Robin M.; Hackett, Tillie-Louise; Willemse, Brigitte W. M.; Holloway, John; Van Roy, Frans; Koppelman, Gerard H.

2015-01-01

Genetic studies have identified Protocadherin-1 (PCDH1) and Mothers against decapentaplegic homolog-3 (SMAD3) as susceptibility genes for asthma. PCDH1 is expressed in bronchial epithelial cells and has been found to interact with SMAD3 in yeast two-hybrid (Y2H) overexpression assays. Here, we test whether PCDH1 and SMAD3 interact at endogenous protein levels in bronchial epithelial cells and evaluate the consequences thereof for transforming growth factor-β1 (TGF-β1)-induced gene transcription. We performed Y2H screens and coimmunoprecipitation (co-IP) experiments of PCDH1 and SMAD3 in HEK293T and 16HBE14o− (16HBE) cell lines. Activity of a SMAD3-driven luciferase reporter gene in response to TGF-β1 was measured in BEAS-2B cells transfected with PCDH1 and in 16HBE cells transfected with PCDH1-small-interfering RNA (siRNA). TGF-β1-induced gene expression was quantified in BEAS-2B clones overexpressing PCDH1 and in human primary bronchial epithelial cells (PBECs) transfected with PCDH1-siRNA. We confirm PCDH1 and SMAD3 interactions by Y2H and by co-IP in HEK293T cells overexpressing both proteins, and at endogenous protein levels in 16HBE cells. TGF-β-induced activation of a SMAD3-driven reporter was reduced by exogenous PCDH1 in BEAS2B cells, whereas it was increased by siRNA-mediated knockdown of endogenous PCDH1 in 16HBE cells. Overexpression of PCDH1 suppressed expression of TGF-β target genes in BEAS-2B cells, whereas knockdown of PCDH1 in human PBECs increased TGF-β-induced gene expression. In conclusion, we demonstrate that PCDH1 binds to SMAD3 and regulates its activation by TGF-β signaling in bronchial epithelial cells. We propose that PCDH1 and SMAD3 act in a single pathway in asthma susceptibility that affects sensitivity of the airway epithelium to TGF-β. PMID:26209277

Partially Redundant Enhancers Cooperatively Maintain Mammalian Pomc Expression Above a Critical Functional Threshold

PubMed Central

Lam, Daniel D.; de Souza, Flavio S. J.; Nasif, Sofia; Yamashita, Miho; López-Leal, Rodrigo; Meece, Kana; Sampath, Harini; Mercer, Aaron J.; Wardlaw, Sharon L.

2015-01-01

Cell-specific expression of many genes is conveyed by multiple enhancers, with each individual enhancer controlling a particular expression domain. In contrast, multiple enhancers drive similar expression patterns of some genes involved in embryonic development, suggesting regulatory redundancy. Work in Drosophila has indicated that functionally overlapping enhancers canalize development by buffering gene expression against environmental and genetic disturbances. However, little is known about regulatory redundancy in vertebrates and in genes mainly expressed during adulthood. Here we study nPE1 and nPE2, two phylogenetically conserved mammalian enhancers that drive expression of the proopiomelanocortin gene (Pomc) to the same set of hypothalamic neurons. The simultaneous deletion of both enhancers abolished Pomc expression at all ages and induced a profound metabolic dysfunction including early-onset extreme obesity. Targeted inactivation of either nPE1 or nPE2 led to very low levels of Pomc expression during early embryonic development indicating that both enhancers function synergistically. In adult mice, however, Pomc expression is controlled additively by both enhancers, with nPE1 being responsible for ∼80% and nPE2 for ∼20% of Pomc transcription. Consequently, nPE1 knockout mice exhibit mild obesity whereas nPE2-deficient mice maintain a normal body weight. These results suggest that nPE2-driven Pomc expression is compensated by nPE1 at later stages of development, essentially rescuing the earlier phenotype of nPE2 deficiency. Together, these results reveal that cooperative interactions between the enhancers confer robustness of Pomc expression against gene regulatory disturbances and preclude deleterious metabolic phenotypes caused by Pomc deficiency in adulthood. Thus, our study demonstrates that enhancer redundancy can be used by genes that control adult physiology in mammals and underlines the potential significance of regulatory sequence mutations in common diseases. PMID:25671638

Relationship of age and body mass index to the expression of obesity and osteoarthritis-related genes in human meniscus.

PubMed

Rai, M F; Sandell, L J; Cheverud, J M; Brophy, R H

2013-09-01

Aging and obesity contribute to the initiation and progression of osteoarthritis with little information on their relation to gene expression in joint tissues, particularly the meniscus. Here, we test the hypothesis that patient age and body mass index (BMI) correlate with the expression of osteoarthritis- and obesity-related gene signatures in the meniscus. Meniscus was obtained from patients (N=68) undergoing arthroscopic partial meniscectomy. The mRNA expression of 24 osteoarthritis-related and 4 obesity-related genes in meniscus was assessed by quantitative real-time PCR. The relationship between gene expression and patient age and BMI was analyzed using Spearman's rank-order correlation. Hierarchical cluster dendrogram and heat map were generated to study inter-gene associations. Age was negatively correlated (P<0.05) with the expression of MMP-1 (r=-0.447), NFκB2 (r=-0.361), NFκBIA (r=-0.312), IκBA (r=-0.308), IL-8 (r=-0.305), ADAMTS-4 (r=-0.294), APLN (apelin) (r=-0.250) and IL-6 (r=-0.244). Similarly, BMI was negatively correlated with the expression of APLN (r=-0.328), ACAN (r=-0.268) and MMP-1 (r=-0.261). After adjusting for the correlation between age and BMI (r=0.310; P=0.008), the only independent effect of BMI on gene expression was for APLN (r=-0.272). However, age had an independent effect on the expression on ADAMTS-4 (r=-0.253), MMP-1 (r=-0.399), IL-8 (r=-0.327), COL1A1 (r=-0.287), NFκBIA (r=-0.278), NFκB2 (r=-0.312) and IκBA (r=-0.299). The gene correlation analysis identified four clusters of potentially relevant genes: transcription factors, matrix-degrading enzymes, cytokines and chemokines, and obesity genes. Age and BMI were negatively correlated with several osteoarthritis- and obesity-related genes. Although the bulk of these changes appeared to be driven by age, expression of APLN was related to BMI. Inter-gene correlation analysis implicated a common role for strongly correlated genes. Although age-related variations in gene expression appear to be more relevant than obesity-related differences for the role of the meniscus in osteoarthritis development, further investigation into the role of APLN in meniscus and joint health is warranted.

Gene Expression Profiling of Liver Cancer Stem Cells by RNA-Sequencing

PubMed Central

Lam, Chi Tat; Ng, Michael N. P.; Yu, Wan Ching; Lau, Joyce; Wan, Timothy; Wang, Xiaoqi; Yan, Zhixiang; Liu, Hang; Fan, Sheung Tat

2012-01-01

Background Accumulating evidence supports that tumor growth and cancer relapse are driven by cancer stem cells. Our previous work has demonstrated the existence of CD90+ liver cancer stem cells (CSCs) in hepatocellular carcinoma (HCC). Nevertheless, the characteristics of these cells are still poorly understood. In this study, we employed a more sensitive RNA-sequencing (RNA-Seq) to compare the gene expression profiling of CD90+ cells sorted from tumor (CD90+CSCs) with parallel non-tumorous liver tissues (CD90+NTSCs) and elucidate the roles of putative target genes in hepatocarcinogenesis. Methodology/Principal Findings CD90+ cells were sorted respectively from tumor and adjacent non-tumorous human liver tissues using fluorescence-activated cell sorting. The amplified RNAs of CD90+ cells from 3 HCC patients were subjected to RNA-Seq analysis. A differential gene expression profile was established between CD90+CSCs and CD90+NTSCs, and validated by quantitative real-time PCR (qRT-PCR) on the same set of amplified RNAs, and further confirmed in an independent cohort of 12 HCC patients. Five hundred genes were differentially expressed (119 up-regulated and 381 down-regulated genes) between CD90+CSCs and CD90+NTSCs. Gene ontology analysis indicated that the over-expressed genes in CD90+CSCs were associated with inflammation, drug resistance and lipid metabolism. Among the differentially expressed genes, glypican-3 (GPC3), a member of glypican family, was markedly elevated in CD90+CSCs compared to CD90+NTSCs. Immunohistochemistry demonstrated that GPC3 was highly expressed in forty-two human liver tumor tissues but absent in adjacent non-tumorous liver tissues. Flow cytometry indicated that GPC3 was highly expressed in liver CD90+CSCs and mature cancer cells in liver cancer cell lines and human liver tumor tissues. Furthermore, GPC3 expression was positively correlated with the number of CD90+CSCs in liver tumor tissues. Conclusions/Significance The identified genes, such as GPC3 that are distinctly expressed in liver CD90+CSCs, may be promising gene candidates for HCC therapy without inducing damages to normal liver stem cells. PMID:22606345

The metastasis suppressor RARRES3 as an endogenous inhibitor of the immunoproteasome expression in breast cancer cells

NASA Astrophysics Data System (ADS)

Anderson, Alison M.; Kalimutho, Murugan; Harten, Sarah; Nanayakkara, Devathri M.; Khanna, Kum Kum; Ragan, Mark A.

2017-01-01

In breast cancer metastasis, the dynamic continuum involving pro- and anti-inflammatory regulators can become compromised. Over 600 genes have been implicated in metastasis to bone, lung or brain but how these genes might contribute to perturbation of immune function is poorly understood. To gain insight, we adopted a gene co-expression network approach that draws on the functional parallels between naturally occurring bone marrow-derived mesenchymal stem cells (BM-MSCs) and cancer stem cells (CSCs). Our network analyses indicate a key role for metastasis suppressor RARRES3, including potential to regulate the immunoproteasome (IP), a specialized proteasome induced under inflammatory conditions. Knockdown of RARRES3 in near-normal mammary epithelial and breast cancer cell lines increases overall transcript and protein levels of the IP subunits, but not of their constitutively expressed counterparts. RARRES3 mRNA expression is controlled by interferon regulatory factor IRF1, an inducer of the IP, and is sensitive to depletion of the retinoid-related receptor RORA that regulates various physiological processes including immunity through modulation of gene expression. Collectively, these findings identify a novel regulatory role for RARRES3 as an endogenous inhibitor of IP expression, and contribute to our evolving understanding of potential pathways underlying breast cancer driven immune modulation.

Heterologous gene expression driven by carbonic anhydrase gene promoter in Dunaliella salina

NASA Astrophysics Data System (ADS)

Chai, Yurong; Lu, Yumin; Wang, Tianyun; Hou, Weihong; Xue, Lexun

2006-12-01

Dunaliella salina, a halotolerant unicellular green alga without a rigid cell wall, can live in salinities ranging from 0.05 to 5 mol/L NaCl. These features of D. salina make it an ideal host for the production of antibodies, oral vaccine, and commercially valuable polypeptides. To produce high level of heterologous proteins from D. salina, highly efficient promoters are required to drive expression of target genes under controlled condition. In the present study, we cloned a 5' franking region of 1.4 kb from the carbonic anhydrase ( CAH) gene of D. salina by genomic walking and PCR. The fragment was ligated to the pMD18-T vector and characterized. Sequence analysis indicated that this region contained conserved motifs, including a TATA- like box and CAAT-box. Tandem (GT)n repeats that had a potential role of transcriptional control, were also found in this region. The transcription start site (TSS) of the CAH gene was determined by 5' RACE and nested PCR method. Transformation assays showed that the 1.4 kb fragment was able to drive expression of the selectable bar (bialaphos resistance) gene when the fusion was transformed into D. salina by biolistics. Northern blotting hybridizations showed that the bar transcript was most abundant in cells grown in 2 mol/L NaCl, and less abundant in 0.5 mol/L NaCl, indicating that expression of the bar gene was induced at high salinity. These results suggest the potential use of the CAH gene promoter to induce the expression of heterologous genes in D. salina under varied salt condition.

Stem cell regulatory gene expression in human adult dental pulp and periodontal ligament cells undergoing odontogenic/osteogenic differentiation.

PubMed

Liu, Lu; Ling, Junqi; Wei, Xi; Wu, Liping; Xiao, Yin

2009-10-01

During development and regeneration, odontogenesis and osteogenesis are initiated by a cascade of signals driven by several master regulatory genes. In this study, we investigated the differential expression of 84 stem cell-related genes in dental pulp cells (DPCs) and periodontal ligament cells (PDLCs) undergoing odontogenic/osteogenic differentiation. Our results showed that, although there was considerable overlap, certain genes had more differential expression in PDLCs than in DPCs. CCND2, DLL1, and MME were the major upregulated genes in both PDLCs and DPCs, whereas KRT15 was the only gene significantly downregulated in PDLCs and DPCs in both odontogenic and osteogenic differentiation. Interestingly, a large number of regulatory genes in odontogenic and osteogenic differentiation interact or crosstalk via Notch, Wnt, transforming growth factor beta (TGF-beta)/bone morphogenic protein (BMP), and cadherin signaling pathways, such as the regulation of APC, DLL1, CCND2, BMP2, and CDH1. Using a rat dental pulp and periodontal defect model, the expression and distribution of both BMP2 and CDH1 have been verified for their spatial localization in dental pulp and periodontal tissue regeneration. This study has generated an overview of stem cell-related gene expression in DPCs and PDLCs during odontogenic/osteogenic differentiation and revealed that these genes may interact through the Notch, Wnt, TGF-beta/BMP, and cadherin signaling pathways to play a crucial role in determining the fate of dental derived cell and dental tissue regeneration. These findings provided a new insight into the molecular mechanisms of the dental tissue mineralization and regeneration.

Transgene-mediated suppression of dengue viruses in the salivary glands of the yellow fever mosquito, Aedes aegypti.

PubMed

Mathur, G; Sanchez-Vargas, I; Alvarez, D; Olson, K E; Marinotti, O; James, A A

2010-12-01

Controlled sex-, stage- and tissue-specific expression of antipathogen effector molecules is important for genetic engineering strategies to control mosquito-borne diseases. Adult female salivary glands are involved in pathogen transmission to human hosts and are target sites for expression of antipathogen effector molecules. The Aedes aegypti 30K a and 30K b genes are expressed exclusively in adult female salivary glands and are transcribed divergently from start sites separated by 263 nucleotides. The intergenic, 5'- and 3'-end untranslated regions of both genes are sufficient to express simultaneously two different transgene products in the distal-lateral lobes of the female salivary glands. An antidengue effector gene, membranes no protein (Mnp), driven by the 30K b promoter, expresses an inverted-repeat RNA with sequences derived from the premembrane protein-encoding region of the dengue virus serotype 2 genome and reduces significantly the prevalence and mean intensities of viral infection in mosquito salivary glands and saliva. © 2010 The Authors. Insect Molecular Biology © 2010 The Royal Entomological Society.

PU.1 is a major transcriptional activator of the tumour suppressor gene LIMD1

PubMed Central

Foxler, Daniel E.; James, Victoria; Shelton, Samuel J.; Vallim, Thomas Q. de A.; Shaw, Peter E.; Sharp, Tyson V.

2011-01-01

LIMD1 is a tumour suppressor gene (TSG) down regulated in ∼80% of lung cancers with loss also demonstrated in breast and head and neck squamous cell carcinomas. LIMD1 is also a candidate TSG in childhood acute lymphoblastic leukaemia. Mechanistically, LIMD1 interacts with pRB, repressing E2F-driven transcription as well as being a critical component of microRNA-mediated gene silencing. In this study we show a CpG island within the LIMD1 promoter contains a conserved binding motif for the transcription factor PU.1. Mutation of the PU.1 consensus reduced promoter driven transcription by 90%. ChIP and EMSA analysis demonstrated that PU.1 specifically binds to the LIMD1 promoter. siRNA depletion of PU.1 significantly reduced endogenous LIMD1 expression, demonstrating that PU.1 is a major transcriptional activator of LIMD1. PMID:21402070

Regulatory Features for Odorant Receptor Genes in the Mouse Genome.

PubMed

Degl'Innocenti, Andrea; D'Errico, Anna

2017-01-01

The odorant receptor genes, seven transmembrane receptor genes constituting the vastest mammalian gene multifamily, are expressed monogenically and monoallelicaly in each sensory neuron in the olfactory epithelium. This characteristic, often referred to as the one neuron-one receptor rule, is driven by mostly uncharacterized molecular dynamics, generally named odorant receptor gene choice . Much attention has been paid by the scientific community to the identification of sequences regulating the expression of odorant receptor genes within their loci , where related genes are usually arranged in genomic clusters. A number of studies identified transcription factor binding sites on odorant receptor promoter sequences. Similar binding sites were also found on a number of enhancers that regulate in cis their transcription, but have been proposed to form interchromosomal networks. Odorant receptor gene choice seems to occur via the local removal of strongly repressive epigenetic markings, put in place during the maturation of the sensory neuron on each odorant receptor locus . Here we review the fast-changing state of art for the study of regulatory features for odorant receptor genes.

Evolutionary origin and functional divergence of totipotent cell homeobox genes in eutherian mammals.

PubMed

Maeso, Ignacio; Dunwell, Thomas L; Wyatt, Chris D R; Marlétaz, Ferdinand; Vető, Borbála; Bernal, Juan A; Quah, Shan; Irimia, Manuel; Holland, Peter W H

2016-06-13

A central goal of evolutionary biology is to link genomic change to phenotypic evolution. The origin of new transcription factors is a special case of genomic evolution since it brings opportunities for novel regulatory interactions and potentially the emergence of new biological properties. We demonstrate that a group of four homeobox gene families (Argfx, Leutx, Dprx, Tprx), plus a gene newly described here (Pargfx), arose by tandem gene duplication from the retinal-expressed Crx gene, followed by asymmetric sequence evolution. We show these genes arose as part of repeated gene gain and loss events on a dynamic chromosomal region in the stem lineage of placental mammals, on the forerunner of human chromosome 19. The human orthologues of these genes are expressed specifically in early embryo totipotent cells, peaking from 8-cell to morula, prior to cell fate restrictions; cow orthologues have similar expression. To examine biological roles, we used ectopic gene expression in cultured human cells followed by high-throughput RNA-seq and uncovered extensive transcriptional remodelling driven by three of the genes. Comparison to transcriptional profiles of early human embryos suggest roles in activating and repressing a set of developmentally-important genes that spike at 8-cell to morula, rather than a general role in genome activation. We conclude that a dynamic chromosome region spawned a set of evolutionarily new homeobox genes, the ETCHbox genes, specifically in eutherian mammals. After these genes diverged from the parental Crx gene, we argue they were recruited for roles in the preimplantation embryo including activation of genes at the 8-cell stage and repression after morula. We propose these new homeobox gene roles permitted fine-tuning of cell fate decisions necessary for specification and function of embryonic and extra-embryonic tissues utilised in mammalian development and pregnancy.

Faster-X evolution of gene expression is driven by recessive adaptive cis-regulatory variation in Drosophila.

PubMed

Llopart, Ana

2018-05-01

The hemizygosity of the X (Z) chromosome fully exposes the fitness effects of mutations on that chromosome and has evolutionary consequences on the relative rates of evolution of X and autosomes. Specifically, several population genetics models predict increased rates of evolution in X-linked loci relative to autosomal loci. This prediction of faster-X evolution has been evaluated and confirmed for both protein coding sequences and gene expression. In the case of faster-X evolution for gene expression divergence, it is often assumed that variation in 5' noncoding sequences is associated with variation in transcript abundance between species but a formal, genomewide test of this hypothesis is still missing. Here, I use whole genome sequence data in Drosophila yakuba and D. santomea to evaluate this hypothesis and report positive correlations between sequence divergence at 5' noncoding sequences and gene expression divergence. I also examine polymorphism and divergence in 9,279 noncoding sequences located at the 5' end of annotated genes and detected multiple signals of positive selection. Notably, I used the traditional synonymous sites as neutral reference to test for adaptive evolution, but I also used bases 8-30 of introns <65 bp, which have been proposed to be a better neutral choice. X-linked genes with high degree of male-biased expression show the most extreme adaptive pattern at 5' noncoding regions, in agreement with faster-X evolution for gene expression divergence and a higher incidence of positively selected recessive mutations. © 2018 The Authors. Molecular Ecology Published by John Wiley & Sons Ltd.

Separate and combined effects of genetic variants and pre-treatment whole blood gene expression on response to exposure-based cognitive behavioural therapy for anxiety disorders.

PubMed

Coleman, Jonathan R I; Lester, Kathryn J; Roberts, Susanna; Keers, Robert; Lee, Sang Hyuck; De Jong, Simone; Gaspar, Héléna; Teismann, Tobias; Wannemüller, André; Schneider, Silvia; Jöhren, Peter; Margraf, Jürgen; Breen, Gerome; Eley, Thalia C

2017-04-01

Exposure-based cognitive behavioural therapy (eCBT) is an effective treatment for anxiety disorders. Response varies between individuals. Gene expression integrates genetic and environmental influences. We analysed the effect of gene expression and genetic markers separately and together on treatment response. Adult participants (n ≤ 181) diagnosed with panic disorder or a specific phobia underwent eCBT as part of standard care. Percentage decrease in the Clinical Global Impression severity rating was assessed across treatment, and between baseline and a 6-month follow-up. Associations with treatment response were assessed using expression data from 3,233 probes, and expression profiles clustered in a data- and literature-driven manner. A total of 3,343,497 genetic variants were used to predict treatment response alone and combined in polygenic risk scores. Genotype and expression data were combined in expression quantitative trait loci (eQTL) analyses. Expression levels were not associated with either treatment phenotype in any analysis. A total of 1,492 eQTLs were identified with q < 0.05, but interactions between genetic variants and treatment response did not affect expression levels significantly. Genetic variants did not significantly predict treatment response alone or in polygenic risk scores. We assessed gene expression alone and alongside genetic variants. No associations with treatment outcome were identified. Future studies require larger sample sizes to discover associations.

Evaluation of biolistic gene transfer methods in vivo using non-invasive bioluminescent imaging techniques.

PubMed

Xia, Jixiang; Martinez, Angela; Daniell, Henry; Ebert, Steven N

2011-06-02

Gene therapy continues to hold great potential for treating many different types of disease and dysfunction. Safe and efficient techniques for gene transfer and expression in vivo are needed to enable gene therapeutic strategies to be effective in patients. Currently, the most commonly used methods employ replication-defective viral vectors for gene transfer, while physical gene transfer methods such as biolistic-mediated ("gene-gun") delivery to target tissues have not been as extensively explored. In the present study, we evaluated the efficacy of biolistic gene transfer techniques in vivo using non-invasive bioluminescent imaging (BLI) methods. Plasmid DNA carrying the firefly luciferase (LUC) reporter gene under the control of the human Cytomegalovirus (CMV) promoter/enhancer was transfected into mouse skin and liver using biolistic methods. The plasmids were coupled to gold microspheres (1 μm diameter) using different DNA Loading Ratios (DLRs), and "shot" into target tissues using a helium-driven gene gun. The optimal DLR was found to be in the range of 4-10. Bioluminescence was measured using an In Vivo Imaging System (IVIS-50) at various time-points following transfer. Biolistic gene transfer to mouse skin produced peak reporter gene expression one day after transfer. Expression remained detectable through four days, but declined to undetectable levels by six days following gene transfer. Maximum depth of tissue penetration following biolistic transfer to abdominal skin was 200-300 μm. Similarly, biolistic gene transfer to mouse liver in vivo also produced peak early expression followed by a decline over time. In contrast to skin, however, liver expression of the reporter gene was relatively stable 4-8 days post-biolistic gene transfer, and remained detectable for nearly two weeks. The use of bioluminescence imaging techniques enabled efficient evaluation of reporter gene expression in vivo. Our results demonstrate that different tissues show different expression kinetics following gene transfer of the same reporter plasmid to different mouse tissues in vivo. We evaluated superficial (skin) and abdominal organ (liver) targets, and found that reporter gene expression peaked within the first two days post-transfer in each case, but declined most rapidly in the skin (3-4 days) compared to liver (10-14 days). This information is essential for designing effective gene therapy strategies in different target tissues.

Epigenetic Control of Cytokine Gene Expression: Regulation of the TNF/LT Locus and T Helper Cell Differentiation

PubMed Central

Falvo, James V.; Jasenosky, Luke D.; Kruidenier, Laurens; Goldfeld, Anne E.

2014-01-01

Epigenetics encompasses transient and heritable modifications to DNA and nucleosomes in the native chromatin context. For example, enzymatic addition of chemical moieties to the N-terminal “tails” of histones, particularly acetylation and methylation of lysine residues in the histone tails of H3 and H4, plays a key role in regulation of gene transcription. The modified histones, which are physically associated with gene regulatory regions that typically occur within conserved noncoding sequences, play a functional role in active, poised, or repressed gene transcription. The “histone code” defined by these modifications, along with the chromatin-binding acetylases, deacetylases, methylases, demethylases, and other enzymes that direct modifications resulting in specific patterns of histone modification, shows considerable evolutionary conservation from yeast to humans. Direct modifications at the DNA level, such as cytosine methylation at CpG motifs that represses promoter activity, are another highly conserved epigenetic mechanism of gene regulation. Furthermore, epigenetic modifications at the nucleosome or DNA level can also be coupled with higher-order intra- or interchromosomal interactions that influence the location of regulatory elements and that can place them in an environment of specific nucleoprotein complexes associated with transcription. In the mammalian immune system, epigenetic gene regulation is a crucial mechanism for a range of physiological processes, including the innate host immune response to pathogens and T cell differentiation driven by specific patterns of cytokine gene expression. Here, we will review current findings regarding epigenetic regulation of cytokine genes important in innate and/or adaptive immune responses, with a special focus upon the tumor necrosis factor/lymphotoxin locus and cytokine-driven CD4+ T cell differentiation into the Th1, Th2, and Th17 lineages. PMID:23683942

Cytotoxic effect of the Her-2/Her-1 inhibitor PKI-166 on renal cancer cells expressing the connexin 32 gene.

PubMed

Fujimoto, Eriko; Yano, Tomohiro; Sato, Hiromi; Hagiwara, Kiyokazu; Yamasaki, Hiroshi; Shirai, Sumiko; Fukumoto, Keiko; Hagiwara, Hiromi; Negishi, Etsuko; Ueno, Koichi

2005-02-01

We have reported that connexin (Cx) 32 acts as a tumor suppressor gene in renal cancer cells partly due to Her-2 inactivation. Here, we determined if a Her-2/Her-1 inhibitor (PKI-166) can enhance the tumor-suppressive effect of Cx32 in Caki-2 cells from human renal cell carcinoma. The expression of Cx32 in Caki-2 cells was required for PKI-166-induced cytotoxic effect at lower doses. The cyctotoxicity was dependent on the occurrence of apoptosis and partly mediated by Cx32-driven gap junction intercellular communications. These results suggest that PKI-166 further supports the tumor-suppressive effect of the Cx32 gene in renal cancer cells through the induction of apoptosis.

Overexpression of genes involved in miRNA biogenesis in medullary thyroid carcinomas with RET mutation.

PubMed

Puppin, Cinzia; Durante, Cosimo; Sponziello, Marialuisa; Verrienti, Antonella; Pecce, Valeria; Lavarone, Elisa; Baldan, Federica; Campese, Antonio Francesco; Boichard, Amelie; Lacroix, Ludovic; Russo, Diego; Filetti, Sebastiano; Damante, Giuseppe

2014-11-01

Abnormal expression of non-coding micro RNA (miRNA) has been described in medullary thyroid carcinoma (MTC). Expression of genes encoding factors involved in miRNA biogenesis results often deregulated in human cancer and correlates with aggressive clinical behavior. In this study, expression of four genes involved in miRNA biogenesis (DICER, DROSHA, DCGR8, and XPO5) was investigated in 54 specimens of MTC. Among them, 33 and 13 harbored RET and RAS mutations, respectively. DICER, DGCR8, and XPO5 mRNA levels were significantly overexpressed in MTC harboring RET mutations, in particular, in the presence of RET634 mutation. When MTCs with RET and RAS mutations were compared, only DGCR8 displayed a significant difference, while MTCs with RAS mutations did not show significant differences with respect to non-mutated tumors. We then attempted to correlate expression of miRNA biogenesis genes with tumor aggressiveness. According to the TNM status, MTCs were divided in two groups and compared (N0 M0 vs. N1 and/or M1): for all four genes no significant difference was detected. Cell line experiments, in which expression of a RET mutation is silenced by siRNA, suggest the existence of a causal relationship between RET mutation and overexpression of DICER, DGCR8, and XPO5 genes. These findings demonstrate that RET- but not RAS-driven tumorigenic alterations include abnormalities in the expression of some important genes involved in miRNA biogenesis that could represent new potential markers for targeted therapies in the treatment of RET-mutated MTCs aimed to restore the normal miRNA expression profile.

Cardiac stem cell genetic engineering using the alphaMHC promoter.

PubMed

Bailey, Brandi; Izarra, Alberto; Alvarez, Roberto; Fischer, Kimberlee M; Cottage, Christopher T; Quijada, Pearl; Díez-Juan, Antonio; Sussman, Mark A

2009-11-01

Cardiac stem cells (CSCs) show potential as a cellular therapeutic approach to blunt tissue damage and facilitate reparative and regenerative processes after myocardial infarction. Despite multiple published reports of improvement, functional benefits remain modest using normal stem cells delivered by adoptive transfer into damaged myocardium. The goal of this study is to enhance survival and proliferation of CSCs that have undergone lineage commitment in early phases as evidenced by expression of proteins driven by the alpha-myosin heavy chain (alphaMHC) promoter. The early increased expression of survival kinases augments expansion of the cardiogenic CSC pool and subsequent daughter progeny. Normal CSCs engineered with fluorescent reporter protein constructs under control of the alphaMHC promoter show transgene protein expression, confirming activity of the promoter in CSCs. Cultured CSCs from both nontransgenic and cardiac-specific transgenic mice expressing survival kinases driven by the alphaMHC promoter were analyzed to characterize transgene expression following treatments to promote differentiation in culture. Therapeutic genes controlled by the alphaMHC promoter can be engineered into and expressed in CSCs and cardiomyocyte progeny with the goal of improving the efficacy of cardiac stem cell therapy.

Gene expression profiling defined pathways correlated with fibroblast cell proliferation induced by Opisthorchis viverrini excretory/secretory product.

PubMed

Thuwajit, Chanitra; Thuwajit, Peti; Uchida, Kazuhiko; Daorueang, Daoyot; Kaewkes, Sasithorn; Wongkham, Sopit; Miwa, Masanao

2006-06-14

To investigate the mechanism of fibroblast cell proliferation stimulated by the Opisthorchis viverrini excretory/secretory (ES) product. NIH-3T3, mouse fibroblast cells were treated with O. viverrini ES product by non-contact co-cultured with the adult parasites. Total RNA from NIH-3T3 treated and untreated with O. viverrini was extracted, reverse transcribed and hybridized with the mouse 15K complementary DNA (cDNA) array. The result was analyzed by ArrayVision version 5 and GeneSpring version 5 softwares. After normalization, the ratios of gene expression of parasite treated to untreated NIH-3T3 cells of 2-and more-fold upregulated was defined as the differentially expressed genes. The expression levels of the signal transduction genes were validated by semi-quantitative SYBR-based real-time RT-PCR. Among a total of 15,000 genes/ESTs, 239 genes with established cell proliferation-related function were 2 fold-and more-up-regulated by O. viverrini ES product compared to those in cells without exposure to the parasitic product. These genes were classified into groups including energy and metabolism, signal transduction, protein synthesis and translation, matrix and structural protein, transcription control, cell cycle and DNA replication. Moreover, the expressions of serine-threonine kinase receptor, receptor tyrosine kinase and collagen production-related genes were up-regulated by O. viverrini ES product. The expression level of signal transduction genes; pkC, pdgfr alpha, jak 1, eps 8, tgf beta 1i4, strap and h ras measured by real-time RT-PCR confirmed their expression levels to those obtained from cDNA array. However, only the up-regulated expression of pkC, eps 8 and tgfbeta 1i4 which are the downstream signaling molecules of either epidermal growth factor (EGF) or transforming growth factor-beta (TGF-beta) showed statistical significance (P < 0.05). O. viverrini ES product stimulates the significant changes of gene expression in several functional categories and these mainly include transcripts related to cell proliferation. The TGF-beta and EGF signal transduction pathways are indicated as the possible pathways of O. viverrini-driven cell proliferation.

Identification of human circadian genes based on time course gene expression profiles by using a deep learning method.

PubMed

Cui, Peng; Zhong, Tingyan; Wang, Zhuo; Wang, Tao; Zhao, Hongyu; Liu, Chenglin; Lu, Hui

2018-06-01

Circadian genes express periodically in an approximate 24-h period and the identification and study of these genes can provide deep understanding of the circadian control which plays significant roles in human health. Although many circadian gene identification algorithms have been developed, large numbers of false positives and low coverage are still major problems in this field. In this study we constructed a novel computational framework for circadian gene identification using deep neural networks (DNN) - a deep learning algorithm which can represent the raw form of data patterns without imposing assumptions on the expression distribution. Firstly, we transformed time-course gene expression data into categorical-state data to denote the changing trend of gene expression. Two distinct expression patterns emerged after clustering of the state data for circadian genes from our manually created learning dataset. DNN was then applied to discriminate the aperiodic genes and the two subtypes of periodic genes. In order to assess the performance of DNN, four commonly used machine learning methods including k-nearest neighbors, logistic regression, naïve Bayes, and support vector machines were used for comparison. The results show that the DNN model achieves the best balanced precision and recall. Next, we conducted large scale circadian gene detection using the trained DNN model for the remaining transcription profiles. Comparing with JTK_CYCLE and a study performed by Möller-Levet et al. (doi: https://doi.org/10.1073/pnas.1217154110), we identified 1132 novel periodic genes. Through the functional analysis of these novel circadian genes, we found that the GTPase superfamily exhibits distinct circadian expression patterns and may provide a molecular switch of circadian control of the functioning of the immune system in human blood. Our study provides novel insights into both the circadian gene identification field and the study of complex circadian-driven biological control. This article is part of a Special Issue entitled: Accelerating Precision Medicine through Genetic and Genomic Big Data Analysis edited by Yudong Cai & Tao Huang. Copyright © 2017. Published by Elsevier B.V.

Domestication-driven Gossypium profilin 1 (GhPRF1) gene transduces early flowering phenotype in tobacco by spatial alteration of apical/floral-meristem related gene expression.

PubMed

Pandey, Dhananjay K; Chaudhary, Bhupendra

2016-05-13

Plant profilin genes encode core cell-wall structural proteins and are evidenced for their up-regulation under cotton domestication. Notwithstanding striking discoveries in the genetics of cell-wall organization in plants, little is explicit about the manner in which profilin-mediated molecular interplay and corresponding networks are altered, especially during cellular signalling of apical meristem determinacy and flower development. Here we show that the ectopic expression of GhPRF1 gene in tobacco resulted in the hyperactivation of apical meristem and early flowering phenotype with increased flower number in comparison to the control plants. Spatial expression alteration in CLV1, a key meristem-determinacy gene, is induced by the GhPRF1 overexpression in a WUS-dependent manner and mediates cell signalling to promote flowering. But no such expression alterations are recorded in the GhPRF1-RNAi lines. The GhPRF1 transduces key positive flowering regulator AP1 gene via coordinated expression of FT4, SOC1, FLC1 and FT1 genes involved in the apical-to-floral meristem signalling cascade which is consistent with our in silico profilin interaction data. Remarkably, these positive and negative flowering regulators are spatially controlled by the Actin-Related Protein (ARP) genes, specifically ARP4 and ARP6 in proximate association with profilins. This study provides a novel and systematic link between GhPRF1 gene expression and the flower primordium initiation via up-regulation of the ARP genes, and an insight into the functional characterization of GhPRF1 gene acting upstream to the flowering mechanism. Also, the transgenic plants expressing GhPRF1 gene show an increase in the plant height, internode length, leaf size and plant vigor. Overexpression of GhPRF1 gene induced early and increased flowering in tobacco with enhanced plant vigor. During apical meristem determinacy and flower development, the GhPRF1 gene directly influences key flowering regulators through ARP-genes, indicating for its role upstream in the apical-to-floral meristem signalling cascade.

Targeted expression of suicide gene by tissue-specific promoter and microRNA regulation for cancer gene therapy.

PubMed

Danda, Ravikanth; Krishnan, Gopinath; Ganapathy, Kalaivani; Krishnan, Uma Maheswari; Vikas, Khetan; Elchuri, Sailaja; Chatterjee, Nivedita; Krishnakumar, Subramanian

2013-01-01

In order to realise the full potential of cancer suicide gene therapy that allows the precise expression of suicide gene in cancer cells, we used a tissue specific Epithelial cell adhesion molecule (EpCAM) promoter (EGP-2) that directs transgene Herpes simplex virus-thymidine kinase (HSV-TK) expression preferentially in EpCAM over expressing cancer cells. EpCAM levels are considerably higher in retinoblastoma (RB), a childhood eye cancer with limited expression in normal cells. Use of miRNA regulation, adjacent to the use of the tissue-specific promoter, would provide the second layer of control to the transgene expression only in the tumor cells while sparing the normal cells. To test this hypothesis we cloned let-7b miRNA targets in the 3'UTR region of HSV-TK suicide gene driven by EpCAM promoter because let-7 family miRNAs, including let-7b, were found to be down regulated in the RB tumors and cell lines. We used EpCAM over expressing and let-7 down regulated RB cell lines Y79, WERI-Rb1 (EpCAM (+ve)/let-7b(down-regulated)), EpCAM down regulated, let-7 over expressing normal retinal Müller glial cell line MIO-M1(EpCAM (-ve)/let-7b(up-regulated)), and EpCAM up regulated, let-7b up-regulated normal thyroid cell line N-Thy-Ori-3.1(EpCAM (+ve)/let-7b(up-regulated)) in the study. The cell proliferation was measured by MTT assay, apoptosis was measured by probing cleaved Caspase3, EpCAM and TK expression were quantified by Western blot. Our results showed that the EGP2-promoter HSV-TK (EGP2-TK) construct with 2 or 4 copies of let-7b miRNA targets expressed TK gene only in Y79, WERI-Rb-1, while the TK gene did not express in MIO-M1. In summary, we have developed a tissue-specific, miRNA-regulated dual control vector, which selectively expresses the suicide gene in EpCAM over expressing cells.

Chronobiology in mammalian health.

PubMed

Liu, Zhihua; Chu, Guiyan

2013-03-01

Circadian rhythms are daily cycles of physiology and behavior that are driven by an endogenous oscillator with a period of approximately one day. In mammals, the hypothalamic suprachiasmatic nuclei are our principal circadian oscillators which influences peripheral tissue clocks via endocrine, autonomic and behavioral cues, and other brain regions and most peripheral tissues contain circadian clocks as well. The circadian molecular machinery comprises a group of circadian genes, namely Clock, Bmal1, Per1, Per2, Per3, Cry1 and Cry2. These circadian genes drive endogenous oscillations which promote rhythmically expression of downstream genes and thereby physiological and behavioral processes. Disruptions in circadian homeostasis have pronounced impact on physiological functioning, overall health and disease susceptibility. This review introduces the general profile of circadian gene expression and tissue-specific circadian regulation, highlights the connection between the circadian rhythms and physiological processes, and discusses the role of circadian rhythms in human disease.

Assessment of the utility of the tomato fruit-specific E8 promoter for driving vaccine antigen expression.

PubMed

He, Zhu-Mei; Jiang, Xiao-Ling; Qi, Yu; Luo, Di-Qing

2008-06-01

To assess the utility of the tomato fruit-specific E8 gene's promoter for driving vaccine antigen expression in plant, the 2.2 kb and 1.1 kb E8 promoters were isolated and sequenced from Lycopersicon esculentum cv. Jinfeng #1. The 1.1 kb promoter was fused to vaccine antigen HBsAg M gene for the transfer to Nicotiana tabacum, and the CaMV 35S promoter was used for comparison. Cholera toxin B (ctb) gene under the control of the 1.1 kb promoter was transformed into both N. tabacum and L. esculentum. Southern blot hybridization confirmed the stable integration of the target genes into the tomato and tobacco genomes. ELISA assay showed that the expression product of HBsAg M gene under the control of the 1.1 kb E8 promoter could not be detected in transgenic tobacco tissues such as leaves, flowers, and seeds. In contrast, the expression of HBsAg M gene driven by CaMV 35S promoter could be detected in transgenic tobacco. ELISA assay for CTB proved that the 1.1 kb E8 promoter was able to direct the expression of exotic gene in ripe fruits of transgenic tomato, but expression was absent in leaf, flower, and unripe fruit of tomato, and CTB protein was not detected in transgenic tobacco tissues such as leaves, flowers, and seeds when the gene was under the control of the 1.1 kb E8 promoter. The results indicated that the E8 promoter acted not only in an organ-specific, but also in a species-specific fashion in plant transformation.

Temporally and spatially controllable gene expression and knockout in mouse urothelium.

PubMed

Zhou, Haiping; Liu, Yan; He, Feng; Mo, Lan; Sun, Tung-Tien; Wu, Xue-Ru

2010-08-01

Urothelium that lines almost the entire urinary tract performs important functions and is prone to assaults by urinary microbials, metabolites, and carcinogens. To improve our understanding of urothelial physiology and disease pathogenesis, we sought to develop two novel transgenic systems, one that would allow inducible and urothelium-specific gene expression, and another that would allow inducible and urothelium-specific knockout. Toward this end, we combined the ability of the mouse uroplakin II promoter (mUPII) to drive urothelium-specific gene expression with a versatile tetracycline-mediated inducible system. We found that, when constructed under the control of mUPII, only a modified, reverse tetracycline trans-activator (rtTA-M2), but not its original version (rtTA), could efficiently trans-activate reporter gene expression in mouse urothelium on doxycycline (Dox) induction. The mUPII/rtTA-M2-inducible system retained its strict urothelial specificity, had no background activity in the absence of Dox, and responded rapidly to Dox administration. Using a reporter gene whose expression was secondarily controlled by histone remodeling, we were able to identify, colocalize with 5-bromo-2-deoxyuridine incorporation, and semiquantify newly divided urothelial cells. Finally, we established that, when combined with a Cre recombinase under the control of the tetracycline operon, the mUPII-driven rtTA-M2 could inducibly inactivate any gene of interest in mouse urothelium. The establishment of these two new transgenic mouse systems enables the manipulation of gene expression and/or inactivation in adult mouse urothelium at any given time, thus minimizing potential compensatory effects due to gene overexpression or loss and allowing more accurate modeling of urothelial diseases than previously reported constitutive systems.

Quercetin manipulates the expression of genes involved in the reactive oxygen species (ROS) processin chicken heterophils.

PubMed

Nambooppha, Boondarika; Photichai, Kornravee; Wongsawan, Kanreuthai; Chuammitri, Phongsakorn

2018-06-06

Chicken heterophils generate reactive oxygen species (ROS) molecules to defend against invading pathogens. The present study examined effects of quercetin on chicken heterophils. Heterophils were stimulated with PBS, 50 μM quercetin (QH), PMA or Escherichia coli (EC) and the resulting intracellular ROS molecules were determined. Flow cytometry results showed that cells stimulated with QH, PMA and EC had a higher ROS production. Increases in intracellular ROS molecules were identified in all treatment groups by fluorescence microscopy. Determination of the ability of quercetin to manipulate mRNA expression of ROS subunits was assessed using real-time RT-PCR. Quercetin and other stimulants up-regulated the majority of genes involved in ROS production: CYBB (NOX2), NCF1 (p47 phox ), NCF2 (p67 phox ), NOX1 and RAC2. The antioxidant property of QH was explored by measuring mRNA expression of CAT and SOD1. The data indicate increased levels of CAT with all treatments; however, only QH attenuated the expression ofthe SOD1 gene. To further investigate the effects of ROS-driven inflammation or cell death, IL6, CASP8, and MCL1 genes were preferentially tested. The inflammatory gene (IL6) was profoundly down-regulated in the QH- and PMA-treated groups while EC induced a strikingly high IL6 expression level. Investigation of the known apoptotic (CASP8) and anti-apoptotic (MCL1) genes found down-regulation of CASP8 in the QH- and PMA-treated groups which were contradicted to the MCL1 gene. In conclusion, quercetin can enhance ROS production by regulating the expression of genes involved in ROS production as well as in subsequent processes.

Phosphite, an analog of phosphate, suppresses the coordinated expression of genes under phosphate starvation.

PubMed

Varadarajan, Deepa K; Karthikeyan, Athikkattuvalasu S; Matilda, Paino Durzo; Raghothama, Kashchandra G

2002-07-01

Phosphate (Pi) and its analog phosphite (Phi) are acquired by plants via Pi transporters. Although the uptake and mobility of Phi and Pi are similar, there is no evidence suggesting that plants can utilize Phi as a sole source of phosphorus. Phi is also known to interfere with many of the Pi starvation responses in plants and yeast (Saccharomyces cerevisiae). In this study, effects of Phi on plant growth and coordinated expression of genes induced by Pi starvation were analyzed. Phi suppressed many of the Pi starvation responses that are commonly observed in plants. Enhanced root growth and root to shoot ratio, a hallmark of Pi stress response, was strongly inhibited by Phi. The negative effects of Phi were not obvious in plants supplemented with Pi. The expression of Pi starvation-induced genes such as LePT1, LePT2, AtPT1, and AtPT2 (high-affinity Pi transporters); LePS2 (a novel acid phosphatase); LePS3 and TPSI1 (novel genes); and PAP1 (purple acid phosphatase) was suppressed by Phi in plants and cell cultures. Expression of luciferase reporter gene driven by the Pi starvation-induced AtPT2 promoter was also suppressed by Phi. These analyses showed that suppression of Pi starvation-induced genes is an early response to addition of Phi. These data also provide evidence that Phi interferes with gene expression at the level of transcription. Synchronized suppression of multiple Pi starvation-induced genes by Phi points to its action on the early molecular events, probably signal transduction, in Pi starvation response.

A Small System—High-Resolution Study of Metabolic Adaptation in the Central Metabolic Pathway to Temperate Climates in Drosophila melanogaster

PubMed Central

Lavington, Erik; Cogni, Rodrigo; Kuczynski, Caitlin; Koury, Spencer; Behrman, Emily L.; O’Brien, Katherine R.; Schmidt, Paul S.; Eanes, Walter F.

2014-01-01

In this article, we couple the geographic variation in 127 single-nucleotide polymorphism (SNP) frequencies in genes of 46 enzymes of central metabolism with their associated cis-expression variation to predict latitudinal or climatic-driven gene expression changes in the metabolic architecture of Drosophila melanogaster. Forty-two percent of the SNPs in 65% of the genes show statistically significant clines in frequency with latitude across the 20 local population samples collected from southern Florida to Ontario. A number of SNPs in the screened genes are also associated with significant expression variation within the Raleigh population from North Carolina. A principal component analysis of the full variance–covariance matrix of latitudinal changes in SNP-associated standardized gene expression allows us to identify those major genes in the pathway and its associated branches that are likely targets of natural selection. When embedded in a central metabolic context, we show that these apparent targets are concentrated in the genes of the upper glycolytic pathway and pentose shunt, those controlling glycerol shuttle activity, and finally those enzymes associated with the utilization of glutamate and pyruvate. These metabolites possess high connectivity and thus may be the points where flux balance can be best shifted. We also propose that these points are conserved points associated with coupling energy homeostasis and energy sensing in mammals. We speculate that the modulation of gene expression at specific points in central metabolism that are associated with shifting flux balance or possibly energy-state sensing plays a role in adaptation to climatic variation. PMID:24770333

Adenovirus-mediated in utero gene transfer in mice and guinea pigs: tissue distribution of recombinant adenovirus determined by quantitative TaqMan-polymerase chain reaction assay.

PubMed

Senoo, M; Matsubara, Y; Fujii, K; Nagasaki, Y; Hiratsuka, M; Kure, S; Uehara, S; Okamura, K; Yajima, A; Narisawa, K

2000-04-01

Fetal somatic cell gene therapy could become an attractive solution for some congenital genetic diseases or the disorders which manifest themselves during the fetal period. We performed adenovirus-mediated gene transfer to mice and guinea pig fetuses in utero and evaluated the efficiency of gene transfer by histochemical analysis and a quantitative TaqMan-polymerase chain reaction (TaqMan-PCR) assay. We first injected a replication-deficient recombinant adenovirus containing the Escherichia coli LacZ gene driven by a CAG promoter (AxCALacZ) into pregnant mice through the amniotic space, placenta, or intraperitoneal space of the fetus. Histochemical analysis showed limited transgene expression in fetal tissues. We then administered AxCALacZ to guinea pig fetuses in the late stage of pregnancy through the umbilical vein. The highest beta-galactosidase expression was observed in liver followed by moderate expression in heart, spleen, and adrenal gland. The transgene expression was also present in kidney, intestine, and placenta to a lesser degree. No positively stained cells were observed in lung, muscle, or pancreas except in the vascular endothelium of these organs. Quantitative measurement of recombinant adenoviral DNA by the TaqMan-PCR assay showed that the vast majority of the injected viruses was present in liver. The current study indicated that adenovirus-mediated gene transfer into guinea pig fetus through the umbilical vein is feasible and results in efficient transgene expression in fetal tissues. The experimental procedures using pregnant guinea pigs might serve as a good experimental model for in utero gene transfer. Since our TaqMan-PCR assay detects the LacZ gene, one of the most widely used reporter genes, it may be generally applicable to adenovirus quantification in various gene transfer experiments.

Two different Bacillus thuringiensis toxin genes confer resistance to beet armyworm (Spodoptera exigua Hübner) in transgenic Bt-shallots (Allium cepa L.).

PubMed

Zheng, Si-Jun; Henken, Betty; de Maagd, Ruud A; Purwito, Agus; Krens, Frans A; Kik, Chris

2005-06-01

Agrobacterium-mediated genetic transformation was applied to produce beet armyworm (Spodoptera exigua Hübner) resistant tropical shallots (Allium cepa L. group Aggregatum). A cry1Ca or a H04 hybrid gene from Bacillus thuringiensis, driven by the chrysanthemum ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit (Rubisco SSU) promoter, along with the hygromycin phosphotransferase gene (hpt) driven by the CaMV 35S promoter, was employed for genetic transformation. An average transformation frequency of 3.68% was obtained from two shallot cultivars, Tropix and Kuning. After transfer of the in vitro plants to the greenhouse 69% of the cry1Ca and 39% of the H04 transgenic shallots survived the first half year. After one year of cultivation in the greenhouse the remaining cry1Ca and H04 transgenic plants grew vigorously and had a normal bulb formation, although the cry1Ca transgenic plants (and controls) had darker green leaves compared to their H04 counterparts. Standard PCR, adaptor ligation PCR and Southern analyses confirmed the integration of T-DNA into the shallot genome. Northern blot and ELISA analyses revealed expression of the cry1Ca or H04 gene in the transgenic plants. The amount of Cry1Ca expressed in transgenic plants was higher than the expression levels of H04 (0.39 vs. 0.16% of the total soluble leaf proteins, respectively). There was a good correlation between protein expression and beet armyworm resistance. Cry1Ca or H04 gene expression of at least 0.22 or 0.08% of the total soluble protein in shallot leaves was sufficient to give a complete resistance against beet armyworm. This confirms earlier observations that the H04 toxin is more toxic to S. exigua than the Cry1Ca toxin. The results from this study suggest that the cry1Ca and H04 transgenic shallots developed could be used for introducing resistance to beet armyworm in (sub) tropical shallot.

Phenotypic Checkpoints Regulate Neuronal Development

PubMed Central

Ben-Ari, Yehezkel; Spitzer, Nicholas C.

2010-01-01

Nervous system development proceeds by sequential gene expression mediated by cascades of transcription factors in parallel with sequences of patterned network activity driven by receptors and ion channels. These sequences are cell type- and developmental stage-dependent and modulated by paracrine actions of substances released by neurons and glia. How and to what extent these sequences interact to enable neuronal network development is not understood. Recent evidence demonstrates that CNS development requires intermediate stages of differentiation providing functional feedback that influences gene expression. We suggest that embryonic neuronal functions constitute a series of phenotypic checkpoint signatures; neurons failing to express these functions are delayed or developmentally arrested. Such checkpoints are likely to be a general feature of neuronal development and may constitute presymptomatic signatures of neurological disorders when they go awry. PMID:20864191

Interaction of Osmotic Stress, Temperature, and Abscisic Acid in the Regulation of Gene Expression in Arabidopsis

PubMed Central

Xiong, Liming; Ishitani, Manabu; Zhu, Jian-Kang

1999-01-01

The impact of simultaneous environmental stresses on plants and how they respond to combined stresses compared with single stresses is largely unclear. By using a transgene (RD29A-LUC) consisting of the firefly luciferase coding sequence (LUC) driven by the stress-responsive RD29A promoter, we investigated the interactive effects of temperature, osmotic stress, and the phytohormone abscisic acid (ABA) in the regulation of gene expression in Arabidopsis seedlings. Results indicated that both positive and negative interactions exist among the studied stress factors in regulating gene expression. At a normal growth temperature (22°C), osmotic stress and ABA act synergistically to induce the transgene expression. Low temperature inhibits the response to osmotic stress or to combined treatment of osmotic stress and ABA, whereas low temperature and ABA treatments are additive in inducing transgene expression. Although high temperature alone does not activate the transgene, it significantly amplifies the effects of ABA and osmotic stress. The effect of multiple stresses in the regulation of RD29A-LUC expression in signal transduction mutants was also studied. The results are discussed in the context of cold and osmotic stress signal transduction pathways. PMID:9880362

The Arabidopsis GASA10 gene encodes a cell wall protein strongly expressed in developing anthers and seeds.

PubMed

Trapalis, Menelaos; Li, Song Feng; Parish, Roger W

2017-07-01

The Arabidopsis GASA10 gene encodes a GAST1-like (Gibberellic Acid-Stimulated) protein. Reporter gene analysis identified consistent expression in anthers and seeds. In anthers expression was developmentally regulated, first appearing at stage 7 of anther development and reaching a maximum at stage 11. Strongest expression was in the tapetum and developing microspores. GASA10 expression also occurred throughout the seed and in root vasculature. GASA10 was shown to be transported to the cell wall. Using GASA1 and GASA6 as positive controls, gibberellic acid was found not to induce GASA10 expression in Arabidopsis suspension cells. Overexpression of GASA10 (35S promoter-driven) resulted in a reduction in silique elongation. GASA10 shares structural similarities to the antimicrobial peptide snakin1, however, purified GASA10 failed to influence the growth of a variety of bacterial and fungal species tested. We propose cell wall associated GASA proteins are involved in regulating the hydroxyl radical levels at specific sites in the cell wall to facilitate wall growth (regulating cell wall elongation). Copyright © 2017 Elsevier B.V. All rights reserved.

Integration of multiple stimuli-sensing systems to regulate HrpS and type III secretion system in Erwinia amylovora.

PubMed

Lee, Jae Hoon; Zhao, Youfu

2018-02-01

The bacterial enhancer binding protein (bEBP) HrpS is essential for Erwinia amylovora virulence by activating the type III secretion system (T3SS). However, how the hrpS gene is regulated remains poorly understood in E. amylovora. In this study, 5' rapid amplification of cDNA ends and promoter deletion analyses showed that the hrpS gene contains two promoters driven by HrpX/HrpY and the Rcs phosphorelay system, respectively. Electrophoretic mobility shift and gene expression assays demonstrated that integration host factor IHF positively regulates hrpS expression through directly binding the hrpX promoter and positively regulating hrpX/hrpY expression. Moreover, hrpX expression was down-regulated in the relA/spoT ((p)ppGpp-deficient) mutant and the dksA mutant, but up-regulated when the wild-type strain was treated with serine hydroxamate, which induced (p)ppGpp-mediated stringent response. Furthermore, the csrA mutant showed significantly reduced transcripts of major hrpS activators, including the hrpX/hrpY, rcsA and rcsB genes, indicating that CsrA is required for full hrpS expression. On the other hand, the csrB mutant exhibited up-regulation of the rcsA and rcsB genes, and hrpS expression was largely diminished in the csrB/rcsB mutant, indicating that the Rcs system is mainly responsible for the increased hrpS expression in the csrB mutant. These findings suggest that E. amylovora recruits multiple stimuli-sensing systems, including HrpX/HrpY, the Rcs phosphorelay system and the Gac-Csr system, to regulate hrpS and T3SS gene expression.

CRISP-3, a protein with homology to plant defense proteins, is expressed in mouse B cells under the control of Oct2.

PubMed

Pfisterer, P; König, H; Hess, J; Lipowsky, G; Haendler, B; Schleuning, W D; Wirth, T

1996-11-01

The Oct2 transcription factor is expressed throughout the B-lymphoid lineage and plays an essential role during the terminal phase of B-cell differentiation. Several genes specifically expressed in B lymphocytes have been identified that contain a functional octamer motif in their regulatory elements. However, expression of only a single gene, the murine CD36 gene, has been shown to date to be dependent on Oct2. Here, we present the identification and characterization of a further gene, coding for cysteine-rich secreted protein 3 (CRISP-3), whose expression in B cells is regulated by Oct2. We show that CRISP-3 is expressed in the B-lymphoid lineage specifically at the pre-B-cell stage. By using different experimental strategies, including nuclear run-on experiments, we demonstrate that this gene is transcriptionally activated by Oct2. Furthermore, analysis of CRISP-3 expression in primary B cells derived from either wild-type or Oct2-deficient mice demonstrates the dependence on Oct2. Two variant octamer motifs were identified in the upstream promoter region of the crisp-3 gene, and Oct2 interacts with both of them in vitro. Cotransfection experiments with expression vectors for Oct1 and Oct2 together with a reporter driven by the crisp-3 promoter showed that transcriptional activation of this promoter can only be achieved with Oct2. The C-terminal transactivation domain of Oct2 is required for this activation. Finally, introducing specific mutations in the two variant octamer motifs revealed that both of them are important for full transcriptional activation by Oct2.

Exercise-driven metabolic pathways in healthy cartilage.

PubMed

Blazek, A D; Nam, J; Gupta, R; Pradhan, M; Perera, P; Weisleder, N L; Hewett, T E; Chaudhari, A M; Lee, B S; Leblebicioglu, B; Butterfield, T A; Agarwal, S

2016-07-01

Exercise is vital for maintaining cartilage integrity in healthy joints. Here we examined the exercise-driven transcriptional regulation of genes in healthy rat articular cartilage to dissect the metabolic pathways responsible for the potential benefits of exercise. Transcriptome-wide gene expression in the articular cartilage of healthy Sprague-Dawley female rats exercised daily (low intensity treadmill walking) for 2, 5, or 15 days was compared to that of non-exercised rats, using Affymetrix GeneChip arrays. Database for Annotation, Visualization and Integrated Discovery (DAVID) was used for Gene Ontology (GO)-term enrichment and Functional Annotation analysis of differentially expressed genes (DEGs). Kyoto Encyclopedia of Genes and Genome (KEGG) pathway mapper was used to identify the metabolic pathways regulated by exercise. Microarray analysis revealed that exercise-induced 644 DEGs in healthy articular cartilage. The DAVID bioinformatics tool demonstrated high prevalence of functional annotation clusters with greater enrichment scores and GO-terms associated with extracellular matrix (ECM) biosynthesis/remodeling and inflammation/immune response. The KEGG database revealed that exercise regulates 147 metabolic pathways representing molecular interaction networks for Metabolism, Genetic Information Processing, Environmental Information Processing, Cellular Processes, Organismal Systems, and Diseases. These pathways collectively supported the complex regulation of the beneficial effects of exercise on the cartilage. Overall, the findings highlight that exercise is a robust transcriptional regulator of a wide array of metabolic pathways in healthy cartilage. The major actions of exercise involve ECM biosynthesis/cartilage strengthening and attenuation of inflammatory pathways to provide prophylaxis against onset of arthritic diseases in healthy cartilage. Copyright © 2016 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

Novel Role of 3’UTR-Embedded Alu Elements as Facilitators of Processed Pseudogene Genesis and Host Gene Capture by Viral Genomes

PubMed Central

Engel, Pablo; Angulo, Ana

2016-01-01

Since the discovery of the high abundance of Alu elements in the human genome, the interest for the functional significance of these retrotransposons has been increasing. Primate Alu and rodent Alu-like elements are retrotransposed by a mechanism driven by the LINE1 (L1) encoded proteins, the same machinery that generates the L1 repeats, the processed pseudogenes (PPs), and other retroelements. Apart from free Alu RNAs, Alus are also transcribed and retrotranscribed as part of cellular gene transcripts, generally embedded inside 3’ untranslated regions (UTRs). Despite different proposed hypotheses, the functional implication of the presence of Alus inside 3’UTRs remains elusive. In this study we hypothesized that Alu elements in 3’UTRs could be involved in the genesis of PPs. By analyzing human genome data we discovered that the existence of 3’UTR-embedded Alu elements is overrepresented in genes source of PPs. In contrast, the presence of other retrotransposable elements in 3’UTRs does not show this PP linked overrepresentation. This research was extended to mouse and rat genomes and the results accordingly reveal overrepresentation of 3’UTR-embedded B1 (Alu-like) elements in PP parent genes. Interestingly, we also demonstrated that the overrepresentation of 3’UTR-embedded Alus is particularly significant in PP parent genes with low germline gene expression level. Finally, we provide data that support the hypothesis that the L1 machinery is also the system that herpesviruses, and possibly other large DNA viruses, use to capture host genes expressed in germline or somatic cells. Altogether our results suggest a novel role for Alu or Alu-like elements inside 3’UTRs as facilitators of the genesis of PPs, particularly in lowly expressed genes. Moreover, we propose that this L1-driven mechanism, aided by the presence of 3’UTR-embedded Alus, may also be exploited by DNA viruses to incorporate host genes to their viral genomes. PMID:28033411

Knowledge Driven Variable Selection (KDVS) – a new approach to enrichment analysis of gene signatures obtained from high–throughput data

PubMed Central

2013-01-01

Background High–throughput (HT) technologies provide huge amount of gene expression data that can be used to identify biomarkers useful in the clinical practice. The most frequently used approaches first select a set of genes (i.e. gene signature) able to characterize differences between two or more phenotypical conditions, and then provide a functional assessment of the selected genes with an a posteriori enrichment analysis, based on biological knowledge. However, this approach comes with some drawbacks. First, gene selection procedure often requires tunable parameters that affect the outcome, typically producing many false hits. Second, a posteriori enrichment analysis is based on mapping between biological concepts and gene expression measurements, which is hard to compute because of constant changes in biological knowledge and genome analysis. Third, such mapping is typically used in the assessment of the coverage of gene signature by biological concepts, that is either score–based or requires tunable parameters as well, limiting its power. Results We present Knowledge Driven Variable Selection (KDVS), a framework that uses a priori biological knowledge in HT data analysis. The expression data matrix is transformed, according to prior knowledge, into smaller matrices, easier to analyze and to interpret from both computational and biological viewpoints. Therefore KDVS, unlike most approaches, does not exclude a priori any function or process potentially relevant for the biological question under investigation. Differently from the standard approach where gene selection and functional assessment are applied independently, KDVS embeds these two steps into a unified statistical framework, decreasing the variability derived from the threshold–dependent selection, the mapping to the biological concepts, and the signature coverage. We present three case studies to assess the usefulness of the method. Conclusions We showed that KDVS not only enables the selection of known biological functionalities with accuracy, but also identification of new ones. An efficient implementation of KDVS was devised to obtain results in a fast and robust way. Computing time is drastically reduced by the effective use of distributed resources. Finally, integrated visualization techniques immediately increase the interpretability of results. Overall, KDVS approach can be considered as a viable alternative to enrichment–based approaches. PMID:23302187

Enhancer activity of Helitron in sericin-1 gene promoter from Bombyx mori.

PubMed

Huang, Ke; Li, Chun-Feng; Wu, Jie; Wei, Jun-Hong; Zou, Yong; Han, Min-Jin; Zhou, Ze-Yang

2016-06-01

Sericin is a kind of water-soluble protein expressed specifically in the middle silk gland of Bombyx mori. When the sericin-1 gene promoter was cloned and a transgenic vector was constructed to express a foreign protein, a specific Helitron, Bmhel-8, was identified in the sericin-1 gene promoter sequence in some genotypes of Bombyx mori and Bombyx mandarina. Given that the Bmhel-8 Helitron transposon was present only in some genotypes, it could be the source of allelic variation in the sericin-1 promoter. The length of the sericin-1 promoter sequence is approximately 1063 or 643 bp. The larger size of the sequence or allele is ascribed to the presence of Bmhel-8. Silkworm genotypes can be homozygous for either the shorter or larger promoter sequence or heterozygous, containing both alleles. Bmhel-8 in the sericin-1 promoter exhibits enhancer activity, as demonstrated by a dual-luciferase reporter system in BmE cell lines. Furthermore, Bmhel-8 displays enhancer activity in a sericin-1 promoter-driven gene expression system but does not regulate the tissue-specific expression of sericin-1. © 2016 Institute of Zoology, Chinese Academy of Sciences.

Ovarian-specific expression of a new gene regulated by the goat PIS region and transcribed by a FOXL2 bidirectional promoter.

PubMed

Pannetier, Maëlle; Renault, Lauriane; Jolivet, Geneviève; Cotinot, Corinne; Pailhoux, Eric

2005-06-01

Studies on XX sex reversal in polled goats (PIS mutation: polled intersex syndrome) have led to the discovery of a female-specific locus crucial for ovarian differentiation. This genomic region is composed of at least two genes, FOXL2 and PISRT1, sharing a common transcriptional regulatory region, PIS. In this paper, we describe a third gene, PFOXic (promoter FOXL2 inverse complementary), located near FOXL2 in the opposite orientation. This gene composed of five exons encodes a 1723-bp cDNA, enclosing two repetitive elements in its 3' end. PFOXic mRNA encodes a putative protein of 163 amino acids with no homologies in any of the databases tested. The transcriptional expression of PFOXic is driven by a bidirectional promoter also enhancing FOXL2 transcription. In goats, PFOXic is expressed in developing ovaries, from 36 days postcoitum until adulthood. Ovarian-specific expression of PFOXic is regulated by the PIS region. PFOXic is found conserved only in Bovidae. But, a human gene located in the opposite orientation relative to FOXL2 can be considered a human PFOXic. Finally, we discuss evidence arguing for regulation of the level of FOXL2 transcription via the bidirectional promoter and the level of transcription of PFOXic.

Temporal profiling of gene networks associated with the late phase of long-term potentiation in vivo.

PubMed

Ryan, Margaret M; Ryan, Brigid; Kyrke-Smith, Madeleine; Logan, Barbara; Tate, Warren P; Abraham, Wickliffe C; Williams, Joanna M

2012-01-01

Long-term potentiation (LTP) is widely accepted as a cellular mechanism underlying memory processes. It is well established that LTP persistence is strongly dependent on activation of constitutive and inducible transcription factors, but there is limited information regarding the downstream gene networks and controlling elements that coalesce to stabilise LTP. To identify these gene networks, we used Affymetrix RAT230.2 microarrays to detect genes regulated 5 h and 24 h (n = 5) after LTP induction at perforant path synapses in the dentate gyrus of awake adult rats. The functional relationships of the differentially expressed genes were examined using DAVID and Ingenuity Pathway Analysis, and compared with our previous data derived 20 min post-LTP induction in vivo. This analysis showed that LTP-related genes are predominantly upregulated at 5 h but that there is pronounced downregulation of gene expression at 24 h after LTP induction. Analysis of the structure of the networks and canonical pathways predicted a regulation of calcium dynamics via G-protein coupled receptors, dendritogenesis and neurogenesis at the 5 h time-point. By 24 h neurotrophin-NFKB driven pathways of neuronal growth were identified. The temporal shift in gene expression appears to be mediated by regulation of protein synthesis, ubiquitination and time-dependent regulation of specific microRNA and histone deacetylase expression. Together this programme of genomic responses, marked by both homeostatic and growth pathways, is likely to be critical for the consolidation of LTP in vivo.

Transcription factor AtTCP14 regulates embryonic growth potential during seed germination in Arabidopsis thaliana.

PubMed

Tatematsu, Kiyoshi; Nakabayashi, Kazumi; Kamiya, Yuji; Nambara, Eiji

2008-01-01

To understand the molecular mechanisms underlying regulation of seed germination, we searched enriched cis elements in the upstream regions of Arabidopsis genes whose transcript levels increased during seed germination. Using available published microarray data, we found that two cis elements, Up1 or Up2, which regulate outgrowth of Arabidopsis axillary shoots, were significantly over-represented. Classification of Up1- and Up2-containing genes by gene ontology revealed that protein synthesis-related genes, especially ribosomal protein genes, were highly over-represented. Expression analysis using a reporter gene driven by a synthetic promoter regulated by these elements showed that the Up1 is necessary and sufficient for germination-associated gene induction, whereas Up2 acts as an enhancer of Up1. Up1-mediated gene expression was suppressed by treatments that blocked germination. Up1 is almost identical to the site II motif, which is the predicted target of TCP transcription factors. Of 24 AtTCP genes, AtTCP14, which showed the highest transcript level just prior to germination, was functionally characterized to test its involvement in the regulation of seed germination. Transposon-tagged lines for AtTCP14 showed delayed germination. In addition, germination of attcp14 mutants exhibited hypersensitivity to exogenously applied abscisic acid and paclobutrazol, an inhibitor of gibberellin biosynthesis. AtTCP14 was predominantly expressed in the vascular tissues of the embryo, and affected gene expression in radicles in a non-cell-autonomous manner. Taken together, these results indicate that AtTCP14 regulates the activation of embryonic growth potential in Arabidopsis seeds.

Integrative Approach to Pain Genetics Identifies Pain Sensitivity Loci across Diseases

PubMed Central

Ruau, David; Dudley, Joel T.; Chen, Rong; Phillips, Nicholas G.; Swan, Gary E.; Lazzeroni, Laura C.; Clark, J. David

2012-01-01

Identifying human genes relevant for the processing of pain requires difficult-to-conduct and expensive large-scale clinical trials. Here, we examine a novel integrative paradigm for data-driven discovery of pain gene candidates, taking advantage of the vast amount of existing disease-related clinical literature and gene expression microarray data stored in large international repositories. First, thousands of diseases were ranked according to a disease-specific pain index (DSPI), derived from Medical Subject Heading (MESH) annotations in MEDLINE. Second, gene expression profiles of 121 of these human diseases were obtained from public sources. Third, genes with expression variation significantly correlated with DSPI across diseases were selected as candidate pain genes. Finally, selected candidate pain genes were genotyped in an independent human cohort and prospectively evaluated for significant association between variants and measures of pain sensitivity. The strongest signal was with rs4512126 (5q32, ABLIM3, P = 1.3×10−10) for the sensitivity to cold pressor pain in males, but not in females. Significant associations were also observed with rs12548828, rs7826700 and rs1075791 on 8q22.2 within NCALD (P = 1.7×10−4, 1.8×10−4, and 2.2×10−4 respectively). Our results demonstrate the utility of a novel paradigm that integrates publicly available disease-specific gene expression data with clinical data curated from MEDLINE to facilitate the discovery of pain-relevant genes. This data-derived list of pain gene candidates enables additional focused and efficient biological studies validating additional candidates. PMID:22685391

Selection-driven evolution of sex-biased genes is consistent with sexual selection in Arabidopsis thaliana.

PubMed

Gossmann, Toni I; Schmid, Marc W; Grossniklaus, Ueli; Schmid, Karl J

2014-03-01

Sex-biased genes are genes with a preferential or specific expression in one sex and tend to show an accelerated rate of evolution in animals. Various hypotheses--which are not mutually exclusive--have been put forth to explain observed patterns of rapid evolution. One possible explanation is positive selection, but this has been shown only in few animal species and mostly for male-specific genes. Here, we present a large-scale study that investigates evolutionary patterns of sex-biased genes in the predominantly self-fertilizing plant Arabidopsis thaliana. Unlike most animal species, A. thaliana does not possess sex chromosomes, its flowers develop both male and female sexual organs, and it is characterized by low outcrossing rates. Using cell-specific gene expression data, we identified genes whose expression is enriched in comparison with all other tissues in the male and female gametes (sperm, egg, and central cell), as well as in synergids, pollen, and pollen tubes, which also play an important role in reproduction. Genes specifically expressed in gametes and synergids show higher rates of protein evolution compared with the genome-wide average and no evidence for positive selection. In contrast, pollen- and pollen tube-specific genes not only have lower rates of protein evolution but also exhibit a higher proportion of adaptive amino acid substitutions. We show that this is the result of increased levels of purifying and positive selection among genes with pollen- and pollen tube-specific expression. The increased proportion of adaptive substitutions cannot be explained by the fact that pollen- and pollen tube-expressed genes are enriched in segmental duplications, are on average older, or have a larger effective population size. Our observations are consistent with prezygotic sexual selection as a result of interactions during pollination and pollen tube growth such as pollen tube competition.

Identification and temporal expression of putative circadian clock transcripts in the amphipod crustacean Talitrus saltator

PubMed Central

O’Grady, Joseph F.; Hoelters, Laura S.; Swain, Martin T.

2016-01-01

Background Talitrus saltator is an amphipod crustacean that inhabits the supralittoral zone on sandy beaches in the Northeast Atlantic and Mediterranean. T. saltator exhibits endogenous locomotor activity rhythms and time-compensated sun and moon orientation, both of which necessitate at least one chronometric mechanism. Whilst their behaviour is well studied, currently there are no descriptions of the underlying molecular components of a biological clock in this animal, and very few in other crustacean species. Methods We harvested brain tissue from animals expressing robust circadian activity rhythms and used homology cloning and Illumina RNAseq approaches to sequence and identify the core circadian clock and clock-related genes in these samples. We assessed the temporal expression of these genes in time-course samples from rhythmic animals using RNAseq. Results We identified a comprehensive suite of circadian clock gene homologues in T. saltator including the ‘core’ clock genes period (Talper), cryptochrome 2 (Talcry2), timeless (Taltim), clock (Talclk), and bmal1 (Talbmal1). In addition we describe the sequence and putative structures of 23 clock-associated genes including two unusual, extended isoforms of pigment dispersing hormone (Talpdh). We examined time-course RNAseq expression data, derived from tissues harvested from behaviourally rhythmic animals, to reveal rhythmic expression of these genes with approximately circadian period in Talper and Talbmal1. Of the clock-related genes, casein kinase IIβ (TalckIIβ), ebony (Talebony), jetlag (Taljetlag), pigment dispensing hormone (Talpdh), protein phosphatase 1 (Talpp1), shaggy (Talshaggy), sirt1 (Talsirt1), sirt7 (Talsirt7) and supernumerary limbs (Talslimb) show temporal changes in expression. Discussion We report the sequences of principle genes that comprise the circadian clock of T. saltator and highlight the conserved structural and functional domains of their deduced cognate proteins. Our sequencing data contribute to the growing inventory of described comparative clocks. Expression profiling of the identified clock genes illuminates tantalising targets for experimental manipulation to elucidate the molecular and cellular control of clock-driven phenotypes in this crustacean. PMID:27761341

In smokers, Sonic hedgehog modulates pulmonary endothelial function through vascular endothelial growth factor.

PubMed

Henno, Priscilla; Grassin-Delyle, Stanislas; Belle, Emeline; Brollo, Marion; Naline, Emmanuel; Sage, Edouard; Devillier, Philippe; Israël-Biet, Dominique

2017-05-23

Tobacco-induced pulmonary vascular disease is partly driven by endothelial dysfunction. The Sonic hedgehog (SHH) pathway is involved in vascular physiology. We sought to establish whether the SHH pathway has a role in pulmonary endothelial dysfunction in smokers. The ex vivo endothelium-dependent relaxation of pulmonary artery rings in response to acetylcholine (Ach) was compared in 34 current or ex-smokers and 8 never-smokers. The results were expressed as a percentage of the contraction with phenylephrine. We tested the effects of SHH inhibitors (GANT61 and cyclopamine), an SHH activator (SAG) and recombinant VEGF on the Ach-induced relaxation. The level of VEGF protein in the pulmonary artery ring was measured in an ELISA. SHH pathway gene expression was quantified in reverse transcriptase-quantitative polymerase chain reactions. Ach-induced relaxation was much less intense in smokers than in never-smokers (respectively 24 ± 6% and 50 ± 7% with 10 -4 M Ach; p = 0.028). All SHH pathway genes were expressed in pulmonary artery rings from smokers. SHH inhibition by GANT61 reduced Ach-induced relaxation and VEGF gene expression in the pulmonary artery ring. Recombinant VEGF restored the ring's endothelial function. VEGF gene and protein expression levels in the pulmonary artery rings were positively correlated with the degree of Ach-induced relaxation and negatively correlated with the number of pack-years. SHH pathway genes and proteins are expressed in pulmonary artery rings from smokers, where they modulate endothelial function through VEGF.

Comparative analysis of cis-regulation following stroke and seizures in subspaces of conserved eigensystems

PubMed Central

2010-01-01

Background It is often desirable to separate effects of different regulators on gene expression, or to identify effects of the same regulator across several systems. Here, we focus on the rat brain following stroke or seizures, and demonstrate how the two tasks can be approached simultaneously. Results We applied SVD to time-series gene expression datasets from the rat experimental models of stroke and seizures. We demonstrate conservation of two eigensystems, reflecting inflammation and/or apoptosis (eigensystem 2) and neuronal synaptic activity (eigensystem 3), between the stroke and seizures. We analyzed cis-regulation of gene expression in the subspaces of the conserved eigensystems. Bayesian networks analysis was performed separately for either experimental model, with cross-system validation of the highest-ranking features. In this way, we correctly re-discovered the role of AP1 in the regulation of apoptosis, and the involvement of Creb and Egr in the regulation of synaptic activity-related genes. We identified a novel antagonistic effect of the motif recognized by the nuclear matrix attachment region-binding protein Satb1 on AP1-driven transcriptional activation, suggesting a link between chromatin loop structure and gene activation by AP1. The effects of motifs binding Satb1 and Creb on gene expression in brain conform to the assumption of the linear response model of gene regulation. Our data also suggest that numerous enhancers of neuronal-specific genes are important for their responsiveness to the synaptic activity. Conclusion Eigensystems conserved between stroke and seizures separate effects of inflammation/apoptosis and neuronal synaptic activity, exerted by different transcription factors, on gene expression in rat brain. PMID:20565733

Interactions of HIPPI, a molecular partner of Huntingtin interacting protein HIP1, with the specific motif present at the putative promoter sequence of the caspase-1, caspase-8 and caspase-10 genes.

PubMed

Majumder, P; Choudhury, A; Banerjee, M; Lahiri, A; Bhattacharyya, N P

2007-08-01

To investigate the mechanism of increased expression of caspase-1 caused by exogenous Hippi, observed earlier in HeLa and Neuro2A cells, in this work we identified a specific motif AAAGACATG (- 101 to - 93) at the caspase-1 gene upstream sequence where HIPPI could bind. Various mutations in this specific sequence compromised the interaction, showing the specificity of the interactions. In the luciferase reporter assay, when the reporter gene was driven by caspase-1 gene upstream sequences (- 151 to - 92) with the mutation G to T at position - 98, luciferase activity was decreased significantly in green fluorescent protein-Hippi-expressing HeLa cells in comparison to that obtained with the wild-type caspase-1 gene 60 bp upstream sequence, indicating the biological significance of such binding. It was observed that the C-terminal 'pseudo' death effector domain of HIPPI interacted with the 60 bp (- 151 to - 92) upstream sequence of the caspase-1 gene containing the motif. We further observed that expression of caspase-8 and caspase-10 was increased in green fluorescent protein-Hippi-expressing HeLa cells. In addition, HIPPI interacted in vitro with putative promoter sequences of these genes, containing a similar motif. In summary, we identified a novel function of HIPPI; it binds to specific upstream sequences of the caspase-1, caspase-8 and caspase-10 genes and alters the expression of the genes. This result showed the motif-specific interaction of HIPPI with DNA, and indicates that it could act as transcription regulator.

Evaluation of viral and mammalian promoters for driving transgene expression in mouse liver

DOE Office of Scientific and Technical Information (OSTI.GOV)

Al-Dosari, Mohammed; Zhang Guisheng; Knapp, Joseph E.

2006-01-13

Fifteen luciferase plasmid constructs driven by various promoters including cytomegalovirus (CMV), Rous sarcoma virus (RSV), human serum albumin (SA), {alpha}-1 antitrypsin (AAT), cytochrome P450 CYP1A2, CYP2C9, CYP2C18, CYP2D6, CYP3A4, mouse CYP2b10, human amyloid precursor protein (APP), chicken {beta} actin (ACT), nuclear factor {kappa} B (NF{kappa}B), and heat shock protein 70 (HS) promoters were hydrodynamically introduced into mouse hepatocytes, and the level and persistence of luciferase gene expression were examined. Eight hours post-gene transfer, the CMV and AAT promoters showed the highest activity, followed by the CYP2D6, HS, and RSV promoters which were slightly less active. The human serum albumin promotermore » exhibited the lowest activity among the promoters examined. The time course of gene expression showed a two-phase decline in luciferase activity with a rapid phase within First 5-7 days and a slower decline thereafter. Results from Southern and Northern blot analyses revealed a good correlation between the decline of luciferase activity and the decrease in mRNA level, suggesting promoter silencing as the possible mechanism for the observed transient luciferase gene expression. Inclusion of EBN1 and oriP sequences of Epstein-Barr virus into the plasmid extended the period of active transcription for about one week. These results provide important information concerning the role of promoters in regulating transgene expression and for the proper design of plasmids for gene expression and gene therapy.« less

Oryza sativa BRASSINOSTEROID UPREGULATED1 LIKE1 Induces the Expression of a Gene Encoding a Small Leucine-Rich-Repeat Protein to Positively Regulate Lamina Inclination and Grain Size in Rice

PubMed Central

Jang, Seonghoe; Li, Hsing-Yi

2017-01-01

Oryza sativa BRASSINOSTEROID UPREGULATED1 LIKE1 (OsBUL1) positively affects lamina inclination and grain size. OsBUL1 knock-out (osbul1) plants as well as transgenic rice with reduced level of OsBUL1 expression produce erect leaves and small grains. Here, we identified a putative downstream gene of OsBUL1, OsBUL1 DOWNSTREAM GENE1 (OsBDG1) encoding a small protein with short leucine-rich-repeats by cDNA microarray analyses in the lamina joint and panicles of wild-type and osbul1 plants. Transgenic rice plants with increased OsBDG1 expression exhibit increased leaf angle and grain size, which is similar to an OsBDG1 activation tagging line whereas double stranded RNA interference (dsRNAi) lines for OsBDG1 knock-down generate erect leaves with smaller grains. Moreover, transgenic rice expressing OsBDG1 under the control of OsBUL1 promoter also shows enlarged leaf bending and grain size phenotypes. Two genes, OsAP2 (OsAPETALA2) and OsWRKY24 were identified as being upregulated transcriptional activators in the lamina joint of pOsBUL1:OsBDG1 plants and induced expression of the two genes driven by OsBUL1 promoter caused increased lamina inclination and grain size in rice. Thus, our work demonstrates that a series of genes showing expression cascades are involved in the promotion of cell elongation in lamina joints and functionally cause increased lamina inclination. PMID:28769958

Entrainment to feeding but not to light: circadian phenotype of VPAC2 receptor-null mice.

PubMed

Sheward, W John; Maywood, Elizabeth S; French, Karen L; Horn, Jacqueline M; Hastings, Michael H; Seckl, Jonathan R; Holmes, Megan C; Harmar, Anthony J

2007-04-18

The master clock driving mammalian circadian rhythms is located in the suprachiasmatic nuclei (SCN) of the hypothalamus and entrained by daily light/dark cycles. SCN lesions abolish circadian rhythms of behavior and result in a loss of synchronized circadian rhythms of clock gene expression in peripheral organs (e.g., the liver) and of hormone secretion (e.g., corticosterone). We examined rhythms of behavior, hepatic clock gene expression, and corticosterone secretion in VPAC2 receptor-null (Vipr2-/-) mice, which lack a functional SCN clock. Unexpectedly, although Vipr2-/- mice lacked robust circadian rhythms of wheel-running activity and corticosterone secretion, hepatic clock gene expression was strongly rhythmic, but advanced in phase compared with that in wild-type mice. The timing of food availability is thought to be an important entrainment signal for circadian clocks outside the SCN. Vipr2-/- mice consumed food significantly earlier in the 24 h cycle than wild-type mice, consistent with the observed timing of peripheral rhythms of circadian gene expression. When restricted to feeding only during the daytime (RF), mice develop rhythms of activity and of corticosterone secretion in anticipation of feeding time, thought to be driven by a food-entrainable circadian oscillator, located outside the SCN. Under RF, mice of both genotypes developed food-anticipatory rhythms of activity and corticosterone secretion, and hepatic gene expression rhythms also became synchronized to the RF stimulus. Thus, food intake is an effective zeitgeber capable of coordinating circadian rhythms of behavior, peripheral clock gene expression, and hormone secretion, even in the absence of a functional SCN clock.

A quantitative study of the benefits of co-regulation using the spoIIA operon as an example

PubMed Central

Iber, Dagmar

2006-01-01

The distribution of most genes is not random, and functionally linked genes are often found in clusters. Several theories have been put forward to explain the emergence and persistence of operons in bacteria. Careful analysis of genomic data favours the co-regulation model, where gene organization into operons is driven by the benefits of coordinated gene expression and regulation. Direct evidence that coexpression increases the individual's fitness enough to ensure operon formation and maintenance is, however, still lacking. Here, a previously described quantitative model of the network that controls the transcription factor σF during sporulation in Bacillus subtilis is employed to quantify the benefits arising from both organization of the sporulation genes into the spoIIA operon and from translational coupling. The analysis shows that operon organization, together with translational coupling, is important because of the inherent stochastic nature of gene expression, which skews the ratios between protein concentrations in the absence of co-regulation. The predicted impact of different forms of gene regulation on fitness and survival agrees quantitatively with published sporulation efficiencies. PMID:16924264

A quantitative study of the benefits of co-regulation using the spoIIA operon as an example.

PubMed

Iber, Dagmar

2006-01-01

The distribution of most genes is not random, and functionally linked genes are often found in clusters. Several theories have been put forward to explain the emergence and persistence of operons in bacteria. Careful analysis of genomic data favours the co-regulation model, where gene organization into operons is driven by the benefits of coordinated gene expression and regulation. Direct evidence that coexpression increases the individual's fitness enough to ensure operon formation and maintenance is, however, still lacking. Here, a previously described quantitative model of the network that controls the transcription factor sigma(F) during sporulation in Bacillus subtilis is employed to quantify the benefits arising from both organization of the sporulation genes into the spoIIA operon and from translational coupling. The analysis shows that operon organization, together with translational coupling, is important because of the inherent stochastic nature of gene expression, which skews the ratios between protein concentrations in the absence of co-regulation. The predicted impact of different forms of gene regulation on fitness and survival agrees quantitatively with published sporulation efficiencies.

Strong positive selection and recombination drive the antigenic variation of the PilE protein of the human pathogen Neisseria meningitidis.

PubMed

Andrews, T Daniel; Gojobori, Takashi

2004-01-01

The PilE protein is the major component of the Neisseria meningitidis pilus, which is encoded by the pilE/pilS locus that includes an expressed gene and eight homologous silent fragments. The silent gene fragments have been shown to recombine through gene conversion with the expressed gene and thereby provide a means by which novel antigenic variants of the PilE protein can be generated. We have analyzed the evolutionary rate of the pilE gene using the nucleotide sequence of two complete pilE/pilS loci. The very high rate of evolution displayed by the PilE protein appears driven by both recombination and positive selection. Within the semivariable region of the pilE and pilS genes, recombination appears to occur within multiple small sequence blocks that lie between conserved sequence elements. Within the hypervariable region, positive selection was identified from comparison of the silent and expressed genes. The unusual gene conversion mechanism that operates at the pilE/pilS locus is a strategy employed by N. meningitidis to enhance mutation of certain regions of the PilE protein. The silent copies of the gene effectively allow "parallelized" evolution of pilE, thus enabling the encoded protein to rapidly explore a large area of sequence space in an effort to find novel antigenic variants.

Use of Attribute Driven Incremental Discretization and Logic Learning Machine to build a prognostic classifier for neuroblastoma patients.

PubMed

Cangelosi, Davide; Muselli, Marco; Parodi, Stefano; Blengio, Fabiola; Becherini, Pamela; Versteeg, Rogier; Conte, Massimo; Varesio, Luigi

2014-01-01

Cancer patient's outcome is written, in part, in the gene expression profile of the tumor. We previously identified a 62-probe sets signature (NB-hypo) to identify tissue hypoxia in neuroblastoma tumors and showed that NB-hypo stratified neuroblastoma patients in good and poor outcome 1. It was important to develop a prognostic classifier to cluster patients into risk groups benefiting of defined therapeutic approaches. Novel classification and data discretization approaches can be instrumental for the generation of accurate predictors and robust tools for clinical decision support. We explored the application to gene expression data of Rulex, a novel software suite including the Attribute Driven Incremental Discretization technique for transforming continuous variables into simplified discrete ones and the Logic Learning Machine model for intelligible rule generation. We applied Rulex components to the problem of predicting the outcome of neuroblastoma patients on the bases of 62 probe sets NB-hypo gene expression signature. The resulting classifier consisted in 9 rules utilizing mainly two conditions of the relative expression of 11 probe sets. These rules were very effective predictors, as shown in an independent validation set, demonstrating the validity of the LLM algorithm applied to microarray data and patients' classification. The LLM performed as efficiently as Prediction Analysis of Microarray and Support Vector Machine, and outperformed other learning algorithms such as C4.5. Rulex carried out a feature selection by selecting a new signature (NB-hypo-II) of 11 probe sets that turned out to be the most relevant in predicting outcome among the 62 of the NB-hypo signature. Rules are easily interpretable as they involve only few conditions. Our findings provided evidence that the application of Rulex to the expression values of NB-hypo signature created a set of accurate, high quality, consistent and interpretable rules for the prediction of neuroblastoma patients' outcome. We identified the Rulex weighted classification as a flexible tool that can support clinical decisions. For these reasons, we consider Rulex to be a useful tool for cancer classification from microarray gene expression data.

Microbiota-driven transcriptional changes in prefrontal cortex override genetic differences in social behavior

PubMed Central

Gacias, Mar; Gaspari, Sevasti; Santos, Patricia-Mae G; Tamburini, Sabrina; Andrade, Monica; Zhang, Fan; Shen, Nan; Tolstikov, Vladimir; Kiebish, Michael A; Dupree, Jeffrey L; Zachariou, Venetia; Clemente, Jose C; Casaccia, Patrizia

2016-01-01

Gene-environment interactions impact the development of neuropsychiatric disorders, but the relative contributions are unclear. Here, we identify gut microbiota as sufficient to induce depressive-like behaviors in genetically distinct mouse strains. Daily gavage of vehicle (dH2O) in nonobese diabetic (NOD) mice induced a social avoidance behavior that was not observed in C57BL/6 mice. This was not observed in NOD animals with depleted microbiota via oral administration of antibiotics. Transfer of intestinal microbiota, including members of the Clostridiales, Lachnospiraceae and Ruminococcaceae, from vehicle-gavaged NOD donors to microbiota-depleted C57BL/6 recipients was sufficient to induce social avoidance and change gene expression and myelination in the prefrontal cortex. Metabolomic analysis identified increased cresol levels in these mice, and exposure of cultured oligodendrocytes to this metabolite prevented myelin gene expression and differentiation. Our results thus demonstrate that the gut microbiota modifies the synthesis of key metabolites affecting gene expression in the prefrontal cortex, thereby modulating social behavior. DOI: http://dx.doi.org/10.7554/eLife.13442.001 PMID:27097105

Biodegradable DNA Nanoparticles that Provide Widespread Gene Delivery in the Brain

PubMed Central

Mastorakos, Panagiotis; Song, Eric; Zhang, Clark; Berry, Sneha; Park, Hee Won; Kim, Young Eun; Park, Jong Sung; Lee, Seulki; Suk, Jung Soo; Hanes, Justin

2016-01-01

Successful gene therapy of neurological disorders is predicated on achieving widespread and uniform transgene expression throughout the affected disease area in the brain. However, conventional gene vectors preferentially travel through low-resistance perivascular spaces and/or are confined to the administration site even with the aid of a pressure-driven flow provided by convection-enhanced delivery. Biodegradable DNA nanoparticles offer a safe gene delivery platform devoid of adverse effects associated with virus-based or synthetic non-biodegradable systems. Using a state-of-the-art biodegradable polymer, poly(β-amino ester), we engineered colloidally stable sub-100 nm DNA nanoparticles coated with a non-adhesive polyethylene glycol corona that are able to avoid the adhesive and steric hindrances imposed by the extracellular matrix. Following convection enhanced delivery, these brain-penetrating nanoparticles were able to homogeneously distribute throughout the rodent striatum and mediate widespread and high-level transgene expression. These nanoparticles provide a biodegradable DNA nanoparticle platform enabling uniform transgene expression patterns in vivo and hold promise for the treatment of neurological diseases. PMID:26680637

TRAM (Transcriptome Mapper): database-driven creation and analysis of transcriptome maps from multiple sources

PubMed Central

2011-01-01

Background Several tools have been developed to perform global gene expression profile data analysis, to search for specific chromosomal regions whose features meet defined criteria as well as to study neighbouring gene expression. However, most of these tools are tailored for a specific use in a particular context (e.g. they are species-specific, or limited to a particular data format) and they typically accept only gene lists as input. Results TRAM (Transcriptome Mapper) is a new general tool that allows the simple generation and analysis of quantitative transcriptome maps, starting from any source listing gene expression values for a given gene set (e.g. expression microarrays), implemented as a relational database. It includes a parser able to assign univocal and updated gene symbols to gene identifiers from different data sources. Moreover, TRAM is able to perform intra-sample and inter-sample data normalization, including an original variant of quantile normalization (scaled quantile), useful to normalize data from platforms with highly different numbers of investigated genes. When in 'Map' mode, the software generates a quantitative representation of the transcriptome of a sample (or of a pool of samples) and identifies if segments of defined lengths are over/under-expressed compared to the desired threshold. When in 'Cluster' mode, the software searches for a set of over/under-expressed consecutive genes. Statistical significance for all results is calculated with respect to genes localized on the same chromosome or to all genome genes. Transcriptome maps, showing differential expression between two sample groups, relative to two different biological conditions, may be easily generated. We present the results of a biological model test, based on a meta-analysis comparison between a sample pool of human CD34+ hematopoietic progenitor cells and a sample pool of megakaryocytic cells. Biologically relevant chromosomal segments and gene clusters with differential expression during the differentiation toward megakaryocyte were identified. Conclusions TRAM is designed to create, and statistically analyze, quantitative transcriptome maps, based on gene expression data from multiple sources. The release includes FileMaker Pro database management runtime application and it is freely available at http://apollo11.isto.unibo.it/software/, along with preconfigured implementations for mapping of human, mouse and zebrafish transcriptomes. PMID:21333005

ABCG5 gene responses to treadmill running with or without administration of Pistachio atlantica in female rats

PubMed Central

Ghanbari-Niaki, Abbass; Zare-Kookandeh, Navabeh; Zare-Kookandeh, Asghar

2014-01-01

Objective(s): ABC transporters comprise a large family of transmembrane proteins that use the energy provided by ATP hydrolysis to translocate a variety of substrates across biological membranes. All members of the human ABCG subfamily, except for ABCG2, are cholesterol-transporter. The aim of this study was to determine the liver, the small intestine and kidney ABCG5 relative gene expression in response to treadmill-running training in female rats. Materials and Methods: Twenty Wistar rats (6-8 weeks old and 125-135 g weight) were used. Animals were randomly assigned to saline-control (SC), saline-training (ST), and Baneh-control (BC), and Baneh-training (BT) groups. Training groups did the exercise on a motor-driven treadmill at 25 m/min (0% grade) for 60 min/day for eight weeks (5 days/week). Rats were fed orally, with Baneh extraction and saline for six weeks. The two-way ANOVA was employed for statistical analysis. ABCG5 relative gene expression was detected by Real-time PCR method. Results: The current findings indicate that the Baneh-treated tissues had significantly lower levels of ABCG5 gene expression in the liver, small intestine, and kidneys (P< 0.001, P< 0.003, P< 0.001, respectively), when compared with saline-treated tissues. However, a higher level of gene expression was observed in exercise groups. A lower level of HDL-c but not triglyceride (TG) and total cholesterol (TC) levels were found in Baneh-treated animals at rest. Conclusion: Exercise training increases ABCG5 relative gene expression in the liver, small intestine and kidney tissues; therefore exercise training may adjust the reduction of ABCG5 relative gene expression in Baneh-training group. PMID:24847418

FLO1 is a variable green beard gene that drives biofilm-like cooperation in budding yeast

PubMed Central

Smukalla, Scott; Caldara, Marina; Pochet, Nathalie; Beauvais, Anne; Guadagnini, Stephanie; Yan, Chen; Vinces, Marcelo D.; Jansen, An; Prevost, Marie Christine; Latgé, Jean-Paul; Fink, Gerald R.; Foster, Kevin R.; Verstrepen, Kevin J.

2008-01-01

Summary The budding yeast, Saccharomyces cerevisiae, has emerged as an archetype of eukaryotic cell biology. Here we show that S. cerevisiae is also a model for the evolution of cooperative behavior by revisiting flocculation, a self-adherence phenotype lacking in most laboratory strains. Expression of the gene FLO1 in the laboratory strain S288C restores flocculation, an altered physiological state, reminiscent of bacterial biofilms. Flocculation protects the FLO1-expressing cells from multiple stresses, including antimicrobials and ethanol. Furthermore, FLO1+ cells avoid exploitation by non-expressing flo1 cells by self/non-self recognition: FLO1+ cells preferentially stick to one another, regardless of genetic relatedness across the rest of the genome. Flocculation, therefore, is driven by one of a few known “green beard genes”, which direct cooperation towards other carriers of the same gene. Moreover, FLO1 is highly variable among strains both in expression and in sequence, suggesting that flocculation in S. cerevisiae is a dynamic, rapidly-evolving social trait. PMID:19013280

Identification and expression pattern analysis of BoMYB51 involved in indolic glucosinolate biosynthesis from broccoli (Brassica oleracea var. italica).

PubMed

Yu, Qingyue; Hao, Guodong; Zhou, Jianxin; Wang, Jingying; Evivie, Ejiroghene Ruona; Li, Jing

2018-06-22

Glucosinolates are a class of amino acid-derived specialized metabolites characteristic of the Brassicales order. Trp derived indolic glucosinolates are essential for the effective plant defense responses to a wide range of pathogens and herbivores. In Arabidopsis, MYB51 is the key transcription factor positively regulates indolic glucosinolate production by activating certain biosynthetic genes. In this study, we report the isolation and identification of a MYB51 from broccoli designated as BoMYB51. Overexpression of BoMYB51 in Arabidopsis increased indolic glucosinolate production by upregulating biosynthetic genes and resulted in enhanced flagellin22 (Flg22) induced callose deposition. The spatial expression pattern and responsive expression of BoMYB51 to several hormones and stress treatments were investigated by expressing the β-glucuronidase (GUS) reporter gene driven by BoMYB51 promotor in Arabidopsis and quantitative real-time PCR analysis in broccoli. Our study provides information on molecular characteristics of BoMYB51 and possible physiological process BoMYB51 may involve. Copyright © 2018 Elsevier Inc. All rights reserved.

An elt-3/elt-5/elt-6 GATA Transcription Circuit Guides Aging in C. elegans

PubMed Central

Budovskaya, Yelena V.; Wu, Kendall; Southworth, Lucinda K.; Jiang, Min; Tedesco, Patricia; Johnson, Thomas E.; Kim, Stuart K.

2016-01-01

SUMMARY To define the C. elegans aging process at the molecular level, we used DNA microarray experiments to identify a set of 1294 age-regulated genes and found that the GATA transcription factors ELT-3, ELT-5, and ELT-6 are responsible for age regulation of a large fraction of these genes. Expression of elt-5 and elt-6 increases during normal aging, and both of these GATA factors repress expression of elt-3, which shows a corresponding decrease in expression in old worms. elt-3 regulates a large number of downstream genes that change expression in old age, including ugt-9, col-144, and sod-3. elt-5(RNAi) and elt-6(RNAi) worms have extended longevity, indicating that elt-3, elt-5, and elt-6 play an important functional role in the aging process. These results identify a transcriptional circuit that guides the rapid aging process in C. elegans and indicate that this circuit is driven by drift of developmental pathways rather than accumulation of damage. PMID:18662544

Distinct gene expression profiles characterize the histopathological stages of disease in Helicobacter-induced mucosa-associated lymphoid tissue lymphoma

PubMed Central

Mueller, Anne; O'Rourke, Jani; Grimm, Jan; Guillemin, Karen; Dixon, Michael F.; Lee, Adrian; Falkow, Stanley

2003-01-01

Long-term colonization of humans with Helicobacter pylori can cause the development of gastric B cell mucosa-associated lymphoid tissue lymphoma, yet little is known about the sequence of molecular steps that accompany disease progression. We used microarray analysis and laser microdissection to identify gene expression profiles characteristic and predictive of the various histopathological stages in a mouse model of the disease. The initial step in lymphoma development is marked by infiltration of reactive lymphocytes into the stomach and the launching of a mucosal immune response. Our analysis uncovered molecular markers of both of these processes, including genes coding for the immunoglobulins and the small proline-rich protein Sprr 2A. The subsequent step is characterized histologically by the antigen-driven proliferation and aggregation of B cells and the gradual appearance of lymphoepithelial lesions. In tissues of this stage, we observed increased expression of genes previously associated with malignancy, including the laminin receptor-1 and the multidrug-resistance channel MDR-1. Finally, we found that the transition to destructive lymphoepithelial lesions and malignant lymphoma is marked by an increase in transcription of a single gene encoding calgranulin A/Mrp-8. PMID:12552104

Epigenetic Heterogeneity of B-Cell Lymphoma: DNA Methylation, Gene Expression and Chromatin States

PubMed Central

Hopp, Lydia; Löffler-Wirth, Henry; Binder, Hans

2015-01-01

Mature B-cell lymphoma is a clinically and biologically highly diverse disease. Its diagnosis and prognosis is a challenge due to its molecular heterogeneity and diverse regimes of biological dysfunctions, which are partly driven by epigenetic mechanisms. We here present an integrative analysis of DNA methylation and gene expression data of several lymphoma subtypes. Our study confirms previous results about the role of stemness genes during development and maturation of B-cells and their dysfunction in lymphoma locking in more proliferative or immune-reactive states referring to B-cell functionalities in the dark and light zone of the germinal center and also in plasma cells. These dysfunctions are governed by widespread epigenetic effects altering the promoter methylation of the involved genes, their activity status as moderated by histone modifications and also by chromatin remodeling. We identified four groups of genes showing characteristic expression and methylation signatures among Burkitt’s lymphoma, diffuse large B cell lymphoma, follicular lymphoma and multiple myeloma. These signatures are associated with epigenetic effects such as remodeling from transcriptionally inactive into active chromatin states, differential promoter methylation and the enrichment of targets of transcription factors such as EZH2 and SUZ12. PMID:26371046

A global RNA-seq-driven analysis of CHO host and production cell lines reveals distinct differential expression patterns of genes contributing to recombinant antibody glycosylation.

PubMed

Könitzer, Jennifer D; Müller, Markus M; Leparc, Germán; Pauers, Martin; Bechmann, Jan; Schulz, Patrick; Schaub, Jochen; Enenkel, Barbara; Hildebrandt, Tobias; Hampel, Martin; Tolstrup, Anne B

2015-09-01

Boehringer Ingelheim uses two CHO-DG44 lines for manufacturing biotherapeutics, BI-HEX-1 and BI-HEX-2, which produce distinct cell type-specific antibody glycosylation patterns. A recently established CHO-K1 descended host, BI-HEX-K1, generates antibodies with glycosylation profiles differing from CHO-DG44. Manufacturing process development is significantly influenced by these unique profiles. To investigate the underlying glycosylation related gene expression, we leveraged our CHO host and production cell RNA-seqtranscriptomics and product quality database together with the CHO-K1 genome. We observed that each BI-HEX host and antibody producing cell line has a unique gene expression fingerprint. CHO-DG44 cells only transcribe Fut10, Gfpt2 and ST8Sia6 when expressing antibodies. BI-HEX-K1 cells express ST8Sia6 at host cell level. We detected a link between BI-HEX-1/BI-HEX-2 antibody galactosylation and mannosylation and the gene expression of the B4galt gene family and genes controlling mannose processing. Furthermore, we found major differences between the CHO-DG44 and CHO-K1 lineages in the expression of sialyl transferases and enzymes synthesizing sialic acid precursors, providing a rationale for the lack of immunogenic NeuGc/NGNA synthesis in CHO. Our study highlights the value of systems biotechnology to understand glycoprotein synthesis and product glycoprofiles. Such data improve future production clone selection and process development strategies for better steering of biotherapeutic product quality. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Complex interactions between cis-regulatory modules in native conformation are critical for Drosophila snail expression

PubMed Central

Dunipace, Leslie; Ozdemir, Anil; Stathopoulos, Angelike

2011-01-01

It has been shown in several organisms that multiple cis-regulatory modules (CRMs) of a gene locus can be active concurrently to support similar spatiotemporal expression. To understand the functional importance of such seemingly redundant CRMs, we examined two CRMs from the Drosophila snail gene locus, which are both active in the ventral region of pre-gastrulation embryos. By performing a deletion series in a ∼25 kb DNA rescue construct using BAC recombineering and site-directed transgenesis, we demonstrate that the two CRMs are not redundant. The distal CRM is absolutely required for viability, whereas the proximal CRM is required only under extreme conditions such as high temperature. Consistent with their distinct requirements, the CRMs support distinct expression patterns: the proximal CRM exhibits an expanded expression domain relative to endogenous snail, whereas the distal CRM exhibits almost complete overlap with snail except at the anterior-most pole. We further show that the distal CRM normally limits the increased expression domain of the proximal CRM and that the proximal CRM serves as a `damper' for the expression levels driven by the distal CRM. Thus, the two CRMs interact in cis in a non-additive fashion and these interactions may be important for fine-tuning the domains and levels of gene expression. PMID:21813571

Incorporating genomic, transcriptomic and clinical data: a prognostic and stem cell-like MYC and PRC imbalance in high-risk neuroblastoma.

PubMed

Yang, Xinan Holly; Tang, Fangming; Shin, Jisu; Cunningham, John M

2017-10-03

Previous studies suggested that cancer cells possess traits reminiscent of the biological mechanisms ascribed to normal embryonic stem cells (ESCs) regulated by MYC and Polycomb repressive complex 2 (PRC2). Several poorly differentiated adult tumors showed preferentially high expression levels in targets of MYC, coincident with low expression levels in targets of PRC2. This paper will reveal this ESC-like cancer signature in high-risk neuroblastoma (HR-NB), the most common extracranial solid tumor in children. We systematically assembled genomic variants, gene expression changes, priori knowledge of gene functions, and clinical outcomes to identify prognostic multigene signatures. First, we assigned a new, individualized prognostic index using the relative expressions between the poor- and good-outcome signature genes. We then characterized HR-NB aggressiveness beyond these prognostic multigene signatures through the imbalanced effects of MYC and PRC2 signaling. We further analyzed Retinoic acid (RA)-induced HR-NB cells to model tumor cell differentiation. Finally, we performed in vitro validation on ZFHX3, a cell differentiation marker silenced by PRC2, and compared cell morphology changes before and after blocking PRC2 in HR-NB cells. A significant concurrence existed between exons with verified variants and genes showing MYCN-dependent expression in HR-NB. From these biomarker candidates, we identified two novel prognostic gene-set pairs with multi-scale oncogenic defects. Intriguingly, MYC targets over-represented an unfavorable component of the identified prognostic signatures while PRC2 targets over-represented a favorable component. The cell cycle arrest and neuronal differentiation marker ZFHX3 was identified as one of PRC2-silenced tumor suppressor candidates. Blocking PRC2 reduced tumor cell growth and increased the mRNA expression levels of ZFHX3 in an early treatment stage. This hypothesis-driven systems bioinformatics work offered novel insights into the PRC2-mediated tumor cell growth and differentiation in neuroblastoma, which may exert oncogenic effects together with MYC regulation. Our results propose a prognostic effect of imbalanced MYC and PRC2 moderations in pediatric HR-NB for the first time. This study demonstrates an incorporation of genomic landscapes and transcriptomic profiles into the hypothesis-driven precision prognosis and biomarker discovery. The application of this approach to neuroblastoma, as well as other cancer more broadly, could contribute to reduced relapse and mortality rates in the long term.

Development of an adenoviral vector with robust expression driven by p53

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bajgelman, Marcio C.; Biotechnology Program, Biomedical Sciences Institute, University of Sao Paulo; Millennium Institute-Gene Therapy Network, Ministry of Science and Technology

2008-02-05

Here we introduce a new adenoviral vector where transgene expression is driven by p53. We first developed a synthetic promoter, referred to as PGTx{beta}, containing a p53-responsive element, a minimal promoter and the first intron of the rabbit {beta}-globin gene. Initial assays using plasmid-based vectors indicated that expression was tightly controlled by p53 and was 5-fold stronger than the constitutive CMV immediate early promoter/enhancer. The adenoviral vector, AdPG, was also shown to offer p53-responsive expression in prostate carcinoma cells LNCaP (wt p53), DU-145 (temperature sensitive mutant of p53) and PC3 (p53-null, but engineered to express temperature-sensitive p53 mutants). AdPG servedmore » as a sensor of p53 activity in LNCaP cells treated with chemotherapeutic agents. Since p53 can be induced by radiotherapy and chemotherapy, this new vector could be further developed for use in combination with conventional therapies to bring about cooperation between the genetic and pharmacologic treatment modalities.« less

Glucocorticoid Signaling Enhances Expression of Glucose-Sensing Molecules in Immature Pancreatic Beta-Like Cells Derived from Murine Embryonic Stem Cells In Vitro.

PubMed

Ghazalli, Nadiah; Wu, Xiaoxing; Walker, Stephanie; Trieu, Nancy; Hsin, Li-Yu; Choe, Justin; Chen, Chialin; Hsu, Jasper; LeBon, Jeanne; Kozlowski, Mark T; Rawson, Jeffrey; Tirrell, David A; Yip, M L Richard; Ku, Hsun Teresa

2018-06-06

Pluripotent stem cells may serve as an alternative source of beta-like cells for replacement therapy of type 1 diabetes; however, the beta-like cells generated in many differentiation protocols are immature. The maturation of endogenous beta cells involves an increase in insulin expression starting in late gestation and a gradual acquisition of the abilities to sense glucose and secrete insulin by week 2 after birth in mice; however, what molecules regulate these maturation processes are incompletely known. In this study, we aim to identify small molecules that affect immature beta cells. A cell-based assay, using pancreatic beta-like cells derived from murine embryonic stem (ES) cells harboring a transgene containing an insulin 1-promoter driven enhanced green fluorescent protein reporter, was used to screen a compound library (NIH Clinical Collection-003). Cortisone, a glucocorticoid, was among five positive hit compounds. Quantitative reverse transcription-polymerase chain reaction analysis revealed that glucocorticoids enhance the gene expression of not only insulin 1 but also glucose transporter-2 (Glut2; Slc2a2) and glucokinase (Gck), two molecules important for glucose sensing. Mifepristone, a pharmacological inhibitor of glucocorticoid receptor (GR) signaling, reduced the effects of glucocorticoids on Glut2 and Gck expression. The effects of glucocorticoids on ES-derived cells were further validated in immature primary islets. Isolated islets from 1-week-old mice had an increased Glut2 and Gck expression in response to a 4-day treatment of exogenous hydrocortisone in vitro. Gene deletion of GR in beta cells using rat insulin 2 promoter-driven Cre crossed with GR flox/flox mice resulted in a reduced gene expression of Glut2, but not Gck, and an abrogation of insulin secretion when islets were incubated in 0.5 mM d-glucose and stimulated by 17 mM d-glucose in vitro. These results demonstrate that glucocorticoids positively regulate glucose sensors in immature murine beta-like cells.

RNA-seq analysis of Drosophila clock and non-clock neurons reveals neuron-specific cycling and novel candidate neuropeptides.

PubMed

Abruzzi, Katharine C; Zadina, Abigail; Luo, Weifei; Wiyanto, Evelyn; Rahman, Reazur; Guo, Fang; Shafer, Orie; Rosbash, Michael

2017-02-01

Locomotor activity rhythms are controlled by a network of ~150 circadian neurons within the adult Drosophila brain. They are subdivided based on their anatomical locations and properties. We profiled transcripts "around the clock" from three key groups of circadian neurons with different functions. We also profiled a non-circadian outgroup, dopaminergic (TH) neurons. They have cycling transcripts but fewer than clock neurons as well as low expression and poor cycling of clock gene transcripts. This suggests that TH neurons do not have a canonical circadian clock and that their gene expression cycling is driven by brain systemic cues. The three circadian groups are surprisingly diverse in their cycling transcripts and overall gene expression patterns, which include known and putative novel neuropeptides. Even the overall phase distributions of cycling transcripts are distinct, indicating that different regulatory principles govern transcript oscillations. This surprising cell-type diversity parallels the functional heterogeneity of the different neurons.

Cloning and Characterization of 5′ Flanking Regulatory Sequences of AhLEC1B Gene from Arachis Hypogaea L.

PubMed Central

Tang, Guiying; Xu, Pingli; Liu, Wei; Liu, Zhanji; Shan, Lei

2015-01-01

LEAFY COTYLEDON1 (LEC1) is a B subunit of Nuclear Factor Y (NF-YB) transcription factor that mainly accumulates during embryo development. We cloned the 5′ flanking regulatory sequence of AhLEC1B gene, a homolog of Arabidopsis LEC1, and analyzed its regulatory elements using online software. To identify the crucial regulatory region, we generated a series of GUS expression frameworks driven by different length promoters with 5′ terminal and/or 3′ terminal deletion. We further characterized the GUS expression patterns in the transgenic Arabidopsis lines. Our results show that both the 65bp proximal promoter region and the 52bp 5′ UTR of AhLEC1B contain the key motifs required for the essential promoting activity. Moreover, AhLEC1B is preferentially expressed in the embryo and is co-regulated by binding of its upstream genes with both positive and negative corresponding cis-regulatory elements. PMID:26426444

Development of tyrosinase-based reporter genes for preclinical photoacoustic imaging of mesenchymal stem cells

NASA Astrophysics Data System (ADS)

Märk, Julia; Ruschke, Karen; Dortay, Hakan; Schreiber, Isabelle; Sass, Andrea; Qazi, Taimoor; Pumberger, Matthias; Laufer, Jan

2014-03-01

The capability to image stem cells in vivo in small animal models over extended periods of time is important to furthering our understanding of the processes involved in tissue regeneration. Photoacoustic imaging is suited to this application as it can provide high resolution (tens of microns) absorption-based images of superficial tissues (cm depths). However, stem cells are rare, highly migratory, and can divide into more specialised cells. Genetic labelling strategies are therefore advantageous for their visualisation. In this study, methods for the transfection and viral transduction of mesenchymal stem cells with reporter genes for the co-expression of tyrosinase and a fluorescent protein (mCherry). Initial photoacoustic imaging experiments of tyrosinase expressing cells in small animal models of tissue regeneration were also conducted. Lentiviral transduction methods were shown to result in stable expression of tyrosinase and mCherry in mesenchymal stem cells. The results suggest that photoacoustic imaging using reporter genes is suitable for the study of stem cell driven tissue regeneration in small animals.

Morpho-functional characterization of the systemic venous pole of the reptile heart.

PubMed

Jensen, Bjarke; Vesterskov, Signe; Boukens, Bastiaan J; Nielsen, Jan M; Moorman, Antoon F M; Christoffels, Vincent M; Wang, Tobias

2017-07-27

Mammals evolved from reptile-like ancestors, and while the mammalian heart is driven by a distinct sinus node, a sinus node is not apparent in reptiles. We characterized the myocardial systemic venous pole, the sinus venosus, in reptiles to identify the dominant pacemaker and to assess whether the sinus venosus remodels and adopts an atrium-like phenotype as observed in mammals. Anolis lizards had an extensive sinus venosus of myocardium expressing Tbx18. A small sub-population of cells encircling the sinuatrial junction expressed Isl1, Bmp2, Tbx3, and Hcn4, homologues of genes marking the mammalian sinus node. Electrical mapping showed that hearts of Anolis lizards and Python snakes were driven from the sinuatrial junction. The electrical impulse was delayed between the sinus venosus and the right atrium, allowing the sinus venosus to contract and aid right atrial filling. In proximity of the systemic veins, the Anolis sinus venosus expressed markers of the atrial phenotype Nkx2-5 and Gja5. In conclusion, the reptile heart is driven by a pacemaker region with an expression signature similar to that of the immature sinus node of mammals. Unlike mammals, reptiles maintain a sinuatrial delay of the impulse, allowing the partly atrialized sinus venosus to function as a chamber.

GPER in CAFs regulates hypoxia-driven breast cancer invasion in a CTGF-dependent manner.

PubMed

Ren, Juan; Guo, Hui; Wu, Huili; Tian, Tao; Dong, Danfeng; Zhang, Yuelang; Sui, Yanxia; Zhang, Yong; Zhao, Dongli; Wang, Shufeng; Li, Zongfang; Zhang, Xiaozhi; Liu, Rui; Qian, Jianshneg; Wei, Hongxia; Jiang, Wenjun; Liu, Ya; Li, Yi

2015-04-01

Recent advances indicate that cancer‑associated fibroblasts (CAFs) play a key role in cancer progression by contributing to invasion, metastasis and angiogenesis. Solid tumors often experience low oxygen tension environments, which induce gene expression changes and biological features leading to poor outcomes. The G-protein estrogen receptor (GPER) exhibits a stimulatory role in diverse types of cancer cells and in CAFs under hypoxic conditions. We investigated the role of CAFs and hypoxia in breast cancer aggressiveness, and examined the effect of GPER in CAFs on hypoxia-driven breast cancer progression. The results showed that hypoxia upregulated HIF-1α, GPER and α-SMA expression in CAFs, and induced the secretion of Interleukin-6 (IL-6), vascular endothelial growth factor (VEGF) and connective tissue growth factor (CTGF) in CAFs. However, GPER silencing abrogated the above hypoxia-driven cytokine expression in CAFs. Moreover, knockdown of GPER in CAFs suppressed breast cancer cell invasion induced by CAF conditioned media (CM). Furthermore, GPER silencing in CAFs inhibited hypoxia-increased CTGF expression in CAFs and breast cancer cells cultured with CM from CAFs under hypoxic conditions. In addition, CTGF is responsible for the observed effects of GPER on CAFs activation and breast cancer invasion. Our findings further extend the molecular mechanisms through which the tumor microenvironment may contribute to cancer progression.

Use of DEAD-box polypeptide-4 (Ddx4) gene promoter-driven fluorescent reporter mice to identify mitotically active germ cells in post-natal mouse ovaries

PubMed Central

Park, Eun-Sil; Tilly, Jonathan L.

2015-01-01

Several laboratories have independently isolated mitotically active germ cells, termed female germline stem cells or oogonial stem cells (OSCs), from adult mouse ovaries. However, a recent study using Ddx4-Cre;Rosa26 reporter mice concluded that such germ cells do not exist. Given the disparity in conclusions drawn in this study compared with others, we felt it was important to re-assess the utility of Ddx4-Cre;Rosa26 reporter mice for identification of OSCs in adult mouse ovaries. Transgenic Ddx4-Cre mice were crossed with Rosa26tdTm/tdTm mice to drive restricted tomato red (tdTm) gene expression in cells in which the Ddx4 gene promoter has been activated. Crude dispersion of ovaries from recombined offspring generated cell fractions containing tdTm-positive immature oocytes, which are incapable of proliferation and thus probably represent the uncharacterized reporter-positive ovarian cells identified in the paper Zhang et al. (2012) as being mitotically inactive. Dispersed ovaries further subjected to fluorescence-activated cell sorting yielded a large population of non-germline tdTm-positive cells, indicative of promoter ‘leakiness’ in the Ddx4-Cre mouse line. Nonetheless, a small percentage of these tdTm-positive cells exhibited externalized (extracellular, ec) expression of Ddx4 protein (ecDdx4-positive), expressed markers of primitive germ cells but not of oocytes, and actively proliferated in culture, all of which are characteristic features of OSCs. Thus, crude dispersion of ovaries collected from Ddx4 gene promoter-driven reporter mice is not, by itself, a reliable approach to identify OSCs, whereas the same ovarian dispersates further subjected to cell sorting strategies yield purified OSCs that can be expanded in culture. PMID:25147160

Decision tree-based method for integrating gene expression, demographic, and clinical data to determine disease endotypes

PubMed Central

2013-01-01

Background Complex diseases are often difficult to diagnose, treat and study due to the multi-factorial nature of the underlying etiology. Large data sets are now widely available that can be used to define novel, mechanistically distinct disease subtypes (endotypes) in a completely data-driven manner. However, significant challenges exist with regard to how to segregate individuals into suitable subtypes of the disease and understand the distinct biological mechanisms of each when the goal is to maximize the discovery potential of these data sets. Results A multi-step decision tree-based method is described for defining endotypes based on gene expression, clinical covariates, and disease indicators using childhood asthma as a case study. We attempted to use alternative approaches such as the Student’s t-test, single data domain clustering and the Modk-prototypes algorithm, which incorporates multiple data domains into a single analysis and none performed as well as the novel multi-step decision tree method. This new method gave the best segregation of asthmatics and non-asthmatics, and it provides easy access to all genes and clinical covariates that distinguish the groups. Conclusions The multi-step decision tree method described here will lead to better understanding of complex disease in general by allowing purely data-driven disease endotypes to facilitate the discovery of new mechanisms underlying these diseases. This application should be considered a complement to ongoing efforts to better define and diagnose known endotypes. When coupled with existing methods developed to determine the genetics of gene expression, these methods provide a mechanism for linking genetics and exposomics data and thereby accounting for both major determinants of disease. PMID:24188919

Transcriptional profiling identifies differentially expressed genes in developing turkey skeletal muscle

PubMed Central

2011-01-01

Background Skeletal muscle growth and development from embryo to adult consists of a series of carefully regulated changes in gene expression. Understanding these developmental changes in agriculturally important species is essential to the production of high quality meat products. For example, consumer demand for lean, inexpensive meat products has driven the turkey industry to unprecedented production through intensive genetic selection. However, achievements of increased body weight and muscle mass have been countered by an increased incidence of myopathies and meat quality defects. In a previous study, we developed and validated a turkey skeletal muscle-specific microarray as a tool for functional genomics studies. The goals of the current study were to utilize this microarray to elucidate functional pathways of genes responsible for key events in turkey skeletal muscle development and to compare differences in gene expression between two genetic lines of turkeys. To achieve these goals, skeletal muscle samples were collected at three critical stages in muscle development: 18d embryo (hyperplasia), 1d post-hatch (shift from myoblast-mediated growth to satellite cell-modulated growth by hypertrophy), and 16wk (market age) from two genetic lines: a randombred control line (RBC2) maintained without selection pressure, and a line (F) selected from the RBC2 line for increased 16wk body weight. Array hybridizations were performed in two experiments: Experiment 1 directly compared the developmental stages within genetic line, while Experiment 2 directly compared the two lines within each developmental stage. Results A total of 3474 genes were differentially expressed (false discovery rate; FDR < 0.001) by overall effect of development, while 16 genes were differentially expressed (FDR < 0.10) by overall effect of genetic line. Ingenuity Pathways Analysis was used to group annotated genes into networks, functions, and canonical pathways. The expression of 28 genes involved in extracellular matrix regulation, cell death/apoptosis, and calcium signaling/muscle function, as well as genes with miscellaneous function was confirmed by qPCR. Conclusions The current study identified gene pathways and uncovered novel genes important in turkey muscle growth and development. Future experiments will focus further on several of these candidate genes and the expression and mechanism of action of their protein products. PMID:21385442

Alteration of gene expression by alcohol exposure at early neurulation.

PubMed

Zhou, Feng C; Zhao, Qianqian; Liu, Yunlong; Goodlett, Charles R; Liang, Tiebing; McClintick, Jeanette N; Edenberg, Howard J; Li, Lang

2011-02-21

We have previously demonstrated that alcohol exposure at early neurulation induces growth retardation, neural tube abnormalities, and alteration of DNA methylation. To explore the global gene expression changes which may underline these developmental defects, microarray analyses were performed in a whole embryo mouse culture model that allows control over alcohol and embryonic variables. Alcohol caused teratogenesis in brain, heart, forelimb, and optic vesicle; a subset of the embryos also showed cranial neural tube defects. In microarray analysis (accession number GSM9545), adopting hypothesis-driven Gene Set Enrichment Analysis (GSEA) informatics and intersection analysis of two independent experiments, we found that there was a collective reduction in expression of neural specification genes (neurogenin, Sox5, Bhlhe22), neural growth factor genes [Igf1, Efemp1, Klf10 (Tieg), and Edil3], and alteration of genes involved in cell growth, apoptosis, histone variants, eye and heart development. There was also a reduction of retinol binding protein 1 (Rbp1), and de novo expression of aldehyde dehydrogenase 1B1 (Aldh1B1). Remarkably, four key hematopoiesis genes (glycophorin A, adducin 2, beta-2 microglobulin, and ceruloplasmin) were absent after alcohol treatment, and histone variant genes were reduced. The down-regulation of the neurospecification and the neurotrophic genes were further confirmed by quantitative RT-PCR. Furthermore, the gene expression profile demonstrated distinct subgroups which corresponded with two distinct alcohol-related neural tube phenotypes: an open (ALC-NTO) and a closed neural tube (ALC-NTC). Further, the epidermal growth factor signaling pathway and histone variants were specifically altered in ALC-NTO, and a greater number of neurotrophic/growth factor genes were down-regulated in the ALC-NTO than in the ALC-NTC embryos. This study revealed a set of genes vulnerable to alcohol exposure and genes that were associated with neural tube defects during early neurulation.

Comparative Genomics Identifies Epidermal Proteins Associated with the Evolution of the Turtle Shell

PubMed Central

Holthaus, Karin Brigit; Strasser, Bettina; Sipos, Wolfgang; Schmidt, Heiko A.; Mlitz, Veronika; Sukseree, Supawadee; Weissenbacher, Anton; Tschachler, Erwin; Alibardi, Lorenzo; Eckhart, Leopold

2016-01-01

The evolution of reptiles, birds, and mammals was associated with the origin of unique integumentary structures. Studies on lizards, chicken, and humans have suggested that the evolution of major structural proteins of the outermost, cornified layers of the epidermis was driven by the diversification of a gene cluster called Epidermal Differentiation Complex (EDC). Turtles have evolved unique defense mechanisms that depend on mechanically resilient modifications of the epidermis. To investigate whether the evolution of the integument in these reptiles was associated with specific adaptations of the sequences and expression patterns of EDC-related genes, we utilized newly available genome sequences to determine the epidermal differentiation gene complement of turtles. The EDC of the western painted turtle (Chrysemys picta bellii) comprises more than 100 genes, including at least 48 genes that encode proteins referred to as beta-keratins or corneous beta-proteins. Several EDC proteins have evolved cysteine/proline contents beyond 50% of total amino acid residues. Comparative genomics suggests that distinct subfamilies of EDC genes have been expanded and partly translocated to loci outside of the EDC in turtles. Gene expression analysis in the European pond turtle (Emys orbicularis) showed that EDC genes are differentially expressed in the skin of the various body sites and that a subset of beta-keratin genes within the EDC as well as those located outside of the EDC are expressed predominantly in the shell. Our findings give strong support to the hypothesis that the evolutionary innovation of the turtle shell involved specific molecular adaptations of epidermal differentiation. PMID:26601937

A light-driven proton pump from Haloterrigena turkmenica: Functional expression in Escherichia coli membrane and coupling with a H{sup +} co-transporter

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kamo, Naoki; Hashiba, Tsuyoshi; Kikukawa, Takashi

2006-03-10

A gene encoding putative retinal protein was cloned from Haloterrigena turkmenica (JCM9743). The deduced amino acid sequence was most closely related to that of deltarhodopsin, which functions as a light-driven H{sup +} pump and was identified in a novel strain Haloterrigena sp. arg-4 (K. Ihara, T. Uemura, I. Katagiri, T. Kitajima-Ihara, Y. Sugiyama, Y. Kimura, Y. Mukohata, Evolution of the archaeal rhodopsins: Evolution rate changes by gene duplication and functional differentiation, J. Mol. Biol. 285 (1999) 163-174. GenBank Accession No. AB009620). Thus, we called the present protein H. turkmenica deltarhodopsin (HtdR) in this report. Differing from the Halobacterium salinarum bacteriorhodopsinmore » (bR), functional expression of HtdR was achieved in Escherichia coli membrane with a high yield of 10-15mg protein/L culture. The photocycle of purified HtdR was similar to that of bR. The photo-induced electrogenic proton pumping activity of HtdR was verified. We co-expressed both HtdR and EmrE, a proton-coupled multi-drug efflux transporter in E. coli, and the cells successfully extruded ethidium, a substrate of EmrE, on illumination.« less

Putative synaptic genes defined from a Drosophila whole body developmental transcriptome by a machine learning approach.

PubMed

Pazos Obregón, Flavio; Papalardo, Cecilia; Castro, Sebastián; Guerberoff, Gustavo; Cantera, Rafael

2015-09-15

Assembly and function of neuronal synapses require the coordinated expression of a yet undetermined set of genes. Although roughly a thousand genes are expected to be important for this function in Drosophila melanogaster, just a few hundreds of them are known so far. In this work we trained three learning algorithms to predict a "synaptic function" for genes of Drosophila using data from a whole-body developmental transcriptome published by others. Using statistical and biological criteria to analyze and combine the predictions, we obtained a gene catalogue that is highly enriched in genes of relevance for Drosophila synapse assembly and function but still not recognized as such. The utility of our approach is that it reduces the number of genes to be tested through hypothesis-driven experimentation.

The physiological role of CTGF/CCN2 in zebrafish notochond development and biological analysis of the proximal promoter region.

PubMed

Chiou, Ming-Jyun; Chao, Tsung-Tai; Wu, Jen-Leih; Kuo, Ching-Ming; Chen, Jyh-Yih

2006-10-20

During mouse embryogenesis, CTGF/CCN2 is expressed in zones containing hypertrophic chondroctyes and calcifying cartilage such as long bones, ribs, vertebral column, and phalanges. But in fish, its expression is yet unclear. Development of the vertebrae is morphologically similar among vertebrates, indicating that the underlying mechanism regulating the process is highly conserved during evolution. Analysis of 3.2kb of the CTGF/CCN2 proximal promoter sequence revealed a consensus TATAA box, putative AP1, Brn-2, CdxA, C/EBP alpha, C/EBP beta, C-Ets-, delta E, HFH-2, and HSF2 binding sites. Transient expression experiments with a 5'-deletion revealed at least 4 regulatory regions in the zebrafish CTGF/CCN2 gene, 2 with a stimulatory effect on transcription and 2 with an apparent inhibitory effect after IGF-I treatment in the ZFL cell line. To study the promoter-specific expression, we constructed a series of CTGF/CCN2 (3.0-, 2.5-, 2.0-, 1.5-, 1.0-, and 0.4-kb) promoter-driven green fluorescent protein (GFP) fragments encoding the GFP cDNA transgene which was microinjected into zebrafish embryos. Morphological studies of transgenic zebrafish indicated that the CTGF/CCN2 promoter-driven GFP transcripts appeared in the notochord. Targeted knockdown of the CTGF/CCN2 gene by two antisense morpholino oligonucleotides resulted in disruptions to notochord development. From a comparative point of view, this study of the CTGF/CCN2 gene in zebrafish may correlate well with those previously published on the mouse. These molecular results suggest that CTGF/CCN2 plays an important role in notochord development and is required for general embryonic development.

Bioengineered 'golden' indica rice cultivars with beta-carotene metabolism in the endosperm with hygromycin and mannose selection systems.

PubMed

Datta, Karabi; Baisakh, Niranjan; Oliva, Norman; Torrizo, Lina; Abrigo, Editha; Tan, Jing; Rai, Mayank; Rehana, Sayda; Al-Babili, Salim; Beyer, Peter; Potrykus, Ingo; Datta, Swapan K

2003-03-01

Vitamin-A deficiency (VAD) is a major malnutrition problem in South Asia, where indica rice is the staple food. Indica-type rice varieties feed more than 2 billion people. Hence, we introduced a combination of transgenes using the biolistic system of transformation enabling biosynthesis of provitamin A in the endosperm of several indica rice cultivars adapted to diverse ecosystems of different countries. The rice seed-specific glutelin promoter (Gt-1 P) was used to drive the expression of phytoene synthase (psy), while lycopene beta-cyclase (lcy) and phytoene desaturase (crtI), fused to the transit peptide sequence of the pea-Rubisco small subunit, were driven by the constitutive cauliflower mosaic virus promoter (CaMV35S P). Transgenic plants were recovered through selection with either CaMV35S P driven hph (hygromycin phosphotransferase) gene or cestrum yellow leaf curling virus promoter (CMP) driven pmi (phophomannose isomerase) gene. Molecular and biochemical analyses demonstrated stable integration and expression of the transgenes. The yellow colour of the polished rice grain evidenced the carotenoid accumulation in the endosperm. The colour intensity correlated with the estimated carotenoid content by spectrophotometric and HPLC analysis. Carotenoid level in cooked polished seeds was comparable (with minor loss of xanthophylls) to that in non-cooked seeds of the same transgenic line. The variable segregation pattern in T1 selfing generation indicated single to multiple loci insertion of the transgenes in the genome. This is the first report of using nonantibiotic pmi driven by a novel promoter in generating transgenic indica rice for possible future use in human nutrition.

Spermatogenic Cell-Specific Gene Mutation in Mice via CRISPR-Cas9.

PubMed

Bai, Meizhu; Liang, Dan; Wang, Yinghua; Li, Qing; Wu, Yuxuan; Li, Jinsong

2016-05-20

Tissue-specific knockout technology enables the analysis of the gene function in specific tissues in adult mammals. However, conventional strategy for producing tissue-specific knockout mice is a time- and labor-consuming process, restricting rapid study of the gene function in vivo. CRISPR-Cas9 system from bacteria is a simple and efficient gene-editing technique, which has enabled rapid generation of gene knockout lines in mouse by direct injection of CRISPR-Cas9 into zygotes. Here, we demonstrate CRISPR-Cas9-mediated spermatogenic cell-specific disruption of Scp3 gene in testes in one step. We first generated transgenic mice by pronuclear injection of a plasmid containing Hspa2 promoter driving Cas9 expression and showed Cas9 specific expression in spermatogenic cells. We then produced transgenic mice carrying Hspa2 promoter driven Cas9 and constitutive expressed sgRNA targeting Scp3 gene. Male founders were infertile due to developmental arrest of spermatogenic cells while female founders could produce progeny normally. Consistently, male progeny from female founders were infertile and females could transmit the transgenes to the next generation. Our study establishes a CRISPR-Cas9-based one-step strategy to analyze the gene function in adult tissues by a temporal-spatial pattern. Copyright © 2016 Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and Genetics Society of China. Published by Elsevier Ltd. All rights reserved.

Generation of marker-free Bt transgenic indica rice and evaluation of its yellow stem borer resistance.

PubMed

Kumar, S; Arul, L; Talwar, D

2010-01-01

We report on generation of marker-free (‘clean DNA’) transgenic rice (Oryza sativa), carrying minimal gene-expression-cassettes of the genes of interest, and evaluation of its resistance to yellow stem borer Scirpophaga incertulas (Lepidoptera: Pyralidae). The transgenic indica rice harbours a translational fusion of 2 different Bacillus thuringiensis (Bt) genes, namely cry1B-1Aa, driven by the green-tissue-specific phosphoenol pyruvate carboxylase (PEPC) promoter. Mature seed-derived calli of an elite indica rice cultivar Pusa Basmati-1 were co-bombarded with gene-expression-cassettes (clean DNA fragments) of the Bt gene and the marker hpt gene, to generate marker-free transgenic rice plants. The clean DNA fragments for bombardment were obtained by restriction digestion and gel extraction. Through biolistic transformation, 67 independent transformants were generated. Transformation frequency reached 3.3%, and 81% of the transgenic plants were co-transformants. Stable integration of the Bt gene was confirmed, and the insert copy number was determined by Southern analysis. Western analysis and ELISA revealed a high level of Bt protein expression in transgenic plants. Progeny analysis confirmed stable inheritance of the Bt gene according to the Mendelian (3:1) ratio. Insect bioassays revealed complete protection of transgenic plants from yellow stem borer infestation. PCR analysis of T2 progeny plants resulted in the recovery of up to 4% marker-free transgenic rice plants.

A small system--high-resolution study of metabolic adaptation in the central metabolic pathway to temperate climates in Drosophila melanogaster.

PubMed

Lavington, Erik; Cogni, Rodrigo; Kuczynski, Caitlin; Koury, Spencer; Behrman, Emily L; O'Brien, Katherine R; Schmidt, Paul S; Eanes, Walter F

2014-08-01

In this article, we couple the geographic variation in 127 single-nucleotide polymorphism (SNP) frequencies in genes of 46 enzymes of central metabolism with their associated cis-expression variation to predict latitudinal or climatic-driven gene expression changes in the metabolic architecture of Drosophila melanogaster. Forty-two percent of the SNPs in 65% of the genes show statistically significant clines in frequency with latitude across the 20 local population samples collected from southern Florida to Ontario. A number of SNPs in the screened genes are also associated with significant expression variation within the Raleigh population from North Carolina. A principal component analysis of the full variance-covariance matrix of latitudinal changes in SNP-associated standardized gene expression allows us to identify those major genes in the pathway and its associated branches that are likely targets of natural selection. When embedded in a central metabolic context, we show that these apparent targets are concentrated in the genes of the upper glycolytic pathway and pentose shunt, those controlling glycerol shuttle activity, and finally those enzymes associated with the utilization of glutamate and pyruvate. These metabolites possess high connectivity and thus may be the points where flux balance can be best shifted. We also propose that these points are conserved points associated with coupling energy homeostasis and energy sensing in mammals. We speculate that the modulation of gene expression at specific points in central metabolism that are associated with shifting flux balance or possibly energy-state sensing plays a role in adaptation to climatic variation. © The Author 2014. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

The genome-wide landscape of DNA methylation and hydroxymethylation in response to sleep deprivation impacts on synaptic plasticity genes.

PubMed

Massart, R; Freyburger, M; Suderman, M; Paquet, J; El Helou, J; Belanger-Nelson, E; Rachalski, A; Koumar, O C; Carrier, J; Szyf, M; Mongrain, V

2014-01-21

Sleep is critical for normal brain function and mental health. However, the molecular mechanisms mediating the impact of sleep loss on both cognition and the sleep electroencephalogram remain mostly unknown. Acute sleep loss impacts brain gene expression broadly. These data contributed to current hypotheses regarding the role for sleep in metabolism, synaptic plasticity and neuroprotection. These changes in gene expression likely underlie increased sleep intensity following sleep deprivation (SD). Here we tested the hypothesis that epigenetic mechanisms coordinate the gene expression response driven by SD. We found that SD altered the cortical genome-wide distribution of two major epigenetic marks: DNA methylation and hydroxymethylation. DNA methylation differences were enriched in gene pathways involved in neuritogenesis and synaptic plasticity, whereas large changes (>4000 sites) in hydroxymethylation where observed in genes linked to cytoskeleton, signaling and neurotransmission, which closely matches SD-dependent changes in the transcriptome. Moreover, this epigenetic remodeling applied to elements previously linked to sleep need (for example, Arc and Egr1) and synaptic partners of Neuroligin-1 (Nlgn1; for example, Dlg4, Nrxn1 and Nlgn3), which we recently identified as a regulator of sleep intensity following SD. We show here that Nlgn1 mutant mice display an enhanced slow-wave slope during non-rapid eye movement sleep following SD but this mutation does not affect SD-dependent changes in gene expression, suggesting that the Nlgn pathway acts downstream to mechanisms triggering gene expression changes in SD. These data reveal that acute SD reprograms the epigenetic landscape, providing a unique molecular route by which sleep can impact brain function and health.

Construction of a β-galactosidase-gene-based fusion is convenient for screening candidate genes involved in regulation of pyrrolnitrin biosynthesis in Pseudomonas chlororaphis G05.

PubMed

Luo, Wangtai; Miao, Jing; Feng, Zhibin; Lu, Ruiyang; Sun, Xiaoqiang; Zhang, Baoshen; Ding, Weiqiu; Lu, Yang; Wang, Yanhua; Chi, Xiaoyan; Ge, Yihe

2018-05-28

In our recent work, we found that pyrrolnitrin, and not phenazines, pyrrolnitrin contributed to the suppression of the mycelia growth of Fusarium graminearum that causes heavy Fusarium head blight (FHB) disease in cereal crops. However, pyrrolnitrin production of Pseudomonas chlororaphis G05 in King's B medium was very low. Although a few regulatory genes mediating the prnABCD (the prn operon, pyrrolnitrin biosynthetic locus) expression have been identified, it is not enough for us to enhance pyrrolnitrin production by systematically constructing a genetically-engineered strain. To obtain new candidate genes involved in regulation of the prn operon expression, we successfully constructed a fusion mutant G05ΔphzΔprn::lacZ, in which most of the coding regions of the prn operon and the phzABCDEFG (the phz operon, phenazine biosynthetic locus) were deleted, and the promoter region plus the first thirty condons of the prnA was in-frame fused with the truncated lacZ gene on its chromosome. The expression of the fused lacZ reporter gene driven by the promoter of the prn operon made it easy for us to detect the level of the prn expression in terms of the color variation of colonies on LB agar plates supplemented with 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-Gal). With this fusion mutant as a recipient strain, mini-Tn5-based random insertional mutagenesis was then conducted. By picking up colonies with color change, it is possible for us to screen and identify new candidate genes involved in regulation of the prn expression. Identification of additional regulatory genes in further work could reasonably be expected to increase pyrrolnitrin production in G05 and to improve its biological control function.

A meiotic gene regulatory cascade driven by alternative fates for newly synthesized transcripts

PubMed Central

Cremona, Nicole; Potter, Kristine; Wise, Jo Ann

2011-01-01

To determine the relative importance of transcriptional regulation versus RNA processing and turnover during the transition from proliferation to meiotic differentiation in the fission yeast Schizosaccharomyces pombe, we analyzed temporal profiles and effects of RNA surveillance factor mutants on expression of 32 meiotic genes. A comparison of nascent transcription with steady-state RNA accumulation reveals that the vast majority of these genes show a lag between maximal RNA synthesis and peak RNA accumulation. During meiosis, total RNA levels parallel 3′ processing, which occurs in multiple, temporally distinct waves that peak from 3 to 6 h after meiotic induction. Most early genes and one middle gene, mei4, share a regulatory mechanism in which a specialized RNA surveillance factor targets newly synthesized transcripts for destruction. Mei4p, a member of the forkhead transcription factor family, in turn regulates a host of downstream genes. Remarkably, a spike in transcription is observed for less than one-third of the genes surveyed, and even these show evidence of RNA-level regulation. In aggregate, our findings lead us to propose that a regulatory cascade driven by changes in processing and stability of newly synthesized transcripts operates alongside the well-known transcriptional cascade as fission yeast cells enter meiosis. PMID:21148298

A novel cold-inducible zinc finger protein from soybean, SCOF-1, enhances cold tolerance in transgenic plants.

PubMed

Kim, J C; Lee, S H; Cheong, Y H; Yoo, C M; Lee, S I; Chun, H J; Yun, D J; Hong, J C; Lee, S Y; Lim, C O; Cho, M J

2001-02-01

Cold stress on plants induces changes in the transcription of cold response genes. A cDNA clone encoding C2H2-type zinc finger protein, SCOF-1, was isolated from soybean. The transcription of SCOF-1 is specifically induced by low temperature and abscisic acid (ABA) but not by dehydration or high salinity. Constitutive overexpression of SCOF-1 induced cold-regulated (COR) gene expression and enhanced cold tolerance of non-acclimated transgenic Arabidopsis and tobacco plants. SCOF-1 localized to the nucleus but did not bind directly to either C-repeat/dehydration (CRT/DRE) or ABA responsive element (ABRE), cis-acting DNA regulatory elements present in COR gene promoters. However, SCOF-1 greatly enhanced the DNA binding activity of SGBF-1, a soybean G-box binding bZIP transcription factor, to ABRE in vitro. SCOF-1 also interacted with SGBF-1 in a yeast two-hybrid system. The SGBF-1 transactivated the beta-glucuronidase reporter gene driven by the ABRE element in Arabidopsis leaf protoplasts. Furthermore, the SCOF-1 enhanced ABRE-dependent gene expression mediated by SGBF-1. These results suggest that SCOF-1 may function as a positive regulator of COR gene expression mediated by ABRE via protein-protein interaction, which in turn enhances cold tolerance of plants.

Integration of high-risk human papillomavirus into cellular cancer-related genes in head and neck cancer cell lines.

PubMed

Walline, Heather M; Goudsmit, Christine M; McHugh, Jonathan B; Tang, Alice L; Owen, John H; Teh, Bin T; McKean, Erin; Glover, Thomas W; Graham, Martin P; Prince, Mark E; Chepeha, Douglas B; Chinn, Steven B; Ferris, Robert L; Gollin, Susanne M; Hoffmann, Thomas K; Bier, Henning; Brakenhoff, Ruud; Bradford, Carol R; Carey, Thomas E

2017-05-01

Human papillomavirus (HPV)-positive oropharyngeal cancer is generally associated with excellent response to therapy, but some HPV-positive tumors progress despite aggressive therapy. The purpose of this study was to evaluate viral oncogene expression and viral integration sites in HPV16- and HPV18-positive squamous cell carcinoma lines. E6/E7 alternate transcripts were assessed by reverse transcriptase-polymerase chain reaction (RT-PCR). Detection of integrated papillomavirus sequences (DIPS-PCR) and sequencing identified viral insertion sites and affected host genes. Cellular gene expression was assessed across viral integration sites. All HPV-positive cell lines expressed alternate HPVE6/E7 splicing indicative of active viral oncogenesis. HPV integration occurred within cancer-related genes TP63, DCC, JAK1, TERT, ATR, ETV6, PGR, PTPRN2, and TMEM237 in 8 head and neck squamous cell carcinoma (HNSCC) lines but UM-SCC-105 and UM-GCC-1 had only intergenic integration. HPV integration into cancer-related genes occurred in 7 of 9 HPV-positive cell lines and of these 6 were from tumors that progressed. HPV integration into cancer-related genes may be a secondary carcinogenic driver in HPV-driven tumors. © 2017 Wiley Periodicals, Inc. Head Neck 39: 840-852, 2017. © 2017 Wiley Periodicals, Inc.

Genome and epigenome engineering CRISPR toolkit for in vivo modulation of cis-regulatory interactions and gene expression in the chicken embryo.

PubMed

Williams, Ruth M; Senanayake, Upeka; Artibani, Mara; Taylor, Gunes; Wells, Daniel; Ahmed, Ahmed Ashour; Sauka-Spengler, Tatjana

2018-02-23

CRISPR/Cas9 genome engineering has revolutionised all aspects of biological research, with epigenome engineering transforming gene regulation studies. Here, we present an optimised, adaptable toolkit enabling genome and epigenome engineering in the chicken embryo, and demonstrate its utility by probing gene regulatory interactions mediated by neural crest enhancers. First, we optimise novel efficient guide-RNA mini expression vectors utilising chick U6 promoters, provide a strategy for rapid somatic gene knockout and establish a protocol for evaluation of mutational penetrance by targeted next-generation sequencing. We show that CRISPR/Cas9-mediated disruption of transcription factors causes a reduction in their cognate enhancer-driven reporter activity. Next, we assess endogenous enhancer function using both enhancer deletion and nuclease-deficient Cas9 (dCas9) effector fusions to modulate enhancer chromatin landscape, thus providing the first report of epigenome engineering in a developing embryo. Finally, we use the synergistic activation mediator (SAM) system to activate an endogenous target promoter. The novel genome and epigenome engineering toolkit developed here enables manipulation of endogenous gene expression and enhancer activity in chicken embryos, facilitating high-resolution analysis of gene regulatory interactions in vivo . © 2018. Published by The Company of Biologists Ltd.

Emergent Self-Organized Criticality in Gene Expression Dynamics: Temporal Development of Global Phase Transition Revealed in a Cancer Cell Line

PubMed Central

Tsuchiya, Masa; Giuliani, Alessandro; Hashimoto, Midori; Erenpreisa, Jekaterina; Yoshikawa, Kenichi

2015-01-01

Background The underlying mechanism of dynamic control of the genome-wide expression is a fundamental issue in bioscience. We addressed it in terms of phase transition by a systemic approach based on both density analysis and characteristics of temporal fluctuation for the time-course mRNA expression in differentiating MCF-7 breast cancer cells. Methodology In a recent work, we suggested criticality as an essential aspect of dynamic control of genome-wide gene expression. Criticality was evident by a unimodal-bimodal transition through flattened unimodal expression profile. The flatness on the transition suggests the existence of a critical transition at which up- and down-regulated expression is balanced. Mean field (averaging) behavior of mRNAs based on the temporal expression changes reveals a sandpile type of transition in the flattened profile. Furthermore, around the transition, a self-similar unimodal-bimodal transition of the whole expression occurs in the density profile of an ensemble of mRNA expression. These singular and scaling behaviors identify the transition as the expression phase transition driven by self-organized criticality (SOC). Principal Findings Emergent properties of SOC through a mean field approach are revealed: i) SOC, as a form of genomic phase transition, consolidates distinct critical states of expression, ii) Coupling of coherent stochastic oscillations between critical states on different time-scales gives rise to SOC, and iii) Specific gene clusters (barcode genes) ranging in size from kbp to Mbp reveal similar SOC to genome-wide mRNA expression and ON-OFF synchronization to critical states. This suggests that the cooperative gene regulation of topological genome sub-units is mediated by the coherent phase transitions of megadomain-scaled conformations between compact and swollen chromatin states. Conclusion and Significance In summary, our study provides not only a systemic method to demonstrate SOC in whole-genome expression, but also introduces novel, physically grounded concepts for a breakthrough in the study of biological regulation. PMID:26067993

Identification of Dlk1, Ptpru and Klhl1 as novel Nurr1 target genes in meso-diencephalic dopamine neurons

PubMed Central

Jacobs, Frank M. J.; van der Linden, Annemarie J. A.; Wang, Yuhui; von Oerthel, Lars; Sul, Hei Sook; Burbach, J. Peter H.; Smidt, Marten P.

2009-01-01

The orphan nuclear receptor Nurr1 is essential for the development of meso-diencephalic dopamine (mdDA) neurons and is required, together with the homeobox transcription factor Pitx3, for the expression of genes involved in dopamine metabolism. In order to elucidate the molecular mechanisms that underlie the neuronal deficits in Nurr1-/- mice, we performed combined gene expression microarrays and ChIP-on-chip analysis and thereby identified Dlk1, Ptpru and Klhl1 as novel Nurr1 target genes in vivo. In line with the previously described cooperativity between Nurr1 and Pitx3, we show that the expression of Ptpru and Klhl1 in mdDA neurons is also dependent on Pitx3. Furthermore, we demonstrate that Nurr1 interacts with the Ptpru promoter directly and requires Pitx3 for full expression of Ptpru in mdDA neurons. By contrast, the expression of Dlk1 is maintained in Pitx3-/- embryos and is even expanded into the rostral part of the mdDA area, suggesting a unique position of Dlk1 in the Nurr1 and Pitx3 transcriptional cascades. Expression analysis in Dlk1-/- embryos reveals that Dlk1 is required to prevent premature expression of Dat in mdDA neuronal precursors as part of the multifaceted process of mdDA neuronal differentiation driven by Nurr1 and Pitx3. Taken together, the involvement of Nurr1 and Pitx3 in the expression of novel target genes involved in important neuronal processes such as neuronal patterning, axon outgrowth and terminal differentiation, opens up new avenues to study the properties of mdDA neurons during development and in neuronal pathology as observed in Parkinson's disease. PMID:19515692

Analysis of IgV gene mutations in B cell chronic lymphocytic leukaemia according to antigen-driven selection identifies subgroups with different prognosis and usage of the canonical somatic hypermutation machinery.

PubMed

Degan, Massimo; Bomben, Riccardo; Bo, Michele Dal; Zucchetto, Antonella; Nanni, Paola; Rupolo, Maurizio; Steffan, Agostino; Attadia, Vincenza; Ballerini, Pier Ferruccio; Damiani, Daniela; Pucillo, Carlo; Poeta, Giovanni Del; Colombatti, Alfonso; Gattei, Valter

2004-07-01

Cases of B-cell chronic lymphocytic leukaemia (B-CLL) with mutated (M) IgV(H) genes have a better prognosis than unmutated (UM) cases. We analysed the IgV(H) mutational status of B-CLL according to the features of a canonical somatic hypermutation (SHM) process, correlating this data with survival. In a series of 141 B-CLLs, 124 cases were examined for IgV(H) gene per cent mutations and skewing of replacement/silent mutations in the framework/complementarity-determining regions as evidence of antigen-driven selection; this identified three B-CLL subsets: significantly mutated (sM), with evidence of antigen-driven selection, not significantly mutated (nsM) and UM, without such evidence and IgV(H) gene per cent mutations above or below the 2% cut-off. sM B-CLL patients had longer survival within the good prognosis subgroup that had more than 2% mutations of IgV(H) genes. sM, nsM and UM B-CLL were also characterized for the biased usage of IgV(H) families, intraclonal IgV(H) gene diversification, preference of mutations to target-specific nucleotides or hotspots, and for the expression of enzymes involved in SHM (translesion DNA polymerase zeta and eta and activation-induced cytidine deaminase). These findings indicate the activation of a canonical SHM process in nsM and sM B-CLLs and underscore the role of the antigen in defining the specific clinical and biological features of B-CLL.

Active FOXO1 is a Key Determinant of Isoform-Specific Progesterone Receptor Transactivation and Senescence Programming

PubMed Central

Diep, Caroline H.; Knutson, Todd P.; Lange, Carol A.

2015-01-01

Progesterone promotes differentiation coupled to proliferation and pro-survival in the breast, but inhibits estrogen-driven growth in the reproductive tract and ovaries. Herein, it is demonstrated, using progesterone receptor (PR) isoform-specific ovarian cancer model systems, that PR-A and PR-B promote distinct gene expression profiles that differ from PR-driven genes in breast cancer cells. In ovarian cancer models, PR-A primarily regulates genes independently of progestin, while PR-B is the dominant ligand-dependent isoform. Notably, FOXO1 and the PR/FOXO1 target-gene p21 (CDKN1A) are repressed by PR-A, but induced by PR-B. In the presence of progestin, PR-B, but not PR-A, robustly induced cellular senescence via FOXO1-dependent induction of p21 and p15 (CDKN2B). Chromatin immunoprecipitation (ChIP) assays performed on PR-isoform specific cells demonstrated that while each isoform is recruited to the same PRE-containing region of the p21 promoter in response to progestin, only PR-B elicits active chromatin marks. Overexpression of constitutively active FOXO1 in PR-A-expressing cells conferred robust ligand-dependent upregulation of the PR-B target genes GZMA, IGFBP1, and p21, and induced cellular senescence. In the presence of endogenous active FOXO1, PR-A was phosphorylated on Ser294 and transactivated PR-B at PR-B target genes; these events were blocked by the FOXO1 inhibitor (AS1842856). PR isoform-specific regulation of the FOXO1/p21 axis recapitulated in human primary ovarian tumor explants treated with progestin; loss of progestin sensitivity correlated with high AKT activity. PMID:26577046

Directed module detection in a large-scale expression compendium.

PubMed

Fu, Qiang; Lemmens, Karen; Sanchez-Rodriguez, Aminael; Thijs, Inge M; Meysman, Pieter; Sun, Hong; Fierro, Ana Carolina; Engelen, Kristof; Marchal, Kathleen

2012-01-01

Public online microarray databases contain tremendous amounts of expression data. Mining these data sources can provide a wealth of information on the underlying transcriptional networks. In this chapter, we illustrate how the web services COLOMBOS and DISTILLER can be used to identify condition-dependent coexpression modules by exploring compendia of public expression data. COLOMBOS is designed for user-specified query-driven analysis, whereas DISTILLER generates a global regulatory network overview. The user is guided through both web services by means of a case study in which condition-dependent coexpression modules comprising a gene of interest (i.e., "directed") are identified.

Green tissue-specific co-expression of chitinase and oxalate oxidase 4 genes in rice for enhanced resistance against sheath blight.

PubMed

Karmakar, Subhasis; Molla, Kutubuddin Ali; Chanda, Palas K; Sarkar, Sailendra Nath; Datta, Swapan K; Datta, Karabi

2016-01-01

Green tissue-specific simultaneous overexpression of two defense-related genes ( OsCHI11 & OsOXO4 ) in rice leads to significant resistance against sheath blight pathogen ( R. solani ) without distressing any agronomically important traits. Overexpressing two defense-related genes (OsOXO4 and OsCHI11) cloned from rice is effective at enhancing resistance against sheath blight caused by Rhizoctonia solani. These genes were expressed under the control of two different green tissue-specific promoters, viz. maize phosphoenolpyruvate carboxylase gene promoter, PEPC, and rice cis-acting 544-bp DNA element, immediately upstream of the D54O translational start site, P D54O-544 . Putative T0 transgenic rice plants were screened by PCR and integration of genes was confirmed by Southern hybridization of progeny (T1) rice plants. Successful expression of OsOXO4 and OsCHI11 in all tested plants was confirmed. Expression of PR genes increased significantly following pathogen infection in overexpressing transgenic plants. Following infection, transgenic plants exhibited elevated hydrogen peroxide levels, significant changes in activity of ROS scavenging enzymes and reduced membrane damage when compared to their wild-type counterpart. In a Rhizoctonia solani toxin assay, a detached leaf inoculation test and an in vivo plant bioassay, transgenic plants showed a significant reduction in disease symptoms in comparison to non-transgenic control plants. This is the first report of overexpression of two different PR genes driven by two green tissue-specific promoters providing enhanced sheath blight resistance in transgenic rice.

A high-throughput quantitative expression analysis of cancer-related genes in human HepG2 cells in response to limonene, a potential anticancer agent.

PubMed

Hafidh, Rand R; Hussein, Saba Z; MalAllah, Mohammed Q; Abdulamir, Ahmed S; Abu Bakar, Fatimah

2017-11-14

Citrus bioactive compounds, as active anticancer agent, have been under focus by several studies worldwide. However, the underlying genes responsible for the anticancer potential have not been sufficiently highlighted. The current study investigated the gene expression profile of hepatocellular carcinoma, HepG2, cells after treatment with Limonene. The concentration that killed 50% of HepG2 cells was used to elucidate the genetic mechanisms of limonene anticancer activity. The apoptotic induction was detected by flow cytometry and confocal fluorescence microscope. Two of pro-apoptotic events, caspase-3 activation and phosphatidylserine translocation were manifested by confocal fluorescence microscopy. High-throughput real-time PCR was used to profile 1023 cancer-related genes in 16 different gene families related to the cancer development. In comparison to untreated cells, limonene increased the percentage of apoptotic cells up to 89.61%, by flow cytometry, and 48.2% by fluorescence microscopy. There was a significant limonene-driven differential gene expression of HepG2 cells in 15 different gene families. Limonene was shown to significantly (>2log) up-regulate and down-regulate 14 and 59 genes, respectively. The affected gene families, from most to least affected, were apoptosis induction, signal transduction, cancer genes augmentation, alteration in kinases expression, inflammation, DNA damage repair, and cell cycle proteins. The current study reveals that limonene could be a promising, cheap, and effective anticancer compound. The broad spectrum of limonene anticancer activity is interesting for anticancer drug development. Further research is needed to confirm the current findings and to examine the anticancer potential of limonene along with underlying mechanisms on different cell lines. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Simvastatin and Dipentyl Phthalate Display Different Mechanisms of Action but Exhibit Dose Additive Effects on Fetal Testicular Testosterone Production in Sprague Dawley Rats

EPA Science Inventory

Sex differentiation of the male reproductive tract in mammals is driven, in part, by fetal androgen production. In utero exposure to some phthalate esters (PEs) alters fetal Leydig cell differentiation, reducing the expression of several genes associated with steroid synthesis/tr...

Live-cell monitoring of periodic gene expression in synchronous human cells identifies Forkhead genes involved in cell cycle control

PubMed Central

Grant, Gavin D.; Gamsby, Joshua; Martyanov, Viktor; Brooks, Lionel; George, Lacy K.; Mahoney, J. Matthew; Loros, Jennifer J.; Dunlap, Jay C.; Whitfield, Michael L.

2012-01-01

We developed a system to monitor periodic luciferase activity from cell cycle–regulated promoters in synchronous cells. Reporters were driven by a minimal human E2F1 promoter with peak expression in G1/S or a basal promoter with six Forkhead DNA-binding sites with peak expression at G2/M. After cell cycle synchronization, luciferase activity was measured in live cells at 10-min intervals across three to four synchronous cell cycles, allowing unprecedented resolution of cell cycle–regulated gene expression. We used this assay to screen Forkhead transcription factors for control of periodic gene expression. We confirmed a role for FOXM1 and identified two novel cell cycle regulators, FOXJ3 and FOXK1. Knockdown of FOXJ3 and FOXK1 eliminated cell cycle–dependent oscillations and resulted in decreased cell proliferation rates. Analysis of genes regulated by FOXJ3 and FOXK1 showed that FOXJ3 may regulate a network of zinc finger proteins and that FOXK1 binds to the promoter and regulates DHFR, TYMS, GSDMD, and the E2F binding partner TFDP1. Chromatin immunoprecipitation followed by high-throughput sequencing analysis identified 4329 genomic loci bound by FOXK1, 83% of which contained a FOXK1-binding motif. We verified that a subset of these loci are activated by wild-type FOXK1 but not by a FOXK1 (H355A) DNA-binding mutant. PMID:22740631

Identification of the subgenomic promoter of the coat protein gene of cucumber fruit mottle mosaic virus and development of a heterologous expression vector.

PubMed

Rhee, Sun-Ju; Jang, Yoon Jeong; Lee, Gung Pyo

2016-06-01

Heterologous gene expression using plant virus vectors enables research on host-virus interactions and the production of useful proteins, but the host range of plant viruses limits the practical applications of such vectors. Here, we aimed to develop a viral vector based on cucumber fruit mottle mosaic virus (CFMMV), a member of the genus Tobamovirus, whose members infect cucurbits. The subgenomic promoter (SGP) in the coat protein (CP) gene, which was used to drive heterologous expression, was mapped by analyzing deletion mutants from a CaMV 35S promoter-driven infectious CFMMV clone. The region from nucleotides (nt) -55 to +160 relative to the start codon of the open reading frame (ORF) of CP was found to be a fully active promoter, and the region from nt -55 to +100 was identified as the active core promoter. Based on these SGPs, we constructed a cloning site in the CFMMV vector and successfully expressed enhanced green fluorescent protein (EGFP) in Nicotiana benthamiana and watermelon (Citrullus lanatus). Co-inoculation with the P19 suppressor increased EGFP expression and viral replication by blocking degradation of the viral genome. Our CFMMV vector will be useful as an expression vector in cucurbits.

Cone-Specific Promoters for Gene Therapy of Achromatopsia and Other Retinal Diseases

PubMed Central

Ye, Guo-Jie; Budzynski, Ewa; Sonnentag, Peter; Nork, T. Michael; Sheibani, Nader; Gurel, Zafer; Boye, Sanford L.; Peterson, James J.; Boye, Shannon E.; Hauswirth, William W.; Chulay, Jeffrey D.

2016-01-01

Adeno-associated viral (AAV) vectors containing cone-specific promoters have rescued cone photoreceptor function in mouse and dog models of achromatopsia, but cone-specific promoters have not been optimized for use in primates. Using AAV vectors administered by subretinal injection, we evaluated a series of promoters based on the human L-opsin promoter, or a chimeric human cone transducin promoter, for their ability to drive gene expression of green fluorescent protein (GFP) in mice and nonhuman primates. Each of these promoters directed high-level GFP expression in mouse photoreceptors. In primates, subretinal injection of an AAV-GFP vector containing a 1.7-kb L-opsin promoter (PR1.7) achieved strong and specific GFP expression in all cone photoreceptors and was more efficient than a vector containing the 2.1-kb L-opsin promoter that was used in AAV vectors that rescued cone function in mouse and dog models of achromatopsia. A chimeric cone transducin promoter that directed strong GFP expression in mouse and dog cone photoreceptors was unable to drive GFP expression in primate cones. An AAV vector expressing a human CNGB3 gene driven by the PR1.7 promoter rescued cone function in the mouse model of achromatopsia. These results have informed the design of an AAV vector for treatment of patients with achromatopsia. PMID:26603570

Cone-Specific Promoters for Gene Therapy of Achromatopsia and Other Retinal Diseases.

PubMed

Ye, Guo-Jie; Budzynski, Ewa; Sonnentag, Peter; Nork, T Michael; Sheibani, Nader; Gurel, Zafer; Boye, Sanford L; Peterson, James J; Boye, Shannon E; Hauswirth, William W; Chulay, Jeffrey D

2016-01-01

Adeno-associated viral (AAV) vectors containing cone-specific promoters have rescued cone photoreceptor function in mouse and dog models of achromatopsia, but cone-specific promoters have not been optimized for use in primates. Using AAV vectors administered by subretinal injection, we evaluated a series of promoters based on the human L-opsin promoter, or a chimeric human cone transducin promoter, for their ability to drive gene expression of green fluorescent protein (GFP) in mice and nonhuman primates. Each of these promoters directed high-level GFP expression in mouse photoreceptors. In primates, subretinal injection of an AAV-GFP vector containing a 1.7-kb L-opsin promoter (PR1.7) achieved strong and specific GFP expression in all cone photoreceptors and was more efficient than a vector containing the 2.1-kb L-opsin promoter that was used in AAV vectors that rescued cone function in mouse and dog models of achromatopsia. A chimeric cone transducin promoter that directed strong GFP expression in mouse and dog cone photoreceptors was unable to drive GFP expression in primate cones. An AAV vector expressing a human CNGB3 gene driven by the PR1.7 promoter rescued cone function in the mouse model of achromatopsia. These results have informed the design of an AAV vector for treatment of patients with achromatopsia.

Naphthazarin protects against glutamate-induced neuronal death via activation of the Nrf2/ARE pathway

DOE Office of Scientific and Technical Information (OSTI.GOV)

Son, Tae Gen; Kawamoto, Elisa M.; Yu, Qian-Sheng

2013-04-19

Highlights: •Naphthazarin activates the Nrf2/ARE pathway. •Naphthazarin induces Nrf2-driven genes in neurons and astrocytes. •Naphthazarin protects neurons against excitotoxicity. -- Abstract: Nuclear factor E2-related factor 2 (Nrf2)/antioxidant response element (ARE) pathway is an important cellular stress response pathway involved in neuroprotection. We previously screened several natural phytochemicals and identified plumbagin as a novel activator of the Nrf2/ARE pathway that can protect neurons against ischemic injury. Here we extended our studies to natural and synthetic derivatives of plumbagin. We found that 5,8-dimethoxy-1,4-naphthoquinone (naphthazarin) is a potent activator of the Nrf2/ARE pathway, up-regulates the expression of Nrf2-driven genes in primary neuronal andmore » glial cultures, and protects neurons against glutamate-induced excitotoxicity.« less

Sequences required for induction of neurotensin receptor gene expression during neuronal differentiation of N1E-115 neuroblastoma cells.

PubMed

Tavares, D; Tully, K; Dobner, P R

1999-10-15

The promoter region of the mouse high affinity neurotensin receptor (Ntr-1) gene was characterized, and sequences required for expression in neuroblastoma cell lines that express high affinity NT-binding sites were characterized. Me(2)SO-induced neuronal differentiation of N1E-115 neuroblastoma cells increased both the expression of the endogenous Ntr-1 gene and reporter genes driven by NTR-1 promoter sequences by 3-4-fold. Deletion analysis revealed that an 83-base pair promoter region containing the transcriptional start site is required for Me(2)SO activation. Detailed mutational analysis of this region revealed that a CACCC box and the central region of a large GC-rich palindrome are the crucial cis-regulatory elements required for Me(2)SO induction. The CACCC box is bound by at least one factor that is induced upon Me(2)SO treatment of N1E-115 cells. The Me(2)SO effect was found to be both selective and cell type-restricted. Basal expression in the neuroblastoma cell lines required a distinct set of sequences, including an Sp1-like sequence, and a sequence resembling an NGFI-A-binding site; however, a more distal 5' sequence was found to repress basal activity in N1E-115 cells. These results provide evidence that Ntr-1 gene regulation involves both positive and negative regulatory elements located in the 5'-flanking region and that Ntr-1 gene activation involves the coordinate activation or induction of several factors, including a CACCC box binding complex.

Gene expression and metabolism preceding soft scald, a chilling injury of 'Honeycrisp' apple fruit.

PubMed

Leisso, Rachel S; Gapper, Nigel E; Mattheis, James P; Sullivan, Nathanael L; Watkins, Christopher B; Giovannoni, James J; Schaffer, Robert J; Johnston, Jason W; Hanrahan, Ines; Hertog, Maarten L A T M; Nicolaï, Bart M; Rudell, David R

2016-10-12

'Honeycrisp' is an apple cultivar that is susceptible to soft scald, a chilling injury expressed as necrotic patches on the peel. Improved understanding of metabolism associated with the disorder would improve our understanding of soft scald and contribute to developing more effective management strategies for apple storage. It was expected that specific gene expression and specific metabolite levels in the peel would be linked with soft scald risk at harvest and/or specific time points during cold storage. Fruit from nine 'Honeycrisp' apple orchards that would eventually develop different incidences of soft scald between 4 and 8 weeks of cold air storage were used to contrast and determine differential transcriptomic and metabolomic changes during storage. Untargeted metabolic profiling revealed changes in a number of distinct pathways preceding and concurrent with soft scald symptom development, including elevated γ-aminobutryic acid (GABA), 1-hexanol, acylated steryl glycosides, and free p-coumaryl acyl esters. At harvest, levels of sesquiterpenoid and triterpenoid acyl esters were relatively higher in peel of fruit that did not later develop the disorder. RNA-seq driven gene expression profiling highlighted possible involvement of genes and associated metabolic processes with soft scald development. These included elevated expression of genes involved in lipid peroxidation and phenolic metabolism in fruit with soft scald, and isoprenoid/brassinosteroid metabolism in fruit that did not develop soft scald. Expression of other stress-related genes in fruit that developed soft scald included chlorophyll catabolism, cell wall loosening, and lipid transport while superoxide dismutases were up-regulated in fruit that did not develop the disorder. This study delineates the sequential transcriptomic and metabolomic changes preceding soft scald symptom development. Changes were differential depending on susceptibility of fruit to the disorder and could be attributed to key stress related and mediating pathways.

[Molecular mechanisms in sex determination: from gene regulation to pathology].

PubMed

Ravel, C; Chantot-Bastaraud, S; Siffroi, J-P

2004-01-01

Testis determination is the complex process by which the bipotential gonad becomes a normal testis during embryo development. As a consequence, this process leads to sexual differentiation corresponding to the masculinization of both genital track and external genitalia. The whole phenomenon is under genetic control and is particularly driven by the presence of the Y chromosome and by the SRY gene, which acts as the key initiator of the early steps of testis determination. However, many other autosomal genes, present in both males and females, are expressed during testis formation in a gene activation pathway, which is far to be totally elucidated. All these genes act in a dosage-sensitive manner by which quantitative gene abnormalities, due to chromosomal deletions, duplications or mosaicism, may lead to testis determination failure and sex reversal.

Investigating the effect of carbon source on rabies virus glycoprotein production in Pichia pastoris by a transcriptomic approach.

PubMed

Ben Azoun, Safa; Kallel, Héla

2017-08-01

Several factors affect protein expression in Pichia pastoris, one among them is the carbon source. In this work, we studied the effect of this factor on the expression level of rabies virus glycoprotein (RABV-G) in two recombinant clones harboring seven copies of the gene of interest. The expression was driven either by the constitutive glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter or the inducible alcohol oxidase1 (AOX1) promoter. Clones were compared in terms of cell physiology and carbon source metabolism. The transcription levels of 16 key genes involved in the central metabolic pathway, the methanol catabolism, and the oxidative stress were investigated in both clones. Cell size, as a parameter reflecting cell physiological changes, was also monitored. Our results showed that when glucose was used as the sole carbon source, large cells were obtained. Transcript levels of the genes of the central metabolic pathway were also upregulated, whereas antioxidative gene transcript levels were low. By contrast, the use of methanol as a carbon source generated small cells and a shift in carbon metabolism toward the dissimilatory pathway by the upregulation of formaldehyde dehydrogenase gene and the downregulation of those of the central metabolic. These observations are in favor of the use of glucose to enhance the expression of RABV-G in P. pastoris. © 2017 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

A quantitative proteomics approach identifies ETV6 and IKZF1 as new regulators of an ERG-driven transcriptional network

PubMed Central

Unnikrishnan, Ashwin; Guan, Yi F.; Huang, Yizhou; Beck, Dominik; Thoms, Julie A. I.; Peirs, Sofie; Knezevic, Kathy; Ma, Shiyong; de Walle, Inge V.; de Jong, Ineke; Ali, Zara; Zhong, Ling; Raftery, Mark J.; Taghon, Tom; Larsson, Jonas; MacKenzie, Karen L.; Van Vlierberghe, Pieter; Wong, Jason W. H.; Pimanda, John E.

2016-01-01

Aberrant stem cell-like gene regulatory networks are a feature of leukaemogenesis. The ETS-related gene (ERG), an important regulator of normal haematopoiesis, is also highly expressed in T-ALL and acute myeloid leukaemia (AML). However, the transcriptional regulation of ERG in leukaemic cells remains poorly understood. In order to discover transcriptional regulators of ERG, we employed a quantitative mass spectrometry-based method to identify factors binding the 321 bp ERG +85 stem cell enhancer region in MOLT-4 T-ALL and KG-1 AML cells. Using this approach, we identified a number of known binders of the +85 enhancer in leukaemic cells along with previously unknown binders, including ETV6 and IKZF1. We confirmed that ETV6 and IKZF1 were also bound at the +85 enhancer in both leukaemic cells and in healthy human CD34+ haematopoietic stem and progenitor cells. Knockdown experiments confirmed that ETV6 and IKZF1 are transcriptional regulators not just of ERG, but also of a number of genes regulated by a densely interconnected network of seven transcription factors. At last, we show that ETV6 and IKZF1 expression levels are positively correlated with expression of a number of heptad genes in AML and high expression of all nine genes confers poorer overall prognosis. PMID:27604872

Antagonistic control of a dual-input mammalian gene switch by food additives.

PubMed

Xie, Mingqi; Ye, Haifeng; Hamri, Ghislaine Charpin-El; Fussenegger, Martin

2014-08-01

Synthetic biology has significantly advanced the design of mammalian trigger-inducible transgene-control devices that are able to programme complex cellular behaviour. Fruit-based benzoate derivatives licensed as food additives, such as flavours (e.g. vanillate) and preservatives (e.g. benzoate), are a particularly attractive class of trigger compounds for orthogonal mammalian transgene control devices because of their innocuousness, physiological compatibility and simple oral administration. Capitalizing on the genetic componentry of the soil bacterium Comamonas testosteroni, which has evolved to catabolize a variety of aromatic compounds, we have designed different mammalian gene expression systems that could be induced and repressed by the food additives benzoate and vanillate. When implanting designer cells engineered for gene switch-driven expression of the human placental secreted alkaline phosphatase (SEAP) into mice, blood SEAP levels of treated animals directly correlated with a benzoate-enriched drinking programme. Additionally, the benzoate-/vanillate-responsive device was compatible with other transgene control systems and could be assembled into higher-order control networks providing expression dynamics reminiscent of a lap-timing stopwatch. Designer gene switches using licensed food additives as trigger compounds to achieve antagonistic dual-input expression profiles and provide novel control topologies and regulation dynamics may advance future gene- and cell-based therapies. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

Rapid Evolution of Ovarian-Biased Genes in the Yellow Fever Mosquito (Aedes aegypti).

PubMed

Whittle, Carrie A; Extavour, Cassandra G

2017-08-01

Males and females exhibit highly dimorphic phenotypes, particularly in their gonads, which is believed to be driven largely by differential gene expression. Typically, the protein sequences of genes upregulated in males, or male-biased genes, evolve rapidly as compared to female-biased and unbiased genes. To date, the specific study of gonad-biased genes remains uncommon in metazoans. Here, we identified and studied a total of 2927, 2013, and 4449 coding sequences (CDS) with ovary-biased, testis-biased, and unbiased expression, respectively, in the yellow fever mosquito Aedes aegypti The results showed that ovary-biased and unbiased CDS had higher nonsynonymous to synonymous substitution rates (dN/dS) and lower optimal codon usage (those codons that promote efficient translation) than testis-biased genes. Further, we observed higher dN/dS in ovary-biased genes than in testis-biased genes, even for genes coexpressed in nonsexual (embryo) tissues. Ovary-specific genes evolved exceptionally fast, as compared to testis- or embryo-specific genes, and exhibited higher frequency of positive selection. Genes with ovary expression were preferentially involved in olfactory binding and reception. We hypothesize that at least two potential mechanisms could explain rapid evolution of ovary-biased genes in this mosquito: (1) the evolutionary rate of ovary-biased genes may be accelerated by sexual selection (including female-female competition or male-mate choice) affecting olfactory genes during female swarming by males, and/or by adaptive evolution of olfactory signaling within the female reproductive system ( e.g. , sperm-ovary signaling); and/or (2) testis-biased genes may exhibit decelerated evolutionary rates due to the formation of mating plugs in the female after copulation, which limits male-male sperm competition. Copyright © 2017 by the Genetics Society of America.

MET expression and amplification in patients with localized gastric cancer

PubMed Central

Janjigian, Yelena Y.; Tang, Laura H.; Coit, Daniel G.; Kelsen, David P.; Francone, Todd D.; Weiser, Martin R.; Jhanwar, Suresh C.; Shah, Manish A.

2013-01-01

Background MET, the receptor for hepatocyte growth factor has been proposed as a therapeutic target in gastric cancer. This study assessed the incidence of MET expression and gene amplification in tumors of Western patients with gastric cancer. Methods Tumor specimens from patients enrolled on a preoperative chemotherapy study (NCI 5700) were examined for presence of MET gene amplification by fluorescence in situ hybridization (FISH), MET mRNA expression by quantitative polymerase chain reaction, MET overexpression by immunohistochemistry (IHC), and for evidence of MET pathway activation by p-MET IHC. Results Although high-level of MET protein and mRNA were commonly encountered (in 63% and 50% of resected tumor specimens, respectively), none of these tumors had MET gene amplification by FISH, and only 6.6% had evidence of MET tyrosine kinase activity by p-MET IHC. Conclusions In this cohort of patients with localized gastric cancer, the presence of high MET protein and RNA expression does not correlate with MET gene amplification or pathway activation as evidenced by the absence of amplification by FISH and negative p-MET IHC analysis. Impact This paper demonstrates a lack of MET amplification and pathway activation in a cohort of 38 patients with localized gastric cancer, suggesting that MET-driven gastric cancers are relatively rare in Western patients. PMID:21393565

Transgene expression patterns indicate that spaceflight affects stress signal perception and transduction in arabidopsis.

PubMed

Paul, A L; Daugherty, C J; Bihn, E A; Chapman, D K; Norwood, K L; Ferl, R J

2001-06-01

The use of plants as integral components of life support systems remains a cornerstone of strategies for long-term human habitation of space and extraterrestrial colonization. Spaceflight experiments over the past few decades have refined the hardware required to grow plants in low-earth orbit and have illuminated fundamental issues regarding spaceflight effects on plant growth and development. Potential incipient hypoxia, resulting from the lack of convection-driven gas movement, has emerged as a possible major impact of microgravity. We developed transgenic Arabidopsis containing the alcohol dehydrogenase (Adh) gene promoter linked to the beta-glucuronidase (GUS) reporter gene to address specifically the possibility that spaceflight induces the plant hypoxia response and to assess whether any spaceflight response was similar to control terrestrial hypoxia-induced gene expression patterns. The staining patterns resulting from a 5-d mission on the orbiter Columbia during mission STS-93 indicate that the Adh/GUS reporter gene was activated in roots during the flight. However, the patterns of expression were not identical to terrestrial control inductions. Moreover, although terrestrial hypoxia induces Adh/GUS expression in the shoot apex, no apex staining was observed in the spaceflight plants. This indicates that either the normal hypoxia response signaling is impaired in spaceflight or that spaceflight inappropriately induces Adh/GUS activity for reasons other than hypoxia.

An interferon regulatory factor binding site in the U5 region of the bovine leukemia virus long terminal repeat stimulates Tax-independent gene expression.

PubMed

Kiermer, V; Van Lint, C; Briclet, D; Vanhulle, C; Kettmann, R; Verdin, E; Burny, A; Droogmans, L

1998-07-01

Bovine leukemia virus (BLV) replication is controlled by both cis- and trans-acting elements. The virus-encoded transactivator, Tax, is necessary for efficient transcription from the BLV promoter, although it is not present during the early stages of infection. Therefore, sequences that control Tax-independent transcription must play an important role in the initiation of viral gene expression. This study demonstrates that the R-U5 sequence of BLV stimulates Tax-independent reporter gene expression directed by the BLV promoter. R-U5 was also stimulatory when inserted immediately downstream from the transcription initiation site of a heterologous promoter. Progressive deletion analysis of this region revealed that a 46-bp element corresponding to the 5' half of U5 is principally responsible for the stimulation. This element exhibited enhancer activity when inserted upstream or downstream from the herpes simplex virus thymidine kinase promoter. This enhancer contains a binding site for the interferon regulatory factors IRF-1 and IRF-2. A 3-bp mutation that destroys the IRF recognition site caused a twofold decrease in Tax-independent BLV long terminal repeat-driven gene expression. These observations suggest that the IRF binding site in the U5 region of BLV plays a role in the initiation of virus replication.

Transgene expression patterns indicate that spaceflight affects stress signal perception and transduction in arabidopsis

NASA Technical Reports Server (NTRS)

Paul, A. L.; Daugherty, C. J.; Bihn, E. A.; Chapman, D. K.; Norwood, K. L.; Ferl, R. J.

2001-01-01

The use of plants as integral components of life support systems remains a cornerstone of strategies for long-term human habitation of space and extraterrestrial colonization. Spaceflight experiments over the past few decades have refined the hardware required to grow plants in low-earth orbit and have illuminated fundamental issues regarding spaceflight effects on plant growth and development. Potential incipient hypoxia, resulting from the lack of convection-driven gas movement, has emerged as a possible major impact of microgravity. We developed transgenic Arabidopsis containing the alcohol dehydrogenase (Adh) gene promoter linked to the beta-glucuronidase (GUS) reporter gene to address specifically the possibility that spaceflight induces the plant hypoxia response and to assess whether any spaceflight response was similar to control terrestrial hypoxia-induced gene expression patterns. The staining patterns resulting from a 5-d mission on the orbiter Columbia during mission STS-93 indicate that the Adh/GUS reporter gene was activated in roots during the flight. However, the patterns of expression were not identical to terrestrial control inductions. Moreover, although terrestrial hypoxia induces Adh/GUS expression in the shoot apex, no apex staining was observed in the spaceflight plants. This indicates that either the normal hypoxia response signaling is impaired in spaceflight or that spaceflight inappropriately induces Adh/GUS activity for reasons other than hypoxia.

Developmental switching in Physarum polycephalum: Petri net analysis of single cell trajectories of gene expression indicates responsiveness and genetic plasticity of the Waddington quasipotential landscape

NASA Astrophysics Data System (ADS)

Werthmann, Britta; Marwan, Wolfgang

2017-11-01

The developmental switch to sporulation in Physarum polycephalum is a phytochrome-mediated far-red light-induced cell fate decision that synchronously encompasses the entire multinucleate plasmodial cell and is associated with extensive reprogramming of the transcriptome. By repeatedly taking samples of single cells after delivery of a light stimulus pulse, we analysed differential gene expression in two mutant strains and in a heterokaryon of the two strains all of which display a different propensity for making the cell fate decision. Multidimensional scaling of the gene expression data revealed individually different single cell trajectories eventually leading to sporulation. Characterization of the trajectories as walks through states of gene expression discretized by hierarchical clustering allowed the reconstruction of Petri nets that model and predict the observed behavior. Structural analyses of the Petri nets indicated stimulus- and genotype-dependence of both, single cell trajectories and of the quasipotential landscape through which these trajectories are taken. The Petri net-based approach to the analysis and decomposition of complex cellular responses and of complex mutant phenotypes may provide a scaffold for the data-driven reconstruction of causal molecular mechanisms that shape the topology of the quasipotential landscape.

Epigenetic dysregulation of the dopamine system in diet-induced obesity.

PubMed

Vucetic, Zivjena; Carlin, Jesse Lea; Totoki, Kathy; Reyes, Teresa M

2012-03-01

Chronic intake of high-fat (HF) diet is known to alter brain neurotransmitter systems that participate in the central regulation of food intake. Dopamine (DA) system changes in response to HF diet have been observed in the hypothalamus, important in the homeostatic control of food intake, as well as within the central reward circuitry [ventral tegmental area (VTA), nucleus accumbens (NAc), and pre-frontal cortex (PFC)], critical for coding the rewarding properties of palatable food and important in hedonically driven feeding behavior. Using a mouse model of diet-induced obesity (DIO), significant alterations in the expression of DA-related genes were documented in adult animals, and the general pattern of gene expression changes was opposite within the hypothalamus versus the reward circuitry (increased vs. decreased, respectively). Differential DNA methylation was identified within the promoter regions of tyrosine hydroxylase (TH) and dopamine transporter (DAT), and the pattern of this response was consistent with the pattern of gene expression. Behaviors consistent with increased hypothalamic DA and decreased reward circuitry DA were observed. These data identify differential DNA methylation as an epigenetic mechanism linking the chronic intake of HF diet with altered DA-related gene expression, and this response varies by brain region and DNA sequence. © 2012 The Authors. Journal of Neurochemistry © 2012 International Society for Neurochemistry.

Evidence of translation efficiency adaptation of the coding regions of the bacteriophage lambda.

PubMed

Goz, Eli; Mioduser, Oriah; Diament, Alon; Tuller, Tamir

2017-08-01

Deciphering the way gene expression regulatory aspects are encoded in viral genomes is a challenging mission with ramifications related to all biomedical disciplines. Here, we aimed to understand how the evolution shapes the bacteriophage lambda genes by performing a high resolution analysis of ribosomal profiling data and gene expression related synonymous/silent information encoded in bacteriophage coding regions.We demonstrated evidence of selection for distinct compositions of synonymous codons in early and late viral genes related to the adaptation of translation efficiency to different bacteriophage developmental stages. Specifically, we showed that evolution of viral coding regions is driven, among others, by selection for codons with higher decoding rates; during the initial/progressive stages of infection the decoding rates in early/late genes were found to be superior to those in late/early genes, respectively. Moreover, we argued that selection for translation efficiency could be partially explained by adaptation to Escherichia coli tRNA pool and the fact that it can change during the bacteriophage life cycle.An analysis of additional aspects related to the expression of viral genes, such as mRNA folding and more complex/longer regulatory signals in the coding regions, is also reported. The reported conclusions are likely to be relevant also to additional viruses. © The Author 2017. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.

NRASG12V oncogene facilitates self-renewal in a murine model of acute myelogenous leukemia

PubMed Central

LaRue, Rebecca S.; Nguyen, Hanh T.; Sachs, Karen; Noble, Klara E.; Mohd Hassan, Nurul Azyan; Diaz-Flores, Ernesto; Rathe, Susan K.; Sarver, Aaron L.; Bendall, Sean C.; Ha, Ngoc A.; Diers, Miechaleen D.; Nolan, Garry P.; Shannon, Kevin M.; Largaespada, David A.

2014-01-01

Mutant RAS oncoproteins activate signaling molecules that drive oncogenesis in multiple human tumors including acute myelogenous leukemia (AML). However, the specific functions of these pathways in AML are unclear, thwarting the rational application of targeted therapeutics. To elucidate the downstream functions of activated NRAS in AML, we used a murine model that harbors Mll-AF9 and a tetracycline-repressible, activated NRAS (NRASG12V). Using computational approaches to explore our gene-expression data sets, we found that NRASG12V enforced the leukemia self-renewal gene-expression signature and was required to maintain an MLL-AF9– and Myb-dependent leukemia self-renewal gene-expression program. NRASG12V was required for leukemia self-renewal independent of its effects on growth and survival. Analysis of the gene-expression patterns of leukemic subpopulations revealed that the NRASG12V-mediated leukemia self-renewal signature is preferentially expressed in the leukemia stem cell–enriched subpopulation. In a multiplexed analysis of RAS-dependent signaling, Mac-1Low cells, which harbor leukemia stem cells, were preferentially sensitive to NRASG12V withdrawal. NRASG12V maintained leukemia self-renewal through mTOR and MEK pathway activation, implicating these pathways as potential targets for cancer stem cell–specific therapies. Together, these experimental results define a RAS oncogene–driven function that is critical for leukemia maintenance and represents a novel mechanism of oncogene addiction. PMID:25316678

Convergent evolution at the pathway level: predictable regulatory changes during flower color transitions.

PubMed

Larter, Maximilian; Dunbar-Wallis, Amy; Berardi, Andrea E; Smith, Stacey D

2018-06-07

The predictability of evolution, or whether lineages repeatedly follow the same evolutionary trajectories during phenotypic convergence remains an open question of evolutionary biology. In this study, we investigate evolutionary convergence at the biochemical pathway level and test the predictability of evolution using floral anthocyanin pigmentation, a trait with a well-understood genetic and regulatory basis. We reconstructed the evolution of floral anthocyanin content across 28 species of the Andean clade Iochrominae (Solanaceae) and investigated how shifts in pigmentation are related to changes in expression of 7 key anthocyanin pathway genes. We used phylogenetic multivariate analysis of gene expression to test for phenotypic and developmental convergence at a macroevolutionary scale. Our results show that the four independent losses of the ancestral pigment delphinidin involved convergent losses of expression of the three late pathway genes (F3'5'h, Dfr and Ans). Transitions between pigment types affecting floral hue (e.g. blue to red) involve changes to the expression of branching genes F3'h and F3'5'h, while the expression levels of early steps of the pathway are strongly conserved in all species. These patterns support the idea that the macroevolution of floral pigmentation follows predictable evolutionary trajectories to reach convergent phenotype space, repeatedly involving regulatory changes. This is likely driven by constraints at the pathway level, such as pleiotropy and regulatory structure.

Amelioration of murine beta-thalassemia through drug selection of hematopoietic stem cells transduced with a lentiviral vector encoding both gamma-globin and the MGMT drug-resistance gene.

PubMed

Zhao, Huifen; Pestina, Tamara I; Nasimuzzaman, Md; Mehta, Perdeep; Hargrove, Phillip W; Persons, Derek A

2009-06-04

Correction of murine models of beta-thalassemia has been achieved through high-level globin lentiviral vector gene transfer into mouse hematopoietic stem cells (HSCs). However, transduction of human HSCs is less robust and may be inadequate to achieve therapeutic levels of genetically modified erythroid cells. We therefore developed a double gene lentiviral vector encoding both human gamma-globin under the transcriptional control of erythroid regulatory elements and methylguanine methyltransferase (MGMT), driven by a constitutive cellular promoter. MGMT expression provides cellular resistance to alkylator drugs, which can be administered to kill residual untransduced, diseased HSCs, whereas transduced cells are protected. Mice transplanted with beta-thalassemic HSCs transduced with a gamma-globin/MGMT vector initially had subtherapeutic levels of red cells expressing gamma-globin. To enrich gamma-globin-expressing cells, transplanted mice were treated with the alkylator agent 1,3-bis-chloroethyl-1-nitrosourea. This resulted in significant increases in the number of gamma-globin-expressing red cells and the amount of fetal hemoglobin, leading to resolution of anemia. Selection of transduced HSCs was also obtained when cells were drug-treated before transplantation. Mice that received these cells demonstrated reconstitution with therapeutic levels of gamma-globin-expressing cells. These data suggest that MGMT-based drug selection holds promise as a modality to improve gene therapy for beta-thalassemia.

Transcriptome responses to temperature, water availability and photoperiod are conserved among mature trees of two divergent Douglas-fir provenances from a coastal and an interior habitat.

PubMed

Hess, Moritz; Wildhagen, Henning; Junker, Laura Verena; Ensminger, Ingo

2016-08-26

Local adaptation and phenotypic plasticity are important components of plant responses to variations in environmental conditions. While local adaptation has been widely studied in trees, little is known about plasticity of gene expression in adult trees in response to ever changing environmental conditions in natural habitats. Here we investigate plasticity of gene expression in needle tissue between two Douglas-fir provenances represented by 25 adult trees using deep RNA sequencing (RNA-Seq). Using linear mixed models we investigated the effect of temperature, soil water availability and photoperiod on the abundance of 59189 detected transcripts. Expression of more than 80 % of all identified transcripts revealed a response to variations in environmental conditions in the field. GO term overrepresentation analysis revealed gene expression responses to temperature, soil water availability and photoperiod that are highly conserved among many plant taxa. However, expression differences between the two Douglas-fir provenances were rather small compared to the expression differences observed between individual trees. Although the effect of environment on global transcript expression was high, the observed genotype by environment (GxE) interaction of gene expression was surprisingly low, since only 21 of all detected transcripts showed a GxE interaction. The majority of the transcriptome responses in plant leaf tissue is driven by variations in environmental conditions. The small variation between individuals and populations suggests strong conservation of this response within Douglas-fir. Therefore we conclude that plastic transcriptome responses to variations in environmental conditions are only weakly affected by local adaptation in Douglas-fir.

Pancreatic Transduction by Helper-Dependent Adenoviral Vectors via Intraductal Delivery

PubMed Central

Morró, Meritxell; Teichenne, Joan; Jimenez, Veronica; Kratzer, Ramona; Marletta, Serena; Maggioni, Luca; Mallol, Cristina; Ruberte, Jesus; Kochanek, Stefan; Bosch, Fatima

2014-01-01

Abstract Pancreatic gene transfer could be useful to treat several diseases, such as diabetes mellitus, cystic fibrosis, chronic pancreatitis, or pancreatic cancer. Helper-dependent adenoviral vectors (HDAds) are promising tools for gene therapy because of their large cloning capacity, high levels of transgene expression, and long-term persistence in immunocompetent animals. Nevertheless, the ability of HDAds to transduce the pancreas in vivo has not been investigated yet. Here, we have generated HDAds carrying pancreas-specific expression cassettes, that is, driven either by the elastase or insulin promoter, using a novel and convenient plasmid family and homologous recombination in bacteria. These HDAds were delivered to the pancreas of immunocompetent mice via intrapancreatic duct injection. HDAds, encoding a CMV-GFP reporter cassette, were able to transduce acinar and islet cells, but transgene expression was lost 15 days postinjection in correlation with severe lymphocytic infiltration. When HDAds encoding GFP under the control of the specific elastase promoter were used, expression was detected in acinar cells, but similarly, the expression almost disappeared 30 days postinjection and lymphocytic infiltration was also observed. In contrast, long-term transgene expression (>8 months) was achieved with HDAds carrying the insulin promoter and the secretable alkaline phosphatase as the reporter gene. Notably, transduction of the liver, the preferred target for adenovirus, was minimal by this route of delivery. These data indicate that HDAds could be used for pancreatic gene therapy but that selection of the expression cassette is of critical importance to achieve long-term expression of the transgene in this tissue. PMID:25046147

Complete regression of human malignant mesothelioma xenografts following local injection of midkine promoter-driven oncolytic adenovirus

PubMed Central

Kubo, Shuji; Kawasaki, Yoshiko; Yamaoka, Norie; Tagawa, Masatoshi; Kasahara, Noriyuki; Terada, Nobuyuki; Okamura, Haruki

2010-01-01

Background Malignant mesothelioma is a highly aggressive tumor with poor prognosis. Conventional therapies for mesothelioma are generally non-curative, and new treatment paradigms are urgently needed. We hypothesized that the tumor-specific midkine (Mdk) promoter could confer transcriptional targeting to oncolytic adenoviruses for effective treatment of malignant mesothelioma. Methods We analyzed Mdk expression by quantitative RT-PCR in six human mesothelioma cell lines, and tested Mdk promoter activity by luciferase reporter assay. Based on these data, we constructed a replication-selective oncolytic adenovirus, designated AdMdk-E1-iresTK, which contains an Mdk promoter-driven adenoviral E1 gene and HSV-thymidine kinase (TK) suicide gene, and CMV promoter-driven green fluorescent protein (GFP) marker gene. Selectivity of viral replication and cytolysis were characterized in normal vs. mesothelioma cells in vitro, and intratumoral spread and antitumor efficacy were investigated in vivo. Results Mdk promoter activity was restricted in normal cells, but highly activated in mesothelioma cell lines. AdMdk-E1-iresTK was seen to efficiently replicate, produce viral progeny, and spread in multiple mesothelioma cell lines. Lytic spread of AdMdk-E1-iresTK mediated efficient killing of these mesothelioma cells, and its in vitro cytocidal effect was significantly enhanced by treatment with the prodrug, ganciclovir. Intratumoral injection of AdMdk-E1-iresTK caused complete regression of MESO4 and MSTO human mesothelioma xenografts in athymic mice. In vivo fluorescence imaging demonstrated intratumoral spread of AdMdk-E1-iresTK-derived signals, which vanished after tumor eradication. Conclusions These data indicate that transcriptional targeting of viral replication by the Mdk promoter represents a promising general strategy for oncolytic virotherapy of cancers with upregulated Mdk expression, including malignant mesothelioma. PMID:20635326

Anti-apoptotic A1 is not essential for lymphoma development in Eµ-Myc mice but helps sustain transplanted Eµ-Myc tumour cells.

PubMed

Mensink, Mark; Anstee, Natasha S; Robati, Mikara; Schenk, Robyn L; Herold, Marco J; Cory, Suzanne; Vandenberg, Cassandra J

2018-03-01

The transcription factor c-MYC regulates a multiplicity of genes involved in cellular growth, proliferation, metabolism and DNA damage response and its overexpression is a hallmark of many tumours. Since MYC promotes apoptosis under conditions of stress, such as limited availability of nutrients or cytokines, MYC-driven cells are very much dependent on signals that inhibit cell death. Stress signals trigger apoptosis via the pathway regulated by opposing fractions of the BCL-2 protein family and previous genetic studies have shown that the development of B lymphoid tumours in Eµ-Myc mice is critically dependent on expression of pro-survival BCL-2 relatives MCL-1, BCL-W and, to a lesser extent, BCL-X L , but not BCL-2 itself, and that sustained growth of these lymphomas is dependent on MCL-1. Using recently developed mice that lack expression of all three functional pro-survival A1 genes, we show here that the kinetics of lymphoma development in Eµ-Myc mice and the competitive repopulation capacity of Eµ-Myc haemopoietic stem and progenitor cells is unaffected by the absence of A1. However, conditional loss of a single remaining functional A1 gene from transplanted A1-a -/- A1-b fl/fl A1-c -/- Eµ-Myc lymphomas slowed their expansion, significantly extending the life of the transplant recipients. Thus, A1 contributes to the survival of malignant Eµ-Myc-driven B lymphoid cells. These results strengthen the case for BFL-1, the human homologue of A1, being a valid target for drug development for MYC-driven tumours.

Rescue of infectious rift valley fever virus entirely from cDNA, analysis of virus lacking the NSs gene, and expression of a foreign gene.

PubMed

Ikegami, Tetsuro; Won, Sungyong; Peters, C J; Makino, Shinji

2006-03-01

Rift Valley fever virus (RVFV) (genus Phlebovirus, family Bunyaviridae) has a tripartite negative-strand genome, causes a mosquito-borne disease that is endemic in sub-Saharan African countries and that also causes large epidemics among humans and livestock. Furthermore, it is a bioterrorist threat and poses a risk for introduction to other areas. In spite of its danger, neither veterinary nor human vaccines are available. We established a T7 RNA polymerase-driven reverse genetics system to rescue infectious clones of RVFV MP-12 strain entirely from cDNA, the first for any phlebovirus. Expression of viral structural proteins from the protein expression plasmids was not required for virus rescue, whereas NSs protein expression abolished virus rescue. Mutants of MP-12 partially or completely lacking the NSs open reading frame were viable. These NSs deletion mutants replicated efficiently in Vero and 293 cells, but not in MRC-5 cells. In the latter cell line, accumulation of beta interferon mRNA occurred after infection by these NSs deletion mutants, but not after infection by MP-12. The NSs deletion mutants formed larger plaques than MP-12 did in Vero E6 cells and failed to shut off host protein synthesis in Vero cells. An MP-12 mutant carrying a luciferase gene in place of the NSs gene replicated as efficiently as MP-12 did, produced enzymatically active luciferase during replication, and stably retained the luciferase gene after 10 virus passages, representing the first demonstration of foreign gene expression in any bunyavirus. This reverse genetics system can be used to study the molecular virology of RVFV, assess current vaccine candidates, produce new vaccines, and incorporate marker genes into animal vaccines.

A tetO Toolkit To Alter Expression of Genes in Saccharomyces cerevisiae.

PubMed

Cuperus, Josh T; Lo, Russell S; Shumaker, Lucia; Proctor, Julia; Fields, Stanley

2015-07-17

Strategies to optimize a metabolic pathway often involve building a large collection of strains, each containing different versions of sequences that regulate the expression of pathway genes. Here, we develop reagents and methods to carry out this process at high efficiency in the yeast Saccharomyces cerevisiae. We identify variants of the Escherichia coli tet operator (tetO) sequence that bind a TetR-VP16 activator with differential affinity and therefore result in different TetR-VP16 activator-driven expression. By recombining these variants upstream of the genes of a pathway, we generate unique combinations of expression levels. Here, we built a tetO toolkit, which includes the I-OnuI homing endonuclease to create double-strand breaks, which increases homologous recombination by 10(5); a plasmid carrying six variant tetO sequences flanked by I-OnuI sites, uncoupling transformation and recombination steps; an S. cerevisiae-optimized TetR-VP16 activator; and a vector to integrate constructs into the yeast genome. We introduce into the S. cerevisiae genome the three crt genes from Erwinia herbicola required for yeast to synthesize lycopene and carry out the recombination process to produce a population of cells with permutations of tetO variants regulating the three genes. We identify 0.7% of this population as making detectable lycopene, of which the vast majority have undergone recombination at all three crt genes. We estimate a rate of ∼20% recombination per targeted site, much higher than that obtained in other studies. Application of this toolkit to medically or industrially important end products could reduce the time and labor required to optimize the expression of a set of metabolic genes.

An agmatine-inducible system for the expression of recombinant proteins in Enterococcus faecalis.

PubMed

Linares, Daniel M; Perez, Marta; Ladero, Victor; Del Rio, Beatriz; Redruello, Begoña; Martin, M Cruz; Fernandez, María; Alvarez, Miguel A

2014-12-04

Scientific interest in Enterococcus faecalis has increased greatly over recent decades. Some strains are involved in food fermentation and offer health benefits, whereas others are vancomycin-resistant and cause infections that are difficult to treat. The limited availability of vectors able to express cloned genes efficiently in E. faecalis has hindered biotechnological studies on the bacterium's regulatory and pathogenicity-related genes. The agmatine deiminase (AGDI) pathway of E. faecalis, involved in the conversion of agmatine into putrescine, is driven by a response inducer gene aguR. This study describes that the exposure to the induction factor (agmatine) results in the transcription of genes under the control of the aguB promoter, including the aguBDAC operon. A novel E. faecalis expression vector, named pAGEnt, combining the aguR inducer gene and the aguB promoter followed by a cloning site and a stop codon was constructed. pAGEnt was designed for the overexpression and purification of a protein fused to a 10-amino-acid His-tag at the C-terminus. The use of GFP as a reporter of gene expression in E. faecalis revealed that under induction with 60 mM agmatine, fluorescence reached 40 arbitrary units compared to 0 in uninduced cells. pAGEnt vector can be used for the overexpression of recombinant proteins under the induction of agmatine in E. faecalis, with a close correlation between agmatine concentration and fluorescence when GFP was used as reporter.

CRHR1 promoter hypomethylation: An epigenetic readout of panic disorder?

PubMed

Schartner, Christoph; Ziegler, Christiane; Schiele, Miriam A; Kollert, Leonie; Weber, Heike; Zwanzger, Peter; Arolt, Volker; Pauli, Paul; Deckert, Jürgen; Reif, Andreas; Domschke, Katharina

2017-04-01

The corticotropin releasing hormone receptor 1 (CRHR1) is crucially involved in the hypothalamic-pituitary-adrenal axis and thus a major regulator of the stress response. CRHR1 gene variation is associated with several mental disorders including anxiety disorders. Studies in rodents have demonstrated epigenetic regulation of CRHR1 gene expression to moderate response to stressful environment. In the present study, we investigated CRHR1 promoter methylation for the first time regarding its role in panic disorder applying a case-control approach (N=131 patients, N=131 controls). In an independent sample of healthy volunteers (N=255), CRHR1 methylation was additionally analyzed for association with the Beck Anxiety Inventory (BAI) score as a dimensional panic-related intermediate phenotype. The functional relevance of altered CRHR1 promoter methylation was investigated by means of luciferase-based reporter gene assays. In panic disorder patients, a significantly decreased CRHR1 methylation was discerned (p<0.001). Accordingly, healthy controls with high BAI scores showed significantly decreased CRHR1 methylation. Functional analyses revealed an increased gene expression in presence of unmethylated as compared to methylated pCpGl_CRHR1 reporter gene vectors. The present study identified a potential role of CRHR1 hypomethylation - conferring increased CRHR1 expression - in panic disorder and a related dimensional intermediate phenotype. This up-regulation of CRHR1 gene expression driven by de-methylation might constitute a link between the stress response and panic disorder risk. Copyright © 2017 Elsevier B.V. and ECNP. All rights reserved.

CHD7 Expression Predicts Survival Outcomes in Patients with Resected Pancreatic Cancer

PubMed Central

Colbert, Lauren E.; Petrova, Aleksandra V.; Fisher, Sarah B.; Pantazides, Brooke G.; Madden, Matthew Z.; Hardy, Claire W.; Warren, Matthew D.; Pan, Yunfeng; Nagaraju, Ganji P.; Liu, Elaine A.; Saka, Burcu; Hall, William A.; Shelton, Joseph W.; Gandhi, Khanjan; Pauly, Rini; Kowalski, Jeanne; Kooby, David A.; El-Rayes, Bassel F.; Staley, Charles A.; Adsay, N. Volkan; Curran, Walter J.; Landry, Jerome C.; Maithel, Shishir K.; Yu, David S.

2014-01-01

Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease with poor outcomes with current therapies. Gemcitabine is the primary adjuvant drug used clinically, but its effectiveness is limited. In this study, our objective was to utilize a rationale-driven approach to identify novel biomarkers for outcome in patients with early-stage resected PDAC treated with adjuvant gemcitabine. Using a synthetic lethal screen in human PDAC cells, we identified 93 genes including 55 genes linked to DNA damage responses (DDR) that demonstrated gemcitabine sensitization when silenced, including CHD7 which functions in chromatin remodeling. CHD7 depletion sensitized PDAC cells to gemcitabine and delayed their growth in tumor xenografts. Moreover, CHD7 silencing impaired ATR-dependent phosphorylation of CHK1 and increased DNA damage induced by gemcitabine. CHD7 was dysregulated, ranking above the 90th percentile in differential expression in a panel of PDAC clinical specimens, highlighting its potential as a biomarker. Immunohistochemical analysis of specimens from 59 resected PDAC patients receiving adjuvant gemcitabine revealed that low CHD7 expression was associated with increased recurrence-free survival (RFS) and overall survival (OS), in univariate and multivariate analyses. Notably, CHD7 expression was not associated with RFS or OS for patients not receiving gemcitabine. Thus, low CHD7 expression was correlated selectively with gemcitabine sensitivity in this patient population. These results supported our rationale-driven strategy to exploit dysregulated DDR pathways in PDAC to identify genetic determinants of gemcitabine sensitivity, identifying CHD7 as a novel biomarker candidate to evaluate further for individualizing PDAC treatment. PMID:24626090

Investigating the Production of Foreign Membrane Proteins in Tobacco Chloroplasts: Expression of an Algal Plastid Terminal Oxidase

PubMed Central

Ahmad, Niaz; Michoux, Franck; Nixon, Peter J.

2012-01-01

Chloroplast transformation provides an inexpensive, easily scalable production platform for expression of recombinant proteins in plants. However, this technology has been largely limited to the production of soluble proteins. Here we have tested the ability of tobacco chloroplasts to express a membrane protein, namely plastid terminal oxidase 1 from the green alga Chlamydomonas reinhardtii (Cr-PTOX1), which is predicted to function as a plastoquinol oxidase. A homoplastomic plant containing a codon-optimised version of the nuclear gene encoding PTOX1, driven by the 16S rRNA promoter and 5′UTR of gene 10 from phage T7, was generated using a particle delivery system. Accumulation of Cr-PTOX1 was shown by immunoblotting and expression in an enzymatically active form was confirmed by using chlorophyll fluorescence to measure changes in the redox state of the plastoquinone pool in leaves. Growth of Cr-PTOX1 expressing plants was, however, more sensitive to high light than WT. Overall our results confirm the feasibility of using plastid transformation as a means of expressing foreign membrane proteins in the chloroplast. PMID:22848578

Development of an Improved Mammalian Overexpression Method for Human CD62L

PubMed Central

Brown, Haley A.; Roth, Gwynne; Holzapfel, Genevieve; Shen, Sarek; Rahbari, Kate; Ireland, Joanna; Zou, Zhongcheng; Sun, Peter D.

2014-01-01

We have previously developed a glutamine synthetase (GS)-based mammalian recombinant protein expression system that is capable of producing 5 to 30 mg/L recombinant proteins. The over expression is based on multiple rounds of target gene amplification driven by methionine sulfoximine (MSX), an inhibitor of glutamine synthetase. However, like other stable mammalian over expression systems, a major shortcoming of the GS-based expression system is its lengthy turn-around time, typically taking 4–6 months to produce. To shorten the construction time, we replaced the muti-round target gene amplifications with single-round in situ amplifications, thereby shortening the cell line construction to 2 months. The single-round in situ amplification method resulted in highest recombinant CD62L expressing CHO cell lines producing ~5mg/L soluble CD62L, similar to those derived from the multi-round amplification and selection method. In addition, we developed a MSX resistance assay as an alternative to utilizing ELISA for evaluating the expression level of stable recombinant CHO cell lines. PMID:25286402

The yeast DNA ligase gene CDC9 is controlled by six orientation specific upstream activating sequences that respond to cellular proliferation but which alone cannot mediate cell cycle regulation.

PubMed Central

White, J H; Johnson, A L; Lowndes, N F; Johnston, L H

1991-01-01

By fusing the CDC9 structural gene to the PGK upstream sequences and the CDC9 upstream to lacZ, we showed that the cell cycle expression of CDC9 is largely due to transcriptional regulation. To investigate the role of six ATGATT upstream repeats in CDC9 regulation, synthetic copies of the sequence were attached to a heterologous gene. The repeats stimulated transcription strongly and additively, but, unlike conventional yeast UAS elements, only when present in one orientation. Transcription driven by the repeats declines in cells held at START of the cell cycle or in stationary phase, as occurs with CDC9. However, the repeats by themselves cannot impart cell cycle regulation to a heterologous gene. CDC9 may therefore be controlled by an activating system operating through the repeats that is sensitive to cellular proliferation and a separate mechanism that governs the periodic expression in the cell cycle. Images PMID:1901644

Genetic engineering of human embryonic stem cells with lentiviral vectors.

PubMed

Xiong, Chen; Tang, Dong-Qi; Xie, Chang-Qing; Zhang, Li; Xu, Ke-Feng; Thompson, Winston E; Chou, Wayne; Gibbons, Gary H; Chang, Lung-Ji; Yang, Li-Jun; Chen, Yuqing E

2005-08-01

Human embryonic stem (hES) cells present a valuable source of cells with a vast therapeutic potential. However, the low efficiency of directed differentiation of hES cells remains a major obstacle in their uses for regenerative medicine. While differentiation may be controlled by the genetic manipulation, effective and efficient gene transfer into hES cells has been an elusive goal. Here, we show stable and efficient genetic manipulations of hES cells using lentiviral vectors. This method resulted in the establishment of stable gene expression without loss of pluripotency in hES cells. In addition, lentiviral vectors were effective in conveying the expression of an U6 promoter-driven small interfering RNA (siRNA), which was effective in silencing its specific target. Taken together, our results suggest that lentiviral gene delivery holds great promise for hES cell research and application.

Transcriptomic analyses reveal rhythmic and CLOCK-driven pathways in human skeletal muscle

PubMed Central

Perrin, Laurent; Hulo, Nicolas; Isenegger, Laura; Weger, Benjamin D; Migliavacca, Eugenia; Charpagne, Aline; Betts, James A; Walhin, Jean-Philippe; Templeman, Iain; Stokes, Keith; Thompson, Dylan; Tsintzas, Kostas; Robert, Maud; Howald, Cedric; Riezman, Howard; Feige, Jerome N; Karagounis, Leonidas G; Johnston, Jonathan D; Dermitzakis, Emmanouil T

2018-01-01

Circadian regulation of transcriptional processes has a broad impact on cell metabolism. Here, we compared the diurnal transcriptome of human skeletal muscle conducted on serial muscle biopsies in vivo with profiles of human skeletal myotubes synchronized in vitro. More extensive rhythmic transcription was observed in human skeletal muscle compared to in vitro cell culture as a large part of the in vivo mRNA rhythmicity was lost in vitro. siRNA-mediated clock disruption in primary myotubes significantly affected the expression of ~8% of all genes, with impact on glucose homeostasis and lipid metabolism. Genes involved in GLUT4 expression, translocation and recycling were negatively affected, whereas lipid metabolic genes were altered to promote activation of lipid utilization. Moreover, basal and insulin-stimulated glucose uptake were significantly reduced upon CLOCK depletion. Our findings suggest an essential role for the circadian coordination of skeletal muscle glucose homeostasis and lipid metabolism in humans. PMID:29658882

Cogena, a novel tool for co-expressed gene-set enrichment analysis, applied to drug repositioning and drug mode of action discovery.

PubMed

Jia, Zhilong; Liu, Ying; Guan, Naiyang; Bo, Xiaochen; Luo, Zhigang; Barnes, Michael R

2016-05-27

Drug repositioning, finding new indications for existing drugs, has gained much recent attention as a potentially efficient and economical strategy for accelerating new therapies into the clinic. Although improvement in the sensitivity of computational drug repositioning methods has identified numerous credible repositioning opportunities, few have been progressed. Arguably the "black box" nature of drug action in a new indication is one of the main blocks to progression, highlighting the need for methods that inform on the broader target mechanism in the disease context. We demonstrate that the analysis of co-expressed genes may be a critical first step towards illumination of both disease pathology and mode of drug action. We achieve this using a novel framework, co-expressed gene-set enrichment analysis (cogena) for co-expression analysis of gene expression signatures and gene set enrichment analysis of co-expressed genes. The cogena framework enables simultaneous, pathway driven, disease and drug repositioning analysis. Cogena can be used to illuminate coordinated changes within disease transcriptomes and identify drugs acting mechanistically within this framework. We illustrate this using a psoriatic skin transcriptome, as an exemplar, and recover two widely used Psoriasis drugs (Methotrexate and Ciclosporin) with distinct modes of action. Cogena out-performs the results of Connectivity Map and NFFinder webservers in similar disease transcriptome analyses. Furthermore, we investigated the literature support for the other top-ranked compounds to treat psoriasis and showed how the outputs of cogena analysis can contribute new insight to support the progression of drugs into the clinic. We have made cogena freely available within Bioconductor or https://github.com/zhilongjia/cogena . In conclusion, by targeting co-expressed genes within disease transcriptomes, cogena offers novel biological insight, which can be effectively harnessed for drug discovery and repositioning, allowing the grouping and prioritisation of drug repositioning candidates on the basis of putative mode of action.

Modular transcriptional repertoire and MicroRNA target analyses characterize genomic dysregulation in the thymus of Down syndrome infants

PubMed Central

Moreira-Filho, Carlos Alberto; Bando, Silvia Yumi; Bertonha, Fernanda Bernardi; Silva, Filipi Nascimento; da Fontoura Costa, Luciano; Ferreira, Leandro Rodrigues; Furlanetto, Glaucio; Chacur, Paulo; Zerbini, Maria Claudia Nogueira; Carneiro-Sampaio, Magda

2016-01-01

Trisomy 21-driven transcriptional alterations in human thymus were characterized through gene coexpression network (GCN) and miRNA-target analyses. We used whole thymic tissue - obtained at heart surgery from Down syndrome (DS) and karyotipically normal subjects (CT) - and a network-based approach for GCN analysis that allows the identification of modular transcriptional repertoires (communities) and the interactions between all the system's constituents through community detection. Changes in the degree of connections observed for hierarchically important hubs/genes in CT and DS networks corresponded to community changes. Distinct communities of highly interconnected genes were topologically identified in these networks. The role of miRNAs in modulating the expression of highly connected genes in CT and DS was revealed through miRNA-target analysis. Trisomy 21 gene dysregulation in thymus may be depicted as the breakdown and altered reorganization of transcriptional modules. Leading networks acting in normal or disease states were identified. CT networks would depict the “canonical” way of thymus functioning. Conversely, DS networks represent a “non-canonical” way, i.e., thymic tissue adaptation under trisomy 21 genomic dysregulation. This adaptation is probably driven by epigenetic mechanisms acting at chromatin level and through the miRNA control of transcriptional programs involving the networks' high-hierarchy genes. PMID:26848775

Effects of cetyltriethylammonium bromide on the replication of Bombyx mori nucleopolyhedrovirus.

PubMed

Zhou, Yajing; Zhang, Zhifang; He, Jialu; Zhang, Yuanxing

2002-05-01

An experimental study was undertaken to quantify the effects of cetyltriethylammonium bromide (CTAB) on the replication of Bombyx mori nucleopolyhedrovirus (BmNPV) and the transcriptionalactivity of BmNPV ie-1 promoter. The results demonstrated that the budded virus (BV) titer rose about 3.7-fold by adding CTAB to the culture media up to 0.1 mu g ml(-1) in infected Bm-N cells with a wild-type BmNPV. The transient expression level of luciferase driven by BmNPV ie-1 promoter was enhanced by more than 3-fold in the presence of 0.1 mu g ml(-1) of CTAB in uninfected insect cells via a transient expression system. Contrary to the rise in BV titer, the polyhedra inside the nucleus of infected cells dropped linearly from 4.0 x 10(6) ml(-1) down to 2.1 x 10(6) ml(-1) with in a range of CTAB concentrations from 0 to 0.25 mu g ml(-1). The same trend in expression level of beta -galactosidase or phytase was given when the Bm-N cells or fifth-instar silkworm larvae infected with a recombinant BmNPV containing the beta -galactosidase or phytase reporter gene driven by the polyhedrin promoter. We deduced that CTAB appeared to affect the virus bi-phasic life cycle stages and production pathways, resulting in an enhancement in BV production and a suppression of occluded virus (OV) production and expression of foreign genes controlled by the polyhedrin promoter.

Ras-Driven Transcriptome Analysis Identifies Aurora Kinase A as a Potential Malignant Peripheral Nerve Sheath Tumor Therapeutic Target

PubMed Central

Patel, Ami V.; Eaves, David; Jessen, Walter J.; Rizvi, Tilat A.; Ecsedy, Jeffrey A.; Qian, Mark G.; Aronow, Bruce J.; Perentesis, John P.; Serra, Eduard; Cripe, Timothy P.; Miller, Shyra J.; Ratner, Nancy

2013-01-01

Purpose Patients with Neurofibromatosis Type 1 (NF1) develop malignant peripheral nerve sheath tumors (MPNST) which are often inoperable and do not respond well to current chemotherapies or radiation. The goal of this study was to utilize comprehensive gene expression analysis to identify novel therapeutic targets. Experimental Design Nerve Schwann cells and/or their precursors are the tumorigenic cell types in MPNST due to the loss of the NF1 gene, which encodes the RasGAP protein neurofibromin. Therefore, we created a transgenic mouse model, CNP-HRas12V, expressing constitutively-active HRas in Schwann cells and defined a Ras-induced gene expression signature to drive a Bayesian factor regression model analysis of differentially expressed genes in mouse and human neurofibromas and MPNSTs. We tested functional significance of Aurora kinase over-expression in MPNST in vitro and in vivo using Aurora kinase shRNAs and compounds that inhibit Aurora kinase. Results We identified 2000 genes with probability of linkage to nerve Ras signaling of which 339 were significantly differentially expressed in mouse and human NF1-related tumor samples relative to normal nerves, including Aurora kinase A (AURKA). AURKA was dramatically over-expressed and genomically amplified in MPNSTs but not neurofibromas. Aurora kinase shRNAs and Aurora kinase inhibitors blocked MPNST cell growth in vitro. Furthermore, an AURKA selective inhibitor, MLN8237, stabilized tumor volume and significantly increased survival of mice with MPNST xenografts. Conclusion Integrative cross-species transcriptome analyses combined with preclinical testing has provided an effective method for identifying candidates for molecular-targeted therapeutics. Blocking Aurora kinases may be a viable treatment platform for MPNST. PMID:22811580

Genetic Variation and Gene Expression in Antioxidant-Related Enzymes and Risk of Chronic Obstructive Pulmonary Disease: A Systematic Review

PubMed Central

Bentley, Amy R; Emrani, Parastu; Cassano, Patricia A

2011-01-01

Observational epidemiologic studies of dietary antioxidant intake, serum antioxidant concentration, and lung outcomes suggest that lower levels of antioxidant defenses are associated with decreased lung function. Another approach to understanding the role of oxidant/antioxidant imbalance in risk of Chronic Obstructive Pulmonary Disease (COPD) is to investigate the role of genetic variation in antioxidant enzymes, and indeed family-based studies suggest a heritable component to lung disease. Many studies of the genes encoding antioxidant enzymes have considered COPD or COPD-related outcomes, and a systematic review is needed to summarise the evidence to date, and to provide insights for further research. Genetic association studies of antioxidant enzymes and COPD/COPD-related traits, and comparative gene expression studies with disease or smoking as the exposure were systematically identified and reviewed. Antioxidant enzymes considered included enzymes involved in glutathione (GSH) metabolism, in the thioredoxin (TXN) system, superoxide dismutases (SOD), and catalase (CAT). A total of 29 genetic association and 15 comparative gene expression studies met the inclusion criteria. The strongest and most consistent effects were in the genes GCL, GSTM1, GSTP1, and SOD3. This review also highlights the lack of studies for genes of interest, particularly GSR, GGT, and those related to TXN. There were limited opportunities to evaluate a gene’s contribution to disease risk through a synthesis of results from different study designs, as the majority of studies considered either association of sequence variants with disease or effect of disease on gene expression. Network-driven approaches that consider potential interaction between genes and amoung genes, smoke exposure, and antioxidant intake are needed to fully characterise the role of oxidant/antioxidant balance in pathogenesis. PMID:18566111

Comparative Genomics Identifies Epidermal Proteins Associated with the Evolution of the Turtle Shell.

PubMed

Holthaus, Karin Brigit; Strasser, Bettina; Sipos, Wolfgang; Schmidt, Heiko A; Mlitz, Veronika; Sukseree, Supawadee; Weissenbacher, Anton; Tschachler, Erwin; Alibardi, Lorenzo; Eckhart, Leopold

2016-03-01

The evolution of reptiles, birds, and mammals was associated with the origin of unique integumentary structures. Studies on lizards, chicken, and humans have suggested that the evolution of major structural proteins of the outermost, cornified layers of the epidermis was driven by the diversification of a gene cluster called Epidermal Differentiation Complex (EDC). Turtles have evolved unique defense mechanisms that depend on mechanically resilient modifications of the epidermis. To investigate whether the evolution of the integument in these reptiles was associated with specific adaptations of the sequences and expression patterns of EDC-related genes, we utilized newly available genome sequences to determine the epidermal differentiation gene complement of turtles. The EDC of the western painted turtle (Chrysemys picta bellii) comprises more than 100 genes, including at least 48 genes that encode proteins referred to as beta-keratins or corneous beta-proteins. Several EDC proteins have evolved cysteine/proline contents beyond 50% of total amino acid residues. Comparative genomics suggests that distinct subfamilies of EDC genes have been expanded and partly translocated to loci outside of the EDC in turtles. Gene expression analysis in the European pond turtle (Emys orbicularis) showed that EDC genes are differentially expressed in the skin of the various body sites and that a subset of beta-keratin genes within the EDC as well as those located outside of the EDC are expressed predominantly in the shell. Our findings give strong support to the hypothesis that the evolutionary innovation of the turtle shell involved specific molecular adaptations of epidermal differentiation. © The Author 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Up-regulation of mismatch repair genes MSH6, PMS2 and MLH1 parallels development of genetic instability and is linked to tumor aggressiveness and early PSA recurrence in prostate cancer.

PubMed

Wilczak, Waldemar; Rashed, Semin; Hube-Magg, Claudia; Kluth, Martina; Simon, Ronald; Büscheck, Franziska; Clauditz, Till Sebastian; Grupp, Katharina; Minner, Sarah; Tsourlakis, Maria Christina; Möller-Koop, Christina; Graefen, Markus; Adam, Meike; Haese, Alexander; Wittmer, Corinna; Sauter, Guido; Izbicki, Jakob Robert; Huland, Hartwig; Schlomm, Thorsten; Steurer, Stefan; Krech, Till; Lebok, Patrick

2017-01-01

DNA mismatch repair (MMR) is integral to the maintenance of genetic stability. We aimed to evaluate the clinical impact of MMR gene expression in prostate cancer. The MMR genes MSH6, MLH1 and PMS2 were analyzed by immunohistochemistry on a tissue microarray containing 11152 prostate cancer specimens. Results were compared with ETS-related gene status and deletions of PTEN, 3p13, 5q21 and 6q15. MSH6, MLH1 and PMS2 expression was detectable in 89.5%, 85.4% and 85.0% of cancers and was particularly strong in cancers with advanced pathological tumor stage (P < 0.0001 each), high Gleason grade (P < 0.0001 each), nodal metastasis (P ≤ 0.0083) and early biochemical recurrence (P < 0.0001). High levels of MMR gene expression paralleled features of genetic instability, such as the number of genomic deletions per cancer; strong expression of all three MMR genes was found in 24%, 29%, 30%, 33% and 42% of cancers with no, one, two, three or four to five deletions (P < 0.0001). The prognostic value of the analyzed MMR genes was largely driven by the subset of cancers lacking ERG fusion (P < 0.0001), while the prognostic impact of MMR gene overexpression was only marginal in ERG-positive cancers. Multivariate analyses suggested an independent prognostic relevance of MMR genes in ERG-negative prostate cancers when compared with prognostic parameters available at the time of initial biopsy. In conclusion, MMR overexpression is common in prostate cancer and is linked to poor outcome as well as features indicating genetic instability. ERG fusion should be analyzed along with MMR gene expression in potential clinical tests. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Transcriptome Analysis of Neisseria gonorrhoeae during Natural Infection Reveals Differential Expression of Antibiotic Resistance Determinants between Men and Women.

PubMed

Nudel, Kathleen; McClure, Ryan; Moreau, Matthew; Briars, Emma; Abrams, A Jeanine; Tjaden, Brian; Su, Xiao-Hong; Trees, David; Rice, Peter A; Massari, Paola; Genco, Caroline A

2018-08-29

Neisseria gonorrhoeae is a bacterial pathogen responsible for the sexually transmitted infection gonorrhea. Emergence of antimicrobial resistance (AMR) of N. gonorrhoeae worldwide has resulted in limited therapeutic choices for this infection. Men who seek treatment often have symptomatic urethritis; in contrast, gonococcal cervicitis in women is usually minimally symptomatic, but may progress to pelvic inflammatory disease. Previously, we reported the first analysis of gonococcal transcriptome expression determined in secretions from women with cervical infection. Here, we defined gonococcal global transcriptional responses in urethral specimens from men with symptomatic urethritis and compared these with transcriptional responses in specimens obtained from women with cervical infections and in vitro -grown N. gonorrhoeae isolates. This is the first comprehensive comparison of gonococcal gene expression in infected men and women. RNA sequencing analysis revealed that 9.4% of gonococcal genes showed increased expression exclusively in men and included genes involved in host immune cell interactions, while 4.3% showed increased expression exclusively in women and included phage-associated genes. Infected men and women displayed comparable antibiotic-resistant genotypes and in vitro phenotypes, but a 4-fold higher expression of the Mtr efflux pump-related genes was observed in men. These results suggest that expression of AMR genes is programed genotypically and also driven by sex-specific environments. Collectively, our results indicate that distinct N. gonorrhoeae gene expression signatures are detected during genital infection in men and women. We propose that therapeutic strategies could target sex-specific differences in expression of antibiotic resistance genes. IMPORTANCE Recent emergence of antimicrobial resistance of Neisseria gonorrhoeae worldwide has resulted in limited therapeutic choices for treatment of infections caused by this organism. We performed global transcriptomic analysis of N. gonorrhoeae in subjects with gonorrhea who attended a Nanjing, China, sexually transmitted infection (STI) clinic, where antimicrobial resistance of N. gonorrhoeae is high and increasing. We found that N. gonorrhoeae transcriptional responses to infection differed in genital specimens taken from men and women, particularly antibiotic resistance gene expression, which was increased in men. These sex-specific findings may provide a new approach to guide therapeutic interventions and preventive measures that are also sex specific while providing additional insight to address antimicrobial resistance of N. gonorrhoeae . Copyright © 2018 Nudel et al.

Mechanisms of Dietary Response in Mice and Primates: A Role for EGR1 in Regulating the Reaction to Human-Specific Nutritional Content

PubMed Central

Weng, Kai; Hu, Haiyang; Xu, Augix Guohua; Khaitovich, Philipp; Somel, Mehmet

2012-01-01

Background Humans have a widely different diet from other primate species, and are dependent on its high nutritional content. The molecular mechanisms responsible for adaptation to the human diet are currently unknown. Here, we addressed this question by investigating whether the gene expression response observed in mice fed human and chimpanzee diets involves the same regulatory mechanisms as expression differences between humans and chimpanzees. Results Using mouse and primate transcriptomic data, we identified the transcription factor EGR1 (early growth response 1) as a putative regulator of diet-related differential gene expression between human and chimpanzee livers. Specifically, we predict that EGR1 regulates the response to the high caloric content of human diets. However, we also show that close to 90% of the dietary response to the primate diet found in mice, is not observed in primates. This might be explained by changes in tissue-specific gene expression between taxa. Conclusion Our results suggest that the gene expression response to the nutritionally rich human diet is partially mediated by the transcription factor EGR1. While this EGR1-driven response is conserved between mice and primates, the bulk of the mouse response to human and chimpanzee dietary differences is not observed in primates. This result highlights the rapid evolution of diet-related expression regulation and underscores potential limitations of mouse models in dietary studies. PMID:22937124

Gene expression based mouse brain parcellation using Markov random field regularized non-negative matrix factorization

NASA Astrophysics Data System (ADS)

Pathak, Sayan D.; Haynor, David R.; Thompson, Carol L.; Lein, Ed; Hawrylycz, Michael

2009-02-01

Understanding the geography of genetic expression in the mouse brain has opened previously unexplored avenues in neuroinformatics. The Allen Brain Atlas (www.brain-map.org) (ABA) provides genome-wide colorimetric in situ hybridization (ISH) gene expression images at high spatial resolution, all mapped to a common three-dimensional 200μm3 spatial framework defined by the Allen Reference Atlas (ARA) and is a unique data set for studying expression based structural and functional organization of the brain. The goal of this study was to facilitate an unbiased data-driven structural partitioning of the major structures in the mouse brain. We have developed an algorithm that uses nonnegative matrix factorization (NMF) to perform parts based analysis of ISH gene expression images. The standard NMF approach and its variants are limited in their ability to flexibly integrate prior knowledge, in the context of spatial data. In this paper, we introduce spatial connectivity as an additional regularization in NMF decomposition via the use of Markov Random Fields (mNMF). The mNMF algorithm alternates neighborhood updates with iterations of the standard NMF algorithm to exploit spatial correlations in the data. We present the algorithm and show the sub-divisions of hippocampus and somatosensory-cortex obtained via this approach. The results are compared with established neuroanatomic knowledge. We also highlight novel gene expression based sub divisions of the hippocampus identified by using the mNMF algorithm.

NATbox: a network analysis toolbox in R.

PubMed

Chavan, Shweta S; Bauer, Michael A; Scutari, Marco; Nagarajan, Radhakrishnan

2009-10-08

There has been recent interest in capturing the functional relationships (FRs) from high-throughput assays using suitable computational techniques. FRs elucidate the working of genes in concert as a system as opposed to independent entities hence may provide preliminary insights into biological pathways and signalling mechanisms. Bayesian structure learning (BSL) techniques and its extensions have been used successfully for modelling FRs from expression profiles. Such techniques are especially useful in discovering undocumented FRs, investigating non-canonical signalling mechanisms and cross-talk between pathways. The objective of the present study is to develop a graphical user interface (GUI), NATbox: Network Analysis Toolbox in the language R that houses a battery of BSL algorithms in conjunction with suitable statistical tools for modelling FRs in the form of acyclic networks from gene expression profiles and their subsequent analysis. NATbox is a menu-driven open-source GUI implemented in the R statistical language for modelling and analysis of FRs from gene expression profiles. It provides options to (i) impute missing observations in the given data (ii) model FRs and network structure from gene expression profiles using a battery of BSL algorithms and identify robust dependencies using a bootstrap procedure, (iii) present the FRs in the form of acyclic graphs for visualization and investigate its topological properties using network analysis metrics, (iv) retrieve FRs of interest from published literature. Subsequently, use these FRs as structural priors in BSL (v) enhance scalability of BSL across high-dimensional data by parallelizing the bootstrap routines. NATbox provides a menu-driven GUI for modelling and analysis of FRs from gene expression profiles. By incorporating readily available functions from existing R-packages, it minimizes redundancy and improves reproducibility, transparency and sustainability, characteristic of open-source environments. NATbox is especially suited for interdisciplinary researchers and biologists with minimal programming experience and would like to use systems biology approaches without delving into the algorithmic aspects. The GUI provides appropriate parameter recommendations for the various menu options including default parameter choices for the user. NATbox can also prove to be a useful demonstration and teaching tool in graduate and undergraduate course in systems biology. It has been tested successfully under Windows and Linux operating systems. The source code along with installation instructions and accompanying tutorial can be found at http://bioinformatics.ualr.edu/natboxWiki/index.php/Main_Page.

Radiation Gene-expression Signatures in Primary Breast Cancer Cells.

PubMed

Minafra, Luigi; Bravatà, Valentina; Cammarata, Francesco P; Russo, Giorgio; Gilardi, Maria C; Forte, Giusi I

2018-05-01

In breast cancer (BC) care, radiation therapy (RT) is an efficient treatment to control localized tumor. Radiobiological research is needed to understand molecular differences that affect radiosensitivity of different tumor subtypes and the response variability. The aim of this study was to analyze gene expression profiling (GEP) in primary BC cells following irradiation with doses of 9 Gy and 23 Gy delivered by intraoperative electron radiation therapy (IOERT) in order to define gene signatures of response to high doses of ionizing radiation. We performed GEP by cDNA microarrays and evaluated cell survival after IOERT treatment in primary BC cell cultures. Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) was performed to validate candidate genes. We showed, for the first time, a 4-gene and a 6-gene signature, as new molecular biomarkers, in two primary BC cell cultures after exposure at 9 Gy and 23 Gy respectively, for which we observed a significantly high survival rate. Gene signatures activated by different doses of ionizing radiation may predict response to RT and contribute to defining a personalized biological-driven treatment plan. Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

Development of marker-free transgenic Jatropha curcas producing curcin-deficient seeds through endosperm-specific RNAi-mediated gene silencing.

PubMed

Gu, Keyu; Tian, Dongsheng; Mao, Huizhu; Wu, Lifang; Yin, Zhongchao

2015-10-08

Jatropha curcas L. is a potential biofuel plant and its seed oil is suitable for biodiesel production. Despite this promising application, jatropha seeds contain two major toxic components, namely phorbol esters and curcins. These compounds would reduce commercial value of seed cake and raise safety and environment concerns on jatropha plantation and processing. Curcins are Type I ribosome inactivating proteins. Several curcin genes have been identified in the jatropha genome. Among which, the Curcin 1 (C1) gene is identified to be specifically expressed in endosperm, whereas the Curcin 2A (C2A) is mainly expressed in young leaves. A marker-free RNAi construct carrying a β-estradiol-regulated Cre/loxP system and a C1 promoter-driven RNAi cassette for C1 gene was made and used to generate marker-free transgenic RNAi plants to specifically silence the C1 gene in the endosperm of J. curcas. Plants of transgenic line L1, derived from T0-1, carry two copies of marker-free RNAi cassette, whereas plants of L35, derived from T0-35, harbored one copy of marker-free RNAi cassette and three copies of closely linked and yet truncated Hpt genes. The C1 protein content in endosperm of L1 and L35 seeds was greatly reduced or undetectable, while the C2A proteins in young leaves of T0-1 and T0-35 plants were unaffected. In addition, the C1 mRNA transcripts were undetectable in the endosperm of T3 seeds of L1 and L35. The results demonstrated that the expression of the C1 gene was specifically down-regulated or silenced by the double-stranded RNA-mediated RNA interference generated from the RNAi cassette. The C1 promoter-driven RNAi cassette for the C1 gene in transgenic plants was functional and heritable. Both C1 transcripts and C1 proteins were greatly down-regulated or silenced in the endosperm of transgenic J. curcas. The marker-free transgenic plants and curcin-deficient seeds developed in this study provided a solution for the toxicity of curcins in jatropha seeds and addressed the safety concerns of the marker genes in transgenic plants on the environments.

Actinobacillus pleuropneumoniae genes expression in biofilms cultured under static conditions and in a drip-flow apparatus

PubMed Central

2013-01-01

Background Actinobacillus pleuropneumoniae is the Gram-negative bacterium responsible for porcine pleuropneumonia. This respiratory infection is highly contagious and characterized by high morbidity and mortality. The objectives of our study were to study the transcriptome of A. pleuropneumoniae biofilms at different stages and to develop a protocol to grow an A. pleuropneumoniae biofilm in a drip-flow apparatus. This biofilm reactor is a system with an air-liquid interface modeling lung-like environment. Bacteria attached to a surface (biofilm) and free floating bacteria (plankton) were harvested for RNA isolation. Labelled cDNA was hybridized to a microarray to compare the expression profiles of planktonic cells and biofilm cells. Results It was observed that 47 genes were differentially expressed (22 up, 25 down) in a 4 h-static growing/maturing biofilm and 117 genes were differentially expressed (49 up, 68 down) in a 6h-static dispersing biofilm. The transcriptomes of a 4 h biofilm and a 6 h biofilm were also compared and 456 genes (235 up, 221 down) were identified as differently expressed. Among the genes identified in the 4 h vs 6h biofilm experiment, several regulators of stress response were down-regulated and energy metabolism associated genes were up-regulated. Biofilm bacteria cultured using the drip-flow apparatus differentially expressed 161 genes (68 up, 93 down) compared to the effluent bacteria. Cross-referencing of differentially transcribed genes in the different assays revealed that drip-flow biofilms shared few differentially expressed genes with static biofilms (4 h or 6 h) but shared several differentially expressed genes with natural or experimental infections in pigs. Conclusion The formation of a static biofilm by A. pleuropneumoniae strain S4074 is a rapid process and transcriptional analysis indicated that dispersal observed at 6 h is driven by nutritional stresses. Furthermore, A. pleuropneumoniae can form a biofilm under low-shear force in a drip-flow apparatus and analyses indicated that the formation of a biofilm under low-shear force requires a different sub-set of genes than a biofilm grown under static conditions. The drip-flow apparatus may represent the better in vitro model to investigate biofilm formation of A. pleuropneumoniae. PMID:23725589

Transcriptional Modulation of Ethylene Response Factor Protein JERF3 in the Oxidative Stress Response Enhances Tolerance of Tobacco Seedlings to Salt, Drought, and Freezing1[C][W][OA

PubMed Central

Wu, Lijun; Zhang, Zhijin; Zhang, Haiwen; Wang, Xue-Chen; Huang, Rongfeng

2008-01-01

Abiotic stresses such as drought, cold, and salinity affect normal growth and development in plants. The production and accumulation of reactive oxygen species (ROS) cause oxidative stress under these abiotic conditions. Recent research has elucidated the significant role of ethylene response factor (ERF) proteins in plant adaptation to abiotic stresses. Our earlier functional analysis of an ERF protein, JERF3, indicated that JERF3-expressing tobacco (Nicotiana tabacum) adapts better to salinity in vitro. This article extends that study by showing that transcriptional regulation of JERF3 in the oxidative stress response modulates the increased tolerance to abiotic stresses. First, we confirm that JERF3-expressing tobacco enhances adaptation to drought, freezing, and osmotic stress during germination and seedling development. Then we demonstrate that JERF3-expressing tobacco imparts not only higher expression of osmotic stress genes compared to wild-type tobacco, but also the activation of photosynthetic carbon assimilation/metabolism and oxidative genes. More importantly, this regulation of the expression of oxidative genes subsequently enhances the activities of superoxide dismutase but reduces the content of ROS in tobacco under drought, cold, salt, and abscisic acid treatments. This indicates that JERF3 also modulates the abiotic stress response via the regulation of the oxidative stress response. Further assays indicate that JERF3 activates the expression of reporter genes driven by the osmotic-responsive GCC box, DRE, and CE1 and by oxidative-responsive as-1 in transient assays, suggesting the transcriptional activation of JERF3 in the expression of genes involved in response to oxidative and osmotic stress. Our results therefore establish that JERF3 activates the expression of such genes through transcription, resulting in decreased accumulation of ROS and, in turn, enhanced adaptation to drought, freezing, and salt in tobacco. PMID:18945933

Molecular Characterization of Copepod Photoreception.

PubMed

Porter, Megan L; Steck, Mireille; Roncalli, Vittoria; Lenz, Petra H

2017-08-01

Copepod crustaceans are an abundant and ecologically significant group whose basic biology is guided by numerous visually guided behaviors. These behaviors are driven by copepod eyes, including naupliar eyes and Gicklhorn's organs, which vary widely in structure and function among species. Yet little is known about the molecular aspects of copepod vision. In this study we present a general overview of the molecular aspects of copepod vision by identifying phototransduction genes from newly generated and publicly available RNA-sequencing data and assemblies from 12 taxonomically diverse copepod species. We identify a set of 10 expressed transcripts that serve as a set of target genes for future studies of copepod phototransduction. Our more detailed evolutionary analyses of the opsin gene responsible for forming visual pigments found that all of the copepod species investigated express two main groups of opsins: middle-wavelength-sensitive (MWS) opsins and pteropsins. Additionally, there is evidence from a few species (e.g., Calanus finmarchicus, Eurytemora affinis, Paracyclopina nana, and Lernaea cyprinacea) for the expression of two additional groups of opsins-the peropsins and rhodopsin 7 (Rh7) opsins-at low levels or distinct developmental stages. An ontogenetic analysis of opsin expression in Calanus finmarchicus found the expression of a single dominant MWS opsin, as well as evidence for differences in expression across development in some MWS, pteropsin, and Rh7 opsins, with expression peaking in early naupliar through early copepodite stages.

Claudin gene expression patterns do not associate with interspecific differences in paracellular nutrient absorption.

PubMed

Price, Edwin R; Rott, Katherine H; Caviedes-Vidal, Enrique; Karasov, William H

2016-01-01

Bats exhibit higher paracellular absorption of glucose-sized molecules than non-flying mammals, a phenomenon that may be driven by higher permeability of the intestinal tight junctions. The various claudins, occludin, and other proteins making up the tight junctions are thought to determine their permeability properties. Here we show that absorption of the paracellular probe l-arabinose is higher in a bat (Eptesicus fuscus) than in a vole (Microtus pennsylvanicus) or a hedgehog (Atelerix albiventris). Furthermore, histological measurements demonstrated that hedgehogs have many more enterocytes in their intestines, suggesting that bats cannot have higher absorption of arabinose simply by having more tight junctions. We therefore investigated the mRNA levels of several claudins and occludin, because these proteins may affect permeability of tight junctions to macronutrients. To assess the expression levels of claudins per tight junction, we normalized the mRNA levels of the claudins to the constitutively expressed tight junction protein ZO-1, and combined these with measurements previously made in a bat and a rodent to determine if there were among-species differences. Although expression ratios of several genes varied among species, there was not a consistent difference between bats and non-flyers in the expression ratio of any particular gene. Protein expression patterns may differ from mRNA expression patterns, and might better explain differences among species in arabinose absorption. Copyright © 2015 Elsevier Inc. All rights reserved.

Transgene expression of green fluorescent protein and germ line transmission in cloned pigs derived from in vitro transfected adult fibroblasts.

PubMed

Brunetti, Dario; Perota, Andrea; Lagutina, Irina; Colleoni, Silvia; Duchi, Roberto; Calabrese, Fiorella; Seveso, Michela; Cozzi, Emanuele; Lazzari, Giovanna; Lucchini, Franco; Galli, Cesare

2008-12-01

The pig represents the xenogeneic donor of choice for future organ transplantation in humans for anatomical and physiological reasons. However, to bypass several immunological barriers, strong and stable human genes expression must occur in the pig's organs. In this study we created transgenic pigs using in vitro transfection of cultured cells combined with somatic cell nuclear transfer (SCNT) to evaluate the ubiquitous transgene expression driven by pCAGGS vector in presence of different selectors. pCAGGS confirmed to be a very effective vector for ubiquitous transgene expression, irrespective of the selector that was used. Green fluorescent protein (GFP) expression observed in transfected fibroblasts was also maintained after nuclear transfer, through pre- and postimplantation development, at birth and during adulthood. Germ line transmission without silencing of the transgene was demonstrated. The ubiquitous expression of GFP was clearly confirmed in several tissues including endothelial cells, thus making it a suitable vector for the expression of multiple genes relevant to xenotransplantation where tissue specificity is not required. Finally cotransfection of green and red fluorescence protein transgenes was performed in fibroblasts and after nuclear transfer blastocysts expressing both fluorescent proteins were obtained.

Constitutive expression of cell wall invertase genes increases grain yield and starch content in maize.

PubMed

Li, Bei; Liu, Hua; Zhang, Yue; Kang, Tao; Zhang, Li; Tong, Jianhua; Xiao, Langtao; Zhang, Hongxia

2013-12-01

Grain size, number and starch content are important determinants of grain yield and quality. One of the most important biological processes that determine these components is the carbon partitioning during the early grain filling, which requires the function of cell wall invertase. Here, we showed the constitutive expression of cell wall invertase-encoding gene from Arabidopsis, rice (Oryza sativa) or maize (Zea mays), driven by the cauliflower mosaic virus (CaMV) 35S promoter, all increased cell wall invertase activities in different tissues and organs, including leaves and developing seeds, and substantially improved grain yield up to 145.3% in transgenic maize plants as compared to the wild-type plants, an effect that was reproduced in our 2-year field trials at different locations. The dramatically increased grain yield is due to the enlarged ears with both enhanced grain size and grain number. Constitutive expression of the invertase-encoding gene also increased total starch content up to 20% in the transgenic kernels. Our results suggest that cell wall invertase gene can be genetically engineered to improve both grain yield and grain quality in crop plants. © 2013 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

Bacterial community evolutions driven by organic matter and powder activated carbon in simultaneous anammox and denitrification (SAD) process.

PubMed

Ge, Cheng-Hao; Sun, Na; Kang, Qi; Ren, Long-Fei; Ahmad, Hafiz Adeel; Ni, Shou-Qing; Wang, Zhibin

2018-03-01

A distinct shift of bacterial community driven by organic matter (OM) and powder activated carbon (PAC) was discovered in the simultaneous anammox and denitrification (SAD) process which was operated in an anti-fouling submerged anaerobic membrane bio-reactor. Based on anammox performance, optimal OM dose (50 mg/L) was advised to start up SAD process successfully. The results of qPCR and high throughput sequencing analysis indicated that OM played a key role in microbial community evolutions, impelling denitrifiers to challenge anammox's dominance. The addition of PAC not only mitigated the membrane fouling, but also stimulated the enrichment of denitrifiers, accounting for the predominant phylum changing from Planctomycetes to Proteobacteria in SAD process. Functional genes forecasts based on KEGG database and COG database showed that the expressions of full denitrification functional genes were highly promoted in R C , which demonstrated the enhanced full denitrification pathway driven by OM and PAC under low COD/N value (0.11). Copyright © 2017 Elsevier Ltd. All rights reserved.

Remote reprogramming of hepatic circadian transcriptome by breast cancer.

PubMed

Hojo, Hiroaki; Enya, Sora; Arai, Miki; Suzuki, Yutaka; Nojiri, Takashi; Kangawa, Kenji; Koyama, Shinsuke; Kawaoka, Shinpei

2017-05-23

Cancers adversely affect organismal physiology. To date, the genes within a patient responsible for systemically spreading cancer-induced physiological disruption remain elusive. To identify host genes responsible for transmitting disruptive, cancer-driven signals, we thoroughly analyzed the transcriptome of a suite of host organs from mice bearing 4T1 breast cancer, and discovered complexly rewired patterns of circadian gene expression in the liver. Our data revealed that 7 core clock transcription factors, represented by Rev-erba and Rorg, exhibited abnormal daily expression rhythm in the liver of 4T1-bearing mice. Accordingly, expression patterns of specific set of downstream circadian genes were compromised. Osgin1, a marker for oxidative stress, was an example. Specific downstream genes, including E2f8, a transcriptional repressor that controls cellular polyploidy, displayed a striking pattern of disruption, "day-night reversal." Meanwhile, we found that the liver of 4T1-bearing mice suffered from increased oxidative stress. The tetraploid hepatocytes population was concomitantly increased in 4T1-bearing mice, which has not been previously appreciated as a cancer-induced phenotype. In summary, the current study provides a comprehensive characterization of the 4T1-affected hepatic circadian transcriptome that possibly underlies cancer-induced physiological alteration in the liver.

The genomic and transcriptomic architecture of 2,000 breast tumours reveals novel subgroups.

PubMed

Curtis, Christina; Shah, Sohrab P; Chin, Suet-Feung; Turashvili, Gulisa; Rueda, Oscar M; Dunning, Mark J; Speed, Doug; Lynch, Andy G; Samarajiwa, Shamith; Yuan, Yinyin; Gräf, Stefan; Ha, Gavin; Haffari, Gholamreza; Bashashati, Ali; Russell, Roslin; McKinney, Steven; Langerød, Anita; Green, Andrew; Provenzano, Elena; Wishart, Gordon; Pinder, Sarah; Watson, Peter; Markowetz, Florian; Murphy, Leigh; Ellis, Ian; Purushotham, Arnie; Børresen-Dale, Anne-Lise; Brenton, James D; Tavaré, Simon; Caldas, Carlos; Aparicio, Samuel

2012-04-18

The elucidation of breast cancer subgroups and their molecular drivers requires integrated views of the genome and transcriptome from representative numbers of patients. We present an integrated analysis of copy number and gene expression in a discovery and validation set of 997 and 995 primary breast tumours, respectively, with long-term clinical follow-up. Inherited variants (copy number variants and single nucleotide polymorphisms) and acquired somatic copy number aberrations (CNAs) were associated with expression in ~40% of genes, with the landscape dominated by cis- and trans-acting CNAs. By delineating expression outlier genes driven in cis by CNAs, we identified putative cancer genes, including deletions in PPP2R2A, MTAP and MAP2K4. Unsupervised analysis of paired DNA–RNA profiles revealed novel subgroups with distinct clinical outcomes, which reproduced in the validation cohort. These include a high-risk, oestrogen-receptor-positive 11q13/14 cis-acting subgroup and a favourable prognosis subgroup devoid of CNAs. Trans-acting aberration hotspots were found to modulate subgroup-specific gene networks, including a TCR deletion-mediated adaptive immune response in the ‘CNA-devoid’ subgroup and a basal-specific chromosome 5 deletion-associated mitotic network. Our results provide a novel molecular stratification of the breast cancer population, derived from the impact of somatic CNAs on the transcriptome.

A systems immunology approach identifies the collective impact of 5 miRs in Th2 inflammation.

PubMed

Kılıç, Ayşe; Santolini, Marc; Nakano, Taiji; Schiller, Matthias; Teranishi, Mizue; Gellert, Pascal; Ponomareva, Yuliya; Braun, Thomas; Uchida, Shizuka; Weiss, Scott T; Sharma, Amitabh; Renz, Harald

2018-06-07

Allergic asthma is a chronic inflammatory disease dominated by a CD4+ T helper 2 (Th2) cell signature. The immune response amplifies in self-enforcing loops, promoting Th2-driven cellular immunity and leaving the host unable to terminate inflammation. Posttranscriptional mechanisms, including microRNAs (miRs), are pivotal in maintaining immune homeostasis. Since an altered expression of various miRs has been associated with T cell-driven diseases, including asthma, we hypothesized that miRs control mechanisms ensuring Th2 stability and maintenance in the lung. We isolated murine CD4+ Th2 cells from allergic inflamed lungs and profiled gene and miR expression. Instead of focusing on the magnitude of miR differential expression, here we addressed the secondary consequences for the set of molecular interactions in the cell, the interactome. We developed the Impact of Differential Expression Across Layers, a network-based algorithm to prioritize disease-relevant miRs based on the central role of their targets in the molecular interactome. This method identified 5 Th2-related miRs (mir27b, mir206, mir106b, mir203, and mir23b) whose antagonization led to a sharp reduction of the Th2 phenotype. Overall, a systems biology tool was developed and validated, highlighting the role of miRs in Th2-driven immune response. This result offers potentially novel approaches for therapeutic interventions.

Development of a new vector using Soybean yellow common mosaic virus for gene function study or heterologous protein expression in soybeans.

PubMed

Lim, Seungmo; Nam, Moon; Kim, Kil Hyun; Lee, Su-Heon; Moon, Jung-Kyung; Lim, Hyoun-Sub; Choung, Myoung-Gun; Kim, Sang-Mok; Moon, Jae Sun

2016-02-01

A new vector using Soybean yellow common mosaic virus (SYCMV) was constructed for gene function study or heterologous protein expression in soybeans. The in vitro transcript with a 5' cap analog m7GpppG from an SYCMV full-length infectious vector driven by a T7 promoter infected soybeans (pSYCMVT7-full). The symptoms observed in the soybeans infected with either the sap from SYCMV-infected leaves or pSYCMVT7-full were indistinguishable, suggesting that the vector exhibits equivalent biological activity as the virus itself. To utilize the vector further, a DNA-based vector driven by the Cauliflower mosaic virus (CaMV) 35S promoter was constructed. The complete sequence of the SYCMV genome was inserted into a binary vector flanked by a CaMV 35S promoter at the 5' terminus of the SYCMV genome and a cis-cleaving ribozyme sequence followed by a nopaline synthase terminator at the 3' terminus of the SYCMV genome (pSYCMV-full). The SYCMV-derived vector was tested for use as a virus-induced gene silencing (VIGS) vector for the functional analysis of soybean genes. VIGS constructs containing either a fragment of the Phytoene desaturase (PDS) gene (pSYCMV-PDS1) or a fragment of the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (RbcS) gene (pSYCMV-RbcS2) were constructed. Plants infiltrated with each vector using the Agrobacterium-mediated inoculation method exhibited distinct symptoms, such as photo-bleaching in plants infiltrated with pSYCMV-PDS1 and yellow or pale green coloring in plants infiltrated with pSYCMV-RbcS2. In addition, down-regulation of the transcripts of the two target genes was confirmed via northern blot analysis. Particle bombardment and direct plasmid DNA rubbing were also confirmed as alternative inoculation methods. To determine if the SYCMV vector can be used for the expression of heterologous proteins in soybean plants, the vector encoding amino acids 135-160 of VP1 of Foot-and-mouth disease virus (FMDV) serotype O1 Campos (O1C) was constructed (pSYCMV-FMDV). Plants infiltrated with pSYCMV-FMDV were only detected via western blotting using the O1C antibody. Based on these results, we propose that the SYCMV-derived vector can be used for gene function study or expression of useful heterologous proteins in soybeans. Copyright © 2015 Elsevier B.V. All rights reserved.

A heterologous hormone response element enhances expression of rat beta-casein promoter-driven chloramphenicol acetyltransferase fusion genes in the mammary gland of transgenic mice.

PubMed

Greenberg, N M; Reding, T V; Duffy, T; Rosen, J M

1991-10-01

Previous studies have demonstrated that the entire rat beta-casein (R beta C) gene and a -524/+490 R beta C fragment-chloramphenicol acetyltransferase (CAT) fusion gene are expressed preferentially in the mammary gland of transgenic mice in a developmentally regulated fashion. However, transgene expression was infrequent, less than 1% of that observed for the endogenous gene, and varied as much as 500-fold, presumably due to the site of chromosomal integration. To determine whether a heterologous hormone-responsive enhancer could be used to increase both the level and frequency of expression in the mammary gland, a fragment derived from the mouse mammary tumor virus long terminal repeat containing four hormone response elements (HREs) was inserted into the R beta C promoter at a site not known to contain transcriptional regulatory elements. Transgenic mice generated which carried HRE-enhanced R beta C-CAT fusion genes expressed CAT activity in the mammary glands of all founder lines examined at levels that were on average 13-fold greater than for lines generated with similar constructs not carrying HREs. In the highest expressing line, the level of HRE-enhanced transgene expression was found to be developmentally regulated, increasing 14-fold in the mammary gland from virgin to day 10 of lactation. In this line, expression was also observed in the thymus and spleen; however, the level of CAT activity was 4-fold lower than in the mammary gland and was not developmentally regulated. In adrenalectomized mice, the administration of dexamethasone stimulated CAT expression in the mammary gland but not in the thymus and spleen. These studies demonstrate that in the context of the R beta C promoter, the HRE functions in the mammary gland to increase both the frequency and level of transgene expression.

Vector design for liver specific expression of multiple interfering RNAs that target hepatitis B virus transcripts

PubMed Central

Snyder, Lindsey L.; Esser, Jonathan M.; Pachuk, Catherine J.; Steel, Laura F.

2008-01-01

RNA interference (RNAi) is a process that can target intracellular RNAs for degradation in a highly sequence specific manner, making it a powerful tool that is being pursued in both research and therapeutic applications. Hepatitis B virus (HBV) is a serious public health problem in need of better treatment options, and aspects of its life cycle make it an excellent target for RNAi-based therapeutics. We have designed a vector that expresses interfering RNAs that target HBV transcripts, including both viral RNA replicative intermediates and mRNAs encoding viral proteins. Our vector design incorporates many features of endogenous microRNA (miRNA) gene organization that are proving useful for the development of reagents for RNAi. In particular, our vector contains an RNA pol II driven gene cassette that leads to tissue specific expression and efficient processing of multiple interfering RNAs from a single transcript, without the co-expression of any protein product. This vector shows potent silencing of HBV targets in cell culture models of HBV infection. The vector design will be applicable to silencing of additional cellular or disease-related genes. PMID:18499277

Integrative Bioinformatics and Functional Analyses of GEO, ENCODE, and TCGA Reveal FADD as a Direct Target of the Tumor Suppressor BRCA1.

PubMed

Nguyen, Dinh-Duc; Lee, Dong Gyu; Kim, Sinae; Kang, Keunsoo; Rhee, Je-Keun; Chang, Suhwan

2018-05-14

BRCA1 is a multifunctional tumor suppressor involved in several essential cellular processes. Although many of these functions are driven by or related to its transcriptional/epigenetic regulator activity, there has been no genome-wide study to reveal the transcriptional/epigenetic targets of BRCA1. Therefore, we conducted a comprehensive analysis of genomics/transcriptomics data to identify novel BRCA1 target genes. We first analyzed ENCODE data with BRCA1 chromatin immunoprecipitation (ChIP)-sequencing results and identified a set of genes with a promoter occupied by BRCA1. We collected 3085 loci with a BRCA1 ChIP signal from four cell lines and calculated the distance between the loci and the nearest gene transcription start site (TSS). Overall, 66.5% of the BRCA1-bound loci fell into a 2-kb region around the TSS, suggesting a role in transcriptional regulation. We selected 45 candidate genes based on gene expression correlation data, obtained from two GEO (Gene Expression Omnibus) datasets and TCGA data of human breast cancer, compared to BRCA1 expression levels. Among them, we further tested three genes ( MEIS2 , CKS1B and FADD ) and verified FADD as a novel direct target of BRCA1 by ChIP, RT-PCR, and a luciferase reporter assay. Collectively, our data demonstrate genome-wide transcriptional regulation by BRCA1 and suggest target genes as biomarker candidates for BRCA1-associated breast cancer.

Circadian and feeding rhythms differentially affect rhythmic mRNA transcription and translation in mouse liver

PubMed Central

Atger, Florian; Gobet, Cédric; Marquis, Julien; Martin, Eva; Wang, Jingkui; Weger, Benjamin; Lefebvre, Grégory; Descombes, Patrick; Naef, Felix; Gachon, Frédéric

2015-01-01

Diurnal oscillations of gene expression are a hallmark of rhythmic physiology across most living organisms. Such oscillations are controlled by the interplay between the circadian clock and feeding rhythms. Although rhythmic mRNA accumulation has been extensively studied, comparatively less is known about their transcription and translation. Here, we quantified simultaneously temporal transcription, accumulation, and translation of mouse liver mRNAs under physiological light–dark conditions and ad libitum or night-restricted feeding in WT and brain and muscle Arnt-like 1 (Bmal1)-deficient animals. We found that rhythmic transcription predominantly drives rhythmic mRNA accumulation and translation for a majority of genes. Comparison of wild-type and Bmal1 KO mice shows that circadian clock and feeding rhythms have broad impact on rhythmic gene expression, Bmal1 deletion affecting surprisingly both transcriptional and posttranscriptional levels. Translation efficiency is differentially regulated during the diurnal cycle for genes with 5′-Terminal Oligo Pyrimidine tract (5′-TOP) sequences and for genes involved in mitochondrial activity, many harboring a Translation Initiator of Short 5′-UTR (TISU) motif. The increased translation efficiency of 5′-TOP and TISU genes is mainly driven by feeding rhythms but Bmal1 deletion also affects amplitude and phase of translation, including TISU genes. Together this study emphasizes the complex interconnections between circadian and feeding rhythms at several steps ultimately determining rhythmic gene expression and translation. PMID:26554015

Integrative analyses of leprosy susceptibility genes indicate a common autoimmune profile.

PubMed

Zhang, Deng-Feng; Wang, Dong; Li, Yu-Ye; Yao, Yong-Gang

2016-04-01

Leprosy is an ancient chronic infection in the skin and peripheral nerves caused by Mycobacterium leprae. The development of leprosy depends on genetic background and the immune status of the host. However, there is no systematic view focusing on the biological pathways, interaction networks and overall expression pattern of leprosy-related immune and genetic factors. To identify the hub genes in the center of leprosy genetic network and to provide an insight into immune and genetic factors contributing to leprosy. We retrieved all reported leprosy-related genes and performed integrative analyses covering gene expression profiling, pathway analysis, protein-protein interaction network, and evolutionary analyses. A list of 123 differentially expressed leprosy related genes, which were enriched in activation and regulation of immune response, was obtained in our analyses. Cross-disorder analysis showed that the list of leprosy susceptibility genes was largely shared by typical autoimmune diseases such as lupus erythematosus and arthritis, suggesting that similar pathways might be affected in leprosy and autoimmune diseases. Protein-protein interaction (PPI) and positive selection analyses revealed a co-evolution network of leprosy risk genes. Our analyses showed that leprosy associated genes constituted a co-evolution network and might undergo positive selection driven by M. leprae. We suggested that leprosy may be a kind of autoimmune disease and the development of leprosy is a matter of defect or over-activation of body immunity. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Increased signaling entropy in cancer requires the scale-free property of protein interaction networks.

PubMed

Teschendorff, Andrew E; Banerji, Christopher R S; Severini, Simone; Kuehn, Reimer; Sollich, Peter

2015-04-28

One of the key characteristics of cancer cells is an increased phenotypic plasticity, driven by underlying genetic and epigenetic perturbations. However, at a systems-level it is unclear how these perturbations give rise to the observed increased plasticity. Elucidating such systems-level principles is key for an improved understanding of cancer. Recently, it has been shown that signaling entropy, an overall measure of signaling pathway promiscuity, and computable from integrating a sample's gene expression profile with a protein interaction network, correlates with phenotypic plasticity and is increased in cancer compared to normal tissue. Here we develop a computational framework for studying the effects of network perturbations on signaling entropy. We demonstrate that the increased signaling entropy of cancer is driven by two factors: (i) the scale-free (or near scale-free) topology of the interaction network, and (ii) a subtle positive correlation between differential gene expression and node connectivity. Indeed, we show that if protein interaction networks were random graphs, described by Poisson degree distributions, that cancer would generally not exhibit an increased signaling entropy. In summary, this work exposes a deep connection between cancer, signaling entropy and interaction network topology.

Increased signaling entropy in cancer requires the scale-free property of protein interaction networks

PubMed Central

Teschendorff, Andrew E.; Banerji, Christopher R. S.; Severini, Simone; Kuehn, Reimer; Sollich, Peter

2015-01-01

One of the key characteristics of cancer cells is an increased phenotypic plasticity, driven by underlying genetic and epigenetic perturbations. However, at a systems-level it is unclear how these perturbations give rise to the observed increased plasticity. Elucidating such systems-level principles is key for an improved understanding of cancer. Recently, it has been shown that signaling entropy, an overall measure of signaling pathway promiscuity, and computable from integrating a sample's gene expression profile with a protein interaction network, correlates with phenotypic plasticity and is increased in cancer compared to normal tissue. Here we develop a computational framework for studying the effects of network perturbations on signaling entropy. We demonstrate that the increased signaling entropy of cancer is driven by two factors: (i) the scale-free (or near scale-free) topology of the interaction network, and (ii) a subtle positive correlation between differential gene expression and node connectivity. Indeed, we show that if protein interaction networks were random graphs, described by Poisson degree distributions, that cancer would generally not exhibit an increased signaling entropy. In summary, this work exposes a deep connection between cancer, signaling entropy and interaction network topology. PMID:25919796

Development of visible light-responsive RNA scissors based on the 10-23 DNAzyme.

PubMed

Kamiya, Yukiko; Arimura, Yu; Ooi, Hideaki; Kato, Kenjiro; Liang, Xingguo; Asanuma, Hiroyuki

2018-04-22

10-23 DNAzyme is an artificially developed functional oligonucleotide, which can cleave RNA in a sequence-specific manner. In this study, we designed a new photo-driven DNAzyme possessing a photo-responsive DNA overhang complementary to the catalytic core region. The photo-responsive overhang region of the DNAzyme included either azobenzenes (Azos) or 2,6-dimethyl-4-(methylthio)azobenzenes (SDM-Azos) introduced via a D-threoninol linker. When the Azos or SDM-Azos were in the trans form, the photo-responsive DNA overhang hybridized with the DNAzyme, and the RNA cleavage activity was suppressed. Cis isomerization of Azos or SDM-Azos induced by 365 or 400 nm light, respectively, destabilized the duplex between the photo-responsive overhang and the catalytic core, and the DNAzyme recovered RNA cleavage activity. Reversible on and off of the DNAzyme activity was achieved by specific light irradiation. Further, light-dependent on and off of protein expression under the DNAzyme-containing condition was demonstrated. Thus, this photo-driven DNAzyme has potential for application in photo-controlled gene silencing system and a photo-activatable gene expression system. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A novel regulatory element (E77) isolated from CHO-K1 genomic DNA enhances stable gene expression in Chinese hamster ovary cells.

PubMed

Kang, Shin-Young; Kim, Yeon-Gu; Kang, Seunghee; Lee, Hong Weon; Lee, Eun Gyo

2016-05-01

Vectors flanked by regulatory DNA elements have been used to generate stable cell lines with high productivity and transgene stability; however, regulatory elements in Chinese hamster ovary (CHO) cells, which are the most widely used mammalian cells in biopharmaceutical production, are still poorly understood. We isolated a novel gene regulatory element from CHO-K1 cells, designated E77, which was found to enhance the stable expression of a transgene. A genomic library was constructed by combining CHO-K1 genomic DNA fragments with a CMV promoter-driven GFP expression vector, and the E77 element was isolated by screening. The incorporation of the E77 regulatory element resulted in the generation of an increased number of clones with high expression, thereby enhancing the expression level of the transgene in the stable transfectant cell pool. Interestingly, the E77 element was found to consist of two distinct fragments derived from different locations in the CHO genome shotgun sequence. High and stable transgene expression was obtained in transfected CHO cells by combining these fragments. Additionally, the function of E77 was found to be dependent on its site of insertion and specific orientation in the vector construct. Our findings demonstrate that stable gene expression mediated by the CMV promoter in CHO cells may be improved by the isolated novel gene regulatory element E77 identified in the present study. © 2016 The Authors. Biotechnology Journal published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Longitudinal Analysis of Whole Blood Transcriptomes to Explore Molecular Signatures Associated With Acute Renal Allograft Rejection

PubMed Central

Shin, Heesun; Günther, Oliver; Hollander, Zsuzsanna; Wilson-McManus, Janet E.; Ng, Raymond T.; Balshaw, Robert; Keown, Paul A.; McMaster, Robert; McManus, Bruce M.; Isbel, Nicole M.; Knoll, Greg; Tebbutt, Scott J.

2014-01-01

In this study, we explored a time course of peripheral whole blood transcriptomes from kidney transplantation patients who either experienced an acute rejection episode or did not in order to better delineate the immunological and biological processes measureable in blood leukocytes that are associated with acute renal allograft rejection. Using microarrays, we generated gene expression data from 24 acute rejectors and 24 nonrejectors. We filtered the data to obtain the most unambiguous and robustly expressing probe sets and selected a subset of patients with the clearest phenotype. We then performed a data-driven exploratory analysis using data reduction and differential gene expression analysis tools in order to reveal gene expression signatures associated with acute allograft rejection. Using a template-matching algorithm, we then expanded our analysis to include time course data, identifying genes whose expression is modulated leading up to acute rejection. We have identified molecular phenotypes associated with acute renal allograft rejection, including a significantly upregulated signature of neutrophil activation and accumulation following transplant surgery that is common to both acute rejectors and nonrejectors. Our analysis shows that this expression signature appears to stabilize over time in nonrejectors but persists in patients who go on to reject the transplanted organ. In addition, we describe an expression signature characteristic of lymphocyte activity and proliferation. This lymphocyte signature is significantly downregulated in both acute rejectors and nonrejectors following surgery; however, patients who go on to reject the organ show a persistent downregulation of this signature relative to the neutrophil signature. PMID:24526836

Transcriptional analysis of product-concentration driven changes in cellular programs of recombinant Clostridium acetobutylicumstrains.

PubMed

Tummala, Seshu B; Junne, Stefan G; Paredes, Carlos J; Papoutsakis, Eleftherios T

2003-12-30

Antisense RNA (asRNA) downregulation alters protein expression without changing the regulation of gene expression. Downregulation of primary metabolic enzymes possibly combined with overexpression of other metabolic enzymes may result in profound changes in product formation, and this may alter the large-scale transcriptional program of the cells. DNA-array based large-scale transcriptional analysis has the potential to elucidate factors that control cellular fluxes even in the absence of proteome data. These themes are explored in the study of large-scale transcriptional analysis programs and the in vivo primary-metabolism fluxes of several related recombinant C. acetobutylicum strains: C. acetobutylicum ATCC 824(pSOS95del) (plasmid control; produces high levels of butanol snd acetone), 824(pCTFB1AS) (expresses antisense RNA against CoA transferase (ctfb1-asRNA); produces very low levels of butanol and acetone), and 824(pAADB1) (expresses ctfb1-asRNA and the alcohol-aldehyde dahydrogenase gene (aad); produce high alcohol and low acetone levels). DNA-array based transcriptional analysis revealed that the large changes in product concentrations (snd notably butanol concentration) due to ctfb1-asRNA expression alone and in combination with aad overexpression resulted in dramatic changes of the cellular transcriptome. Cluster analysis and gene expression patterns of established and putative operons involved in stress response, motility, sporulation, and fatty-acid biosynthesis indicate that these simple genetic changes dramatically alter the cellular programs of C. acetobutylicum. Comparison of gene expression and flux analysis data may point to possible flux-controling steps and suggest unknown regulatory mechanisms. Copyright 2003; Wiley Periodicals, Inc.

Intraspecies differences in cold hardiness, carbohydrate content and β-amylase gene expression of Vaccinium corymbosum during cold acclimation and deacclimation.

PubMed

Lee, Jun Hyung; Yu, Duk Jun; Kim, Su Jin; Choi, Doil; Lee, Hee Jae

2012-12-01

Changes in cold hardiness, carbohydrate content and β-amylase gene expression were monitored in the shoots of the highbush blueberry (Vaccinium corymbosum L.) cultivars 'Sharpblue' and 'Jersey' during cold acclimation (CA) and deacclimation (DA). The seasonal patterns were similar in both cultivars, but the levels of cold hardiness determined by electrolyte leakage analysis were significantly different; 'Jersey' was hardier than 'Sharpblue'. Cold hardiness was closely related to total soluble sugar content (r = -0.98** and -0.99** for 'Sharpblue' and 'Jersey', respectively). In 'Jersey', more soluble sugars accumulated during CA. Of the detected soluble sugars, glucose, fructose and raffinose contents were significantly associated with cold hardiness in both cultivars. Sucrose was abundant in both cultivars, and stachyose content changed significantly during CA and DA. However, they were not associated with cold hardiness. A sharp decrease in starch contents in the middle of CA coincided with β-amylase gene (VcBMY) expression, indicating the conversion of starch into soluble sugars. During CA, VcBMY was expressed up to twofold higher in 'Jersey' than in 'Sharpblue'. These results suggest that intraspecies differences in the cold hardiness of highbush blueberries are associated with total soluble sugar content, which is driven partly by differential expression of VcBMY.

Genome-wide characterization and expression analysis enables identification of abiotic stress-responsive MYB transcription factors in cassava (Manihot esculenta).

PubMed

Ruan, Meng-Bin; Guo, Xin; Wang, Bin; Yang, Yi-Ling; Li, Wen-Qi; Yu, Xiao-Ling; Zhang, Peng; Peng, Ming

2017-06-15

The myeloblastosis (MYB) transcription factor superfamily is the largest transcription factor family in plants, playing different roles during stress response. However, abiotic stress-responsive MYB transcription factors have not been systematically studied in cassava (Manihot esculenta), an important tropical tuber root crop. In this study, we used a genome-wide transcriptome analysis to predict 299 putative MeMYB genes in the cassava genome. Under drought and cold stresses, many MeMYB genes exhibited different expression patterns in cassava leaves, indicating that these genes might play a role in abiotic stress responses. We found that several stress-responsive MeMYB genes responded to abscisic acid (ABA) in cassava leaves. We characterize four MeMYBs, namely MeMYB1, MeMYB2, MeMYB4, and MeMYB9, as R2R3-MYB transcription factors. Furthermore, RNAi-driven repression of MeMYB2 resulted in drought and cold tolerance in transgenic cassava. Gene expression assays in wild-type and MeMYB2-RNAi cassava plants revealed that MeMYB2 may affect other MeMYBs as well as MeWRKYs under drought and cold stress, suggesting crosstalk between MYB and WRKY family genes under stress conditions in cassava. © The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Oxidized Guanine Base Lesions Function in 8-Oxoguanine DNA Glycosylase-1-mediated Epigenetic Regulation of Nuclear Factor κB-driven Gene Expression*

PubMed Central

Pan, Lang; Hao, Wenjing; Ba, Xueqing

2016-01-01

A large percentage of redox-responsive gene promoters contain evolutionarily conserved guanine-rich clusters; guanines are the bases most susceptible to oxidative modification(s). Consequently, 7,8-dihydro-8-oxoguanine (8-oxoG) is one of the most abundant base lesions in promoters and is primarily repaired via the 8-oxoguanine DNA glycosylase-1 (OOG1)-initiated base excision repair pathway. In view of a prompt cellular response to oxidative challenge, we hypothesized that the 8-oxoG lesion and the cognate repair protein OGG1 are utilized in transcriptional gene activation. Here, we document TNFα-induced enrichment of both 8-oxoG and OGG1 in promoters of pro-inflammatory genes, which precedes interaction of NF-κB with its DNA-binding motif. OGG1 bound to 8-oxoG upstream from the NF-κB motif increased its DNA occupancy by promoting an on-rate of both homodimeric and heterodimeric forms of NF-κB. OGG1 depletion decreased both NF-κB binding and gene expression, whereas Nei-like glycosylase-1 and -2 had a marginal effect. These results are the first to document a novel paradigm wherein the DNA repair protein OGG1 bound to its substrate is coupled to DNA occupancy of NF-κB and functions in epigenetic regulation of gene expression. PMID:27756845

Efficiency of VIGS and gene expression in a novel bipartite potexvirus vector delivery system as a function of strength of TGB1 silencing suppression.

PubMed

Lim, Hyoun-Sub; Vaira, Anna Maria; Domier, Leslie L; Lee, Sung Chul; Kim, Hong Gi; Hammond, John

2010-06-20

We have developed plant virus-based vectors for virus-induced gene silencing (VIGS) and protein expression, based on Alternanthera mosaic virus (AltMV), for infection of a wide range of host plants including Nicotiana benthamiana and Arabidopsis thaliana by either mechanical inoculation of in vitro transcripts or via agroinfiltration. In vivo transcripts produced by co-agroinfiltration of bacteriophage T7 RNA polymerase resulted in T7-driven AltMV infection from a binary vector in the absence of the Cauliflower mosaic virus 35S promoter. An artificial bipartite viral vector delivery system was created by separating the AltMV RNA-dependent RNA polymerase and Triple Gene Block (TGB)123-Coat protein (CP) coding regions into two constructs each bearing the AltMV 5' and 3' non-coding regions, which recombined in planta to generate a full-length AltMV genome. Substitution of TGB1 L(88)P, and equivalent changes in other potexvirus TGB1 proteins, affected RNA silencing suppression efficacy and suitability of the vectors from protein expression to VIGS. Published by Elsevier Inc.

Population transcriptomics with single-cell resolution: a new field made possible by microfluidics: a technology for high throughput transcript counting and data-driven definition of cell types.

PubMed

Plessy, Charles; Desbois, Linda; Fujii, Teruo; Carninci, Piero

2013-02-01

Tissues contain complex populations of cells. Like countries, which are comprised of mixed populations of people, tissues are not homogeneous. Gene expression studies that analyze entire populations of cells from tissues as a mixture are blind to this diversity. Thus, critical information is lost when studying samples rich in specialized but diverse cells such as tumors, iPS colonies, or brain tissue. High throughput methods are needed to address, model and understand the constitutive and stochastic differences between individual cells. Here, we describe microfluidics technologies that utilize a combination of molecular biology and miniaturized labs on chips to study gene expression at the single cell level. We discuss how the characterization of the transcriptome of each cell in a sample will open a new field in gene expression analysis, population transcriptomics, that will change the academic and biomedical analysis of complex samples by defining them as quantified populations of single cells. Copyright © 2013 WILEY Periodicals, Inc.

Whole-Genome Positive Selection and Habitat-Driven Evolution in a Shallow and a Deep-Sea Urchin

PubMed Central

Oliver, Thomas A.; Garfield, David A.; Manier, Mollie K.; Haygood, Ralph; Wray, Gregory A.; Palumbi, Stephen R.

2010-01-01

Comparisons of genomic sequence between divergent species can provide insight into the action of natural selection across many distinct classes of proteins. Here, we examine the extent of positive selection as a function of tissue-specific and stage-specific gene expression in two closely-related sea urchins, the shallow-water Strongylocentrotus purpuratus and the deep-sea Allocentrotus fragilis, which have diverged greatly in their adult but not larval habitats. Genes that are expressed specifically in adult somatic tissue have significantly higher dN/dS ratios than the genome-wide average, whereas those in larvae are indistinguishable from the genome-wide average. Testis-specific genes have the highest dN/dS values, whereas ovary-specific have the lowest. Branch-site models involving the outgroup S. franciscanus indicate greater selection (ωFG) along the A. fragilis branch than along the S. purpuratus branch. The A. fragilis branch also shows a higher proportion of genes under positive selection, including those involved in skeletal development, endocytosis, and sulfur metabolism. Both lineages are approximately equal in enrichment for positive selection of genes involved in immunity, development, and cell–cell communication. The branch-site models further suggest that adult-specific genes have experienced greater positive selection than those expressed in larvae and that ovary-specific genes are more conserved (i.e., experienced greater negative selection) than those expressed specifically in adult somatic tissues and testis. Our results chart the patterns of protein change that have occurred after habitat divergence in these two species and show that the developmental or functional context in which a gene acts can play an important role in how divergent species adapt to new environments. PMID:20935062

Expression of organophosphorus-degradation gene ( opd) in aggregating and non-aggregating filamentous nitrogen-fixing cyanobacteria

NASA Astrophysics Data System (ADS)

Li, Qiong; Tang, Qing; Xu, Xudong; Gao, Hong

2010-11-01

Genetic engineering in filamentous N2-fixing cyanobacteria usually involves Anabaena sp. PCC 7120 and several other non-aggregating species. Mass culture and harvest of such species are more energy consuming relative to aggregating species. To establish a gene transfer system for aggregating species, we tested many species of Anabaena and Nostoc, and identified Nostoc muscorum FACHB244 as a species that can be genetically manipulated using the conjugative gene transfer system. To promote biodegradation of organophosphorus pollutants in aquatic environments, we introduced a plasmid containing the organophosphorus-degradation gene ( opd) into Anabaena sp. PCC 7120 and Nostoc muscorum FACHB244 by conjugation. The opd gene was driven by a strong promoter, P psbA . From both species, we obtained transgenic strains having organophosphorus-degradation activities. At 25°C, the whole-cell activities of the transgenic Anabaena and Nostoc strains were 0.163±0.001 and 0.289±0.042 unit/μg Chl a, respectively. However, most colonies resulting from the gene transfer showed no activity. PCR and DNA sequencing revealed deletions or rearrangements in the plasmid in some of the colonies. Expression of the green fluorescent protein gene from the same promoter in Anabaena sp. PCC 7120 showed similar results. These results suggest that there is the potential to promote the degradation of organophosphorus pollutants with transgenic cyanobacteria and that selection of high-expression transgenic colonies is important for genetic engineering of Anabaena and Nostoc species. For the first time, we established a gene transfer and expression system in an aggregating filamentous N2-fixing cyanobacterium. The genetic manipulation system of Nostoc muscorum FACHB244 could be utilized in the elimination of pollutants and large-scale production of valuable proteins or metabolites.

Transposon-driven transcription is a conserved feature of vertebrate spermatogenesis and transcript evolution.

PubMed

Davis, Matthew P; Carrieri, Claudia; Saini, Harpreet K; van Dongen, Stijn; Leonardi, Tommaso; Bussotti, Giovanni; Monahan, Jack M; Auchynnikava, Tania; Bitetti, Angelo; Rappsilber, Juri; Allshire, Robin C; Shkumatava, Alena; O'Carroll, Dónal; Enright, Anton J

2017-07-01

Spermatogenesis is associated with major and unique changes to chromosomes and chromatin. Here, we sought to understand the impact of these changes on spermatogenic transcriptomes. We show that long terminal repeats (LTRs) of specific mouse endogenous retroviruses (ERVs) drive the expression of many long non-coding transcripts (lncRNA). This process occurs post-mitotically predominantly in spermatocytes and round spermatids. We demonstrate that this transposon-driven lncRNA expression is a conserved feature of vertebrate spermatogenesis. We propose that transposon promoters are a mechanism by which the genome can explore novel transcriptional substrates, increasing evolutionary plasticity and allowing for the genesis of novel coding and non-coding genes. Accordingly, we show that a small fraction of these novel ERV-driven transcripts encode short open reading frames that produce detectable peptides. Finally, we find that distinct ERV elements from the same subfamilies act as differentially activated promoters in a tissue-specific context. In summary, we demonstrate that LTRs can act as tissue-specific promoters and contribute to post-mitotic spermatogenic transcriptome diversity. © 2017 The Authors. Published under the terms of the CC BY 4.0 license.

DDB1-Mediated CRY1 Degradation Promotes FOXO1-Driven Gluconeogenesis in Liver.

PubMed

Tong, Xin; Zhang, Deqiang; Charney, Nicholas; Jin, Ethan; VanDommelen, Kyle; Stamper, Kenneth; Gupta, Neil; Saldate, Johnny; Yin, Lei

2017-10-01

Targeted protein degradation through ubiquitination is an important step in the regulation of glucose metabolism. Here, we present evidence that the DDB1-CUL4A ubiquitin E3 ligase functions as a novel metabolic regulator that promotes FOXO1-driven hepatic gluconeogenesis. In vivo, hepatocyte-specific Ddb1 deletion leads to impaired hepatic gluconeogenesis in the mouse liver but protects mice from high-fat diet-induced hyperglycemia. Lack of Ddb1 downregulates FOXO1 protein expression and impairs FOXO1-driven gluconeogenic response. Mechanistically, we discovered that DDB1 enhances FOXO1 protein stability via degrading the circadian protein cryptochrome 1 (CRY1), a known target of DDB1 E3 ligase. In the Cry1 depletion condition, insulin fails to reduce the nuclear FOXO1 abundance and suppress gluconeogenic gene expression. Chronic depletion of Cry1 in the mouse liver not only increases FOXO1 protein but also enhances hepatic gluconeogenesis. Thus, we have identified the DDB1-mediated CRY1 degradation as an important target of insulin action on glucose homeostasis. © 2017 by the American Diabetes Association.

Rapid evolution and copy number variation of primate RHOXF2, an X-linked homeobox gene involved in male reproduction and possibly brain function.

PubMed

Niu, Ao-lei; Wang, Yin-qiu; Zhang, Hui; Liao, Cheng-hong; Wang, Jin-kai; Zhang, Rui; Che, Jun; Su, Bing

2011-10-12

Homeobox genes are the key regulators during development, and they are in general highly conserved with only a few reported cases of rapid evolution. RHOXF2 is an X-linked homeobox gene in primates. It is highly expressed in the testicle and may play an important role in spermatogenesis. As male reproductive system is often the target of natural and/or sexual selection during evolution, in this study, we aim to dissect the pattern of molecular evolution of RHOXF2 in primates and its potential functional consequence. We studied sequences and copy number variation of RHOXF2 in humans and 16 nonhuman primate species as well as the expression patterns in human, chimpanzee, white-browed gibbon and rhesus macaque. The gene copy number analysis showed that there had been parallel gene duplications/losses in multiple primate lineages. Our evidence suggests that 11 nonhuman primate species have one RHOXF2 copy, and two copies are present in humans and four Old World monkey species, and at least 6 copies in chimpanzees. Further analysis indicated that the gene duplications in primates had likely been mediated by endogenous retrovirus (ERV) sequences flanking the gene regions. In striking contrast to non-human primates, humans appear to have homogenized their two RHOXF2 copies by the ERV-mediated non-allelic recombination mechanism. Coding sequence and phylogenetic analysis suggested multi-lineage strong positive selection on RHOXF2 during primate evolution, especially during the origins of humans and chimpanzees. All the 8 coding region polymorphic sites in human populations are non-synonymous, implying on-going selection. Gene expression analysis demonstrated that besides the preferential expression in the reproductive system, RHOXF2 is also expressed in the brain. The quantitative data suggests expression pattern divergence among primate species. RHOXF2 is a fast-evolving homeobox gene in primates. The rapid evolution and copy number changes of RHOXF2 had been driven by Darwinian positive selection acting on the male reproductive system and possibly also on the central nervous system, which sheds light on understanding the role of homeobox genes in adaptive evolution.

Periodic regulation of expression of genes for kisspeptin, gonadotropin-inhibitory hormone and their receptors in the grass puffer: Implications in seasonal, daily and lunar rhythms of reproduction.

PubMed

Ando, Hironori; Shahjahan, Md; Kitahashi, Takashi

2018-04-03

The seasonal, daily and lunar control of reproduction involves photoperiodic, circadian and lunar changes in the activity of kisspeptin, gonadotropin-inhibitory hormone (GnIH) and gonadotropin-releasing hormone (GnRH) neurons. These changes are brought through complex networks of light-, time- and non-photic signal-dependent control mechanisms, which are mostly unknown at present. The grass puffer, Takifugu alboplumbeus, a semilunar spawner, provides a unique and excellent animal model to assess this question because its spawning is synchronized with seasonal, daily and lunar cycles. In the diencephalon, the genes for kisspeptin, GnIH and their receptors showed similar expression patterns with clear seasonal and daily oscillations, suggesting that they are regulated by common mechanisms involving melatonin, circadian clock and water temperature. For implications in semilunar-synchronized spawning rhythm, melatonin receptor genes showed ultradian oscillations in expression with the period of 14.0-15.4 h in the pineal gland. This unique ultradian rhythm might be driven by circatidal clock. The possible circatidal clock and circadian clock in the pineal gland may cooperate to drive circasemilunar rhythm to regulate the expression of the kisspeptin, GnIH and their receptor genes. On the other hand, high temperature (over 28 °C) conditions, under which the expression of the kisspeptin and its receptor genes is markedly suppressed, may provide an environmental signal that terminates reproduction at the end of breeding period. Taken together, the periodic regulation of the kisspeptin, GnIH and their receptor genes by melatonin, circadian clock and water temperature may be important in the precisely-timed spawning of the grass puffer. Copyright © 2018 Elsevier Inc. All rights reserved.

Histone deacetylase 3 regulates the inflammatory gene expression programme of rheumatoid arthritis fibroblast-like synoviocytes

PubMed Central

Angiolilli, Chiara; Kabala, Pawel A; Van Baarsen, Iris M; Ferguson, Bradley S; García, Samuel; Malvar Fernandez, Beatriz; McKinsey, Timothy A; Tak, Paul P; Fossati, Gianluca; Mascagni, Paolo; Baeten, Dominique L; Reedquist, Kris A

2017-01-01

Objectives Non-selective histone deacetylase (HDAC) inhibitors (HDACi) have demonstrated anti-inflammatory properties in both in vitro and in vivo models of rheumatoid arthritis (RA). Here, we investigated the potential contribution of specific class I and class IIb HDACs to inflammatory gene expression in RA fibroblast-like synoviocytes (FLS). Methods RA FLS were incubated with pan-HDACi (ITF2357, givinostat) or selective HDAC1/2i, HDAC3/6i, HDAC6i and HDAC8i. Alternatively, FLS were transfected with HDAC3, HDAC6 or interferon (IFN)-α/β receptor alpha chain (IFNAR1) siRNA. mRNA expression of interleukin (IL)-1β-inducible genes was measured by quantitative PCR (qPCR) array and signalling pathway activation by immunoblotting and DNA-binding assays. Results HDAC3/6i, but not HDAC1/2i and HDAC8i, significantly suppressed the majority of IL-1β-inducible genes targeted by pan-HDACi in RA FLS. Silencing of HDAC3 expression reproduced the effects of HDAC3/6i on gene regulation, contrary to HDAC6-specific inhibition and HDAC6 silencing. Screening of the candidate signal transducers and activators of transcription (STAT)1 transcription factor revealed that HDAC3/6i abrogated STAT1 Tyr701 phosphorylation and DNA binding, but did not affect STAT1 acetylation. HDAC3 activity was required for type I IFN production and subsequent STAT1 activation in FLS. Suppression of type I IFN release by HDAC3/6i resulted in reduced expression of a subset of IFN-dependent genes, including the chemokines CXCL9 and CXCL11. Conclusions Inhibition of HDAC3 in RA FLS largely recapitulates the effects of pan-HDACi in suppressing inflammatory gene expression, including type I IFN production in RA FLS. Our results identify HDAC3 as a potential therapeutic target in the treatment of RA and type I IFN-driven autoimmune diseases. PMID:27457515

Dietary tomato and lycopene impact androgen signaling- and carcinogenesis-related gene expression during early TRAMP prostate carcinogenesis

PubMed Central

Wan, Lei; Tan, Hsueh-Li; Thomas-Ahner, Jennifer M.; Pearl, Dennis K.; Erdman, John W.; Moran, Nancy E.; Clinton, Steven K.

2014-01-01

Consumption of tomato products containing the carotenoid lycopene is associated with a reduced risk of prostate cancer. To identify gene expression patterns associated with early testosterone-driven prostate carcinogenesis, which are impacted by dietary tomato and lycopene, wild type (WT) and transgenic adenocarcinoma of the mouse prostate (TRAMP) mice were fed control or tomato- or lycopene-containing diets from 4-10 wk-of-age. Eight-week-old mice underwent sham surgery, castration, or castration followed by testosterone-repletion (2.5 mg/kg/d initiated 1 wk after castration). Ten-wk-old intact TRAMP mice exhibit early multifocal prostatic intraepithelial neoplasia (PIN). Of the 200 prostate cancer-related genes measured by quantitative NanoString®, 189 are detectable, 164 significantly differ by genotype, 179 by testosterone status, and 30 by diet type (P<0.05). In TRAMP, expression of Birc5, Mki67, Aurkb, Ccnb2, Foxm1, and Ccne2 is greater compared to WT and is decreased by castration. In parallel, castration reduces Ki67-positive staining (P<0.0001) compared to intact and testosterone-repleted TRAMP mice. Expression of genes involved in androgen metabolism/signaling pathways are reduced by lycopene feeding (Srd5a1) and by tomato-feeding (Srd5a2, Pxn, and Srebf1). Additionally, tomato-feeding significantly reduced expression of genes associated with stem cell features, Aldh1a and Ly6a, while lycopene-feeding significantly reduced expression of neuroendocrine differentiation-related genes, Ngfr and Syp. Collectively, these studies demonstrate a profile of testosterone-regulated genes associated with early stages of prostate carcinogenesis that are potential mechanistic targets of dietary tomato components. Future studies on androgen signaling/metabolism, stem cell features, and neuroendocrine differentiation pathways may elucidate the mechanisms by which dietary tomato and lycopene impact prostate cancer risk. PMID:25315431

Elevated toll-like receptor 4 expression and signaling in muscle from insulin-resistant subjects.

PubMed

Reyna, Sara M; Ghosh, Sangeeta; Tantiwong, Puntip; Meka, C S Reddy; Eagan, Phyllis; Jenkinson, Christopher P; Cersosimo, Eugenio; Defronzo, Ralph A; Coletta, Dawn K; Sriwijitkamol, Apiradee; Musi, Nicolas

2008-10-01

OBJECTIVE- Tall-like receptor (TLR)4 has been implicated in the pathogenesis of free fatty acid (FFA)-induced insulin resistance by activating inflammatory pathways, including inhibitor of kappaB (IkappaB)/nuclear factor kappaB (NFkappaB). However, it is not known whether insulin-resistant subjects have abnormal TLR4 signaling. We examined whether insulin-resistant subjects have abnormal TLR4 expression and TLR4-driven (IkappaB/NFkappaB) signaling in skeletal muscle. RESEARCH DESIGN AND METHODS- TLR4 gene expression and protein content were measured in muscle biopsies in 7 lean, 8 obese, and 14 type 2 diabetic subjects. A primary human myotube culture system was used to examine whether FFAs stimulate IkappaB/NFkappaB via TLR4 and whether FFAs increase TLR4 expression/content in muscle. RESULTS- Obese and type 2 diabetic subjects had significantly elevated TLR4 gene expression and protein content in muscle. TLR4 muscle protein content correlated with the severity of insulin resistance. Obese and type 2 diabetic subjects also had lower IkappaBalpha content, an indication of elevated IkappaB/NFkappaB signaling. The increase in TLR4 and NFkappaB signaling was accompanied by elevated expression of the NFkappaB-regulated genes interleukin (IL)-6 and superoxide dismutase (SOD)2. In primary human myotubes, acute palmitate treatment stimulated IkappaB/NFkappaB, and blockade of TLR4 prevented the ability of palmitate to stimulate the IkappaB/NFkappaB pathway. Increased TLR4 content and gene expression observed in muscle from insulin-resistant subjects were reproduced by treating myotubes from lean, normal-glucose-tolerant subjects with palmitate. Palmitate also increased IL-6 and SOD2 gene expression, and this effect was prevented by inhibiting NFkappaB. CONCLUSIONS- Abnormal TLR4 expression and signaling, possibly caused by elevated plasma FFA levels, may contribute to the pathogenesis of insulin resistance in humans.

Expression of phytoene synthase1 and carotene desaturase crtI genes result in an increase in the total carotenoids content in transgenic elite wheat (Triticum aestivum L.).

PubMed

Cong, Ling; Wang, Cheng; Chen, Ling; Liu, Huijuan; Yang, Guangxiao; He, Guangyuan

2009-09-23

Dietary micronutrient deficiencies, such as the lack of vitamin A, are a major source of morbidity and mortality worldwide. Carotenoids in food can function as provitamin A in humans, while grains of Chinese elite wheat cultivars generally have low carotenoid contents. To increase the carotenoid contents in common wheat endosperm, transgenic wheat has been generated by expressing the maize y1 gene encoding phytoene synthase driven by a endosperm-specific 1Dx5 promoter in the elite wheat (Triticum aestivum L.) variety EM12, together with the bacterial phytoene desaturase crtI gene from Erwinia uredovora under the constitutive CaMV 35S promoter control. A clear increase of the carotenoid content was detected in the endosperms of transgenic wheat that visually showed a light yellow color. The total carotenoids content was increased up to 10.8-fold as compared with the nontransgenic EM12 cultivar. To test whether the variability of total carotenoid content in different transgenic lines was due to differences in the transgene copy number or expression pattern, Southern hybridization and semiquantitative reverse transcriptase polymerase chain reaction analyses were curried out. The results showed that transgene copy numbers and transcript levels did not associate well with carotenoid contents. The expression patterns of endogenous carotenoid genes, such as the phytoene synthases and carotene desaturases, were also investigated in wild-type and transgenic wheat lines. No significant changes in expression levels of these genes were detected in the transgenic endosperms, indicating that the increase in carotenoid transgenic wheat endosperms resulted from the expression of transgenes.

Expression of metastasis suppressor gene AES driven by a Yin Yang (YY) element in a CpG island promoter and transcription factor YY2.

PubMed

Kakizaki, Fumihiko; Sonoshita, Masahiro; Miyoshi, Hiroyuki; Itatani, Yoshiro; Ito, Shinji; Kawada, Kenji; Sakai, Yoshiharu; Taketo, M Mark

2016-11-01

We recently found that the product of the AES gene functions as a metastasis suppressor of colorectal cancer (CRC) in both humans and mice. Expression of amino-terminal enhancer of split (AES) protein is significantly decreased in liver metastatic lesions compared with primary colon tumors. To investigate its downregulation mechanism in metastases, we searched for transcriptional regulators of AES in human CRC and found that its expression is reduced mainly by transcriptional dysregulation and, in some cases, by additional haploidization of its coding gene. The AES promoter-enhancer is in a typical CpG island, and contains a Yin-Yang transcription factor recognition sequence (YY element). In human epithelial cells of normal colon and primary tumors, transcription factor YY2, a member of the YY family, binds directly to the YY element, and stimulates expression of AES. In a transplantation mouse model of liver metastases, however, expression of Yy2 (and therefore of Aes) is downregulated. In human CRC metastases to the liver, the levels of AES protein are correlated with those of YY2. In addition, we noticed copy-number reduction for the AES coding gene in chromosome 19p13.3 in 12% (5/42) of human CRC cell lines. We excluded other mechanisms such as point or indel mutations in the coding or regulatory regions of the AES gene, CpG methylation in the AES promoter enhancer, expression of microRNAs, and chromatin histone modifications. These results indicate that Aes may belong to a novel family of metastasis suppressors with a CpG-island promoter enhancer, and it is regulated transcriptionally. © 2016 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

Mechanisms of allele-selective down-regulation of HLA class I in Burkitt's lymphoma.

PubMed

Imreh, M P; Zhang, Q J; de Campos-Lima, P O; Imreh, S; Krausa, P; Browning, M; Klein, G; Masucci, M G

1995-07-04

Burkitt lymphomas (BL) that arise in HLA-AII-positive individuals are characterized by selective loss/down-regulation of the HLA AII polypeptide. We have investigated the molecular basis of such down-regulation by comparing 5 pairs of BL lines and Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines (LCL) derived from the normal B cells of the same individuals. The presence of apparently intact HLA AII genes was confirmed in all 5 BL/LCL pairs by polymerase chain reaction (PCR) typing and by Southern-blot hybridization with HLA A locus-specific probes. Northern-blot analysis with locus- and allele-specific probes revealed a significantly lower expression or absence of AII-specific mRNA in all 5 BL lines compared to the corresponding LCLs. Up-regulation of AII-specific mRNA was achieved by IFN alpha treatment of 2 BL lines with low HLA AII expression (BL-28 and BL-72) while the treatment had no effect in 3 BL lines (WWI-BL, WW2-BL and BL41) that did not express the endogenous gene. HLA AII expression was restored by transfection of the gene in WWI-BL whereas transfectants of BL-41 remained AII-negative. An HLA-AII-promoter-driven chloramphenicol acetyl transferase reporter gene (pAIICAT) was active in WWI-BL but not in BL-41. HLA-AII was expressed in hybrids of BL-41 with an AII-positive LCL, while expression of the endogenous HLA AII gene could not be restored by fusion of BL-41 with an AII-negative LCL, although an adequate set of transcription factors was present in the hybrid. Our results suggest that genetic defects and lack of transcription factors may contribute to the selective down-regulation of HLA AII in BL cells.

A Celiac Diasease Associated lncRNA Named HCG14 Regulates NOD1 Expression in Intestinal Cells.

PubMed

Santin, Izortze; Jauregi-Miguel, Amaia; Velayos, Teresa; Castellanos-Rubio, Ainara; Garcia-Etxebarria, Koldo; Romero-Garmendia, Irati; Fernandez-Jimenez, Nora; Irastorza, Iñaki; Castaño, Luis; Bilbao, Jose Ramón

2018-03-29

To identify additional celiac disease associated loci in the Major Histocompatibility Complex independent from classical HLA risk alleles (HLA-DR3-DQ2) and to characterize their potential functional impact in celiac disease pathogenesis at the intestinal level. We performed a high resolution SNP genotyping of the MHC region, comparing HLA-DR3 homozygous celiac patients and non-celiac controls carrying a single copy of the B8-DR3-DQ2 conserved extended haplotype. Expression level of potential novel risk genes was determined by RT-PCR in intestinal biopsies and in intestinal and immune cells isolated from control and celiac individuals. Small interfering RNA-driven silencing of selected genes was performed in the intestinal cell line T84. MHC genotyping revealed two associated SNPs, one located in TRIM27 gene and another in the non-coding gene HCG14. After stratification analysis, only HCG14 showed significant association independent from HLA-DR-DQ loci Expression of HCG14 was slightly downregulated in epithelial cells isolated from duodenal biopsies of celiac patients, and eQTL analysis revealed that polymorphisms in HCG14 region were associated with decreased NOD1 expression in duodenal intestinal cells. We have sucessfully employed a conserved extended haplotype-matching strategy and identified a novel additional celiac disease risk variant in the lncRNA HGC14. This lncRNA seems to regulate the expression of NOD1 in an allele-specific manner. Further functional studies are needed to clarify the role of HCG14 in the regulation of gene expression and to determine the molecular mechanisms by which the risk variant in HCG14 contributes to celiac disease pathogenesis.

KRAS driven expression signature has prognostic power superior to mutation status in non-small cell lung cancer.

PubMed

Nagy, Ádám; Pongor, Lőrinc Sándor; Szabó, András; Santarpia, Mariacarmela; Győrffy, Balázs

2017-02-15

KRAS is the most frequently mutated oncogene in non-small cell lung cancer (NSCLC). However, the prognostic role of KRAS mutation status in NSCLC still remains controversial. We hypothesize that the expression changes of genes affected by KRAS mutation status will have the most prominent effect and could be used as a prognostic signature in lung cancer. We divided NSCLC patients with mutation and RNA-seq data into KRAS mutated and wild type groups. Mann-Whitney test was used to identify genes showing altered expression between these cohorts. Mean expression of the top five genes was designated as a "transcriptomic fingerprint" of the mutation. We evaluated the effect of this signature on clinical outcome in 2,437 NSCLC patients using univariate and multivariate Cox regression analysis. Mutation of KRAS was most common in adenocarcinoma. Mutation status and KRAS expression were not correlated to prognosis. The transcriptomic fingerprint of KRAS include FOXRED2, KRAS, TOP1, PEX3 and ABL2. The KRAS signature had a high prognostic power. Similar results were achieved when using the second and third set of strongest genes. Moreover, all cutoff values delivered significant prognostic power (p < 0.01). The KRAS signature also remained significant (p < 0.01) in a multivariate analysis including age, gender, smoking history and tumor stage. We generated a "surrogate signature" of KRAS mutation status in NSCLC patients by computationally linking genotype and gene expression. We show that secondary effects of a mutation can have a higher prognostic relevance than the primary genetic alteration itself. © 2016 The Authors International Journal of Cancer published by John Wiley & Sons Ltd on behalf of UICC.

Lack of promoter IV-driven BDNF transcription results in depression-like behavior.

PubMed

Sakata, K; Jin, L; Jha, S

2010-10-01

Transcription of Bdnf is controlled by multiple promoters, in which promoter IV contributes significantly to activity-dependent Bdnf transcription. We have generated promoter IV mutant mice [brain-derived neurotrophic factor (BDNF)-KIV] in which promoter IV-driven expression of BDNF is selectively disrupted by inserting a green fluorescent protein (GFP)-STOP cassette within the Bdnf exon IV locus. BDNF-KIV animals exhibited depression-like behavior as shown by the tail suspension test (TST), sucrose preference test (SPT) and learned helplessness test (LHT). In addition, BDNF-KIV mice showed reduced activity in the open field test (OFT) and reduced food intake in the novelty-suppressed feeding test (NSFT). The mutant mice did not display anxiety-like behavior in the light and dark box test and elevated plus maze tests. Interestingly, the mutant mice showed defective response inhibition in the passive avoidance test (PAT) even though their learning ability was intact when measured with the active avoidance test (AAT). These results suggest that promoter IV-dependent BDNF expression plays a critical role in the control of mood-related behaviors. This is the first study that directly addressed the effects of endogenous promoter-driven expression of BDNF in depression-like behavior. © 2010 The Authors. Genes, Brain and Behavior © 2010 Blackwell Publishing Ltd and International Behavioural and Neural Genetics Society.

Mutagenesis of diploid mammalian genes by gene entrapment

PubMed Central

Lin, Qing; Donahue, Sarah L.; Moore-Jarrett, Tracy; Cao, Shang; Osipovich, Anna B.; Ruley, H. Earl

2006-01-01

The present study describes a genome-wide method for biallelic mutagenesis in mammalian cells. Novel poly(A) gene trap vectors, which contain features for direct cloning vector–cell fusion transcripts and for post-entrapment genome engineering, were used to generate a library of 979 mutant ES cells. The entrapment mutations generally disrupted gene expression and were readily transmitted through the germline, establishing the library as a resource for constructing mutant mice. Cells homozygous for most entrapment loci could be isolated by selecting for enhanced expression of an inserted neomycin-resistance gene that resulted from losses of heterozygosity (LOH). The frequencies of LOH measured at 37 sites in the genome ranged from 1.3 × 10−5 to 1.2 × 10−4 per cell and increased with increasing distance from the centromere, implicating mitotic recombination in the process. The ease and efficiency of obtaining homozygous mutations will (i) facilitate genetic studies of gene function in cultured cells, (ii) permit genome-wide studies of recombination events that result in LOH and mediate a type of chromosomal instability important in carcinogenesis, and (iii) provide new strategies for phenotype-driven mutagenesis screens in mammalian cells. PMID:17062627

Overproduction of recombinant laccase using a homologous expression system in Coriolus versicolor.

PubMed

Kajita, Shinya; Sugawara, Shinsuke; Miyazaki, Yasumasa; Nakamura, Masaya; Katayama, Yoshihiro; Shishido, Kazuo; Iimura, Yosuke

2004-12-01

One of the major extracellular enzymes of the white-rot fungus Coriolus versicolor is laccase, which is involved in the degradation of lignin. We constructed a homologous system for the expression of a gene for laccase III (cvl3) in C. versicolor, using a chimeric laccase gene driven by the promoter of a gene for glyceraldehyde-3-phosphate dehydrogenase (gpd) from this fungus. We transformed C. versicolor successfully by introducing both a gene for hygromycin B phosphotransferase (hph) and the chimeric laccase gene. In three independent experiments, we recovered 47 hygromycin-resistant transformants at a transformation frequency of 13 transformants microg(-1) of plasmid DNA. We confirmed the introduction of the chimeric laccase gene into the mycelia of transformants by a polymerase chain reaction in nine randomly selected transformants. Overproduction of extracellular laccase by the transformants was revealed by a colorimetric assay for laccase activity. We examined the transformant (T2) that had the highest laccase activity and found that its activity was significantly higher than that of the wild type, particularly in the presence of copper (II). Our transformation system should contribute to the efficient production of the extracellular proteins of C. versicolor for the accelerated degradation of lignin and aromatic pollutants.

Wnt5a suppresses inflammation-driven intervertebral disc degeneration via a TNF-α/NF-κB-Wnt5a negative-feedback loop.

PubMed

Li, Z; Zhang, K; Li, X; Pan, H; Li, S; Chen, F; Zhang, J; Zheng, Z; Wang, J; Liu, H

2018-04-12

This study was to investigate the molecular role of Wnt5a on inflammation-driven intervertebral disc degeneration (IVDD). The expression of Wnt5a was analyzed in human nucleus pulposus (NP) tissues with immunohistochemical staining. The effects of Wnt5a on matrix production were assessed by RT-qPCR and western blotting. Small interfering RNAs (siRNAs), promoter deletion assay, and promoter binding site mutant were used to reveal the molecular role of Wnt5a in TNF-α-induced matrix metalloproteinase (MMP) expression. The regulatory effects of TNF-α on Wnt5a were investigated with pharmachemical inhibitors and siRNA experiment. The expression of Wnt5a was elevated in moderately degenerated human NP tissue with similar expression pattern of TNF-α. In NP cells, Wnt5a significantly increased aggrecan and collagen II expression. Inhibition of JNK or interfering Sox9 gene expression significantly suppressed Wnt5a-induced matrix production. AP-1(JunB) binding sites were located in Sox9 promoter and mutation of these sites sabotaged Wnt5a-induced Sox9 up-regulation and subsequent matrix genes expression. Notably, Wnt5a, which was induced by TNF-α, on the other way round suppressed TNF-α-NF-κB (p65) signaling and subsequent MMPs expression. In vivo studies with MR imaging confirmed the protective role of Wnt5a in IVDD. Wnt5a, which can be induced by TNF-α, increased matrix production in a Sox9-dependent manner through the activation of JNK-AP1 (JunB) signaling, and antagonized TNF-α-induced up-regulation of MMPs through the inhibition of NF-κB signaling. It indicates that Wnt5a suppresses IVDD through a TNF-α/NF-κB-Wnt5a negative-feedback loop. Copyright © 2018 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

Association between expression of random gene sets and survival is evident in multiple cancer types and may be explained by sub-classification.

PubMed

Shimoni, Yishai

2018-02-01

One of the goals of cancer research is to identify a set of genes that cause or control disease progression. However, although multiple such gene sets were published, these are usually in very poor agreement with each other, and very few of the genes proved to be functional therapeutic targets. Furthermore, recent findings from a breast cancer gene-expression cohort showed that sets of genes selected randomly can be used to predict survival with a much higher probability than expected. These results imply that many of the genes identified in breast cancer gene expression analysis may not be causal of cancer progression, even though they can still be highly predictive of prognosis. We performed a similar analysis on all the cancer types available in the cancer genome atlas (TCGA), namely, estimating the predictive power of random gene sets for survival. Our work shows that most cancer types exhibit the property that random selections of genes are more predictive of survival than expected. In contrast to previous work, this property is not removed by using a proliferation signature, which implies that proliferation may not always be the confounder that drives this property. We suggest one possible solution in the form of data-driven sub-classification to reduce this property significantly. Our results suggest that the predictive power of random gene sets may be used to identify the existence of sub-classes in the data, and thus may allow better understanding of patient stratification. Furthermore, by reducing the observed bias this may allow more direct identification of biologically relevant, and potentially causal, genes.

Association between expression of random gene sets and survival is evident in multiple cancer types and may be explained by sub-classification

PubMed Central

2018-01-01

One of the goals of cancer research is to identify a set of genes that cause or control disease progression. However, although multiple such gene sets were published, these are usually in very poor agreement with each other, and very few of the genes proved to be functional therapeutic targets. Furthermore, recent findings from a breast cancer gene-expression cohort showed that sets of genes selected randomly can be used to predict survival with a much higher probability than expected. These results imply that many of the genes identified in breast cancer gene expression analysis may not be causal of cancer progression, even though they can still be highly predictive of prognosis. We performed a similar analysis on all the cancer types available in the cancer genome atlas (TCGA), namely, estimating the predictive power of random gene sets for survival. Our work shows that most cancer types exhibit the property that random selections of genes are more predictive of survival than expected. In contrast to previous work, this property is not removed by using a proliferation signature, which implies that proliferation may not always be the confounder that drives this property. We suggest one possible solution in the form of data-driven sub-classification to reduce this property significantly. Our results suggest that the predictive power of random gene sets may be used to identify the existence of sub-classes in the data, and thus may allow better understanding of patient stratification. Furthermore, by reducing the observed bias this may allow more direct identification of biologically relevant, and potentially causal, genes. PMID:29470520

Asialofetuin liposome-mediated human alpha1-antitrypsin gene transfer in vivo results in stationary long-term gene expression.

PubMed

Dasí, F; Benet, M; Crespo, J; Crespo, A; Aliño, S F

2001-05-01

The development of nonviral vectors for in vivo gene delivery to hepatocytes is an interesting topic in view of their safety and tremendous gene therapy potential. Since cationic liposomes and liposome uptake by receptor-mediated mechanisms could offer advantages in the efficacy of liposome-mediated gene transfer, we studied the effect of liposome charge (anionic vs. cationic) and the covalently coupled asialofetuin ligand on the liposome surface in mediating human alpha1-antitrypsin (hAAT) gene transfer to mice in vivo. The changes in liposome charge were made by adding the following lipids to the backbone liposomes: anionic phosphatidylserine, cationic N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethyl-ammonium methylsulfate or a lipopeptide synthesized from dipalmitoylphosphatidylethanolamine and covalently coupled to the cationic nuclear localization signal peptide. Two plasmids containing the hAAT gene were used: pTG7101, containing the complete genomic sequence of the human gene driven by the natural promoter, and p216, containing the human hAAT cDNA under the control of the CMV promoter. The results indicate that both untargeted anionic and cationic liposomes mediate plasma levels of hAAT that decline over time. However, asialofetuin liposomes increase the plasma levels of hAAT and can mediate long-term gene expression (>12 months) with stationary plasma levels of protein. Results from quantitative and qualitative reverse transcriptase polymerase chain reaction match those from protein plasma levels and confirm both the human origin of the message and the liver as source of the protein. The use of asialofetuin liposomes in hepatic gene therapy may both increase and prolong in vivo gene expression of hAAT and other clinically important genes.

Involvement of astrocyte metabolic coupling in Tourette syndrome pathogenesis.

PubMed

de Leeuw, Christiaan; Goudriaan, Andrea; Smit, August B; Yu, Dongmei; Mathews, Carol A; Scharf, Jeremiah M; Verheijen, Mark H G; Posthuma, Danielle

2015-11-01

Tourette syndrome is a heritable neurodevelopmental disorder whose pathophysiology remains unknown. Recent genome-wide association studies suggest that it is a polygenic disorder influenced by many genes of small effect. We tested whether these genes cluster in cellular function by applying gene-set analysis using expert curated sets of brain-expressed genes in the current largest available Tourette syndrome genome-wide association data set, involving 1285 cases and 4964 controls. The gene sets included specific synaptic, astrocytic, oligodendrocyte and microglial functions. We report association of Tourette syndrome with a set of genes involved in astrocyte function, specifically in astrocyte carbohydrate metabolism. This association is driven primarily by a subset of 33 genes involved in glycolysis and glutamate metabolism through which astrocytes support synaptic function. Our results indicate for the first time that the process of astrocyte-neuron metabolic coupling may be an important contributor to Tourette syndrome pathogenesis.

Involvement of astrocyte metabolic coupling in Tourette syndrome pathogenesis

PubMed Central

de Leeuw, Christiaan; Goudriaan, Andrea; Smit, August B; Yu, Dongmei; Mathews, Carol A; Scharf, Jeremiah M; Scharf, J M; Pauls, D L; Yu, D; Illmann, C; Osiecki, L; Neale, B M; Mathews, C A; Reus, V I; Lowe, T L; Freimer, N B; Cox, N J; Davis, L K; Rouleau, G A; Chouinard, S; Dion, Y; Girard, S; Cath, D C; Posthuma, D; Smit, J H; Heutink, P; King, R A; Fernandez, T; Leckman, J F; Sandor, P; Barr, C L; McMahon, W; Lyon, G; Leppert, M; Morgan, J; Weiss, R; Grados, M A; Singer, H; Jankovic, J; Tischfield, J A; Heiman, G A; Verheijen, Mark H G; Posthuma, Danielle

2015-01-01

Tourette syndrome is a heritable neurodevelopmental disorder whose pathophysiology remains unknown. Recent genome-wide association studies suggest that it is a polygenic disorder influenced by many genes of small effect. We tested whether these genes cluster in cellular function by applying gene-set analysis using expert curated sets of brain-expressed genes in the current largest available Tourette syndrome genome-wide association data set, involving 1285 cases and 4964 controls. The gene sets included specific synaptic, astrocytic, oligodendrocyte and microglial functions. We report association of Tourette syndrome with a set of genes involved in astrocyte function, specifically in astrocyte carbohydrate metabolism. This association is driven primarily by a subset of 33 genes involved in glycolysis and glutamate metabolism through which astrocytes support synaptic function. Our results indicate for the first time that the process of astrocyte-neuron metabolic coupling may be an important contributor to Tourette syndrome pathogenesis. PMID:25735483

Overexpression of blueberry FLOWERING LOCUS T is associated with changes in the expression of phytohormone-related genes in blueberry plants.

PubMed

Gao, Xuan; Walworth, Aaron E; Mackie, Charity; Song, Guo-Qing

2016-01-01

Flowering locus T ( FT ) is a primary integrator in the regulation of plant flowering. Overexpressing a blueberry ( Vaccinium corymbosum L.) FT gene ( VcFT ) (herein VcFT -OX) resulted in early flowering and dwarfing in 'Aurora' plants (herein 'VcFT-Aurora'). In this study, we found that VcFT -OX reduced shoot regeneration from leaf explants. To investigate the potential roles of the phytohormone pathway genes associated with VcFT -OX, differentially expressed ( DE ) genes in leaf tissues of 'VcFT-Aurora' plants were annotated and analyzed using non-transgenic 'Aurora' plants as a control. Three DE floral genes, including the blueberry SUPPRESSOR of Overexpression of constans 1 ( VcSOC1 ) (gibberellin related), Abscisic acid responsive elements-binding factor 2 ( VcABF2 ) and protein related to ABI3/VP1 ( VcABI3/VP1 ) (ethylene-related), are present under both the phytohormone-responsive and the dwarfing-related Gene Ontology terms. The gene networks of the DE genes overall showed the molecular basis of the multifunctional aspects of VcFT overexpression beyond flowering promotion and suggested that phytohormone changes could be signaling molecules with important roles in the phenotypic changes driven by VcFT -OX.

Overexpression of blueberry FLOWERING LOCUS T is associated with changes in the expression of phytohormone-related genes in blueberry plants

PubMed Central

Gao, Xuan; Walworth, Aaron E; Mackie, Charity; Song, Guo-qing

2016-01-01

Flowering locus T (FT) is a primary integrator in the regulation of plant flowering. Overexpressing a blueberry (Vaccinium corymbosum L.) FT gene (VcFT) (herein VcFT-OX) resulted in early flowering and dwarfing in ‘Aurora’ plants (herein ‘VcFT-Aurora’). In this study, we found that VcFT-OX reduced shoot regeneration from leaf explants. To investigate the potential roles of the phytohormone pathway genes associated with VcFT-OX, differentially expressed (DE) genes in leaf tissues of ‘VcFT-Aurora’ plants were annotated and analyzed using non-transgenic ‘Aurora’ plants as a control. Three DE floral genes, including the blueberry SUPPRESSOR of Overexpression of constans 1 (VcSOC1) (gibberellin related), Abscisic acid responsive elements-binding factor 2 (VcABF2) and protein related to ABI3/VP1 (VcABI3/VP1) (ethylene-related), are present under both the phytohormone-responsive and the dwarfing-related Gene Ontology terms. The gene networks of the DE genes overall showed the molecular basis of the multifunctional aspects of VcFT overexpression beyond flowering promotion and suggested that phytohormone changes could be signaling molecules with important roles in the phenotypic changes driven by VcFT-OX. PMID:27818778

Unprecedented enhancement of transient gene expression from minimal cassettes using a double terminator.

PubMed

Beyene, Getu; Buenrostro-Nava, Marco T; Damaj, Mona B; Gao, San-Ji; Molina, Joe; Mirkov, T Erik

2011-01-01

The potential of using vector-free minimal gene cassettes (MGCs) with a double terminator for the enhancement and stabilization of transgene expression was tested in sugarcane biolistic transformation. The MGC system used consisted of the enhanced yellow fluorescent protein (EYFP) reporter gene driven by the maize ubiquitin-1 (Ubi) promoter and a single or double terminator from nopaline synthase (Tnos) or/and Cauliflower mosaic virus 35S (35ST). Transient EYFP expression from Tnos or 35ST single terminator MGC was very low and unstable, typically peaking early (8-16 h) and diminishing rapidly (48-72 h) after bombardment. Addition of a ~260 bp vector sequence (VS) to the single MGC downstream of Tnos (Tnos + VS) or 35ST (35ST + VS) enhanced EYFP expression by 1.25- to 25-fold. However, a much more significant increase in EYFP expression was achieved when the VS in 35ST + VS was replaced by Tnos to generate a 35ST-Tnos double terminator MGC, reaching its maximum at 24 h post-bombardment. The enhanced EYFP expression from the double terminator MGC was maintained for a long period of time (168 h), resulting in an overall increase of 5- to 65-fold and 10- to 160-fold as compared to the 35ST and Tnos single terminator MGCs, respectively. The efficiency of the double terminator MGC in enhancing EYFP expression was also demonstrated in sorghum and tobacco, suggesting that the underlying mechanism is highly conserved among monocots and dicots. Our results also suggest the involvement of posttranscriptional gene silencing in the reduced and unstable transgene expression from single terminator MGCs in plants.

Central genomic regulation of the expression of oestrous behaviour in dairy cows: a review.

PubMed

Woelders, H; van der Lende, T; Kommadath, A; te Pas, M F W; Smits, M A; Kaal, L M T E

2014-05-01

The expression of oestrous behaviour in Holstein Friesian dairy cows has progressively decreased over the past 50 years. Reduced oestrus expression is one of the factors contributing to the current suboptimal reproductive efficiency in dairy farming. Variation between and within cows in the expression of oestrous behaviour is associated with variation in peripheral blood oestradiol concentrations during oestrus. In addition, there is evidence for a priming role of progesterone for the full display of oestrous behaviour. A higher rate of metabolic clearance of ovarian steroids could be one of the factors leading to lower peripheral blood concentrations of oestradiol and progesterone in high-producing dairy cows. Oestradiol acts on the brain by genomic, non-genomic and growth factor-dependent mechanisms. A firm base of understanding of the ovarian steroid-driven central genomic regulation of female sexual behaviour has been obtained from studies on rodents. These studies have resulted in the definition of five modules of oestradiol-activated genes in the brain, referred to as the GAPPS modules. In a recent series of studies, gene expression in the anterior pituitary and four brain areas (amygdala, hippocampus, dorsal hypothalamus and ventral hypothalamus) in oestrous and luteal phase cows, respectively, has been measured, and the relation with oestrous behaviour of these cows was analysed. These studies identified a number of genes of which the expression was associated with the intensity of oestrous behaviour. These genes could be grouped according to the GAPPS modules, suggesting close similarity of the regulation of oestrous behaviour in cows and female sexual behaviour in rodents. A better understanding of the central genomic regulation of the expression of oestrous behaviour in dairy cows may in due time contribute to improved (genomic) selection strategies for appropriate oestrus expression in high-producing dairy cows.

The FOXP2-Driven Network in Developmental Disorders and Neurodegeneration

PubMed Central

Oswald, Franz; Klöble, Patricia; Ruland, André; Rosenkranz, David; Hinz, Bastian; Butter, Falk; Ramljak, Sanja; Zechner, Ulrich; Herlyn, Holger

2017-01-01

The transcription repressor FOXP2 is a crucial player in nervous system evolution and development of humans and songbirds. In order to provide an additional insight into its functional role we compared target gene expression levels between human neuroblastoma cells (SH-SY5Y) stably overexpressing FOXP2 cDNA of either humans or the common chimpanzee, Rhesus monkey, and marmoset, respectively. RNA-seq led to identification of 27 genes with differential regulation under the control of human FOXP2, which were previously reported to have FOXP2-driven and/or songbird song-related expression regulation. RT-qPCR and Western blotting indicated differential regulation of additional 13 new target genes in response to overexpression of human FOXP2. These genes may be directly regulated by FOXP2 considering numerous matches of established FOXP2-binding motifs as well as publicly available FOXP2-ChIP-seq reads within their putative promoters. Ontology analysis of the new and reproduced targets, along with their interactors in a network, revealed an enrichment of terms relating to cellular signaling and communication, metabolism and catabolism, cellular migration and differentiation, and expression regulation. Notably, terms including the words “neuron” or “axonogenesis” were also enriched. Complementary literature screening uncovered many connections to human developmental (autism spectrum disease, schizophrenia, Down syndrome, agenesis of corpus callosum, trismus-pseudocamptodactyly, ankyloglossia, facial dysmorphology) and neurodegenerative diseases and disorders (Alzheimer’s, Parkinson’s, and Huntington’s diseases, Lewy body dementia, amyotrophic lateral sclerosis). Links to deafness and dyslexia were detected, too. Such relations existed for single proteins (e.g., DCDC2, NURR1, PHOX2B, MYH8, and MYH13) and groups of proteins which conjointly function in mRNA processing, ribosomal recruitment, cell–cell adhesion (e.g., CDH4), cytoskeleton organization, neuro-inflammation, and processing of amyloid precursor protein. Conspicuously, many links pointed to an involvement of the FOXP2-driven network in JAK/STAT signaling and the regulation of the ezrin–radixin–moesin complex. Altogether, the applied phylogenetic perspective substantiated FOXP2’s importance for nervous system development, maintenance, and functioning. However, the study also disclosed new regulatory pathways that might prove to be useful for understanding the molecular background of the aforementioned developmental disorders and neurodegenerative diseases. PMID:28798667

The FOXP2-Driven Network in Developmental Disorders and Neurodegeneration.

PubMed

Oswald, Franz; Klöble, Patricia; Ruland, André; Rosenkranz, David; Hinz, Bastian; Butter, Falk; Ramljak, Sanja; Zechner, Ulrich; Herlyn, Holger

2017-01-01

The transcription repressor FOXP2 is a crucial player in nervous system evolution and development of humans and songbirds. In order to provide an additional insight into its functional role we compared target gene expression levels between human neuroblastoma cells (SH-SY5Y) stably overexpressing FOXP2 cDNA of either humans or the common chimpanzee, Rhesus monkey, and marmoset, respectively. RNA-seq led to identification of 27 genes with differential regulation under the control of human FOXP2 , which were previously reported to have FOXP2-driven and/or songbird song-related expression regulation. RT-qPCR and Western blotting indicated differential regulation of additional 13 new target genes in response to overexpression of human FOXP2. These genes may be directly regulated by FOXP2 considering numerous matches of established FOXP2-binding motifs as well as publicly available FOXP2-ChIP-seq reads within their putative promoters. Ontology analysis of the new and reproduced targets, along with their interactors in a network, revealed an enrichment of terms relating to cellular signaling and communication, metabolism and catabolism, cellular migration and differentiation, and expression regulation. Notably, terms including the words "neuron" or "axonogenesis" were also enriched. Complementary literature screening uncovered many connections to human developmental (autism spectrum disease, schizophrenia, Down syndrome, agenesis of corpus callosum, trismus-pseudocamptodactyly, ankyloglossia, facial dysmorphology) and neurodegenerative diseases and disorders (Alzheimer's, Parkinson's, and Huntington's diseases, Lewy body dementia, amyotrophic lateral sclerosis). Links to deafness and dyslexia were detected, too. Such relations existed for single proteins (e.g., DCDC2, NURR1, PHOX2B, MYH8, and MYH13) and groups of proteins which conjointly function in mRNA processing, ribosomal recruitment, cell-cell adhesion (e.g., CDH4), cytoskeleton organization, neuro-inflammation, and processing of amyloid precursor protein. Conspicuously, many links pointed to an involvement of the FOXP2-driven network in JAK/STAT signaling and the regulation of the ezrin-radixin-moesin complex. Altogether, the applied phylogenetic perspective substantiated FOXP2's importance for nervous system development, maintenance, and functioning. However, the study also disclosed new regulatory pathways that might prove to be useful for understanding the molecular background of the aforementioned developmental disorders and neurodegenerative diseases.

Nrf2-driven CD36 and HO-1 gene expression in circulating monocytes correlates with favourable clinical outcome in pregnancy-associated malaria.

PubMed

Aubouy, Agnès; Olagnier, David; Bertin, Gwladys; Ezinmegnon, Sem; Majorel, Clarisse; Mimar, Saliha; Massougbodji, Achille; Deloron, Philippe; Pipy, Bernard; Coste, Agnès

2015-09-18

Pregnancy-associated malaria (PAM) constitutes one of the most severe forms of malaria infection leading to fetal growth restriction and high risk of infant death. The severity of the pathology is largely attributed to the recruitment of monocytes and macrophages in the placenta which is evidenced by dysregulated inflammation found in placental blood. Importantly, CD36(+) monocytes/macrophages are also thought to participate in the tight control of the pro- and anti-inflammatory responses following Plasmodium detection through elimination of apoptotic cells and malaria-infected erythrocytes, internalization and recycling of oxidized forms of low-density lipoprotein and collaboration with TLR2 in pro-inflammatory response. Interestingly, previous work demonstrated that CD36 expression was upregulated on inflammatory macrophages following stimulation of the Nrf2 transcription factor, whilst the PPARγ pathway was inhibited and non-functional in the same inflammatory conditions. This current study examined the possible role of Nrf2-driven gene expression, CD36 and Haem-Oxygenase-1 (HO-1), in PAM clinical outcomes. Clinical data and biological samples including peripheral blood mononuclear cells were collected from 27 women presenting PAM. Polychromatic flow cytometry was used to characterize innate immune cell subpopulations and quantify CD36 protein expression level on monocytes. mRNA levels of CD36, PPARγ, Nrf2 and HO-1 were determined by qPCR and related to clinical outcomes. Finally, the capacity of monocytes to modulate CD36 expression upon rosiglitazone or sulforaphane treatment, two respective PPARγ or Nrf2 activators, was also investigated. The CD36 receptor, mostly expressed by CD14(+) circulating monocytes, statistically correlated with increased infant birth weights. Interestingly, mRNA levels of the transcription factor Nrf2 and the enzyme HO-1 also correlated with lower parasitaemia and increased infant birth weight, while PPARγ mRNA levels did not. Finally, monocytes isolated from low infant birth weight pregnant women were capable of up-regulating CD36 via the Nrf2 pathway ex vivo. Altogether these results suggest that Nrf2-driven CD36 and HO-1 expression on innate immune cells could contribute to a protective and detoxifying mechanism during PAM. More powered and mechanistical studies are however needed to strengthen the conclusions of this study.

Intraclonal Cell Expansion and Selection Driven by B Cell Receptor in Chronic Lymphocytic Leukemia

PubMed Central

Colombo, Monica; Cutrona, Giovanna; Reverberi, Daniele; Fabris, Sonia; Neri, Antonino; Fabbi, Marina; Quintana, Giovanni; Quarta, Giovanni; Ghiotto, Fabio; Fais, Franco; Ferrarini, Manlio

2011-01-01

The mutational status of the immunoglobulin heavy-chain variable region (IGHV) genes utilized by chronic lymphocytic leukemia (CLL) clones defines two disease subgroups. Patients with unmutated IGHV have a more aggressive disease and a worse outcome than patients with cells having somatic IGHV gene mutations. Moreover, up to 30% of the unmutated CLL clones exhibit very similar or identical B cell receptors (BcR), often encoded by the same IG genes. These “stereotyped” BcRs have been classified into defined subsets. The presence of an IGHV gene somatic mutation and the utilization of a skewed gene repertoire compared with normal B cells together with the expression of stereotyped receptors by unmutated CLL clones may indicate stimulation/selection by antigenic epitopes. This antigenic stimulation may occur prior to or during neoplastic transformation, but it is unknown whether this stimulation/selection continues after leukemogenesis has ceased. In this study, we focused on seven CLL cases with stereotyped BcR Subset #8 found among a cohort of 700 patients; in six, the cells expressed IgG and utilized IGHV4-39 and IGKV1-39/IGKV1D-39 genes, as reported for Subset #8 BcR. One case exhibited special features, including expression of IgM or IgG by different subclones consequent to an isotype switch, allelic inclusion at the IGH locus in the IgM-expressing cells and a particular pattern of cytogenetic lesions. Collectively, the data indicate a process of antigenic stimulation/selection of the fully transformed CLL cells leading to the expansion of the Subset #8 IgG-bearing subclone. PMID:21541442

The transcriptomic responses of the eastern oyster, Crassostrea virginica, to environmental conditions.

PubMed

Chapman, Robert W; Mancia, Annalaura; Beal, Marion; Veloso, Artur; Rathburn, Charles; Blair, Anne; Holland, A F; Warr, G W; Didinato, Guy; Sokolova, Inna M; Wirth, Edward F; Duffy, Edward; Sanger, Denise

2011-04-01

Understanding the mechanisms by which organisms adapt to environmental conditions is a fundamental question for ecology and evolution. In this study, we evaluate changes in gene expression of a marine mollusc, the eastern oyster Crassostrea virginica, associated with the physico-chemical conditions and the levels of metals and other contaminants in their environment. The results indicate that transcript signatures can effectively disentangle the complex interactive gene expression responses to the environment and are also capable of disentangling the complex dynamic effects of environmental factors on gene expression. In this context, the mapping of environment to gene and gene to environment is reciprocal and mutually reinforcing. In general, the response of transcripts to the environment is driven by major factors known to affect oyster physiology such as temperature, pH, salinity, and dissolved oxygen, with pollutant levels playing a relatively small role, at least within the range of concentrations found in the studied oyster habitats. Further, the two environmental factors that dominate these effects (temperature and pH) interact in a dynamic and nonlinear fashion to impact gene expression. Transcriptomic data obtained in our study provide insights into the mechanisms of physiological responses to temperature and pH in oysters that are consistent with the known effects of these factors on physiological functions of ectotherms and indicate important linkages between transcriptomics and physiological outcomes. Should these linkages hold in further studies and in other organisms, they may provide a novel integrated approach for assessing the impacts of climate change, ocean acidification and anthropogenic contaminants on aquatic organisms via relatively inexpensive microarray platforms. © 2011 Blackwell Publishing Ltd.

Quantitative determination of adenovirus-mediated gene delivery to rat cardiac myocytes in vitro and in vivo.

PubMed Central

Kass-Eisler, A; Falck-Pedersen, E; Alvira, M; Rivera, J; Buttrick, P M; Wittenberg, B A; Cipriani, L; Leinwand, L A

1993-01-01

To optimize the use of modified adenoviruses as vectors for gene delivery to the myocardium, we have characterized infection of cultured fetal and adult rat cardiac myocytes in vitro and of adult cardiac myocytes in vivo by using a replication-defective adenovirus carrying the chloramphenicol acetyltransferase (CAT) reporter gene driven by the cytomegalovirus promoter (AdCMVCATgD). In vitro, virtually all fetal or adult cardiocytes express the CAT gene when infected with 1 plaque-forming unit of virus per cell. CAT enzymatic activity can be detected in these cells as early as 4 hr after infection, reaching near-maximal levels at 48 hr. In fetal cells, CAT expression was maintained without a loss in activity for at least 1 week. Using in vitro studies as a guide, we introduced the AdCMVCATgD virus directly into adult rat myocardium and compared the expression results obtained from virus injection with those obtained by direct injection of pAdCMVCATgD plasmid DNA. The amount of CAT activity resulting from adenovirus infection of the myocardium was orders of magnitude higher than that seen from DNA injection and was proportional to the amount of input virus. Immunostaining for CAT protein in cardiac tissue sections following adenovirus injection demonstrated large numbers of positive cells, reaching nearly 100% of the myocytes in many regions of the heart. Expression of genes introduced by adenovirus peaked at 5 days but was still detectable 55 days following infection. Adenoviruses are therefore a very useful tool for high-efficiency gene transfer into the cardiovascular system. Images Fig. 1 Fig. 5 PMID:8265580

The expression of Per1 and Aa-nat genes in the pineal gland of postnatal rats.

PubMed

Wongchitrat, Prapimpun; Govitrapong, Piyarat; Phansuwan-Pujito, Pansiri

2012-12-01

The circadian rhythm of melatonin synthesis is controlled by the master clock, suprachiasmatic nucleus (SCN). The level of melatonin changes throughout the aging process. The SCN's rhythm is driven by autoregulatory feedback loop composed of a set of clock genes families and their corresponding proteins. The Period (Per1), one of clock gene develops gradually during postnatal ontogenesis in the rat SCN and is also expressed in the pineal gland. It is of interest to study the relationship between the postnatal development of Per1 and Aa-nat, genes that produce the rate-limiting enzyme in melatonin synthesis, in the pineal. Daily profiles of mRNA expression of Per1 and Aa-nat were analyzed in the pineal gland of pups at postnatal ages 4 (P4), P8, P16 and P32, at puberty age of 6 weeks; and in 8 week-old adult rats by real-time PCR. As early as P4, Per1 and Aa-nat mRNAs were expressed and existed at relatively high levels during the nighttime. They gradually increased until puberty and decreased at 8 weeks of age. Additionally, the nocturnal changes of Per1 and Aa-nat mRNA levels in the rat pineal gland from P4 to adults were strongly correlated at r = 0.97 (p < 0.01). The present data indicate that there is a close relationship between the expression pattern of Per1 and that of melatonin synthesis during the development of postnatal rats.

Network-directed cis-mediator analysis of normal prostate tissue expression profiles reveals downstream regulatory associations of prostate cancer susceptibility loci.

PubMed

Larson, Nicholas B; McDonnell, Shannon K; Fogarty, Zach; Larson, Melissa C; Cheville, John; Riska, Shaun; Baheti, Saurabh; Weber, Alexandra M; Nair, Asha A; Wang, Liang; O'Brien, Daniel; Davila, Jaime; Schaid, Daniel J; Thibodeau, Stephen N

2017-10-17

Large-scale genome-wide association studies have identified multiple single-nucleotide polymorphisms associated with risk of prostate cancer. Many of these genetic variants are presumed to be regulatory in nature; however, follow-up expression quantitative trait loci (eQTL) association studies have to-date been restricted largely to cis -acting associations due to study limitations. While trans -eQTL scans suffer from high testing dimensionality, recent evidence indicates most trans -eQTL associations are mediated by cis -regulated genes, such as transcription factors. Leveraging a data-driven gene co-expression network, we conducted a comprehensive cis -mediator analysis using RNA-Seq data from 471 normal prostate tissue samples to identify downstream regulatory associations of previously identified prostate cancer risk variants. We discovered multiple trans -eQTL associations that were significantly mediated by cis -regulated transcripts, four of which involved risk locus 17q12, proximal transcription factor HNF1B , and target trans -genes with known HNF response elements ( MIA2 , SRC , SEMA6A , KIF12 ). We additionally identified evidence of cis -acting down-regulation of MSMB via rs10993994 corresponding to reduced co-expression of NDRG1 . The majority of these cis -mediator relationships demonstrated trans -eQTL replicability in 87 prostate tissue samples from the Gene-Tissue Expression Project. These findings provide further biological context to known risk loci and outline new hypotheses for investigation into the etiology of prostate cancer.

Intrinsic and Extrinsic Regulation of PD-L2 Expression in Oncogene-Driven Non-Small Cell Lung Cancer.

PubMed

Shibahara, Daisuke; Tanaka, Kentaro; Iwama, Eiji; Kubo, Naoki; Ota, Keiichi; Azuma, Koichi; Harada, Taishi; Fujita, Jiro; Nakanishi, Yoichi; Okamoto, Isamu

2018-03-27

The interaction of programmed cell death ligand 2 (PD-L2) with programmed cell death 1 is implicated in tumor immune escape. The regulation of PD-L2 expression in tumor cells has remained unclear, however. We here examined intrinsic and extrinsic regulation of PD-L2 expression in NSCLC. PD-L2 expression was evaluated by reverse transcription and real-time polymerase chain reaction analysis and by flow cytometry. BEAS-2B cells stably expressing an activated mutant form of EGFR or the echinoderm microtubule associated protein like 4 (EML4)-ALK receptor tyrosine kinase fusion oncoprotein manifested increased expression of PD-L2 at both the mRNA and protein levels. Furthermore, treatment of NSCLC cell lines that harbor such driver oncogenes with corresponding EGFR or ALK tyrosine kinase inhibitors or depletion of EGFR or ALK by small interfering RNA transfection suppressed expression of PD-L2, demonstrating that activating EGFR mutations or echinoderm microtubule associated protein like 4 gene (EML4)-ALK receptor tyrosine kinase gene (ALK) fusion intrinsically induce PD-L2 expression. We also found that interferon gamma (IFN-γ) extrinsically induced expression of PD-L2 through signal transducer and activator of transcription 1 signaling in NSCLC cells. Oncogene-driven expression of PD-L2 in NSCLC cells was inhibited by knockdown of the transcription factors signal transducer and activator of transcription 3 (STAT3) or c-FOS. IFN-γ also activated STAT3 and c-FOS, suggesting that these proteins may also contribute to the extrinsic induction of PD-L2 expression. Expression of PD-L2 is induced intrinsically by activating EGFR mutations or EML4-ALK fusion and extrinsically by IFN-γ, with STAT3 and c-FOS possibly contributing to both intrinsic and extrinsic pathways. Our results thus provide insight into the complexity of tumor immune escape in NSCLC. Copyright © 2018 International Association for the Study of Lung Cancer. Published by Elsevier Inc. All rights reserved.

Transgene Expression Patterns Indicate That Spaceflight Affects Stress Signal Perception and Transduction in Arabidopsis1

PubMed Central

Paul, Anna-Lisa; Daugherty, Christine J.; Bihn, Elizabeth A.; Chapman, David K.; Norwood, Kelly L.L.; Ferl, Robert J.

2001-01-01

The use of plants as integral components of life support systems remains a cornerstone of strategies for long-term human habitation of space and extraterrestrial colonization. Spaceflight experiments over the past few decades have refined the hardware required to grow plants in low-earth orbit and have illuminated fundamental issues regarding spaceflight effects on plant growth and development. Potential incipient hypoxia, resulting from the lack of convection-driven gas movement, has emerged as a possible major impact of microgravity. We developed transgenic Arabidopsis containing the alcohol dehydrogenase (Adh) gene promoter linked to the β-glucuronidase (GUS) reporter gene to address specifically the possibility that spaceflight induces the plant hypoxia response and to assess whether any spaceflight response was similar to control terrestrial hypoxia-induced gene expression patterns. The staining patterns resulting from a 5-d mission on the orbiter Columbia during mission STS-93 indicate that the Adh/GUS reporter gene was activated in roots during the flight. However, the patterns of expression were not identical to terrestrial control inductions. Moreover, although terrestrial hypoxia induces Adh/GUS expression in the shoot apex, no apex staining was observed in the spaceflight plants. This indicates that either the normal hypoxia response signaling is impaired in spaceflight or that spaceflight inappropriately induces Adh/GUS activity for reasons other than hypoxia. PMID:11402191

Regime shifts driven by dynamic correlations in gene expression noise

NASA Astrophysics Data System (ADS)

Sharma, Yogita; Dutta, Partha Sharathi

2017-08-01

Gene expression is a noisy process that leads to regime shifts between alternative steady states among individual living cells, inducing phenotypic variability. The effects of white noise on the regime shift in bistable systems have been well characterized, however little is known about such effects of colored noise (noise with nonzero correlation time). Here, we show that noise correlation time, by considering a genetic circuit of autoactivation, can have a significant effect on the regime shift between distinct phenotypic states in gene expression. We demonstrate this theoretically, using stochastic potential, stationary probability density function, and first-passage time based on the Fokker-Planck description, where the Ornstein-Uhlenbeck process is used to model colored noise. We find that an increase in noise correlation time in the degradation rate can induce a regime shift from a low to a high protein concentration state and enhance the bistable regime, while an increase in noise correlation time in the basal rate retains the bimodal distribution. We then show how cross-correlated colored noises in basal and degradation rates can induce regime shifts from a low to a high protein concentration state, but reduce the bistable regime. We also validate these results through direct numerical simulations of the stochastic differential equation. In gene expression understanding the causes of regime shift to a harmful phenotype could improve early therapeutic intervention in complex human diseases.

A plant EPF-type zinc-finger protein, CaPIF1, involved in defence against pathogens.

PubMed

Oh, Sang-Keun; Park, Jeong Mee; Joung, Young Hee; Lee, Sanghyeob; Chung, Eunsook; Kim, Soo-Yong; Yu, Seung Hun; Choi, Doil

2005-05-01

SUMMARY To understand better the defence responses of plants to pathogen attack, we challenged hot pepper plants with bacterial pathogens and identified transcription factor-encoding genes whose expression patterns were altered during the subsequent hypersensitive response. One of these genes, CaPIF1 (Capsicum annuum Pathogen-Induced Factor 1), was characterized further. This gene encodes a plant-specific EPF-type protein that contains two Cys(2)/His(2) zinc fingers. CaPIF1 expression was rapidly and specifically induced when pepper plants were challenged with bacterial pathogens to which they are resistant. In contrast, challenge with a pathogen to which the plants are susceptible only generated weak CaPIF1 expression. CaPIF1 expression was also strongly induced in pepper leaves by the exogenous application of ethephon, an ethylene-releasing compound, and salicylic acid, whereas methyl jasmonate had only moderate effects. CaPIF1 localized to the nuclei of onion epidermis when expressed as a CaPIF1-smGFP fusion protein. Transgenic tobacco plants over-expressing CaPIF1 driven by the CaMV 35S promoter showed increased resistance to challenge with a tobacco-specific pathogen or non-host bacterial pathogens. These plants also showed constitutive up-regulation of multiple defence-related genes. Moreover, virus-induced silencing of the CaPIF1 orthologue in Nicotiana benthamiana enhanced susceptibility to the same host or non-host bacterial pathogens. These observations provide evidence that an EPF-type Cys(2)/His(2) zinc-finger protein plays a crucial role in the activation of the pathogen defence response in plants.

CX3CR1 is dysregulated in blood and brain from schizophrenia patients.

PubMed

Bergon, Aurélie; Belzeaux, Raoul; Comte, Magali; Pelletier, Florence; Hervé, Mylène; Gardiner, Erin J; Beveridge, Natalie J; Liu, Bing; Carr, Vaughan; Scott, Rodney J; Kelly, Brian; Cairns, Murray J; Kumarasinghe, Nishantha; Schall, Ulrich; Blin, Olivier; Boucraut, José; Tooney, Paul A; Fakra, Eric; Ibrahim, El Chérif

2015-10-01

The molecular mechanisms underlying schizophrenia remain largely unknown. Although schizophrenia is a mental disorder, there is increasing evidence to indicate that inflammatory processes driven by diverse environmental factors play a significant role in its development. With gene expression studies having been conducted across a variety of sample types, e.g., blood and postmortem brain, it is possible to investigate convergent signatures that may reveal interactions between the immune and nervous systems in schizophrenia pathophysiology. We conducted two meta-analyses of schizophrenia microarray gene expression data (N=474) and non-psychiatric control (N=485) data from postmortem brain and blood. Then, we assessed whether significantly dysregulated genes in schizophrenia could be shared between blood and brain. To validate our findings, we selected a top gene candidate and analyzed its expression by RT-qPCR in a cohort of schizophrenia subjects stabilized by atypical antipsychotic monotherapy (N=29) and matched controls (N=31). Meta-analyses highlighted inflammation as the major biological process associated with schizophrenia and that the chemokine receptor CX3CR1 was significantly down-regulated in schizophrenia. This differential expression was also confirmed in our validation cohort. Given both the recent data demonstrating selective CX3CR1 expression in subsets of neuroimmune cells, as well as behavioral and neuropathological observations of CX3CR1 deficiency in mouse models, our results of reduced CX3CR1 expression adds further support for a role played by monocyte/microglia in the neurodevelopment of schizophrenia. Copyright © 2015 Elsevier B.V. All rights reserved.

Bromodomain and Extra-terminal (BET) Protein Inhibitors Suppress Chondrocyte Differentiation and Restrain Bone Growth.

PubMed

Niu, Ningning; Shao, Rui; Yan, Guang; Zou, Weiguo

2016-12-23

Small molecule inhibitors for bromodomain and extra-terminal (BET) proteins have recently emerged as potential therapeutic agents in clinical trials for various cancers. However, to date, it is unknown whether these inhibitors have side effects on bone structures. Here, we report that inhibition of BET bromodomain proteins may suppress chondrocyte differentiation and restrain bone growth. We generated a luciferase reporter system using the chondrogenic cell line ATDC5 in which the luciferase gene was driven by the promoter of Col2a1, an elementary collagen of the chondrocyte. The Col2a1-luciferase ATDC5 system was used for rapidly screening both activators and repressors of human collagen Col2a1 gene expression, and we found that BET bromodomain inhibitors reduce the Col2a1-luciferase. Consistent with the luciferase assay, BET inhibitors decrease the expression of Col2a1 Furthermore, we constructed a zebrafish line in which the enhanced green fluorescent protein (EGFP) expression was driven by col2a1 promoter. The transgenic (col2a1-EGFP) zebrafish line demonstrated that BET inhibitors I-BET151 and (+)-JQ1 may affect EGFP expression in zebrafish. Furthermore, we found that I-BET151 and (+)-JQ1 may affect chondrocyte differentiation in vitro and inhibit zebrafish growth in vivo Mechanistic analysis revealed that BET inhibitors influenced the depletion of RNA polymerase II from the Col2a1 promoter. Collectively, these results suggest that BET bromodomain inhibition may have side effects on skeletal bone structures. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Distinct transcriptional MYCN/c-MYC activities are associated with spontaneous regression or malignant progression in neuroblastomas

PubMed Central

Westermann, Frank; Muth, Daniel; Benner, Axel; Bauer, Tobias; Henrich, Kai-Oliver; Oberthuer, André; Brors, Benedikt; Beissbarth, Tim; Vandesompele, Jo; Pattyn, Filip; Hero, Barbara; König, Rainer; Fischer, Matthias; Schwab, Manfred

2008-01-01

Background Amplified MYCN oncogene resulting in deregulated MYCN transcriptional activity is observed in 20% of neuroblastomas and identifies a highly aggressive subtype. In MYCN single-copy neuroblastomas, elevated MYCN mRNA and protein levels are paradoxically associated with a more favorable clinical phenotype, including disseminated tumors that subsequently regress spontaneously (stage 4s-non-amplified). In this study, we asked whether distinct transcriptional MYCN or c-MYC activities are associated with specific neuroblastoma phenotypes. Results We defined a core set of direct MYCN/c-MYC target genes by applying gene expression profiling and chromatin immunoprecipitation (ChIP, ChIP-chip) in neuroblastoma cells that allow conditional regulation of MYCN and c-MYC. Their transcript levels were analyzed in 251 primary neuroblastomas. Compared to localized-non-amplified neuroblastomas, MYCN/c-MYC target gene expression gradually increases from stage 4s-non-amplified through stage 4-non-amplified to MYCN amplified tumors. This was associated with MYCN activation in stage 4s-non-amplified and predominantly c-MYC activation in stage 4-non-amplified tumors. A defined set of MYCN/c-MYC target genes was induced in stage 4-non-amplified but not in stage 4s-non-amplified neuroblastomas. In line with this, high expression of a subset of MYCN/c-MYC target genes identifies a patient subtype with poor overall survival independent of the established risk markers amplified MYCN, disease stage, and age at diagnosis. Conclusions High MYCN/c-MYC target gene expression is a hallmark of malignant neuroblastoma progression, which is predominantly driven by c-MYC in stage 4-non-amplified tumors. In contrast, moderate MYCN function gain in stage 4s-non-amplified tumors induces only a restricted set of target genes that is still compatible with spontaneous regression. PMID:18851746

In vitro and in vivo characterization of a dual-function green fluorescent protein--HSV1-thymidine kinase reporter gene driven by the human elongation factor 1 alpha promoter.

PubMed

Luker, Gary D; Luker, Kathryn E; Sharma, Vijay; Pica, Christina M; Dahlheimer, Julie L; Ocheskey, Joe A; Fahrner, Timothy J; Milbrandt, Jeffrey; Piwnica-Worms, David

2002-01-01

Toward the goal of monitoring activity of native mammalian promoters with molecular imaging techniques, we stably transfected DU145 prostate carcinoma cells with a fusion construct of enhanced green fluorescent protein (EGFP) and wild-type herpes simplex virus-1 thymidine kinase (HSV1-TK) as a reporter gene driven by the promoter for human elongation factor 1 alpha (EF-1 alpha-EGFP-TK). Using this model system, expression of EGFP was quantified by flow cytometry and fluorescence microscopy, while the HSV1-TK component of the reporter was quantified with 8-[3H]ganciclovir (8-[3H]GCV). As analyzed by flow cytometry, passage of EGFP-TK-DU145 transfected cells (ETK) in vitro resulted in populations of cells with high and low expression of EGFP over time. High and low ETK cells retained 23-fold and 5-fold more GCV, respectively, than control. While differences in uptake and retention of GCV corresponded to relative expression of the reporter gene in each subpopulation of cells as determined by both flow cytometry (EGFP) and quantitative RT-PCR, the correlation was not linear. Furthermore, in high ETK cells, net retention of various radiolabeled nucleoside analogues varied; the rank order was 8-[3H]GCV < 9-(4-fluoro-3-hydroxymethylbutyl)guanine ([18F]FHBG) approximately 8-[3H]penciclovir (8-[3H]PCV) < 2'-fluoro-2'-deoxy-5-iodouracil-beta-D-arabinofuranoside (2-[14C]FIAU). Xenograft tumors of ETK cells in vivo accumulated 2.5-fold more 8-[3H]GCV per gram of tissue and showed greater fluorescence from EGFP than control DU145 cells, demonstrating that the reporter gene functioned in vivo. These data extend previous reports by showing that a human promoter can be detected in vitro and in vivo with a dual-function reporter exploiting optical and radiotracer techniques.

Auxin synthesis gene tms1 driven by tuber-specific promoter alters hormonal status of transgenic potato plants and their responses to exogenous phytohormones.

PubMed

Kolachevskaya, Oksana O; Sergeeva, Lidiya I; Floková, Kristyna; Getman, Irina A; Lomin, Sergey N; Alekseeva, Valeriya V; Rukavtsova, Elena B; Buryanov, Yaroslav I; Romanov, Georgy A

2017-03-01

Ectopic auxin overproduction in transgenic potato leads to enhanced productivity accompanied with concerted and occasional changes in hormonal status, and causing altered response of transformants to exogenous auxin or cytokinin. Previously, we generated potato transformants expressing Agrobacterium-derived auxin synthesis gene tms1 driven by tuber-specific patatin gene promoter (B33-promoter). Here, we studied the endogenous hormonal status and the response to exogenous phytohormones in tms1 transformants cultured in vitro. Adding indole-3-acetic acid (IAA) or kinetin to culture medium affected differently tuberization of tms1-transformed and control plants, depending also on sucrose content in the medium. Exogenous phytohormones ceased to stimulate the tuber initiation in transformants at high (5-8%) sucrose concentration, while in control plants the stimulation was observed in all experimental settings. Furthermore, exogenous auxin partly inhibited the tuber initiation, and exogenous cytokinin reduced the average tuber weight in most transformants at high sucrose content. The elevated auxin level in tubers of the transformants was accompanied with a decrease in content of cytokinin bases and their ribosides in tubers and most shoots. No concerted changes in contents of abscisic, jasmonic, salicylic acids and gibberellins in tubers were detected. The data on hormonal status indicated that the enhanced productivity of tms1 transformants was due to auxin and not mediated by other phytohormones. In addition, exogenous cytokinin was shown to upregulate the expression of genes encoding orthologs of auxin receptors. Overall, the results showed that tms1 expression and local increase in IAA level in transformants affect both the balance of endogenous cytokinins and the dynamics of tuberization in response to exogenous hormones (auxin, cytokinin), the latter reaction depending also on the carbohydrate supply. We introduce a basic model for the hormonal network controlling tuberization.

Use of DEAD-box polypeptide-4 (Ddx4) gene promoter-driven fluorescent reporter mice to identify mitotically active germ cells in post-natal mouse ovaries.

PubMed

Park, Eun-Sil; Tilly, Jonathan L

2015-01-01

Several laboratories have independently isolated mitotically active germ cells, termed female germline stem cells or oogonial stem cells (OSCs), from adult mouse ovaries. However, a recent study using Ddx4-Cre;Rosa26 reporter mice concluded that such germ cells do not exist. Given the disparity in conclusions drawn in this study compared with others, we felt it was important to re-assess the utility of Ddx4-Cre;Rosa26 reporter mice for identification of OSCs in adult mouse ovaries. Transgenic Ddx4-Cre mice were crossed with Rosa26(tdTm/tdTm) mice to drive restricted tomato red (tdTm) gene expression in cells in which the Ddx4 gene promoter has been activated. Crude dispersion of ovaries from recombined offspring generated cell fractions containing tdTm-positive immature oocytes, which are incapable of proliferation and thus probably represent the uncharacterized reporter-positive ovarian cells identified in the paper Zhang et al. (2012) as being mitotically inactive. Dispersed ovaries further subjected to fluorescence-activated cell sorting yielded a large population of non-germline tdTm-positive cells, indicative of promoter 'leakiness' in the Ddx4-Cre mouse line. Nonetheless, a small percentage of these tdTm-positive cells exhibited externalized (extracellular, ec) expression of Ddx4 protein (ecDdx4-positive), expressed markers of primitive germ cells but not of oocytes, and actively proliferated in culture, all of which are characteristic features of OSCs. Thus, crude dispersion of ovaries collected from Ddx4 gene promoter-driven reporter mice is not, by itself, a reliable approach to identify OSCs, whereas the same ovarian dispersates further subjected to cell sorting strategies yield purified OSCs that can be expanded in culture. © The Author 2014. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Trypanosoma cruzi H+-ATPase 1 (TcHA1) and 2 (TcHA2) genes complement yeast mutants defective in H+ pumps and encode plasma membrane P-type H+-ATPases with different enzymatic properties.

PubMed

Luo, Shuhong; Scott, David A; Docampo, Roberto

2002-11-15

Previous studies in Trypanosoma cruzi have shown that intracellular pH homeostasis requires ATP and is affected by H(+)-ATPase inhibitors, indicating a major role for ATP-driven proton pumps in intracellular pH control. In the present study, we report the cloning and sequencing of a pair of genes linked in tandem (TcHA1 and TcHA2) in T. cruzi which encode proteins with homology to fungal and plant P-type proton-pumping ATPases. The genes are expressed at the mRNA level in different developmental stages of T. cruzi: TcHA1 is expressed maximally in epimastigotes, whereas TcHA2 is expressed predominantly in trypomastigotes. The proteins predicted from the nucleotide sequence of the genes have 875 and 917 amino acids and molecular masses of 96.3 and 101.2 kDa, respectively. Full-length TcHA1 and an N-terminal truncated version of TcHA2 complemented a Saccharomyces cerevisiae strain deficient in P-type H(+)-ATPase activity, the proteins localized to the yeast plasma membrane, and ATP-driven proton pumping could be detected in proteoliposomes reconstituted from plasma membrane purified from transfected yeast. The reconstituted proton transport activity was reduced by inhibitors of P-type H(+)-ATPases. C-terminal truncation did not affect complementation of mutant yeast, suggesting the lack of C-terminal autoinhibitory domains in these proteins. ATPase activity in plasma membrane from TcHA1- and (N-terminal truncated) TcHA2-transfected yeast was inhibited to different extents by vanadate, whereas the latter yeast strain was more resistant to extremes of pH, suggesting that the native proteins may serve different functions at different stages in the T. cruzi life cycle.

Developmental regulation of ecdysone receptor (EcR) and EcR-controlled gene expression during pharate-adult development of honeybees (Apis mellifera)

PubMed Central

Mello, Tathyana R. P.; Aleixo, Aline C.; Pinheiro, Daniel G.; Nunes, Francis M. F.; Bitondi, Márcia M. G.; Hartfelder, Klaus; Barchuk, Angel R.; Simões, Zilá L. P.

2014-01-01

Major developmental transitions in multicellular organisms are driven by steroid hormones. In insects, these, together with juvenile hormone (JH), control development, metamorphosis, reproduction and aging, and are also suggested to play an important role in caste differentiation of social insects. Here, we aimed to determine how EcR transcription and ecdysteroid titers are related during honeybee postembryonic development and what may actually be the role of EcR in caste development of this social insect. In addition, we expected that knocking-down EcR gene expression would give us information on the participation of the respective protein in regulating downstream targets of EcR. We found that in Apis mellifera females, EcR-A is the predominantly expressed variant in postembryonic development, while EcR-B transcript levels are higher in embryos, indicating an early developmental switch in EcR function. During larval and pupal stages, EcR-B expression levels are very low, while EcR-A transcripts are more variable and abundant in workers compared to queens. Strikingly, these transcript levels are opposite to the ecdysteroid titer profile. 20-hydroxyecdysone (20E) application experiments revealed that low 20E levels induce EcR expression during development, whereas high ecdysteroid titers seem to be repressive. By means of RNAi-mediated knockdown (KD) of both EcR transcript variants we detected the differential expression of 234 poly-A+ transcripts encoding genes such as CYPs, MRJPs and certain hormone response genes (Kr-h1 and ftz-f1). EcR-KD also promoted the differential expression of 70 miRNAs, including highly conserved ones (e.g., miR-133 and miR-375), as well honeybee-specific ones (e.g., miR-3745 and miR-3761). Our results put in evidence a broad spectrum of EcR-controlled gene expression during postembryonic development of honeybees, revealing new facets of EcR biology in this social insect. PMID:25566327

Eye drop delivery of nano-polymeric micelle formulated genes with cornea-specific promoters.

PubMed

Tong, Yaw-Chong; Chang, Shwu-Fen; Liu, Chia-Yang; Kao, Winston W-Y; Huang, Chong Heng; Liaw, Jiahorng

2007-11-01

This study evaluates the eye drop delivery of genes with cornea-specific promoters, i.e., keratin 12 (K12) and keratocan (Kera3.2) promoters, by non-ionic poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO) polymeric micelles (PM) to mouse and rabbit eyes, and investigates the underlying mechanisms. Three PM-formulated plasmids (pCMV-Lac Z, pK12-Lac Z and pKera3.2-Lac Z) containing the Lac Z gene for beta-galactosidase (beta-Gal) whose expression was driven by the promoter of either the cytomegalovirus early gene, the keratin 12 gene or the keratocan gene, were characterized by critical micelle concentration (CMC), dynamic light scattering (DLS), and atomic force microscopy (AFM). Transgene expression in ocular tissue after gene delivery was analyzed by 5-bromo-4-chloro-3-indolyl-beta-D-galactoside (X-Gal) color staining, 1,2-dioxetane beta-Gal enzymatic activity measurement, and real-time polymerase chain reaction (PCR) analysis. The delivery mechanisms of plasmid-PM on mouse and rabbit corneas were evaluated by EDTA and RGD (arginine-glycine-aspartic acid) peptide. The sizes of the three plasmid-PM complexes were around 150-200 nm with unimodal distribution. Enhanced stability was found for three plasmid-PM formulations after DNase I treatment. After six doses of eye drop delivery of pK12-Lac Z-PM three times a day, beta-Gal activity was significantly increased in both mouse and rabbit corneas. Stroma-specific Lac Z expression was only found in pKera3.2-Lac Z-PM-treated animals with pretreatment by 5 mM EDTA, an opener of junctions. Lac Z gene expression in both pK12-Lac Z-PM and pKera3.2-Lac Z-PM delivery groups was decreased by RGD peptide pretreatment. Cornea epithelium- and stroma-specific gene expression could be achieved using cornea-specific promoters of keratin 12 and keratocan genes, and the gene was delivered with PM formulation through non-invasive, eye drop in mice and rabbits. The transfection mechanism of plasmid-PM may involve endocytosis and particle size dependent paracellular transport. 2007 John Wiley & Sons, Ltd

AAV-Mediated Clarin-1 Expression in the Mouse Retina: Implications for USH3A Gene Therapy.

PubMed

Dinculescu, Astra; Stupay, Rachel M; Deng, Wen-Tao; Dyka, Frank M; Min, Seok-Hong; Boye, Sanford L; Chiodo, Vince A; Abrahan, Carolina E; Zhu, Ping; Li, Qiuhong; Strettoi, Enrica; Novelli, Elena; Nagel-Wolfrum, Kerstin; Wolfrum, Uwe; Smith, W Clay; Hauswirth, William W

2016-01-01

Usher syndrome type III (USH3A) is an autosomal recessive disorder caused by mutations in clarin-1 (CLRN1) gene, leading to progressive retinal degeneration and sensorineural deafness. Efforts to develop therapies for preventing photoreceptor cell loss are hampered by the lack of a retinal phenotype in the existing USH3 mouse models and by conflicting reports regarding the endogenous retinal localization of clarin-1, a transmembrane protein of unknown function. In this study, we used an AAV-based approach to express CLRN1 in the mouse retina in order to determine the pattern of its subcellular localization in different cell types. We found that all major classes of retinal cells express AAV-delivered CLRN1 driven by the ubiquitous, constitutive small chicken β-actin promoter, which has important implications for the design of future USH3 gene therapy studies. Within photoreceptor cells, AAV-expressed CLRN1 is mainly localized at the inner segment region and outer plexiform layer, similar to the endogenous expression of other usher proteins. Subretinal delivery using a full strength viral titer led to significant loss of retinal function as evidenced by ERG analysis, suggesting that there is a critical limit for CLRN1 expression in photoreceptor cells. Taken together, these results suggest that CLRN1 expression is potentially supported by a variety of retinal cells, and the right combination of AAV vector dose, promoter, and delivery method needs to be selected to develop safe therapies for USH3 disorder.

Cancer cell specific cytotoxic gene expression mediated by ARF tumor suppressor promoter constructs

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kurayoshi, Kenta; Ozono, Eiko; Iwanaga, Ritsuko

Highlights: • ARF promoter showed higher responsiveness to deregulated E2F activity than the E2F1 promoter. • ARF promoter showed higher cancer cell-specificity than E2F1 promoter to drive gene expression. • HSV-TK driven by ARF promoter showed higher cancer cell-specific cytotoxicity than that driven by E2F1 promoter. - Abstract: In current cancer treatment protocols, such as radiation and chemotherapy, side effects on normal cells are major obstacles to radical therapy. To avoid these side effects, a cancer cell-specific approach is needed. One way to specifically target cancer cells is to utilize a cancer specific promoter to express a cytotoxic gene (suicidemore » gene therapy) or a viral gene required for viral replication (oncolytic virotherapy). For this purpose, the selected promoter should have minimal activity in normal cells to avoid side effects, and high activity in a wide variety of cancers to obtain optimal therapeutic efficacy. In contrast to the AFP, CEA and PSA promoters, which have high activity only in a limited spectrum of tumors, the E2F1 promoter exhibits high activity in wide variety of cancers. This is based on the mechanism of carcinogenesis. Defects in the RB pathway and activation of the transcription factor E2F, the main target of the RB pathway, are observed in almost all cancers. Consequently, the E2F1 promoter, which is mainly regulated by E2F, has high activity in wide variety of cancers. However, E2F is also activated by growth stimulation in normal growing cells, suggesting that the E2F1 promoter may also be highly active in normal growing cells. In contrast, we found that the tumor suppressor ARF promoter is activated by deregulated E2F activity, induced by forced inactivation of pRB, but does not respond to physiological E2F activity induced by growth stimulation. We also found that the deregulated E2F activity, which activates the ARF promoter, is detected only in cancer cell lines. These observations suggest that ARF promoter is activated by E2F only in cancer cells and therefore may be more cancer cell-specific than E2F1 promoter to drive gene expression. We show here that the ARF promoter has lower activity in normal growing fibroblasts and shows higher cancer cell-specificity compared to the E2F1 promoter. We also demonstrate that adenovirus expressing HSV-TK under the control of the ARF promoter shows lower cytotoxicity than that of the E2F1 promoter, in normal growing fibroblasts but has equivalent cytotoxicity in cancer cell lines. These results suggest that the ARF promoter, which is specifically activated by deregulated E2F activity, is an excellent candidate to drive therapeutic cytotoxic gene expression, specifically in cancer cells.« less

Adaptation to acidic soil is achieved by increased numbers of cis-acting elements regulating ALMT1 expression in Holcus lanatus.

PubMed

Chen, Zhi Chang; Yokosho, Kengo; Kashino, Miho; Zhao, Fang-Jie; Yamaji, Naoki; Ma, Jian Feng

2013-10-01

Yorkshire fog (Holcus lanatus), which belongs to the Poaceae family and is a close relative of the agronomic crop oat (Avena sativa), is a widely adaptable grass species that is able to grow on highly acidic soils with high levels of Al, but the mechanism underlying the high Al tolerance is unknown. Here, we characterized two accessions of H. lanatus collected from an acid plot (soil pH 3.6, HL-A) and a neutral plot (pH 7.1, HL-N) in terms of Al tolerance, organic acid anion secretion and related gene expression. In response to Al (pH 4.5), the HL-A roots secreted approximately twice as much malate as the HL-N roots, but there was no difference in citrate secretion. Cloning of the gene HlALMT1 responsible for malate secretion showed that the encoded amino acid sequence did not differ between two accessions, but the expression level in the outer cell layers of the HL-A roots was twice as high as in the HL-N roots. This difference was not due to the genomic copy number, but was due to the number of cis-acting elements for an Al-responsive transcription factor (HlART1) in the promoter region of HlALMT1, as demonstrated by both a yeast one-hybrid assay and a transient assay in tobacco protoplasts. Furthermore, introduction of HlALMT1 driven by the HL-A promoter into rice resulted in significantly more Al-induced malate secretion than introduction of HlALMT1 driven by the HL-N promoter. These findings indicate that the adaptation of H. lanatus to acidic soils may be achieved by increasing number of cis-acting elements for ART1 in the promoter region of the HlALMT1 gene, enhancing the expression of HlALMT1 and the secretion of malate. © 2013 The Authors The Plant Journal © 2013 John Wiley & Sons Ltd.

The expression of dominant negative TCF7L2 in pancreatic beta cells during the embryonic stage causes impaired glucose homeostasis.

PubMed

Shao, Weijuan; Xiong, Xiaoquan; Ip, Wilfred; Xu, Fenghao; Song, Zhuolun; Zeng, Kejing; Hernandez, Marcela; Liang, Tao; Weng, Jianping; Gaisano, Herbert; Nostro, M Cristina; Jin, Tianru

2015-04-01

Disruption of TCF7L2 in mouse pancreatic β-cells has generated different outcomes in several investigations. Here we aim to clarify role of β-cell TCF7L2 and Wnt signaling using a functional-knockdown approach. Adenovirus-mediated dominant negative TCF7L2 (TCF7L2DN) expression was conducted in Ins-1 cells. The fusion gene in which TCF7L2DN expression is driven by P TRE3G was utilized to generate the transgenic mouse line TCF7L2DN Tet . The double transgenic line was created by mating TCF7L2DN Tet with Ins2-rtTA, designated as βTCFDN. β-cell specific TCF7L2DN expression was induced in βTCFDN by doxycycline feeding. TCF7L2DN expression in Ins-1 cells reduced GSIS, cell proliferation and expression of a battery of genes including incretin receptors and β-cell transcription factors. Inducing TCF7L2DN expression in βTCFDN during adulthood or immediately after weaning generated no or very modest metabolic defect, while its expression during embryonic development by doxycycline feeding in pregnant mothers resulted in significant glucose intolerance associated with altered β-cell gene expression and reduced β-cell mass. Our observations support a cell autonomous role for TCF7L2 in pancreatic β-cells suggested by most, though not all, investigations. βTCFDN is a novel model for further exploring the role of TCF7L2 in β-cell genesis and metabolic homeostasis.

Dynamic Maternal Gradients Control Timing and Shift-Rates for Drosophila Gap Gene Expression

PubMed Central

Verd, Berta; Crombach, Anton

2017-01-01

Pattern formation during development is a highly dynamic process. In spite of this, few experimental and modelling approaches take into account the explicit time-dependence of the rules governing regulatory systems. We address this problem by studying dynamic morphogen interpretation by the gap gene network in Drosophila melanogaster. Gap genes are involved in segment determination during early embryogenesis. They are activated by maternal morphogen gradients encoded by bicoid (bcd) and caudal (cad). These gradients decay at the same time-scale as the establishment of the antero-posterior gap gene pattern. We use a reverse-engineering approach, based on data-driven regulatory models called gene circuits, to isolate and characterise the explicitly time-dependent effects of changing morphogen concentrations on gap gene regulation. To achieve this, we simulate the system in the presence and absence of dynamic gradient decay. Comparison between these simulations reveals that maternal morphogen decay controls the timing and limits the rate of gap gene expression. In the anterior of the embyro, it affects peak expression and leads to the establishment of smooth spatial boundaries between gap domains. In the posterior of the embryo, it causes a progressive slow-down in the rate of gap domain shifts, which is necessary to correctly position domain boundaries and to stabilise the spatial gap gene expression pattern. We use a newly developed method for the analysis of transient dynamics in non-autonomous (time-variable) systems to understand the regulatory causes of these effects. By providing a rigorous mechanistic explanation for the role of maternal gradient decay in gap gene regulation, our study demonstrates that such analyses are feasible and reveal important aspects of dynamic gene regulation which would have been missed by a traditional steady-state approach. More generally, it highlights the importance of transient dynamics for understanding complex regulatory processes in development. PMID:28158178

Feedback inhibition by thiols outranks glutathione depletion: a luciferase-based screen reveals glutathione-deficient γ -ECS and glutathione synthetase mutants impaired in cadmium-induced sulfate assimilation

PubMed Central

Jobe, Timothy O.; Sung, Dong-Yul; Akmakjian, Garo; Pham, Allis; Komives, Elizabeth A.; Mendoza-Cózatl, David G.; Schroeder, Julian I.

2015-01-01

Summary Plants exposed to heavy metals rapidly induce changes in gene expression that activate and enhance detoxification mechanisms, including toxic-metal chelation and the scavenging of reactive oxygen species. However, the mechanisms mediating toxic heavy metal-induced gene expression remain largely unknown. To genetically elucidate cadmium-specific transcriptional responses in Arabidopsis, we designed a genetic screen based on the activation of a cadmium-inducible reporter gene. Microarray studies identified a high-affinity sulfate transporter (SULTR1;2) among the most robust and rapid cadmium-inducible transcripts. The SULTR1;2 promoter (2.2 kb) was fused with the firefly luciferase reporter gene to quantitatively report the transcriptional response of plants exposed to cadmium. Stably transformed luciferase reporter lines were ethyl methanesulfonate (EMS) mutagenized, and stable M2 seedlings were screened for an abnormal luciferase response during exposure to cadmium. The screen identified non-allelic mutant lines that fell into one of three categories: (i) super response to cadmium (SRC) mutants; (ii) constitutive response to cadmium (CRC) mutants; or (iii) non-response and reduced response to cadmium (NRC) mutants. Two nrc mutants, nrc1 and nrc2, were mapped, cloned and further characterized. The nrc1 mutation was mapped to the γ-glutamylcysteine synthetase gene and the nrc2 mutation was identified as the first viable recessive mutant allele in the glutathione synthetase gene. Moreover, genetic, HPLC mass spectrometry, and gene expression analysis of the nrc1 and nrc2 mutants, revealed that intracellular glutathione depletion alone would be insufficient to induce gene expression of sulfate uptake and assimilation mechanisms. Our results modify the glutathione-depletion driven model for sulfate assimilation gene induction during cadmium stress, and suggest that an enhanced oxidative state and depletion of upstream thiols, in addition to glutathione depletion, are necessary to induce the transcription of sulfate assimilation genes during early cadmium stress. PMID:22283708

Dynamic Maternal Gradients Control Timing and Shift-Rates for Drosophila Gap Gene Expression.

PubMed

Verd, Berta; Crombach, Anton; Jaeger, Johannes

2017-02-01

Pattern formation during development is a highly dynamic process. In spite of this, few experimental and modelling approaches take into account the explicit time-dependence of the rules governing regulatory systems. We address this problem by studying dynamic morphogen interpretation by the gap gene network in Drosophila melanogaster. Gap genes are involved in segment determination during early embryogenesis. They are activated by maternal morphogen gradients encoded by bicoid (bcd) and caudal (cad). These gradients decay at the same time-scale as the establishment of the antero-posterior gap gene pattern. We use a reverse-engineering approach, based on data-driven regulatory models called gene circuits, to isolate and characterise the explicitly time-dependent effects of changing morphogen concentrations on gap gene regulation. To achieve this, we simulate the system in the presence and absence of dynamic gradient decay. Comparison between these simulations reveals that maternal morphogen decay controls the timing and limits the rate of gap gene expression. In the anterior of the embyro, it affects peak expression and leads to the establishment of smooth spatial boundaries between gap domains. In the posterior of the embryo, it causes a progressive slow-down in the rate of gap domain shifts, which is necessary to correctly position domain boundaries and to stabilise the spatial gap gene expression pattern. We use a newly developed method for the analysis of transient dynamics in non-autonomous (time-variable) systems to understand the regulatory causes of these effects. By providing a rigorous mechanistic explanation for the role of maternal gradient decay in gap gene regulation, our study demonstrates that such analyses are feasible and reveal important aspects of dynamic gene regulation which would have been missed by a traditional steady-state approach. More generally, it highlights the importance of transient dynamics for understanding complex regulatory processes in development.

Transgenic rice plants harboring an introduced potato proteinase inhibitor II gene are insect resistant.

PubMed

Duan, X; Li, X; Xue, Q; Abo-el-Saad, M; Xu, D; Wu, R

1996-04-01

We introduced the potato proteinase inhibitor II (PINII) gene (pin2) into several Japonica rice varieties, and regenerated a large number of transgenic rice plants. Wound-inducible expression of the pin2 gene driven by its own promoter, together with the first intron of the rice actin 1 gene (act1), resulted in high-level accumulation of the PINII protein in the transgenic plants. The introduced pin2 gene was stably inherited in the second, third, and fourth generations, as shown by molecular analyses. Based on data from the molecular analyses, several homozygous transgenic lines were obtained. Bioassay for insect resistance with the fifth-generation transgenic rice plants showed that transgenic rice plants had increased resistance to a major rice insect pest, pink stem borer (Sesamia inferens). Thus, introduction of an insecticidal proteinase inhibitor gene into cereal plants can be used as a general strategy for control of insect pests.

Chronic Postnatal Stress Induces Depressive-like Behavior in Male Mice and Programs second-Hit Stress-Induced Gene Expression Patterns of OxtR and AvpR1a in Adulthood.

PubMed

Lesse, Alexandra; Rether, Kathy; Gröger, Nicole; Braun, Katharina; Bock, Jörg

2017-08-01

Chronic stress (CS) during early life represents a major risk factor for the development of mental disorders, including depression. According to the Two/Multiple-Hit hypothesis, the etiology of neuropsychiatric disorders usually involves multiple stressors experienced subsequently during different phases of life. However, the molecular and cellular mechanisms modulating neuronal and behavioral changes induced by multiple stress experiences are just poorly understood. Since the oxytocinergic and vasopressinergic systems are neuroendocrine modulators involved in environmentally driven adaptations of stress sensitivity we hypothesized that postnatal CS programs oxytocinergic and vasopressinergic receptor expression changes in response to a second stress exposure in young adulthood. First we investigated if postnatal CS (maternal separation + social isolation) induces depressive-like behavior and alters oxytocin receptor (OxtR) and arginine vasopressin receptor type 1a (AvpR1a) gene expression in the hippocampus (HC) of male mice and (2) if a second single stressor (forced swimming, FS) in young adulthood affects gene expression of OxtR and AvpR1a at adulthood dependent on CS pre-experience. We found that postnatal CS induced depressive-like behavior and enhanced AvpR1a expression in HC at young adulthood. Moreover, in line with our hypothesis, only combined stress exposure (CS + FS), but not CS or FS alone, resulted in increased gene expression of OxtR in HC at adulthood. In contrast, AvpR1a expression was decreased in both adult FS and CS + FS animals. Overall, our results provide evidence that CS programs neuroendocrine systems and thereby influences stress responses in later life periods.

Hierarchy within the mammary STAT5-driven Wap super-enhancer.

PubMed

Shin, Ha Youn; Willi, Michaela; HyunYoo, Kyung; Zeng, Xianke; Wang, Chaochen; Metser, Gil; Hennighausen, Lothar

2016-08-01

Super-enhancers comprise dense transcription factor platforms highly enriched for active chromatin marks. A paucity of functional data led us to investigate the role of super-enhancers in the mammary gland, an organ characterized by exceptional gene regulatory dynamics during pregnancy. ChIP-seq analysis for the master regulator STAT5A, the glucocorticoid receptor, H3K27ac and MED1 identified 440 mammary-specific super-enhancers, half of which were associated with genes activated during pregnancy. We interrogated the Wap super-enhancer, generating mice carrying mutations in STAT5-binding sites within its constituent enhancers. Individually, the most distal site displayed the greatest enhancer activity. However, combinatorial mutation analysis showed that the 1,000-fold induction in gene expression during pregnancy relied on all enhancers. Disabling the binding sites of STAT5, NFIB and ELF5 in the proximal enhancer incapacitated the entire super-enhancer. Altogether, these data suggest a temporal and functional enhancer hierarchy. The identification of mammary-specific super-enhancers and the mechanistic exploration of the Wap locus provide insights into the regulation of cell-type-specific expression of hormone-sensing genes.

Targeting MOG expression to dendritic cells delays onset of experimental autoimmune disease.

PubMed

Ko, Hyun-Ja; Chung, Jie-Yu; Nasa, Zeyad; Chan, James; Siatskas, Christopher; Toh, Ban-Hock; Alderuccio, Frank

2011-05-01

Haematopoietic stem cell (HSC) transfer coupled with gene therapy is a powerful approach to treating fatal diseases such as X-linked severe combined immunodeficiency. This ability to isolate and genetically manipulate HSCs also offers a strategy for inducing immune tolerance through ectopic expression of autoantigens. We have previously shown that retroviral transduction of bone marrow (BM) with vectors encoding the autoantigen, myelin oligodendrocyte glycoprotein (MOG), can prevent the induction of experimental autoimmune encephalomyelitis (EAE). However, ubiquitous cellular expression of autoantigen driven by retroviral promoters may not be the best approach for clinical translation and a targeted expression approach may be more acceptable. As BM-derived dendritic cells (DCs) play a major role in tolerance induction, we asked whether targeted expression of MOG, a target autoantigen in EAE, to DCs can promote tolerance induction and influence the development of EAE. Self-inactivating retroviral vectors incorporating the mouse CD11c promoter were generated and used to transduce mouse BM cells. Transplantation of gene-modified cells into irradiated recipients resulted in the generation of chimeric mice with transgene expression limited to DCs. Notably, chimeric mice transplanted with MOG-expressing BM cells manifest a significant delay in the development of EAE suggesting that targeted antigen expression to tolerogenic cell types may be a feasible approach to inducing antigen-specific tolerance.

Unique Physiological and Transcriptional Shifts under Combinations of Salinity, Drought, and Heat.

PubMed

Shaar-Moshe, Lidor; Blumwald, Eduardo; Peleg, Zvi

2017-05-01

Climate-change-driven stresses such as extreme temperatures, water deficit, and ion imbalance are projected to exacerbate and jeopardize global food security. Under field conditions, these stresses usually occur simultaneously and cause damages that exceed single stresses. Here, we investigated the transcriptional patterns and morpho-physiological acclimations of Brachypodium dystachion to single salinity, drought, and heat stresses, as well as their double and triple stress combinations. Hierarchical clustering analysis of morpho-physiological acclimations showed that several traits exhibited a gradually aggravating effect as plants were exposed to combined stresses. On the other hand, other morphological traits were dominated by salinity, while some physiological traits were shaped by heat stress. Response patterns of differentially expressed genes, under single and combined stresses (i.e. common stress genes), were maintained only among 37% of the genes, indicating a limited expression consistency among partially overlapping stresses. A comparison between common stress genes and genes that were uniquely expressed only under combined stresses (i.e. combination unique genes) revealed a significant shift from increased intensity to antagonistic responses, respectively. The different transcriptional signatures imply an alteration in the mode of action under combined stresses and limited ability to predict plant responses as different stresses are combined. Coexpression analysis coupled with enrichment analysis revealed that each gene subset was enriched with different biological processes. Common stress genes were enriched with known stress response pathways, while combination unique-genes were enriched with unique processes and genes with unknown functions that hold the potential to improve stress tolerance and enhance cereal productivity under suboptimal field conditions. © 2017 American Society of Plant Biologists. All Rights Reserved.

Dose additive effects of simvastatin and dipentyl phthalate on ...

EPA Pesticide Factsheets

Sex differentiation of the mammalian reproductive tract is a highly regulated process that is driven, in part, by fetal testosterone (T) production. In utero exposure to phthalate esters (PE) during sex differentiation can cause reproductive tract malformations in rats. PE alter the expression of genes associated with steroid synthesis/transport and cholesterol biosynthesis. Simvastatin (SMV) is a cholesterol-lowering drug that inhibits HMG-CoA reductase. As cholesterol is a precursor for steroid biosynthesis, we proposed that maternal exposure to SMV during the critical period of sex differentiation would lower fetal T and result in corresponding alterations in cholesterol- and androgenmediated gene expression. Timed pregnant SD rats were dosed orally with SMV from GD14-GD18. T production on GD18 was measured by RIA, and changes in gene expression in maternal and fetal tissues were assessed by quantitative rt-PCR. Circulating lipids were also measured in dams and fetuses. SMV lowered fetal T production, altered several genes involved in cholesterol biosynthesis in the maternal liver, and lowered lipids in the fetus but not in the dam. Unlike PE, SMV did not alter genes associated with sex differentiation. In a second experiment, dams were dosed with SMV, dipentyl phthalate (DPeP, a PE), or both. SMV and DPeP alone reduced fetal T production to 44.3 and 37.5% of control values, respectively, but the mixture reduced T production to 19.9% of control. These studies

New potential markers of in vitro tomato morphogenesis identified by mRNA differential display.

PubMed

Torelli, A; Soragni, E; Bolchi, A; Petrucco, S; Ottonello, S; Branca, C

1996-12-01

The identification of plant genes involved in early phases of in vitro morphogenesis can not only contribute to our understanding of the processes underlying growth regulator-controlled determination, but also provide novel markers for evaluating the outcome of in vitro regeneration experiments. To search for such genes and to monitor changes in gene expression accompanying in vitro regeneration, we have adapted the mRNA differential display technique to the comparative analysis of a model system of tomato cotyledons that can be driven selectively toward either shoot or callus formation by means of previously determined growth regulator supplementations. Hormone-independent transcriptional modulation (mainly down-regulation) has been found to be the most common event, indicating that a non-specific reprogramming of gene expression quantitatively predominates during the early phases of in vitro culture. However, cDNA fragments representative of genes that are either down-regulated or induced in a programme-specific manner could also be identified, and two of them (G35, G36) were further characterized. One of these cDNA fragments, G35, corresponds to an mRNA that is down-regulated much earlier in callus- (day 2) than in shoot-determined explants (day 6). The other, G36, identifies an mRNA that is transiently expressed in shoot-determined explants only, well before any macroscopic signs of differentiation become apparent, and thus exhibits typical features of a morphogenetic marker.

Assessment of Chitosan-Affected Metabolic Response by Peroxisome Proliferator-Activated Receptor Bioluminescent Imaging-Guided Transcriptomic Analysis

PubMed Central

Kao, Chia-Hung; Hsiang, Chien-Yun; Ho, Tin-Yun

2012-01-01

Chitosan has been widely used in food industry as a weight-loss aid and a cholesterol-lowering agent. Previous studies have shown that chitosan affects metabolic responses and contributes to anti-diabetic, hypocholesteremic, and blood glucose-lowering effects; however, the in vivo targeting sites and mechanisms of chitosan remain to be clarified. In this study, we constructed transgenic mice, which carried the luciferase genes driven by peroxisome proliferator-activated receptor (PPAR), a key regulator of fatty acid and glucose metabolism. Bioluminescent imaging of PPAR transgenic mice was applied to report the organs that chitosan acted on, and gene expression profiles of chitosan-targeted organs were further analyzed to elucidate the mechanisms of chitosan. Bioluminescent imaging showed that constitutive PPAR activities were detected in brain and gastrointestinal tract. Administration of chitosan significantly activated the PPAR activities in brain and stomach. Microarray analysis of brain and stomach showed that several pathways involved in lipid and glucose metabolism were regulated by chitosan. Moreover, the expression levels of metabolism-associated genes like apolipoprotein B (apoB) and ghrelin genes were down-regulated by chitosan. In conclusion, these findings suggested the feasibility of PPAR bioluminescent imaging-guided transcriptomic analysis on the evaluation of chitosan-affected metabolic responses in vivo. Moreover, we newly identified that downregulated expression of apoB and ghrelin genes were novel mechanisms for chitosan-affected metabolic responses in vivo. PMID:22496881

The genomic and transcriptomic architecture of 2,000 breast tumours reveals novel subgroups

PubMed Central

Curtis, Christina; Shah, Sohrab P.; Chin, Suet-Feung; Turashvili, Gulisa; Rueda, Oscar M.; Dunning, Mark J.; Speed, Doug; Lynch, Andy G.; Samarajiwa, Shamith; Yuan, Yinyin; Gräf, Stefan; Ha, Gavin; Haffari, Gholamreza; Bashashati, Ali; Russell, Roslin; McKinney, Steven; Langerød, Anita; Green, Andrew; Provenzano, Elena; Wishart, Gordon; Pinder, Sarah; Watson, Peter; Markowetz, Florian; Murphy, Leigh; Ellis, Ian; Purushotham, Arnie; Børresen-Dale, Anne-Lise; Brenton, James D.; Tavaré, Simon; Caldas, Carlos; Aparicio, Samuel

2012-01-01

The elucidation of breast cancer subgroups and their molecular drivers requires integrated views of the genome and transcriptome from representative numbers of patients. We present an integrated analysis of copy number and gene expression in a discovery and validation set of 997 and 995 primary breast tumours, respectively, with long-term clinical follow-up. Inherited variants (copy number variants and single nucleotide polymorphisms) and acquired somatic copy number aberrations (CNAs) were associated with expression in ~40% of genes, with the landscape dominated by cis- and trans-acting CNAs. By delineating expression outlier genes driven in cis by CNAs, we identified putative cancer genes, including deletions in PPP2R2A, MTAP and MAP2K4. Unsupervised analysis of paired DNA–RNA profiles revealed novel subgroups with distinct clinical outcomes, which reproduced in the validation cohort. These include a high-risk, oestrogen-receptor-positive 11q13/14 cis-acting subgroup and a favourable prognosis subgroup devoid of CNAs. Trans-acting aberration hotspots were found to modulate subgroup-specific gene networks, including a TCR deletion-mediated adaptive immune response in the ‘CNA-devoid’ subgroup and a basal-specific chromosome 5 deletion-associated mitotic network. Our results provide a novel molecular stratification of the breast cancer population, derived from the impact of somatic CNAs on the transcriptome. PMID:22522925

Comparison of CRISPR/Cas9 expression constructs for efficient targeted mutagenesis in rice.

PubMed

Mikami, Masafumi; Toki, Seiichi; Endo, Masaki

2015-08-01

The CRISPR/Cas9 system is an efficient tool used for genome editing in a variety of organisms. Despite several recent reports of successful targeted mutagenesis using the CRISPR/Cas9 system in plants, in each case the target gene of interest, the Cas9 expression system and guide-RNA (gRNA) used, and the tissues used for transformation and subsequent mutagenesis differed, hence the reported frequencies of targeted mutagenesis cannot be compared directly. Here, we evaluated mutation frequency in rice using different Cas9 and/or gRNA expression cassettes under standardized experimental conditions. We introduced Cas9 and gRNA expression cassettes separately or sequentially into rice calli, and assessed the frequency of mutagenesis at the same endogenous targeted sequences. Mutation frequencies differed significantly depending on the Cas9 expression cassette used. In addition, a gRNA driven by the OsU6 promoter was superior to one driven by the OsU3 promoter. Using an all-in-one expression vector harboring the best combined Cas9/gRNA expression cassette resulted in a much improved frequency of targeted mutagenesis in rice calli, and bi-allelic mutant plants were produced in the T0 generation. The approach presented here could be adapted to optimize the construction of Cas9/gRNA cassettes for genome editing in a variety of plants.

Singing activity-driven Arc expression associated with vocal acoustic plasticity in juvenile songbird.

PubMed

Hayase, Shin; Wada, Kazuhiro

2018-06-23

Learned vocalization, including birdsong and human speech, is acquired through self-motivated vocal practice during the sensitive period of vocal learning. The zebra finch (Taeniopygia guttata) develops a song characterized by vocal variability and crystalizes a defined song pattern as adulthood. However, it remains unknown how vocal variability is regulated with diurnal singing during the sensorimotor learning period. Here, we investigated the expression of activity-dependent neuroplasticity-related gene Arc during the early plastic song phase to examine its potential association with vocal plasticity. We first confirmed that multiple acoustic features of syllables in the plastic song were dramatically and simultaneously modulated during the first 3 hours of singing in a day and the altered features were maintained until sleep. Concurrently, Arc was intensely induced during morning singing and a subsequent attenuation during afternoon singing in the robust nucleus of the arcopallium (RA) and the interfacial nucleus of the nidopallium (NIf). The singing-driven Arc expression was not altered by circadian rhythm, but rather reduced during the day as juveniles produced more songs. Song stabilization accelerated by testosterone administration in juveniles was accompanied with attenuation of Arc induction in RA and NIf. In contrast, although early-deafened birds produced highly unstable song even at adulthood, singing-driven Arc expression was not different between intact and early-deafened adults. These results suggest a potential functional link between Arc expression in RA and NIf and vocal plasticity during the sensorimotor phase of song learning. Nonetheless, Arc expression did not reflect the quality of bird's own song or auditory feedback. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Self-association of Gata1 enhances transcriptional activity in vivo in zebra fish embryos.

PubMed

Nishikawa, Keizo; Kobayashi, Makoto; Masumi, Atsuko; Lyons, Susan E; Weinstein, Brant M; Liu, P Paul; Yamamoto, Masayuki

2003-11-01

Gata1 is a prototype transcription factor that regulates hematopoiesis, yet the molecular mechanisms by which Gata1 transactivates its target genes in vivo remain unclear. We previously showed, in transgenic zebra fish, that Gata1 autoregulates its own expression. In this study, we characterized the molecular mechanisms for this autoregulation by using mutations in the Gata1 protein which impair autoregulation. Of the tested mutations, replacement of six lysine residues with alanine (Gata1KA6), which inhibited self-association activity of Gata1, reduced the Gata1-dependent induction of reporter gene expression driven by the zebra fish gata1 hematopoietic regulatory domain (gata1 HRD). Furthermore, overexpression of wild-type Gata1 but not Gata1KA6 rescued the expression of Gata1 downstream genes in vlad tepes, a germ line gata1 mutant fish. Interestingly, both GATA sites in the double GATA motif in gata1 HRD were critical for the promoter activity and for binding of the self-associated Gata1 complex, whereas only the 3'-GATA site was required for Gata1 monomer binding. These results thus provide the first in vivo evidence that the ability of Gata1 to self-associate critically contributes to the autoregulation of the gata1 gene.

Global survey of genomic imprinting by transcriptome sequencing.

PubMed

Babak, Tomas; Deveale, Brian; Armour, Christopher; Raymond, Christopher; Cleary, Michele A; van der Kooy, Derek; Johnson, Jason M; Lim, Lee P

2008-11-25

Genomic imprinting restricts gene expression to a paternal or maternal allele. To date, approximately 90 imprinted transcripts have been identified in mouse, of which the majority were detected after intense interrogation of clusters of imprinted genes identified by phenotype-driven assays in mice with uniparental disomies [1]. Here we use selective priming and parallel sequencing to measure allelic bias in whole transcriptomes. By distinguishing parent-of-origin bias from strain-specific bias in embryos derived from a reciprocal cross of mice, we constructed a genome-wide map of imprinted transcription. This map was able to objectively locate over 80% of known imprinted loci and allowed the detection and confirmation of six novel imprinted genes. Even in the intensely studied embryonic day 9.5 developmental stage that we analyzed, more than half of all imprinted single-nucleotide polymorphisms did not overlap previously discovered imprinted transcripts; a large fraction of these represent novel noncoding RNAs within known imprinted loci. For example, a previously unnoticed, maternally expressed antisense transcript was mapped within the Grb10 locus. This study demonstrates the feasibility of using transcriptome sequencing for mapping of imprinted gene expression in physiologically normal animals. Such an approach will allow researchers to study imprinting without restricting themselves to individual loci or specific transcripts.

Synthetic phytochelatin surface display in Cupriavidus metallidurans CH34 for enhanced metals bioremediation.

PubMed

Biondo, Ronaldo; da Silva, Felipe Almeida; Vicente, Elisabete José; Souza Sarkis, Jorge Eduardo; Schenberg, Ana Clara Guerrini

2012-08-07

This work describes the effects of the cell surface display of a synthetic phytochelatin in the highly metal tolerant bacterium Cupriavidus metallidurans CH34. The EC20sp synthetic phytochelatin gene was fused between the coding sequences of the signal peptide (SS) and of the autotransporter β-domain of the Neisseria gonorrhoeae IgA protease precursor (IgAβ), which successfully targeted the hybrid protein toward the C. metallidurans outer membrane. The expression of the SS-EC20sp-IgAβ gene fusion was driven by a modified version of the Bacillus subtilis mrgA promoter showing high level basal gene expression that is further enhanced by metal presence in C. metallidurans. The recombinant strain showed increased ability to immobilize Pb(2+), Zn(2+), Cu(2+), Cd(2+), Mn(2+), and Ni(2+) ions from the external medium when compared to the control strain. To ensure plasmid stability and biological containment, the MOB region of the plasmid was replaced by the E. coli hok/sok coding sequence.

Functional Role of PPARs in Ruminants: Potential Targets for Fine-Tuning Metabolism during Growth and Lactation

PubMed Central

Chen, Shuowen; Khan, Muhammad J.; Loor, Juan J.

2013-01-01

Characterization and biological roles of the peroxisome proliferator-activated receptor (PPAR) isotypes are well known in monogastrics, but not in ruminants. However, a wealth of information has accumulated in little more than a decade on ruminant PPARs including isotype tissue distribution, response to synthetic and natural agonists, gene targets, and factors affecting their expression. Functional characterization demonstrated that, as in monogastrics, the PPAR isotypes control expression of genes involved in lipid metabolism, anti-inflammatory response, development, and growth. Contrary to mouse, however, the PPARγ gene network appears to controls milk fat synthesis in lactating ruminants. As in monogastrics, PPAR isotypes in ruminants are activated by long-chain fatty acids, therefore, making them ideal candidates for fine-tuning metabolism in this species via nutrients. In this regard, using information accumulated in ruminants and monogastrics, we propose a model of PPAR isotype-driven biological functions encompassing key tissues during the peripartal period in dairy cattle. PMID:23737762

The dyskerin ribonucleoprotein complex as an OCT4/SOX2 coactivator in embryonic stem cells

PubMed Central

Fong, Yick W; Ho, Jaclyn J; Inouye, Carla; Tjian, Robert

2014-01-01

Acquisition of pluripotency is driven largely at the transcriptional level by activators OCT4, SOX2, and NANOG that must in turn cooperate with diverse coactivators to execute stem cell-specific gene expression programs. Using a biochemically defined in vitro transcription system that mediates OCT4/SOX2 and coactivator-dependent transcription of the Nanog gene, we report the purification and identification of the dyskerin (DKC1) ribonucleoprotein complex as an OCT4/SOX2 coactivator whose activity appears to be modulated by a subset of associated small nucleolar RNAs (snoRNAs). The DKC1 complex occupies enhancers and regulates the expression of key pluripotency genes critical for self-renewal in embryonic stem (ES) cells. Depletion of DKC1 in fibroblasts significantly decreased the efficiency of induced pluripotent stem (iPS) cell generation. This study thus reveals an unanticipated transcriptional role of the DKC1 complex in stem cell maintenance and somatic cell reprogramming. DOI: http://dx.doi.org/10.7554/eLife.03573.001 PMID:25407680

Group 2 Innate Lymphoid Cells Exhibit a Dynamic Phenotype in Allergic Airway Inflammation

PubMed Central

Li, Bobby W. S.; Stadhouders, Ralph; de Bruijn, Marjolein J. W.; Lukkes, Melanie; Beerens, Dior M. J. M.; Brem, Maarten D.; KleinJan, Alex; Bergen, Ingrid; Vroman, Heleen; Kool, Mirjam; van IJcken, Wilfred F. J.; Rao, Tata Nageswara; Fehling, Hans Jörg; Hendriks, Rudi W.

2017-01-01

Group 2 innate lymphoid cells (ILC2) are implicated in allergic asthma as an early innate source of the type 2 cytokines IL-5 and IL-13. However, their induction in house dust mite (HDM)-mediated airway inflammation additionally requires T cell activation. It is currently unknown whether phenotypic differences exist between ILC2s that are activated in a T cell-dependent or T cell-independent fashion. Here, we compared ILC2s in IL-33- and HDM-driven airway inflammation. Using flow cytometry, we found that surface expression levels of various markers frequently used to identify ILC2s were dependent on their mode of activation, highly variable over time, and differed between tissue compartments, including bronchoalveolar lavage (BAL) fluid, lung, draining lymph nodes, and spleen. Whereas in vivo IL-33-activated BAL fluid ILC2s exhibited an almost uniform CD25+CD127+T1/ST2+ICOS+KLRG1+ phenotype, at a comparable time point after HDM exposure BAL fluid ILC2s had a very heterogeneous surface marker phenotype. A major fraction of HDM-activated ILC2s were CD25lowCD127+T1/ST2low ICOSlowKLRG1low, but nevertheless had the capacity to produce large amounts of type 2 cytokines. HDM-activated CD25low ILC2s in BAL fluid and lung rapidly reverted to CD25high ILC2s upon in vivo stimulation with IL-33. Genome-wide transcriptional profiling of BAL ILC2s revealed ~1,600 differentially expressed genes: HDM-stimulated ILC2s specifically expressed genes involved in the regulation of adaptive immunity through B and T cell interactions, whereas IL-33-stimulated ILC2s expressed high levels of proliferation-related and cytokine genes. In both airway inflammation models ILC2s were present in the lung submucosa close to epithelial cells, as identified by confocal microscopy. In chronic HDM-driven airway inflammation ILC2s were also found inside organized cellular infiltrates near T cells. Collectively, our findings show that ILC2s are phenotypically more heterogeneous than previously thought, whereby their surface marker and gene expression profile are highly dynamic. PMID:29250067

SPBP Is a Sulforaphane Induced Transcriptional Coactivator of NRF2 Regulating Expression of the Autophagy Receptor p62/SQSTM1

PubMed Central

Darvekar, Sagar Ramesh; Elvenes, Julianne; Brenne, Hanne Britt; Johansen, Terje; Sjøttem, Eva

2014-01-01

Organisms exposed to oxidative stress respond by orchestrating a stress response to prevent further damage. Intracellular levels of antioxidant agents increase, and damaged components are removed by autophagy induction. The KEAP1-NRF2 signaling pathway is the main pathway responsible for cell defense against oxidative stress and for maintaining the cellular redox balance at physiological levels. Sulforaphane, an isothiocyanate derived from cruciferous vegetables, is a potent inducer of KEAP1-NRF2 signaling and antioxidant response element driven gene expression. In this study, we show that sulforaphane enhances the expression of the transcriptional coregulator SPBP. The expression curve peaks 6–8 hours post stimulation, and parallels the sulforaphane-induced expression of NRF2 and the autophagy receptor protein p62/SQSTM1. Reporter gene assays show that SPBP stimulates the expression of p62/SQSTM1 via ARE elements in the promoter region, and siRNA mediated knock down of SPBP significantly decreases the expression of p62/SQSTM1 and the formation of p62/SQSTM1 bodies in HeLa cells. Furthermore, SPBP siRNA reduces the sulforaphane induced expression of NRF2, and the expression of the autophagy marker protein LC3B. Both these proteins contain ARE-like elements in their promoter regions. Over-expressed SPBP and NRF2 acts synergistically on the p62/SQSTM1 promoter and colocalize in nuclear speckles in HeLa cells. Collectively, these results suggest that SPBP is a coactivator of NRF2, and hence may be important for securing enhanced and sustained expression of NRF2 induced genes such as proteins involved in selective autophagy. PMID:24416372

PromoterCAD: data-driven design of plant regulatory DNA

PubMed Central

Cox, Robert Sidney; Nishikata, Koro; Shimoyama, Sayoko; Yoshida, Yuko; Matsui, Minami; Makita, Yuko; Toyoda, Tetsuro

2013-01-01

Synthetic promoters can control the timing, location and amount of gene expression for any organism. PromoterCAD is a web application for designing synthetic promoters with altered transcriptional regulation. We use a data-first approach, using published high-throughput expression and motif data from for Arabidopsis thaliana to guide DNA design. We demonstrate data mining tools for finding motifs related to circadian oscillations and tissue-specific expression patterns. PromoterCAD is built on the LinkData open platform for data publication and rapid web application development, allowing new data to be easily added, and the source code modified to add new functionality. PromoterCAD URL: http://promotercad.org. LinkData URL: http://linkdata.org. PMID:23766287

Ubiquitin promoter-terminator cassette promotes genetically stable expression of the taste-modifying protein miraculin in transgenic lettuce.

PubMed

Hirai, Tadayoshi; Shohael, Abdullah Mohammad; Kim, You-Wang; Yano, Megumu; Ezura, Hiroshi

2011-12-01

Lettuce is a commercially important leafy vegetable that is cultivated worldwide, and it is also a target crop for plant factories. In this study, lettuce was selected as an alternative platform for recombinant miraculin production because of its fast growth, agronomic value, and wide availability. The taste-modifying protein miraculin is a glycoprotein extracted from the red berries of the West African native shrub Richadella dulcifica. Because of its limited natural availability, many attempts have been made to produce this protein in suitable alternative hosts. We produced transgenic lettuce with miraculin gene driven either by the ubiquitin promoter/terminator cassette from lettuce or a 35S promoter/nos terminator cassette. Miraculin gene expression and miraculin accumulation in both cassettes were compared by quantitative real-time PCR analysis, Western blotting, and enzyme-linked immunosorbent assay. The expression level of the miraculin gene and protein in transgenic lettuce was higher and more genetically stable in the ubiquitin promoter/terminator cassette than in the 35S promoter/nos terminator cassette. These results demonstrated that the ubiquitin promoter/terminator cassette is an efficient platform for the genetically stable expression of the miraculin protein in lettuce and hence this platform is of benefit for recombinant miraculin production on a commercial scale.

The ubiquitin ligase Siah2 regulates obesity-induced adipose tissue inflammation.

PubMed

Kilroy, Gail; Carter, Lauren E; Newman, Susan; Burk, David H; Manuel, Justin; Möller, Andreas; Bowtell, David D; Mynatt, Randall L; Ghosh, Sujoy; Floyd, Z Elizabeth

2015-11-01

Chronic, low-grade adipose tissue inflammation associated with adipocyte hypertrophy is an important link in the relationship between obesity and insulin resistance. Although ubiquitin ligases regulate inflammatory processes, the role of these enzymes in metabolically driven adipose tissue inflammation is relatively unexplored. Herein, the effect of the ubiquitin ligase Siah2 on obesity-related adipose tissue inflammation was examined. Wild-type and Siah2KO mice were fed a low- or high-fat diet for 16 weeks. Indirect calorimetry, body composition, and glucose and insulin tolerance were assayed along with glucose and insulin levels. Gene and protein expression, immunohistochemistry, adipocyte size distribution, and lipolysis were also analyzed. Enlarged adipocytes in obese Siah2KO mice were not associated with obesity-induced insulin resistance. Proinflammatory gene expression, stress kinase signaling, fibrosis, and crown-like structures were reduced in the Siah2KO adipose tissue, and Siah2KO adipocytes were more responsive to insulin-dependent inhibition of lipolysis. Loss of Siah2 increased expression of PPARγ target genes involved in lipid metabolism and decreased expression of proinflammatory adipokines regulated by PPARγ. Siah2 links adipocyte hypertrophy with adipocyte dysfunction and recruitment of proinflammatory immune cells to adipose tissue. Selective regulation of PPARγ activity is a Siah2-mediated mechanism contributing to obesity-induced adipose tissue inflammation. © 2015 The Obesity Society.

Progressive histological damage in renal allografts is associated with expression of innate and adaptive immunity genes

PubMed Central

Naesens, Maarten; Khatri, Purvesh; Li, Li; Sigdel, Tara K.; Vitalone, Matthew J.; Chen, Rong; Butte, Atul J.; Salvatierra, Oscar; Sarwal, Minnie M.

2015-01-01

The degree of progressive chronic histological damage is associated with long-term renal allograft survival. In order to identify promising molecular targets for timely intervention, we examined renal allograft protocol and indication biopsies from 120 low-risk pediatric and adolescent recipients by whole-genome microarray expression profiling. In data-driven analysis, we found a highly regulated pattern of adaptive and innate immune gene expression that correlated with established or ongoing histological chronic injury, and also with development of future chronic histological damage, even in histologically pristine kidneys. Hence, histologically unrecognized immunological injury at a molecular level sets the stage for the development of chronic tissue injury, while the same molecular response is accentuated during established and worsening chronic allograft damage. Irrespective of the hypothesized immune or nonimmune trigger for chronic allograft injury, a highly orchestrated regulation of innate and adaptive immune responses was found in the graft at the molecular level. This occurred months before histologic lesions appear, and quantitatively below the diagnostic threshold of classic T-cell or antibody-mediated rejection. Thus, measurement of specific immune gene expression in protocol biopsies may be warranted to predict the development of subsequent chronic injury in histologically quiescent grafts and as a means to titrate immunosuppressive therapy. PMID:21881554

Progressive histological damage in renal allografts is associated with expression of innate and adaptive immunity genes.

PubMed

Naesens, Maarten; Khatri, Purvesh; Li, Li; Sigdel, Tara K; Vitalone, Matthew J; Chen, Rong; Butte, Atul J; Salvatierra, Oscar; Sarwal, Minnie M

2011-12-01

The degree of progressive chronic histological damage is associated with long-term renal allograft survival. In order to identify promising molecular targets for timely intervention, we examined renal allograft protocol and indication biopsies from 120 low-risk pediatric and adolescent recipients by whole-genome microarray expression profiling. In data-driven analysis, we found a highly regulated pattern of adaptive and innate immune gene expression that correlated with established or ongoing histological chronic injury, and also with development of future chronic histological damage, even in histologically pristine kidneys. Hence, histologically unrecognized immunological injury at a molecular level sets the stage for the development of chronic tissue injury, while the same molecular response is accentuated during established and worsening chronic allograft damage. Irrespective of the hypothesized immune or nonimmune trigger for chronic allograft injury, a highly orchestrated regulation of innate and adaptive immune responses was found in the graft at the molecular level. This occurred months before histologic lesions appear, and quantitatively below the diagnostic threshold of classic T-cell or antibody-mediated rejection. Thus, measurement of specific immune gene expression in protocol biopsies may be warranted to predict the development of subsequent chronic injury in histologically quiescent grafts and as a means to titrate immunosuppressive therapy.

Development of Cre-loxP technology in zebrafish to study the regulation of fish reproduction.

PubMed

Lin, Heng-Ju; Lee, Shu-Hua; Wu, Jen-Leih; Duann, Yeh-Fang; Chen, Jyh-Yih

2013-12-01

One cannot seek permission to market transgenic fish mainly because there is no field test or any basic research on technological developments for evaluating their biosafety. Infertility is a necessary adjunct to exploiting transgenic fish unless completely secure land-locked facilities are available. In this study, we report the generation of a Cre transgenic zebrafish line using a cytomegalovirus promoter. We also produced fish carrying the Bax1 and Bax2 plasmids; these genes were separated by two loxP sites under a zona pellucida C promoter or were driven by an anti-Müllerian hormone promoter. We inserted a red fluorescent protein gene between the two loxP sites. After obtaining transgenic lines with the two transgenic fish crossed with each other (Cre transgenic zebrafish x loxP transgenic zebrafish), the floxed DNA was found to be specifically eliminated from the female or male zebrafish, and apoptosis gene expressions caused ovarian and testicular growth cessation and degeneration. Overexpression of the Bax1 and Bax2 genes caused various expression levels of apoptosis-related genes. Accordingly, this transgenic zebrafish model system provides a method to produce infertile fish and may be useful for application to genetically modified fish.

Digital transcriptome profiling of normal and glioblastoma-derived neural stem cells identifies genes associated with patient survival

PubMed Central

2012-01-01

Background Glioblastoma multiforme, the most common type of primary brain tumor in adults, is driven by cells with neural stem (NS) cell characteristics. Using derivation methods developed for NS cells, it is possible to expand tumorigenic stem cells continuously in vitro. Although these glioblastoma-derived neural stem (GNS) cells are highly similar to normal NS cells, they harbor mutations typical of gliomas and initiate authentic tumors following orthotopic xenotransplantation. Here, we analyzed GNS and NS cell transcriptomes to identify gene expression alterations underlying the disease phenotype. Methods Sensitive measurements of gene expression were obtained by high-throughput sequencing of transcript tags (Tag-seq) on adherent GNS cell lines from three glioblastoma cases and two normal NS cell lines. Validation by quantitative real-time PCR was performed on 82 differentially expressed genes across a panel of 16 GNS and 6 NS cell lines. The molecular basis and prognostic relevance of expression differences were investigated by genetic characterization of GNS cells and comparison with public data for 867 glioma biopsies. Results Transcriptome analysis revealed major differences correlated with glioma histological grade, and identified misregulated genes of known significance in glioblastoma as well as novel candidates, including genes associated with other malignancies or glioma-related pathways. This analysis further detected several long non-coding RNAs with expression profiles similar to neighboring genes implicated in cancer. Quantitative PCR validation showed excellent agreement with Tag-seq data (median Pearson r = 0.91) and discerned a gene set robustly distinguishing GNS from NS cells across the 22 lines. These expression alterations include oncogene and tumor suppressor changes not detected by microarray profiling of tumor tissue samples, and facilitated the identification of a GNS expression signature strongly associated with patient survival (P = 1e-6, Cox model). Conclusions These results support the utility of GNS cell cultures as a model system for studying the molecular processes driving glioblastoma and the use of NS cells as reference controls. The association between a GNS expression signature and survival is consistent with the hypothesis that a cancer stem cell component drives tumor growth. We anticipate that analysis of normal and malignant stem cells will be an important complement to large-scale profiling of primary tumors. PMID:23046790

Identification of a cis-regulatory region of a gene in Arabidopsis thaliana whose induction by dehydration is mediated by abscisic acid and requires protein synthesis.

PubMed

Iwasaki, T; Yamaguchi-Shinozaki, K; Shinozaki, K

1995-05-20

In Arabidopsis thaliana, the induction of a dehydration-responsive gene, rd22, is mediated by abscisic acid (ABA) but the gene does not include any sequence corresponding to the consensus ABA-responsive element (ABRE), RYACGTGGYR, in its promoter region. The cis-regulatory region of the rd22 promoter was identified by monitoring the expression of beta-glucuronidase (GUS) activity in leaves of transgenic tobacco plants transformed with chimeric gene fusions constructed between 5'-deleted promoters of rd22 and the coding region of the GUS reporter gene. A 67-bp nucleotide fragment corresponding to positions -207 to -141 of the rd22 promoter conferred responsiveness to dehydration and ABA on a non-responsive promoter. The 67-bp fragment contains the sequences of the recognition sites for some transcription factors, such as MYC, MYB, and GT-1. The fact that accumulation of rd22 mRNA requires protein synthesis raises the possibility that the expression of rd22 might be regulated by one of these trans-acting protein factors whose de novo synthesis is induced by dehydration or ABA. Although the structure of the RD22 protein is very similar to that of a non-storage seed protein, USP, of Vicia faba, the expression of the GUS gene driven by the rd22 promoter in non-stressed transgenic Arabidopsis plants was found mainly in flowers and bolted stems rather than in seeds.

Matrix metalloproteinase-9 plays a role in protecting zebrafish from lethal infection with Listeria monocytogenes by enhancing macrophage migration.

PubMed

Shan, Ying; Zhang, Yikai; Zhuo, Xunhui; Li, Xiaoliang; Peng, Jinrong; Fang, Weihuan

2016-07-01

Zebrafish could serve as an alternative animal model for pathogenic bacteria in multiple infectious routes. Our previous study showed that immersion infection in zebrafish with Listeria monocytogenes did not cause lethality but induced transient expression of several immune response genes. We used an Affymetrix gene chip to examine the expression profiles of genes of zebrafish immersion-infected with L. monocytogenes. A total of 239 genes were up-regulated and 56 genes down-regulated compared with uninfected fish. Highest expression (>20-fold) was seen with the mmp-9 gene encoding the matrix metalloproteinase-9 (Mmp-9) known to degrade the extracellular matrix proteins. By morpholino knockdown of mmp-9, we found that the morphants showed rapid death with much higher bacterial load after intravenous or intraventricular (brain ventricle) infection with L. monocytogenes. Macrophages in mmp-9-knockdown morphants had significant defect in migrating to the brain cavity upon intraventricular infection. Decreased migration of murine macrophages with knockdown of mmp-9 and cd44 was also seen in transwell inserts with 8-μm pore polycarbonate membrane, as compared with the scrambled RNA. These findings suggest that Mmp-9 is a protective molecule against infection by L. monocytogenes by engaging in migration of zebrafish macrophages to the site of infection via a non-proteolytic role. Further work is required on the molecular mechanisms governing Mmp-9-driven macrophage migration in zebrafish. Copyright © 2016 Elsevier Ltd. All rights reserved.

Genomic characterization, phylogenetic comparison and differential expression of the cyclic nucleotide-gated channels gene family in pear (Pyrus bretchneideri Rehd.).

PubMed

Chen, Jianqing; Yin, Hao; Gu, Jinping; Li, Leiting; Liu, Zhe; Jiang, Xueting; Zhou, Hongsheng; Wei, Shuwei; Zhang, Shaoling; Wu, Juyou

2015-01-01

The cyclic nucleotide-gated channel (CNGC) family is involved in the uptake of various cations, such as Ca(2+), to regulate plant growth and respond to biotic and abiotic stresses. However, there is far less information about this family in woody plants such as pear. Here, we provided a genome-wide identification and analysis of the CNGC gene family in pear. Phylogenetic analysis showed that the 21 pear CNGC genes could be divided into five groups (I, II, III, IVA and IVB). The majority of gene duplications in pear appeared to have been caused by segmental duplication and occurred 32.94-39.14 million years ago. Evolutionary analysis showed that positive selection had driven the evolution of pear CNGCs. Motif analyses showed that Group I CNGCs generally contained 26 motifs, which was the greatest number of motifs in all CNGC groups. Among these, eight motifs were shared by each group, suggesting that these domains play a conservative role in CNGC activity. Tissue-specific expression analysis indicated that functional diversification of the duplicated CNGC genes was a major feature of long-term evolution. Our results also suggested that the P-S6 and PBC & hinge domains had co-evolved during the evolution. These results provide valuable information to increase our understanding of the function, evolution and expression analyses of the CNGC gene family in higher plants. Copyright © 2014 Elsevier Inc. All rights reserved.

Engineering species-like barriers to sexual reproduction.

PubMed

Maselko, Maciej; Heinsch, Stephen C; Chacón, Jeremy M; Harcombe, William R; Smanski, Michael J

2017-10-12

Controlling the exchange of genetic information between sexually reproducing populations has applications in agriculture, eradication of disease vectors, control of invasive species, and the safe study of emerging biotechnology applications. Here we introduce an approach to engineer a genetic barrier to sexual reproduction between otherwise compatible populations. Programmable transcription factors drive lethal gene expression in hybrid offspring following undesired mating events. As a proof of concept, we target the ACT1 promoter of the model organism Saccharomyces cerevisiae using a dCas9-based transcriptional activator. Lethal overexpression of actin results from mating this engineered strain with a strain containing the wild-type ACT1 promoter.Genetic isolation of a genetically modified organism represents a useful strategy for biocontainment. Here the authors use dCas9-VP64-driven gene expression to construct a 'species-like' barrier to reproduction between two otherwise compatible populations.

In vitro differentiation of HT-29 M6 mucus-secreting colon cancer cells involves a trychostatin A and p27(KIP1)-inducible transcriptional program of gene expression.

PubMed

Mayo, Clara; Lloreta, Josep; Real, Francisco X; Mayol, Xavier

2007-07-01

Tumor cell dedifferentiation-such as the loss of cell-to-cell adhesion in epithelial tumors-is associated with tumor progression. To better understand the mechanisms that maintain carcinoma cells in a differentiated state, we have dissected in vitro differentiation pathways in the mucus-secretor HT-29 M6 colon cancer cell line, which spontaneously differentiates in postconfluent cultures. By lowering the extracellular calcium concentration to levels that prevent intercellular adhesion and epithelial polarization, our results reveal that differentiation is calcium-dependent and involves: (i) a process of cell cycle exit to G(0) and (ii) the induction of a transcriptional program of differentiation gene expression (i.e., mucins MUC1 and MUC5AC, and the apical membrane peptidase DPPIV). In calcium-deprived, non-differentiated postconfluent cultures, differentiation gene promoters are repressed by a trichostatin A (TSA)-sensitive mechanism, indicating that loss of gene expression by dedifferentiation is driven by histone deacetylases (HDAC). Since TSA treatment or extracellular calcium restoration allow gene promoter activation to similar levels, we suggest that induction of differentiation is one mechanism of HDAC inhibitor antitumor action. Moreover, transcriptional de-repression can also be induced in non-differentiating culture conditions by overexpressing the cyclin-dependent kinase inhibitor p27(KIP1), which is normally induced during spontaneous differentiation. Since p27(KIP1) downregulation in colon cancer is associated with poor prognosis independently of tumor cell division rates, we propose that p27 (KIP1) may prevent tumor progression by, at least in part, enhancing the expression of some differentiation genes. Therefore, the HT-29 M6 model allows the identification of some basic mechanisms of cancer cell differentiation control, so far revealing HDAC and p27(KIP1) as key regulatory factors of differentiation gene expression.

A plasmid-based reverse genetics system for influenza A virus.

PubMed Central

Pleschka, S; Jaskunas, R; Engelhardt, O G; Zürcher, T; Palese, P; García-Sastre, A

1996-01-01

A reverse genetics system for negative-strand RNA viruses was first successfully developed for influenza viruses. This technology involved the transfection of in vitro-reconstituted ribonucleoprotein (RNP) complexes into influenza virus-infected cells. We have now developed a method that allows intracellular reconstitution of RNP complexes from plasmid-based expression vectors. Expression of a viral RNA-like transcript is achieved from a plasmid containing a truncated human polymerase I (polI) promoter and a ribozyme sequence that generates the desired 3' end by autocatalytic cleavage. The polI-driven plasmid is cotransfected into human 293 cells with polII-responsive plasmids that express the viral PB1, PB2, PA, and NP proteins. This exclusively plasmid-driven system results in the efficient transcription and replication of the viral RNA-like reporter and allows the study of cis- and trans-acting signals involved in the transcription and replication of influenza virus RNAs. Using this system, we have also been able to rescue a synthetic neuraminidase gene into a recombinant influenza virus. This method represents a convenient alternative to the previously established RNP transfection system. PMID:8648766

p53 functional impairment and high p21waf1/cip1 expression in human T-cell lymphotropic/leukemia virus type I-transformed T cells.

PubMed

Cereseto, A; Diella, F; Mulloy, J C; Cara, A; Michieli, P; Grassmann, R; Franchini, G; Klotman, M E

1996-09-01

Human T-cell lymphotropic/leukemia virus type I (HTLV-I) is associated with T-cell transformation both in vivo and in vitro. Although some of the mechanisms responsible for transformation remain unknown, increasing evidence supports a direct role of viral as well as dysregulated cellular proteins in transformation. We investigated the potential role of the tumor suppressor gene p53 and of the p53-regulated gene, p21waf1/cip1 (wild-type p53 activated fragment 1/cycling dependent kinases [cdks] interacting protein 1), in HTLV-I-infected T cells. We have found that the majority of HTLV-I-infected T cells have the wild-type p53 gene. However, its function in HTLV-I-transformed cells appears to be impaired, as shown by the lack of appropriate p53-mediated responses to ionizing radiation (IR). Interestingly, the expression of the p53 inducible gene, p21waf1/cip1, is elevated at the messenger ribonucleic acid and protein levels in all HTLV-I-infected T-cell lines examined as well as in Taxl-1, a human T-cell line stably expressing Tax. Additionally, Tax induces upregulation of a p21waf1/cip1 promoter-driven luciferase gene in p53 null cells, and increases p21waf1/cip1 expression in Jurkat T cells. These findings suggest that the Tax protein is at least partially responsible for the p53-independent expression of p21waf1/cip1 in HTLV-I-infected cells. Dysregulation of p53 and p21waf1/cip1 proteins regulating cell-cycle progression, may represent an important step in HTLV-I-induced T-cell transformation.

Lentivirus-mediated bifunctional cell labeling for in vivo melanoma study

PubMed Central

Day, Chi-Ping; Carter, John; Bonomi, Carrie; Esposito, Dominic; Crise, Bruce; Ortiz-Conde, Betty; Hollingshead, Melinda; Merlino, Glenn

2009-01-01

SUMMARY Lentiviral vectors (LVs) are capable of labeling a broad spectrum of cell types, achieving stable expression of transgenes. However, for in vivo studies, the duration of marker gene expression has been highly variable. We have developed a series of LVs harboring different promoters for expressing reporter gene in mouse cells. Long-term culture and colony formation of several LV-labeled mouse melanoma cells showed that promoters derived from mammalian house-keeping genes, especially those encoding RNA polymerase II (Pol2) and ferritin (FerH), provided the highest consistency for reporter expression. For in vivo studies, primary B16BL6 mouse melanoma were infected with LVs whose luciferase-GFP fusion gene (Luc/GFP) was driven by either Pol2 or FerH promoters. When transplanted into syngeneic C57BL/6 mice, Luc/GFP-labeled B16BL6 mouse melanoma cells can be monitored by bioluminescence imaging in vivo, and GFP-positive cells can be isolated from the tumors by FACS. Pol2-Luc/GFP labeling, while lower in activity, was more sustainable than FerH-Luc/GFP labeling in B16BL6 over consecutive passages into mice. We conclude that Pol-2-Luc/GFP labeling allows long-term in vivo monitoring and tumor cell isolation in immunocompetent mouse melanoma models. SIGNIFICANCE In this study we have developed and identified lentiviral vectors that allow labeled mouse melanoma cells to maintain long-term and consistent expression of a bifunctional luciferase-GFP marker gene, even in syngeneic mice with an intact immune function. This cell-labeling system can be used to build immunocompetent mouse melanoma models that permit both tumor monitoring and FACS-based tumor cell isolation from tissues, greatly facilitating the in vivo study of melanoma. PMID:19175523

Gene Transfer to Chicks Using Lentiviral Vectors Administered via the Embryonic Chorioallantoic Membrane

PubMed Central

Hen, Gideon; Yosefi, Sara; Shinder, Dmitry; Or, Adi; Mygdal, Sivan; Condiotti, Reba; Galun, Eithan; Bor, Amir; Sela-Donenfeld, Dalit; Friedman-Einat, Miriam

2012-01-01

The lack of affordable techniques for gene transfer in birds has inhibited the advancement of molecular studies in avian species. Here we demonstrate a new approach for introducing genes into chicken somatic tissues by administration of a lentiviral vector, derived from the feline immunodeficiency virus (FIV), into the chorioallantoic membrane (CAM) of chick embryos on embryonic day 11. The FIV-derived vectors carried yellow fluorescent protein (YFP) or recombinant alpha-melanocyte-stimulating hormone (α-MSH) genes, driven by the cytomegalovirus (CMV) promoter. Transgene expression, detected in chicks 2 days after hatch by quantitative real-time PCR, was mostly observed in the liver and spleen. Lower expression levels were also detected in the brain, kidney, heart and breast muscle. Immunofluorescence and flow cytometry analyses confirmed transgene expression in chick tissues at the protein level, demonstrating a transduction efficiency of ∼0.46% of liver cells. Integration of the viral vector into the chicken genome was demonstrated using genomic repetitive (CR1)-PCR amplification. Viability and stability of the transduced cells was confirmed using terminal deoxynucleotidyl transferase (dUTP) nick end labeling (TUNEL) assay, immunostaining with anti-proliferating cell nuclear antigen (anti-PCNA), and detection of transgene expression 51 days post transduction. Our approach led to only 9% drop in hatching efficiency compared to non-injected embryos, and all of the hatched chicks expressed the transgenes. We suggest that the transduction efficiency of FIV vectors combined with the accessibility of the CAM vasculature as a delivery route comprise a new powerful and practical approach for gene delivery into somatic tissues of chickens. Most relevant is the efficient transduction of the liver, which specializes in the production and secretion of proteins, thereby providing an optimal target for prolonged study of secreted hormones and peptides. PMID:22606269

Pseudomonas syringae effector avrB confers soybean cultivar-specific avirulence on Soybean mosaic virus adapted for transgene expression but effector avrPto does not.

PubMed

Wang, Li; Eggenberger, Alan; Hill, John; Bogdanove, Adam J

2006-03-01

Soybean mosaic virus (SMV) was adapted for transgene expression in soybean and used to examine the function of avirulence genes avrB and avrPto of Pseudomonas syringae pvs. glycinea and tomato, respectively. A cloning site was introduced between the P1 and HC-Pro genes in 35S-driven infectious cDNAs of strains SMV-N and SMV-G7. Insertion of the uidA gene or the green fluorescent protein gene into either modified cDNA and bombardment into primary leaves resulted in systemic expression that reflected the pattern of viral movement into uninoculated leaves. Insertion of avrB blocked symptom development and detectable viral movement in cv. Harosoy, which carries the Rpg1-b resistance gene corresponding to avrB, but not in cvs. Keburi or Hurrelbrink, which lack Rpg1-b. In Keburi and Hurrelbrink, symptoms caused by SMV carrying avrB appeared more quickly and were more severe than those caused by the virus without avrB. Insertion of avrPto enhanced symptoms in Harosoy, Hurrelbrink, and Keburi. This result was unexpected because avrPto was reported to confer avirulence on P. syringae pv. glycinea inoculated to Harosoy. We inoculated Harosoy with P syringae pv. glycinea expressing avrPto, but observed no hypersensitive reaction, avrPto-dependent induction of pathogenesis-related protein la, or limitation of bacterial population growth. In Hurrelbrink, avrPto enhanced bacterial multiplication and exacerbated symptoms. Our results establish SMV as an expression vector for soybean. They demonstrate that resistance triggered by avrB is effective against SMV, and that avrB and avrPto have general virulence effects in soybean. The results also led to a reevaluation of the reported avirulence activity of avrPto in this plant.

Merlin, the product of NF2 gene, is associated with aromatase expression and estrogen formation in human liver tissues and liver cancer cells.

PubMed

Cocciadiferro, Letizia; Miceli, Vitale; Granata, Orazia M; Carruba, Giuseppe

2017-09-01

The product of neurofibromatosis type 2 (NF2) gene, also known as Merlin/neurofibromin 2, homeostatically regulates liver stem cells by controlling abundance and signaling of epidermal growth factor receptor (EGFR), with a mechanism independent of the Hippo pathway. We have reported that locally elevated estrogen formation, driven by abnormally high expression and function of aromatase, may be implicated in development and progression of human hepatocellular carcinoma (HCC) through activation of a rapid signaling pathway mediated by amphiregulin (AREG) and EGFR. We have recently presented a model by which the aromatase-estrogen-amphiregulin-EGFR axis is activated in response to tissue injury and/or inflammatory disease, with its alteration eventually leading to development of major human tumors (liver, breast, prostate) and other chronic diseases (diabetes, obesity, Alzheimer's and heart disease). In this study, we investigated NF2 expression in liver cancer cells and tissues in relation to aromatase expression/function, estrogen receptor (ER) status and amphiregulin. Our data indicate that NF2 expression is associated with aromatase and AREG expression, being elevated in HCC tissues and HepG2 cells, intermediate in cirrhotic tissues and Huh7 cells, and lower in nontumoral liver and HA22T cells. In addition, NF2 expression is inversely related to wild type hERα66 and proportional to the expression of the membrane-associated hERα36 splice variant, as measured by exon-specific RT-PCR analysis, both in vivo and in vitro. Furthermore, incubation with estradiol induced a significant decrease of NF2 expression in both HA22T and Huh7 cells (over 54% and 22%, respectively), while no change could be observed in HepG2 cells, this effect being inversely related to aromatase expression and activity in HCC cell lines. Based on the above combined evidence, we hypothesize that NF2 behaves as a protein sensing tissue damage and aromatase-driven local estrogen formation, eventually leading to regulation of stem cells differentiation and tissue repair. Copyright © 2016 Elsevier Ltd. All rights reserved.

Imprinted expression of UBE3A in non-neuronal cells from a Prader–Willi syndrome patient with an atypical deletion

PubMed Central

Martins-Taylor, Kristen; Hsiao, Jack S.; Chen, Pin-Fang; Glatt-Deeley, Heather; De Smith, Adam J.; Blakemore, Alexandra I.F.; Lalande, Marc; Chamberlain, Stormy J.

2014-01-01

Prader–Willi syndrome (PWS) and Angelman syndrome (AS) are two neurodevelopmental disorders most often caused by deletions of the same region of paternally inherited and maternally inherited human chromosome 15q, respectively. AS is a single gene disorder, caused by the loss of function of the ubiquitin ligase E3A (UBE3A) gene, while PWS is still considered a contiguous gene disorder. Rare individuals with PWS who carry atypical microdeletions on chromosome 15q have narrowed the critical region for this disorder to a 108 kb region that includes the SNORD116 snoRNA cluster and the Imprinted in Prader–Willi (IPW) non-coding RNA. Here we report the derivation of induced pluripotent stem cells (iPSCs) from a PWS patient with an atypical microdeletion that spans the PWS critical region. We show that these iPSCs express brain-specific portions of the transcripts driven by the PWS imprinting center, including the UBE3A antisense transcript (UBE3A-ATS). Furthermore, UBE3A expression is imprinted in most of these iPSCs. These data suggest that UBE3A imprinting in neurons only requires UBE3A-ATS expression, and no other neuron-specific factors. These data also suggest that a boundary element lying within the PWS critical region prevents UBE3A-ATS expression in non-neural tissues. PMID:24363065

P1 promoter-driven HNF4α isoforms are specifically repressed by β-catenin signaling in colorectal cancer cells.

PubMed

Babeu, Jean-Philippe; Jones, Christine; Geha, Sameh; Carrier, Julie C; Boudreau, François

2018-06-13

HNF4α is a key nuclear receptor for regulating gene expression in the gut. While both P1 and P2 isoform classes of HNF4α are expressed in colonic epithelium, specific inhibition of P1 isoforms is commonly found in colorectal cancer. Previous studies have suggested that P1 and P2 isoforms may regulate different cellular functions. Despite these advances, it remains unclear whether these isoform classes are functionally divergent in the context of human biology. Here, the consequences of specific inhibition of P1 or P2 isoform expression was measured in a human colorectal cancer cell transcriptome. Results indicate that P1 isoforms were specifically associated with the control of cell metabolism while P2 isoforms globally supported aberrant oncogenic signalization, promoting cancer cell survival and progression. P1 promoter-driven isoform expression was found to be repressed by β-catenin, one of the earliest oncogenic pathways to be activated during colon tumorigenesis. These findings identify a novel cascade by which the expression of P1 isoforms are rapidly shut down in the early stages of colon tumorigenesis, allowing a change in HNF4α-dependent transcriptome thereby promoting colorectal cancer progression. © 2018. Published by The Company of Biologists Ltd.

Sox2 promotes tamoxifen resistance in breast cancer cells

PubMed Central

Piva, Marco; Domenici, Giacomo; Iriondo, Oihana; Rábano, Miriam; Simões, Bruno M; Comaills, Valentine; Barredo, Inmaculada; López-Ruiz, Jose A; Zabalza, Ignacio; Kypta, Robert; Vivanco, Maria d M

2014-01-01

Development of resistance to therapy continues to be a serious clinical problem in breast cancer management. Cancer stem/progenitor cells have been shown to play roles in resistance to chemo- and radiotherapy. Here, we examined their role in the development of resistance to the oestrogen receptor antagonist tamoxifen. Tamoxifen-resistant cells were enriched for stem/progenitors and expressed high levels of the stem cell marker Sox2. Silencing of the SOX2 gene reduced the size of the stem/progenitor cell population and restored sensitivity to tamoxifen. Conversely, ectopic expression of Sox2 reduced tamoxifen sensitivity in vitro and in vivo. Gene expression profiling revealed activation of the Wnt signalling pathway in Sox2-expressing cells, and inhibition of Wnt signalling sensitized resistant cells to tamoxifen. Examination of patient tumours indicated that Sox2 levels are higher in patients after endocrine therapy failure, and also in the primary tumours of these patients, compared to those of responders. Together, these results suggest that development of tamoxifen resistance is driven by Sox2-dependent activation of Wnt signalling in cancer stem/progenitor cells. PMID:24178749

Human Immunodeficiency Virus Tat-Activated Expression of Poliovirus Protein 2A Inhibits mRNA Translation

NASA Astrophysics Data System (ADS)

Sun, Xiao-Hong; Baltimore, David

1989-04-01

To study the effect of poliovirus protein 2A on cellular RNA translation, the tat control system of human immunodeficiency virus (HIV) was used. Protein 2A was expressed from a plasmid construct (pHIV/2A) incorporating the HIV long terminal repeat. Protein synthesis was measured by using chloramphenicol acetyltransferase as a reporter gene driven by the Rous sarcoma virus long terminal repeat. When HIV/2A was contransfected with the reporter, addition of a tat-producing plasmid caused at least a 50-fold drop in chloramphenicol acetyltransferase synthesis. A HeLa cell line carrying HIV/2A was established. In it, tat expression caused more than a 10-fold drop in chloramphenicol acetyltransferase synthesis from the reporter plasmid. Furthermore, 2A induction by tat caused cleavage of the cellular translation factor P220, a part of eukaryotic translation initiation factor 4F. Thus protein 2A can, by itself, carry out the inhibition of cellular protein synthesis characteristic of a poliovirus infection. Also, the HIV tat activation provides a very effective method to control gene expression in mammalian cells.

Temporal Expression of Peripheral Blood Leukocyte Biomarkers in a Macaca fascicularis Infection Model of Tuberculosis; Comparison with Human Datasets and Analysis with Parametric/Non-parametric Tools for Improved Diagnostic Biomarker Identification

PubMed Central

Wareham, Alice; Lewandowski, Kuiama S.; Williams, Ann; Dennis, Michael J.; Sharpe, Sally; Vipond, Richard; Silman, Nigel; Ball, Graham

2016-01-01

A temporal study of gene expression in peripheral blood leukocytes (PBLs) from a Mycobacterium tuberculosis primary, pulmonary challenge model Macaca fascicularis has been conducted. PBL samples were taken prior to challenge and at one, two, four and six weeks post-challenge and labelled, purified RNAs hybridised to Operon Human Genome AROS V4.0 slides. Data analyses revealed a large number of differentially regulated gene entities, which exhibited temporal profiles of expression across the time course study. Further data refinements identified groups of key markers showing group-specific expression patterns, with a substantial reprogramming event evident at the four to six week interval. Selected statistically-significant gene entities from this study and other immune and apoptotic markers were validated using qPCR, which confirmed many of the results obtained using microarray hybridisation. These showed evidence of a step-change in gene expression from an ‘early’ FOS-associated response, to a ‘late’ predominantly type I interferon-driven response, with coincident reduction of expression of other markers. Loss of T-cell-associate marker expression was observed in responsive animals, with concordant elevation of markers which may be associated with a myeloid suppressor cell phenotype e.g. CD163. The animals in the study were of different lineages and these Chinese and Mauritian cynomolgous macaque lines showed clear evidence of differing susceptibilities to Tuberculosis challenge. We determined a number of key differences in response profiles between the groups, particularly in expression of T-cell and apoptotic makers, amongst others. These have provided interesting insights into innate susceptibility related to different host `phenotypes. Using a combination of parametric and non-parametric artificial neural network analyses we have identified key genes and regulatory pathways which may be important in early and adaptive responses to TB. Using comparisons between data outputs of each analytical pipeline and comparisons with previously published Human TB datasets, we have delineated a subset of gene entities which may be of use for biomarker diagnostic test development. PMID:27228113

Cdk1 activity acts as a quantitative platform for coordinating cell cycle progression with periodic transcription

PubMed Central

Banyai, Gabor; Baïdi, Feriel; Coudreuse, Damien; Szilagyi, Zsolt

2016-01-01

Cell proliferation is regulated by cyclin-dependent kinases (Cdks) and requires the periodic expression of particular gene clusters in different cell cycle phases. However, the interplay between the networks that generate these transcriptional oscillations and the core cell cycle machinery remains largely unexplored. In this work, we use a synthetic regulable Cdk1 module to demonstrate that periodic expression is governed by quantitative changes in Cdk1 activity, with different clusters directly responding to specific activity levels. We further establish that cell cycle events neither participate in nor interfere with the Cdk1-driven transcriptional program, provided that cells are exposed to the appropriate Cdk1 activities. These findings contrast with current models that propose self-sustained and Cdk1-independent transcriptional oscillations. Our work therefore supports a model in which Cdk1 activity serves as a quantitative platform for coordinating cell cycle transitions with the expression of critical genes to bring about proper cell cycle progression. PMID:27045731

Identification of SR1078, a synthetic agonist for the orphan nuclear receptors RORα and RORγ.

PubMed

Wang, Yongjun; Kumar, Naresh; Nuhant, Philippe; Cameron, Michael D; Istrate, Monica A; Roush, William R; Griffin, Patrick R; Burris, Thomas P

2010-11-19

The retinoic acid receptor-related receptors (RORs) are members of the nuclear receptor (NR) superfamily of transcription factors. Several NRs are still characterized as orphan receptors because ligands have not yet been identified for these proteins. Here, we describe the identification of a synthetic RORα/RORγ ligand, SR1078. SR1078 modulates the conformation of RORγ in a biochemical assay and activates RORα and RORγ driven transcription. Furthermore, SR1078 stimulates expression of endogenous ROR target genes in HepG2 cells that express both RORα and RORγ. Pharmacokinetic studies indicate that SR1078 displays reasonable exposure following injection into mice, and consistent with SR1078 functioning as a RORα/RORγ agonist, expression of two ROR target genes, glucose-6-phosphatase and fibroblast growth factor 21, were stimulated in the liver. Thus, we have identified the first synthetic RORα/γ agonist, and this compound can be utilized as a chemical tool to probe the function of these receptors both in vitro and in vivo.

Identification of a Synthetic Agonist for the Orphan Nuclear Receptors RORα and RORγ, SR1078

PubMed Central

Wang, Yongjun; Kumar, Naresh; Nuhant, Philippe; Cameron, Michael D.; Istrate, Monica A.; Roush, William R.; Griffin, Patrick R.; Burris, Thomas P.

2010-01-01

The retinoic acid receptor-related receptors (RORs) are members of the nuclear receptor (NR) superfamily of transcription factors. Several NRs are still characterized as orphan receptors since ligands have not yet been identified for these proteins. Here, we describe the identification of a synthetic RORα/RORγ ligand, SR1078. SR1078 modulates the conformation of RORγ in a biochemical assay and activates RORα and RORγ driven transcription. Furthermore, SR1078 stimulates expression of endogenous ROR target genes in HepG2 cells that express both RORα and RORγ. Pharmacokinetic studies indicate that SR1078 displays reasonable exposure following injection into mice and consistent with SR1078 functioning as a RORα/RORγ agonist, expression of two ROR target genes, glucose-6-phosphatase and fibroblast growth factor 21, were stimulated in the liver. Thus, we have identified the first synthetic RORα/γ agonist and this compound can be utilized as a chemical tool to probe the function of these receptors both in vitro and in vivo. PMID:20735016

Aurora A regulates expression of AR-V7 in models of castrate resistant prostate cancer.

PubMed

Jones, Dominic; Noble, Martin; Wedge, Steve R; Robson, Craig N; Gaughan, Luke

2017-02-16

Androgen receptor variants (AR-Vs) provide a mechanism of therapy evasion in castrate-resistant prostate cancer (CRPC), yet mechanisms of regulation remain largely unknown. Here we investigate the role of Aurora A kinase on AR-Vs in models of CRPC and show depletion of Aurora A reduces AR-V target gene expression. Importantly, knockdown of Aurora A reconfigures splicing of AR pre-mRNA to discriminately down-regulate synthesis of AR-V transcripts, including AR-V7, without effecting full-length AR mRNA; and as a consequence, AR-V-driven proliferation and survival of CRPC cells is markedly reduced. Critically, these effects are reproduced by Aurora A inhibition. We show that Aurora A levels increase in advanced disease and AURKA is an AR-V target gene demonstrating a positive feedback mechanism of androgenic signalling in CRPC. In all, our data suggests that Aurora A plays a pivotal role in regulation of AR-V7 expression and represents a new therapeutic target in CRPC.

Aurora A regulates expression of AR-V7 in models of castrate resistant prostate cancer

PubMed Central

Jones, Dominic; Noble, Martin; Wedge, Steve R.; Robson, Craig N.; Gaughan, Luke

2017-01-01

Androgen receptor variants (AR-Vs) provide a mechanism of therapy evasion in castrate-resistant prostate cancer (CRPC), yet mechanisms of regulation remain largely unknown. Here we investigate the role of Aurora A kinase on AR-Vs in models of CRPC and show depletion of Aurora A reduces AR-V target gene expression. Importantly, knockdown of Aurora A reconfigures splicing of AR pre-mRNA to discriminately down-regulate synthesis of AR-V transcripts, including AR-V7, without effecting full-length AR mRNA; and as a consequence, AR-V-driven proliferation and survival of CRPC cells is markedly reduced. Critically, these effects are reproduced by Aurora A inhibition. We show that Aurora A levels increase in advanced disease and AURKA is an AR-V target gene demonstrating a positive feedback mechanism of androgenic signalling in CRPC. In all, our data suggests that Aurora A plays a pivotal role in regulation of AR-V7 expression and represents a new therapeutic target in CRPC. PMID:28205582

Negative effect of the 5'-untranslated leader sequence on Ac transposon promoter expression.

PubMed

Scortecci, K C; Raina, R; Fedoroff, N V; Van Sluys, M A

1999-08-01

Transposable elements are used in heterologous plant hosts to clone genes by insertional mutagenesis. The Activator (Ac) transposable element has been cloned from maize, and introduced into a variety of plants. However, differences in regulation and transposition frequency have been observed between different host plants. The cause of this variability is still unknown. To better understand the activity of the Ac element, we analyzed the Ac promoter region and its 5'-untranslated leader sequence (5' UTL). Transient assays in tobacco NT1 suspension cells showed that the Ac promoter is a weak promoter and its activity was localized by deletion analyses. The data presented here indicate that the core of the Ac promoter is contained within 153 bp fragment upstream to transcription start sites. An important inhibitory effect (80%) due to the presence of the 5' UTL was found on the expression of LUC reporter gene. Here we demonstrate that the presence of the 5' UTL in the constructs reduces the expression driven by either strong or weak promoters.