5′ and 3′ Untranslated Regions Strongly Enhance Performance of Geminiviral Replicons in Nicotiana benthamiana Leaves

PubMed Central

Diamos, Andrew G.; Rosenthal, Sun H.; Mason, Hugh S.

2016-01-01

We previously reported a recombinant protein production system based on a geminivirus replicon that yields high levels of vaccine antigens and monoclonal antibodies in plants. The bean yellow dwarf virus (BeYDV) replicon generates massive amounts of DNA copies, which engage the plant transcription machinery. However, we noticed a disparity between transcript level and protein production, suggesting that mRNAs could be more efficiently utilized. In this study, we systematically evaluated genetic elements from human, viral, and plant sources for their potential to improve the BeYDV system. The tobacco extensin terminator enhanced transcript accumulation and protein production compared to other commonly used terminators, indicating that efficient transcript processing plays an important role in recombinant protein production. Evaluation of human-derived 5′ untranslated regions (UTRs) indicated that many provided high levels of protein production, supporting their cross-kingdom function. Among the viral 5′ UTRs tested, we found the greatest enhancement with the tobacco mosaic virus omega leader. An analysis of the 5′ UTRs from the Arabidopsis thaliana and Nicotinana benthamiana photosystem I K genes found that they were highly active when truncated to include only the near upstream region, providing a dramatic enhancement of transgene production that exceeded that of the tobacco mosaic virus omega leader. The tobacco Rb7 matrix attachment region inserted downstream from the gene of interest provided significant enhancement, which was correlated with a reduction in plant cell death. Evaluation of Agrobacterium strains found that EHA105 enhanced protein production and reduced cell death compared to LBA4301 and GV3101. We used these improvements to produce Norwalk virus capsid protein at >20% total soluble protein, corresponding to 1.8 mg/g leaf fresh weight, more than twice the highest level ever reported in a plant system. We also produced the monoclonal antibody rituximab at 1 mg/g leaf fresh weight. PMID:26941764

Suppression and enhancement of transcriptional noise by DNA looping

NASA Astrophysics Data System (ADS)

Vilar, Jose M. G.; Saiz, Leonor

2014-06-01

DNA looping has been observed to enhance and suppress transcriptional noise but it is uncertain which of these two opposite effects is to be expected for given conditions. Here, we derive analytical expressions for the main quantifiers of transcriptional noise in terms of the molecular parameters and elucidate the role of DNA looping. Our results rationalize paradoxical experimental observations and provide the first quantitative explanation of landmark individual-cell measurements at the single molecule level on the classical lac operon genetic system [Choi, L. Cai, K. Frieda, and X. S. Xie, Science 322, 442 (2008), 10.1126/science.1161427].

Enhanced somatic embryogenesis in Theobroma cacao using the homologous BABY BOOM transcription factor.

PubMed

Florez, Sergio L; Erwin, Rachel L; Maximova, Siela N; Guiltinan, Mark J; Curtis, Wayne R

2015-05-16

Theobroma cacao, the chocolate tree, is an important economic crop in East Africa, South East Asia, and South and Central America. Propagation of elite varieties has been achieved through somatic embryogenesis (SE) but low efficiencies and genotype dependence still presents a significant limitation for its propagation at commercial scales. Manipulation of transcription factors has been used to enhance the formation of SEs in several other plant species. This work describes the use of the transcription factor Baby Boom (BBM) to promote the transition of somatic cacao cells from the vegetative to embryonic state. An ortholog of the Arabidopsis thaliana BBM gene (AtBBM) was characterized in T. cacao (TcBBM). TcBBM expression was observed throughout embryo development and was expressed at higher levels during SE as compared to zygotic embryogenesis (ZE). TcBBM overexpression in A. thaliana and T. cacao led to phenotypes associated with SE that did not require exogenous hormones. While transient ectopic expression of TcBBM provided only moderate enhancements in embryogenic potential, constitutive overexpression dramatically increased SE proliferation but also appeared to inhibit subsequent development. Our work provides validation that TcBBM is an ortholog to AtBBM and has a specific role in both somatic and zygotic embryogenesis. Furthermore, our studies revealed that TcBBM transcript levels could serve as a biomarker for embryogenesis in cacao tissue. Results from transient expression of TcBBM provide confirmation that transcription factors can be used to enhance SE without compromising plant development and avoiding GMO plant production. This strategy could compliment a hormone-based method of reprogramming somatic cells and lead to more precise manipulation of SE at the regulatory level of transcription factors. The technology would benefit the propagation of elite varieties with low regeneration potential as well as the production of transgenic plants, which similarly requires somatic cell reprogramming.

Overexpression of the transcription activator Msn2 enhances the fermentation ability of industrial baker's yeast in frozen dough.

PubMed

Sasano, Yu; Haitani, Yutaka; Hashida, Keisuke; Ohtsu, Iwao; Shima, Jun; Takagi, Hiroshi

2012-01-01

We constructed a self-cloning diploid baker's yeast strain that overexpressed the transcription activator Msn2. It showed higher tolerance to freeze-thaw stress and higher intracellular trehalose level than observed in the wild-type strain. Overexpression of Msn2 also enhanced the fermentation ability of baker's yeast cells in frozen dough. Hence, Msn2-overexpressing baker's yeast should be useful in frozen-dough baking.

Occupancy by key transcription factors is a more accurate predictor of enhancer activity than histone modifications or chromatin accessibility

DOE PAGES

Dogan, Nergiz; Wu, Weisheng; Morrissey, Christapher S.; ...

2015-04-23

Regulated gene expression controls organismal development, and variation in regulatory patterns has been implicated in complex traits. Thus accurate prediction of enhancers is important for further understanding of these processes. Genome-wide measurement of epigenetic features, such as histone modifications and occupancy by transcription factors, is improving enhancer predictions, but the contribution of these features to prediction accuracy is not known. Given the importance of the hematopoietic transcription factor TAL1 for erythroid gene activation, we predicted candidate enhancers based on genomic occupancy by TAL1 and measured their activity. Contributions of multiple features to enhancer prediction were evaluated based on the resultsmore » of these and other studies. Results: TAL1-bound DNA segments were active enhancers at a high rate both in transient transfections of cultured cells (39 of 79, or 56%) and transgenic mice (43 of 66, or 65%). The level of binding signal for TAL1 or GATA1 did not help distinguish TAL1-bound DNA segments as active versus inactive enhancers, nor did the density of regulation-related histone modifications. A meta-analysis of results from this and other studies (273 tested predicted enhancers) showed that the presence of TAL1, GATA1, EP300, SMAD1, H3K4 methylation, H3K27ac, and CAGE tags at DNase hypersensitive sites gave the most accurate predictors of enhancer activity, with a success rate over 80% and a median threefold increase in activity. Chromatin accessibility assays and the histone modifications H3K4me1 and H3K27ac were sensitive for finding enhancers, but they have high false positive rates unless transcription factor occupancy is also included. Conclusions: Occupancy by key transcription factors such as TAL1, GATA1, SMAD1, and EP300, along with evidence of transcription, improves the accuracy of enhancer predictions based on epigenetic features.« less

LBH Gene Transcription Regulation by the Interplay of an Enhancer Risk Allele and DNA Methylation in Rheumatoid Arthritis.

PubMed

Hammaker, Deepa; Whitaker, John W; Maeshima, Keisuke; Boyle, David L; Ekwall, Anna-Karin H; Wang, Wei; Firestein, Gary S

2016-11-01

To identify nonobvious therapeutic targets for rheumatoid arthritis (RA), we performed an integrative analysis incorporating multiple "omics" data and the Encyclopedia of DNA Elements (ENCODE) database for potential regulatory regions. This analysis identified the limb bud and heart development (LBH) gene, which has risk alleles associated with RA/celiac disease and lupus, and can regulate cell proliferation in RA. We identified a novel LBH transcription enhancer with an RA risk allele (rs906868 G [Ref]/T) 6 kb upstream of the LBH gene with a differentially methylated locus. The confluence of 3 regulatory elements, rs906868, an RA differentially methylated locus, and a putative enhancer, led us to investigate their effects on LBH regulation in fibroblast-like synoviocytes (FLS). We cloned the 1.4-kb putative enhancer with either the rs906868 Ref allele or single-nucleotide polymorphism (SNP) variant into reporter constructs. The constructs were methylated in vitro and transfected into cultured FLS by nucleofection. We found that both variants increased transcription, thereby confirming the region's enhancer function. Unexpectedly, the transcriptional activity of the Ref risk allele was significantly lower than that of the SNP variant and is consistent with low LBH levels as a risk factor for aggressive FLS behavior. Using RA FLS lines with a homozygous Ref or SNP allele, we confirmed that homozygous Ref lines expressed lower LBH messenger RNA levels than did the SNP lines. Methylation significantly reduced enhancer activity for both alleles, indicating that enhancer function is dependent on its methylation status. This study shows how the interplay between genetics and epigenetics can affect expression of LBH in RA. © 2016, American College of Rheumatology.

Transcriptional "silencer" element in rat repetitive sequences associated with the rat insulin 1 gene locus.

PubMed Central

Laimins, L; Holmgren-König, M; Khoury, G

1986-01-01

The enhancer elements from either simian virus 40 or murine sarcoma virus activate the expression of a transfected rat insulin 1 (rI1) gene when placed within 2.0 kilobases or less of the rI1 gene cap site. Inclusion of 4.0 kilobases of upstream rI1 sequence, however, results in a substantial reduction in the enhancer-dependent insulin gene expression. These observations suggested that a negative transcriptional regulatory element was present between 2.0 and 4.0 kilobases of the rI1 sequence. To test this notion, we employed a heterologous enhancer-dependent transcription assay in which the simian virus 40 72-base-pair repeat is linked to a human beta-globin gene. Addition of the upstream rI1 element to this system decreased the level of enhancer-dependent beta-globin transcription by a factor of 5 to 15. This rI1 "silencer" element functions in a manner relatively independent of position and orientation and requires a cis-dependent relationship to the transcription unit on which it acts. Thus, the silencer sequence seems to have a number of the characteristics of enhancer elements, and we suggest that it may function by the converse of the enhancer mechanism. The rI1 silencer sequence was identified as a member of a long interspersed rat repetitive family. Thus, a potential role for certain repetitive sequences interspersed throughout the eukaryotic genome may be to regulate gene expression by retaining transcriptional activity within defined domains. Images PMID:3010279

Negative modulation of the chicken infectious anemia virus promoter by COUP-TF1 and an E box-like element at the transcription start site binding deltaEF1.

PubMed

Miller, Myrna M; Jarosinski, Keith W; Schat, Karel A

2008-12-01

Expression of enhanced green fluorescent protein (EGFP) under control of the promoter-enhancer of chicken infectious anemia virus (CAV) is increased in an oestrogen receptor-enhanced cell line when treated with oestrogen and the promoter-enhancer binds unidentified proteins that recognize a consensus oestrogen response element (ERE). Co-transfection assays with the CAV promoter and the nuclear receptor chicken ovalbumin upstream promoter transcription factor 1 (COUP-TF1) showed that expression of EGFP was decreased by 50 to 60 % in DF-1 and LMH cells. The CAV promoter that included sequences at and downstream of the transcription start point had less expression than a short promoter construct. Mutation of a putative E box at this site restored expression levels. Electromobility shift assays showed that the transcription regulator delta-EF1 (deltaEF1) binds to this E box region. These findings indicate that the CAV promoter activity can be affected directly or indirectly by COUP-TF1 and deltaEF1.

Differential regulation of HIF-1α and HIF-2α in neuroblastoma: Estrogen-related receptor alpha (ERRα) regulates HIF2A transcription and correlates to poor outcome

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

Hamidian, Arash; Stedingk, Kristoffer von; Munksgaard Thorén, Matilda

2015-06-05

Hypoxia-inducible factors (HIFs) are differentially regulated in tumor cells. While the current paradigm supports post-translational regulation of the HIF-α subunits, we recently showed that hypoxic HIF-2α is also transcriptionally regulated via insulin-like growth factor (IGF)-II in the childhood tumor neuroblastoma. Here, we demonstrate that transcriptional regulation of HIF-2α seems to be restricted to neural cell-derived tumors, while HIF-1α is canonically regulated at the post-translational level uniformly across different tumor forms. Enhanced expression of HIF2A mRNA at hypoxia is due to de novo transcription rather than increased mRNA stability, and chemical stabilization of the HIF-α proteins at oxygen-rich conditions unexpectedly leadsmore » to increased HIF2A transcription. The enhanced HIF2A levels do not seem to be dependent on active HIF-1. Using a transcriptome array approach, we identified members of the Peroxisome proliferator-activated receptor gamma coactivator (PGC)/Estrogen-related receptor (ERR) complex families as potential regulators of HIF2A. Knockdown or inhibition of one of the members, ERRα, leads to decreased expression of HIF2A, and high expression of the ERRα gene ESRRA correlates with poor overall and progression-free survival in a clinical neuroblastoma material consisting of 88 tumors. Thus, targeting of ERRα and pathways regulating transcriptional HIF-2α are promising therapeutic avenues in neuroblastoma. - Highlights: • Transcriptional control of HIF-2α is restricted to neural cell-derived tumors. • Enhanced transcription of HIF2A is not due to increased mRNA stability. • Chemical stabilization of the HIF-α subunits leads to increased HIF2A transcription. • ERRα regulates HIF2A mRNA expression in neuroblastoma. • High expression of ESRRA correlates to poor outcome in neuroblastoma.« less

Folate deficiency facilitates recruitment of upstream binding factor to hot spots of DNA double-strand breaks of rRNA genes and promotes its transcription.

PubMed

Xie, Qiu; Li, Caihua; Song, Xiaozhen; Wu, Lihua; Jiang, Qian; Qiu, Zhiyong; Cao, Haiyan; Yu, Kaihui; Wan, Chunlei; Li, Jianting; Yang, Feng; Huang, Zebing; Niu, Bo; Jiang, Zhengwen; Zhang, Ting

2017-03-17

The biogenesis of ribosomes in vivo is an essential process for cellular functions. Transcription of ribosomal RNA (rRNA) genes is the rate-limiting step in ribosome biogenesis controlled by environmental conditions. Here, we investigated the role of folate antagonist on changes of DNA double-strand breaks (DSBs) landscape in mouse embryonic stem cells. A significant DSB enhancement was detected in the genome of these cells and a large majority of these DSBs were found in rRNA genes. Furthermore, spontaneous DSBs in cells under folate deficiency conditions were located exclusively within the rRNA gene units, representing a H3K4me1 hallmark. Enrichment H3K4me1 at the hot spots of DSB regions enhanced the recruitment of upstream binding factor (UBF) to rRNA genes, resulting in the increment of rRNA genes transcription. Supplement of folate resulted in a restored UBF binding across DNA breakage sites of rRNA genes, and normal rRNA gene transcription. In samples from neural tube defects (NTDs) with low folate level, up-regulation of rRNA gene transcription was observed, along with aberrant UBF level. Our results present a new view by which alterations in folate levels affects DNA breakage through epigenetic control leading to the regulation of rRNA gene transcription during the early stage of development. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Regulation of muscle GLUT-4 transcription by AMP-activated protein kinase.

PubMed

Zheng, D; MacLean, P S; Pohnert, S C; Knight, J B; Olson, A L; Winder, W W; Dohm, G L

2001-09-01

Skeletal muscle GLUT-4 transcription in response to treatment with 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR), a known activator of AMP-activated protein kinase (AMPK), was studied in rats and mice. The increase in GLUT-4 mRNA levels in response to a single subcutaneous injection of AICAR, peaked at 13 h in white and red quadriceps muscles but not in the soleus muscle. The mRNA level of chloramphenicol acyltransferase reporter gene which is driven by 1,154 or 895 bp of the human GLUT-4 proximal promoter was increased in AICAR-treated transgenic mice, demonstrating the transcriptional upregulation of the GLUT-4 gene by AICAR. However, this induction of transcription was not apparent with 730 bp of the promoter. In addition, nuclear extracts from AICAR-treated mice bound to the consensus sequence of myocyte enhancer factor-2 (from -473 to -464) to a greater extent than from saline-injected mice. Thus AMP-activated protein kinase activation by AICAR increases GLUT-4 transcription by a mechanism that requires response elements within 895 bp of human GLUT-4 proximal promoter and that may be cooperatively mediated by myocyte enhancer factor-2.

Regulation of RNA polymerase III transcription during transformation of human IMR90 fibroblasts with defined genetic elements.

PubMed

Durrieu-Gaillard, Stéphanie; Dumay-Odelot, Hélène; Boldina, Galina; Tourasse, Nicolas J; Allard, Delphine; André, Fabrice; Macari, Françoise; Choquet, Armelle; Lagarde, Pauline; Drutel, Guillaume; Leste-Lasserre, Thierry; Petitet, Marion; Lesluyes, Tom; Lartigue-Faustin, Lydia; Dupuy, Jean-William; Chibon, Frédéric; Roeder, Robert G; Joubert, Dominique; Vagner, Stéphan; Teichmann, Martin

2018-01-01

RNA polymerase (Pol) III transcribes small untranslated RNAs that are essential for cellular homeostasis and growth. Its activity is regulated by inactivation of tumor suppressor proteins and overexpression of the oncogene c-MYC, but the concerted action of these tumor-promoting factors on Pol III transcription has not yet been assessed. In order to comprehensively analyse the regulation of Pol III transcription during tumorigenesis we employ a model system that relies on the expression of five genetic elements to achieve cellular transformation. Expression of these elements in six distinct transformation intermediate cell lines leads to the inactivation of TP53, RB1, and protein phosphatase 2A, as well as the activation of RAS and the protection of telomeres by TERT, thereby conducting to full tumoral transformation of IMR90 fibroblasts. Transformation is accompanied by moderately enhanced levels of a subset of Pol III-transcribed RNAs (7SK; MRP; H1). In addition, mRNA and/or protein levels of several Pol III subunits and transcription factors are upregulated, including increased protein levels of TFIIIB and TFIIIC subunits, of SNAPC1 and of Pol III subunits. Strikingly, the expression of POLR3G and of SNAPC1 is strongly enhanced during transformation in this cellular transformation model. Collectively, our data indicate that increased expression of several components of the Pol III transcription system accompanied by a 2-fold increase in steady state levels of a subset of Pol III RNAs is sufficient for sustaining tumor formation.

Oral Quercetin Supplementation Enhances Adiponectin Receptor Transcript Expression in Polycystic Ovary Syndrome Patients: A Randomized Placebo-Controlled Double-Blind Clinical Trial

PubMed Central

Rezvan, Neda; Moini, Ashraf; Gorgani-Firuzjaee, Sattar; Hosseinzadeh-Attar, Mohammad Javad

2018-01-01

Objective Polycystic ovary syndrome (PCOS), an ovarian-pituitary axis androgen disorder, is a common endocrine disease in women. Obesity-induced androgenesis and imbalance of adipokine secretion may lead to some metabolic features of PCOS. The beneficial effects of polyphenolic compounds such as quercetin have been reported, however, the underlying molecular mechanism is not entirely understood. In the present study, we investigated the effect of quercetin supplementation on the expression of adiponectin receptors at the transcript level in peripheral blood mononuclear cells (PBMC) samples of PCOS patients. Materials and Methods In this randomized clinical trial, 84 PCOS subjects were randomly assigned to two groups; the treatment group received 1 g quercetin (two 500 mg capsules) daily for 12 weeks and the control group received placebo. To examine the effect of quercetin supplementation on PCOS patients in addition to biochemical and anthropometric assessments, the expression of ADIPOR1 and ADIPOR2 at the transcript level and AMPK level were determined by quantitative reverse transcription-polymerase chain reaction (RT-qPCR) and ELISA assays respectively. Results Oral quercetin supplementation significantly increased ADIPOR1 and ADIPOR2 transcript expression by 1.32- and 1.46-fold respecetively (P<0.01). In addition, quercetin supplementation enhanced AMPK level by 12.3% compared with the control group (P<0.05). Conclusion Oral quercetin supplementation improves the metabolic features of PCOS patients by upregulating the expression of adiponectin receptors and AMPK (Registration Number: IRCT2013112515536N1). PMID:29105398

Elongin B-mediated epigenetic alteration of viral chromatin correlates with efficient human cytomegalovirus gene expression and replication.

PubMed

Hwang, Jiwon; Saffert, Ryan T; Kalejta, Robert F

2011-01-01

Elongins B and C are members of complexes that increase the efficiency of transcriptional elongation by RNA polymerase II (RNAPII) and enhance the monoubiquitination of histone H2B, an epigenetic mark of actively transcribed genes. Here we show that, in addition to its role in facilitating transcription of the cellular genome, elongin B also enhances gene expression from the double-stranded DNA genome of human cytomegalovirus (HCMV), a pathogenic herpesvirus. Reducing the level of elongin B by small interfering RNA- or short hairpin RNA-mediated knockdown decreased viral mRNA expression, viral protein accumulation, viral DNA replication, and infectious virion production. Chromatin immunoprecipitation analysis indicated viral genome occupancy of the elongating form of RNAPII, and monoubiquitinated histone H2B was reduced in elongin B-deficient cells. These data suggest that, in addition to the previously documented epigenetic regulation of transcriptional initiation, HCMV also subverts cellular elongin B-mediated epigenetic mechanisms for enhancing transcriptional elongation to enhance viral gene expression and virus replication. The genetic and epigenetic control of transcription initiation at both cellular and viral promoters is well documented. Recently, the epigenetic modification of histone H2B monoubiquitination throughout the bodies of cellular genes has been shown to enhance the elongation of RNA polymerase II-initiated transcripts. Mechanisms that might control the elongation of viral transcripts are less well studied. Here we show that, as with cellular genes, elongin B-mediated monoubiquitination of histone H2B also facilitates the transcriptional elongation of human cytomegalovirus genes. This and perhaps other epigenetic markings of actively transcribed regions may help in identifying viral genes expressed during in vitro latency or during natural infections of humans. Furthermore, this work identifies a novel, tractable model system to further study the regulation of transcriptional elongation in living cells.

Enhanced osteoprogenitor elongated collagen fiber matrix formation by bioactive glass ionic silicon dependent on Sp7 (osterix) transcription.

PubMed

Varanasi, Venu G; Odatsu, Tetsurou; Bishop, Timothy; Chang, Joyce; Owyoung, Jeremy; Loomer, Peter M

2016-10-01

Bioactive glasses release ions, those enhance osteoblast collagen matrix synthesis and osteogenic marker expression during bone healing. Collagen matrix density and osteogenic marker expression depend on osteogenic transcription factors, (e.g., Osterix (OSX)). We hypothesize that enhanced expression and formation of collagen by Si(4+) depends on enhanced expression of OSX transcription. Experimental bioactive glass (6P53-b) and commercial Bioglass(TM) (45S5) were dissolved in basal medium to make glass conditioned medium (GCM). ICP-MS analysis was used to measure bioactive glass ion release rates. MC3T3-E1 cells were cultured for 20 days, and gene expression and extracellular matrix collagen formation was analyzed. In a separate study, siRNA was used to determine the effect of OSX knockdown on impacting the effect of Si(4+) on osteogenic markers and matrix collagen formation. Each bioactive glass exhibited similar ion release rates for all ions, except Mg(2+) released by 6P53-b. Gene expression results showed that GCM markedly enhanced many osteogenic markers, and 45S5 GCM showed higher levels of expression and collagen matrix fiber bundle density than 6P53-b GCM. Upon knockdown of OSX transcription, collagen type 5, alkaline phosphatase, and matrix density were not enhanced as compared to wild type cells. This study illustrates that the enhancement of elongated collagen fiber matrix formation by Si(±) depends on OSX transcription. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2604-2615, 2016. © 2016 Wiley Periodicals, Inc.

Enhancer and Transcription Factor Dynamics during Myeloid Differentiation Reveal an Early Differentiation Block in Cebpa null Progenitors.

PubMed

Pundhir, Sachin; Bratt Lauridsen, Felicia Kathrine; Schuster, Mikkel Bruhn; Jakobsen, Janus Schou; Ge, Ying; Schoof, Erwin Marten; Rapin, Nicolas; Waage, Johannes; Hasemann, Marie Sigurd; Porse, Bo Torben

2018-05-29

Transcription factors PU.1 and CEBPA are required for the proper coordination of enhancer activity during granulocytic-monocytic (GM) lineage differentiation to form myeloid cells. However, precisely how these factors control the chronology of enhancer establishment during differentiation is not known. Through integrated analyses of enhancer dynamics, transcription factor binding, and proximal gene expression during successive stages of murine GM-lineage differentiation, we unravel the distinct kinetics by which PU.1 and CEBPA coordinate GM enhancer activity. We find no evidence of a pioneering function of PU.1 during late GM-lineage differentiation. Instead, we delineate a set of enhancers that gain accessibility in a CEBPA-dependent manner, suggesting a pioneering function of CEBPA. Analyses of Cebpa null bone marrow demonstrate that CEBPA controls PU.1 levels and, unexpectedly, that the loss of CEBPA results in an early differentiation block. Taken together, our data provide insights into how PU.1 and CEBPA functionally interact to drive GM-lineage differentiation. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

Defining Transcriptional Regulatory Mechanisms for Primary let-7 miRNAs

PubMed Central

Gaeta, Xavier; Le, Luat; Lin, Ying; Xie, Yuan; Lowry, William E.

2017-01-01

The let-7 family of miRNAs have been shown to control developmental timing in organisms from C. elegans to humans; their function in several essential cell processes throughout development is also well conserved. Numerous studies have defined several steps of post-transcriptional regulation of let-7 production; from pri-miRNA through pre-miRNA, to the mature miRNA that targets endogenous mRNAs for degradation or translational inhibition. Less-well defined are modes of transcriptional regulation of the pri-miRNAs for let-7. let-7 pri-miRNAs are expressed in polycistronic fashion, in long transcripts newly annotated based on chromatin-associated RNA-sequencing. Upon differentiation, we found that some let-7 pri-miRNAs are regulated at the transcriptional level, while others appear to be constitutively transcribed. Using the Epigenetic Roadmap database, we further annotated regulatory elements of each polycistron identified putative promoters and enhancers. Probing these regulatory elements for transcription factor binding sites identified factors that regulate transcription of let-7 in both promoter and enhancer regions, and identified novel regulatory mechanisms for this important class of miRNAs. PMID:28052101

Enhanced expression of the urokinase-type plasminogen activator gene and reduced colony formation in soft agar by ectopic expression of PU.1 in HT1080 human fibrosarcoma cells.

PubMed Central

Kondoh, N.; Yamada, T.; Kihara-Negishi, F.; Yamamoto, M.; Oikawa, T.

1998-01-01

To investigate the cell biological function of PU.1, a member of the Ets family of transcription factors, a vector capable of expressing the protein was transfected into HT1080 human fibrosarcoma cells. Exogenous expression of PU.1 in HT1080 cells reduced colony-forming efficiency but stimulated cell migration in soft agar, although it did not affect cell growth in adherent culture. Expression of the urokinase-type plasminogen activator (uPA) mRNA, which is known to be correlated with cell migration and invasion, was enhanced in PU.1 transfectants compared with mock transfectants. Run-on analysis demonstrated that uPA transcription was unaffected by PU.1, suggesting that this enhancement mainly occurs at a post-transcriptional level. On the other hand, treatment of HT1080 cells with the synthetic glucocorticoid dexamethasone (DEX; 10(-7) M) significantly reduced uPA gene expression at a transcriptional level. Furthermore, DEX inhibited cell migration in soft agar without affecting cell growth. These negative effects of DEX on uPA expression and cell migration were alleviated by the expression of PU.1 in HT1080 cells, whereas expression of the N-ras oncogene, which is responsible for maintenance of the transformed phenotypes in HT1080 cells, was unaffected by PU.1 expression or DEX treatment in the cells. Our results suggest that expression of PU.1 can stimulate uPA gene expression at the post-transcriptional level, which may subsequently lead to activation of cell motility and/or reduced cell-cell adhesion, but reduces anchorage-independent growth of HT1080 cells. Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:9743289

Sucrose-induced anthocyanin accumulation in vegetative tissue of Petunia plants requires anthocyanin regulatory transcription factors.

PubMed

Ai, Trinh Ngoc; Naing, Aung Htay; Arun, Muthukrishnan; Lim, Sun-Hyung; Kim, Chang Kil

2016-11-01

The effects of three different sucrose concentrations on plant growth and anthocyanin accumulation were examined in non-transgenic (NT) and transgenic (T 2 ) specimens of the Petunia hybrida cultivar 'Mirage rose' that carried the anthocyanin regulatory transcription factors B-Peru+mPAP1 or RsMYB1. Anthocyanin accumulation was not observed in NT plants in any treatments, whereas a range of anthocyanin accumulation was observed in transgenic plants. The anthocyanin content detected in transgenic plants expressing the anthocyanin regulatory transcription factors (B-Peru+mPAP1 or RsMYB1) was higher than that in NT plants. In addition, increasing sucrose concentration strongly enhanced anthocyanin content as shown by quantitative real-time polymerase chain reaction (qRT-PCR) analysis, wherein increased concentrations of sucrose enhanced transcript levels of the transcription factors that are responsible for the induction of biosynthetic genes involved in anthocyanin synthesis; this pattern was not observed in NT plants. In addition, sucrose affected plant growth, although the effects were different between NT and transgenic plants. Taken together, the application of sucrose could enhance anthocyanin production in vegetative tissue of transgenic Petunia carrying anthocyanin regulatory transcription factors, and this study provides insights about interactive effects of sucrose and transcription factors in anthocyanin biosynthesis in the transgenic plant. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

N-acetylcysteine augments adenovirus-mediated gene expression in human endothelial cells by enhancing transgene transcription and virus entry.

PubMed

Jornot, L; Morris, M A; Petersen, H; Moix, I; Rochat, T

2002-01-01

It has previously been shown that oxidants reduce the efficiency of adenoviral transduction in human umbilical vein endothelial cells (HUVECs). In this study, the effect of the antioxidant N-acetylcysteine (NAC) in adenovirus-mediated gene transfer has been investigated. HUVECs were pretreated or not with NAC, and infected with E1E3-deleted adenovirus (Ad) containing the LacZ gene expressed from the RSV-LTR promoter/enhancer in the presence and absence of NAC. Transgene expression was assessed at the protein level (histochemical staining, measurement of beta-Gal activity, and western blot), mRNA level (real-time RT-PCR) and gene level (nuclear run on) 24 h and 48 h after infection. Adenoviral DNA was quantitated by real-time PCR, and cell surface expression of Coxsackie/adenovirus receptors (CAR) was determined by FACS analysis. Pretreatment of cells with NAC prior to Ad infection enhanced beta-Gal activity by two-fold due to an increase in viral DNA, which was related to increased CAR expression. When NAC was present only during the post-infection period, a five-fold increase in beta-Gal activity and LacZ gene transcriptional activity was observed. When NAC was present during both the pretreatment and the post-infection period, beta-Gal activity was further enhanced, by 15-fold. Augmentation of beta-Gal activity was paralleled by an increase in beta-Gal protein and mRNA levels. NAC did not affect the half-life of LacZ mRNA. Pretreatment with NAC prior to Ad infection enhances virus entry, while treatment with NAC post-infection increases transgene transcription. This strategy permits the use of lower adenoviral loads and thus might be helpful for gene therapy of vascular diseases. Copyright 2001 John Wiley & Sons, Ltd.

Transcription factor assisted loading and enhancer dynamics dictate the hepatic fasting response

PubMed Central

Goldstein, Ido; Baek, Songjoon; Presman, Diego M.; Paakinaho, Ville; Swinstead, Erin E.; Hager, Gordon L.

2017-01-01

Fasting elicits transcriptional programs in hepatocytes leading to glucose and ketone production. This transcriptional program is regulated by many transcription factors (TFs). To understand how this complex network regulates the metabolic response to fasting, we aimed at isolating the enhancers and TFs dictating it. Measuring chromatin accessibility revealed that fasting massively reorganizes liver chromatin, exposing numerous fasting-induced enhancers. By utilizing computational methods in combination with dissecting enhancer features and TF cistromes, we implicated four key TFs regulating the fasting response: glucocorticoid receptor (GR), cAMP responsive element binding protein 1 (CREB1), peroxisome proliferator activated receptor alpha (PPARA), and CCAAT/enhancer binding protein beta (CEBPB). These TFs regulate fuel production by two distinctly operating modules, each controlling a separate metabolic pathway. The gluconeogenic module operates through assisted loading, whereby GR doubles the number of sites occupied by CREB1 as well as enhances CREB1 binding intensity and increases accessibility of CREB1 binding sites. Importantly, this GR-assisted CREB1 binding was enhancer-selective and did not affect all CREB1-bound enhancers. Single-molecule tracking revealed that GR increases the number and DNA residence time of a portion of chromatin-bound CREB1 molecules. These events collectively result in rapid synergistic gene expression and higher hepatic glucose production. Conversely, the ketogenic module operates via a GR-induced TF cascade, whereby PPARA levels are increased following GR activation, facilitating gradual enhancer maturation next to PPARA target genes and delayed ketogenic gene expression. Our findings reveal a complex network of enhancers and TFs that dynamically cooperate to restore homeostasis upon fasting. PMID:28031249

Enhancer Activation Requires Trans-Recruitment of a Mega Transcription Factor Complex

PubMed Central

Liu, Zhijie; Merkurjev, Daria; Yang, Feng; Li, Wenbo; Oh, Soohwan; Friedman, Meyer J.; Song, Xiaoyuan; Zhang, Feng; Ma, Qi; Ohgi, Kenneth; Krones, Anna; Rosenfeld, Michael G.

2014-01-01

Summary Enhancers provide critical information directing cell-type specific transcriptional programs, regulated by binding of signal-dependent transcription factors and their associated cofactors. Here we report that the most strongly activated estrogen (E2)-responsive enhancers are characterized by trans-recruitment and in situ assembly of a large 1-2 MDa complex of diverse DNA-binding transcription factors by ERα at ERE-containing enhancers. We refer to enhancers recruiting these factors as mega transcription factor-bound in trans (MegaTrans) enhancers. The MegaTrans complex is a signature of the most potent functional enhancers and is required for activation of enhancer RNA transcription and recruitment of coactivators, including p300 and Med1. The MegaTrans complex functions, in part, by recruiting specific enzymatic machinery, exemplified by DNA-dependent protein kinase. Thus, MegaTrans-containing enhancers represent a cohort of functional enhancers that mediate a broad and important transcriptional program and provide a molecular explanation for transcription factor clustering and hotspots noted in the genome. PMID:25303530

Low levels of aflatoxin B1, ricin and milk enhance recombinant protein production in mammalian cells

USDA-ARS?s Scientific Manuscript database

Changing the optimal tissue culture medium by adding low levels of environmental stress such as 1 µM of the fungal toxin aflatoxin B1 (AFB1), 1 ng of the castor bean protein toxin ricin in transduced mammalian cells or 1% reconstituted milk enhances transcription and increases production of the foll...

Nitrogen treatment enhances sterols and withaferin A through transcriptional activation of jasmonate pathway, WRKY transcription factors, and biosynthesis genes in Withania somnifera (L.) Dunal.

PubMed

Pal, Shaifali; Yadav, Akhilesh Kumar; Singh, Anup Kumar; Rastogi, Shubhra; Gupta, Madan Mohan; Verma, Rajesh Kumar; Nagegowda, Dinesh A; Pal, Anirban; Shasany, Ajit Kumar

2017-01-01

The medicinal plant Withania somnifera is researched extensively to increase the quantity of withanolides and specifically withaferin A, which finds implications in many pharmacological activities. Due to insufficient knowledge on biosynthesis and unacceptability of transgenic approach, it is preferred to follow alternative physiological methods to increase the yield of withanolides. Prior use of elicitors like salicylic acid, methyl jasmonate, fungal extracts, and even mechanical wounding have shown to increase the withanolide biosynthesis with limited success; however, the commercial viability and logistics of application are debatable. In this investigation, we tested the simple nitrogeneous fertilizers pertaining to the enhancement of withaferin A biosynthesis. Application of ammonium sulfate improved the sterol contents required for the withanolide biosynthesis and correlated to higher expression of pathway genes like FPPS, SMT1, SMT2, SMO1, SMO2, and ODM. Increased expression of a gene homologous to allene oxide cyclase, crucial in jasmonic acid biosynthetic pathway, suggested the involvement of jasmonate signaling. High levels of WRKY gene transcripts indicated transcriptional regulation of the pathway genes. Increase in transcript level could be correlated with a corresponding increase in the protein levels for WsSMT1 and WsWRKY1. The withaferin A increase was also demonstrated in the potted plants growing in the glasshouse and in the open field. These results implicated simple physiological management of nitrogen fertilizer signal to improve the yield of secondary metabolite through probable involvement of jasmonate signal and WRKY transcription factor for the first time, in W. somnifera besides improving the foliage.

Analysis of Ly49 gene transcripts in mature NK cells supports a role for the Pro1 element in gene activation, not gene expression.

PubMed

McCullen, M V; Li, H; Cam, M; Sen, S K; McVicar, D W; Anderson, S K

2016-09-01

The variegated expression of murine Ly49 loci has been associated with the probabilistic behavior of an upstream promoter active in immature cells, the Pro1 element. However, recent data suggest that Pro1 may be active in mature natural killer (NK) cells and function as an enhancer element. To assess directly if Pro1 transcripts are present in mature Ly49-expressing NK cells, RNA-sequencing of the total transcript pool was performed on freshly isolated splenic NK cells sorted for expression of either Ly49G or Ly49I. No Pro1 transcripts were detected from the Ly49a, Ly49c or Ly49i genes in mature Ly49(+) NK cells that contained high levels of Pro2 transcripts. Low levels of Ly49g Pro1 transcripts were found in both Ly49G(+) and Ly49G(-) populations, consistent with the presence of a small population of mature NK cells undergoing Ly49g gene activation, as previously demonstrated by culture of splenic NK cells in interleukin-2. Ly49 gene reporter constructs containing Pro1 failed to show any enhancer activity of Pro1 on Pro2 in a mature Ly49-expressing cell line. Taken together, the results are consistent with Pro1 transcription having a role in gene activation in developing NK, and argue against a role for Pro1 in Ly49 gene transcription by mature NK cells.

Phloretin enhances adipocyte differentiation and adiponectin expression in 3T3-L1 cells.

PubMed

Hassan, Meryl; El Yazidi, Claire; Landrier, Jean-François; Lairon, Denis; Margotat, Alain; Amiot, Marie-Josèphe

2007-09-14

Adipocyte dysfunction is strongly associated with the development of cardiovascular risk factors and diabetes. It is accepted that the regulation of adipogenesis or adipokines expression, notably adiponectin, is able to prevent these disorders. In this report, we show that phloretin, a dietary flavonoid, enhances 3T3-L1 adipocyte differentiation as evidenced by increased triglyceride accumulation and GPDH activity. At a molecular level, mRNA expression levels of both PPARgamma and C/EBPalpha, the master adipogenic transcription factors, are markedly increased by phloretin. Moreover, mRNA levels of PPARgamma target genes such as LPL, aP2, CD36 and LXRalpha are up-regulated by phloretin. We also show that phloretin enhances the expression and secretion of adiponectin. Co-transfection studies suggest the induction of PPARgamma transcriptional activity as a possible mechanism underlying the phloretin-mediated effects. Taken together, these results suggest that phloretin may be beneficial for reducing insulin resistance through its potency to regulate adipocyte differentiation and function.

Dual transcriptional-translational cascade permits cellular level tuneable expression control

PubMed Central

Morra, Rosa; Shankar, Jayendra; Robinson, Christopher J.; Halliwell, Samantha; Butler, Lisa; Upton, Mathew; Hay, Sam; Micklefield, Jason; Dixon, Neil

2016-01-01

The ability to induce gene expression in a small molecule dependent manner has led to many applications in target discovery, functional elucidation and bio-production. To date these applications have relied on a limited set of protein-based control mechanisms operating at the level of transcription initiation. The discovery, design and reengineering of riboswitches offer an alternative means by which to control gene expression. Here we report the development and characterization of a novel tunable recombinant expression system, termed RiboTite, which operates at both the transcriptional and translational level. Using standard inducible promoters and orthogonal riboswitches, a multi-layered modular genetic control circuit was developed to control the expression of both bacteriophage T7 RNA polymerase and recombinant gene(s) of interest. The system was benchmarked against a number of commonly used E. coli expression systems, and shows tight basal control, precise analogue tunability of gene expression at the cellular level, dose-dependent regulation of protein production rates over extended growth periods and enhanced cell viability. This novel system expands the number of E. coli expression systems for use in recombinant protein production and represents a major performance enhancement over and above the most widely used expression systems. PMID:26405200

Conserved epigenomic signals in mice and humans reveal immune basis of Alzheimer's disease.

PubMed

Gjoneska, Elizabeta; Pfenning, Andreas R; Mathys, Hansruedi; Quon, Gerald; Kundaje, Anshul; Tsai, Li-Huei; Kellis, Manolis

2015-02-19

Alzheimer's disease (AD) is a severe age-related neurodegenerative disorder characterized by accumulation of amyloid-β plaques and neurofibrillary tangles, synaptic and neuronal loss, and cognitive decline. Several genes have been implicated in AD, but chromatin state alterations during neurodegeneration remain uncharacterized. Here we profile transcriptional and chromatin state dynamics across early and late pathology in the hippocampus of an inducible mouse model of AD-like neurodegeneration. We find a coordinated downregulation of synaptic plasticity genes and regulatory regions, and upregulation of immune response genes and regulatory regions, which are targeted by factors that belong to the ETS family of transcriptional regulators, including PU.1. Human regions orthologous to increasing-level enhancers show immune-cell-specific enhancer signatures as well as immune cell expression quantitative trait loci, while decreasing-level enhancer orthologues show fetal-brain-specific enhancer activity. Notably, AD-associated genetic variants are specifically enriched in increasing-level enhancer orthologues, implicating immune processes in AD predisposition. Indeed, increasing enhancers overlap known AD loci lacking protein-altering variants, and implicate additional loci that do not reach genome-wide significance. Our results reveal new insights into the mechanisms of neurodegeneration and establish the mouse as a useful model for functional studies of AD regulatory regions.

Chromatin potentiates transcription

PubMed Central

Nagai, Shigeki; Davis, Ralph E.; Mattei, Pierre Jean; Eagen, Kyle Patrick; Kornberg, Roger D.

2017-01-01

Chromatin isolated from the chromosomal locus of the PHO5 gene of yeast in a transcriptionally repressed state was transcribed with 12 pure proteins (80 polypeptides): RNA polymerase II, six general transcription factors, TFIIS, the Pho4 gene activator protein, and the SAGA, SWI/SNF, and Mediator complexes. Contrary to expectation, a nucleosome occluding the TATA box and transcription start sites did not impede transcription but rather, enhanced it: the level of chromatin transcription was at least sevenfold greater than that of naked DNA, and chromatin gave patterns of transcription start sites closely similar to those occurring in vivo, whereas naked DNA gave many aberrant transcripts. Both histone acetylation and trimethylation of H3K4 (H3K4me3) were important for chromatin transcription. The nucleosome, long known to serve as a general gene repressor, thus also performs an important positive role in transcription. PMID:28137832

Chromatin reprogramming in breast cancer.

PubMed

Swinstead, Erin E; Paakinaho, Ville; Hager, Gordon

2018-04-24

Reprogramming of the chromatin landscape is a critical component to the transcriptional response in breast cancer. Effects of sex hormones such as estrogens and progesterone have been well described to have a critical impact on breast cancer proliferation. However, the complex network of the chromatin landscape, enhancer regions, and mode of function of steroid receptors (SRs) and other transcription factors (TFs), is an intricate web of signaling and functional processes that is still largely misunderstood at the mechanistic level. In this review, we describe what is currently known about the dynamic interplay between TFs with chromatin and the reprogramming of enhancer elements. Emphasis has been placed on characterizing the different modes of action of TFs in regulating enhancer activity, specifically, how different SRs target enhancer regions and reprogram chromatin in breast cancer cells. In addition, we discuss current techniques employed to study enhancer function at a genome-wide level. Further, we have noted recent advances in live cell imaging technology. These single cell approaches enable the coupling of population based assays with real-time studies to address many unsolved questions about SRs and chromatin dynamics in breast cancer.

Transcription factor assisted loading and enhancer dynamics dictate the hepatic fasting response.

PubMed

Goldstein, Ido; Baek, Songjoon; Presman, Diego M; Paakinaho, Ville; Swinstead, Erin E; Hager, Gordon L

2017-03-01

Fasting elicits transcriptional programs in hepatocytes leading to glucose and ketone production. This transcriptional program is regulated by many transcription factors (TFs). To understand how this complex network regulates the metabolic response to fasting, we aimed at isolating the enhancers and TFs dictating it. Measuring chromatin accessibility revealed that fasting massively reorganizes liver chromatin, exposing numerous fasting-induced enhancers. By utilizing computational methods in combination with dissecting enhancer features and TF cistromes, we implicated four key TFs regulating the fasting response: glucocorticoid receptor (GR), cAMP responsive element binding protein 1 (CREB1), peroxisome proliferator activated receptor alpha (PPARA), and CCAAT/enhancer binding protein beta (CEBPB). These TFs regulate fuel production by two distinctly operating modules, each controlling a separate metabolic pathway. The gluconeogenic module operates through assisted loading, whereby GR doubles the number of sites occupied by CREB1 as well as enhances CREB1 binding intensity and increases accessibility of CREB1 binding sites. Importantly, this GR-assisted CREB1 binding was enhancer-selective and did not affect all CREB1-bound enhancers. Single-molecule tracking revealed that GR increases the number and DNA residence time of a portion of chromatin-bound CREB1 molecules. These events collectively result in rapid synergistic gene expression and higher hepatic glucose production. Conversely, the ketogenic module operates via a GR-induced TF cascade, whereby PPARA levels are increased following GR activation, facilitating gradual enhancer maturation next to PPARA target genes and delayed ketogenic gene expression. Our findings reveal a complex network of enhancers and TFs that dynamically cooperate to restore homeostasis upon fasting. Published by Cold Spring Harbor Laboratory Press.

The Wheat NAC Transcription Factor TaNAC2L Is Regulated at the Transcriptional and Post-Translational Levels and Promotes Heat Stress Tolerance in Transgenic Arabidopsis.

PubMed

Guo, Weiwei; Zhang, Jinxia; Zhang, Ning; Xin, Mingming; Peng, Huiru; Hu, Zhaorong; Ni, Zhongfu; Du, Jinkun

2015-01-01

Heat stress poses a serious threat to global crop production. In efforts that aim to mitigate the adverse effects of heat stress on crops, a variety of genetic tools are being used to develop plants with improved thermotolerance. The characterization of important regulators of heat stress tolerance provides essential information for this aim. In this study, we examine the wheat (Triticum aestivum) NAC transcription factor gene TaNAC2L. High temperature induced TaNAC2L expression in wheat and overexpression of TaNAC2L in Arabidopsis thaliana enhanced acquired heat tolerance without causing obvious alterations in phenotype compared with wild type under normal conditions. TaNAC2L overexpression also activated the expression of heat-related genes in the transgenic Arabidopsis plants, suggesting that TaNAC2L may improve heat tolerance by regulating the expression of stress-responsive genes. Notably, TaNAC2L is also regulated at the post-translational level and might be degraded via a proteasome-mediated pathway. Thus, this wheat transcription factor may have potential uses in enhancing thermotolerance in crops.

tortuga refines Notch pathway gene expression in the zebrafish presomitic mesoderm at the post-transcriptional level.

PubMed

Dill, Kariena K; Amacher, Sharon L

2005-11-15

We have identified the zebrafish tortuga (tor) gene by an ENU-induced mutation that disrupts the presomitic mesoderm (PSM) expression of Notch pathway genes. In tor mutants, Notch pathway gene expression persists in regions of the PSM where expression is normally off in wild type embryos. The expression of hairy/Enhancer of split-related 1 (her1) is affected first, followed by the delta genes deltaC and deltaD, and finally, by another hairy/Enhancer of split-related gene, her7. In situ hybridization with intron-specific probes for her1 and deltaC indicates that transcriptional bursts of expression are normal in tor mutants, suggesting that tor normally functions to refine her1 and deltaC message levels downstream of transcription. Despite the striking defects in Notch pathway gene expression, somite boundaries form normally in tor mutant embryos, although somitic mesoderm defects are apparent later, when cells mature to form muscle fibers. Thus, while the function of Notch pathway genes is required for proper somite formation, the tor mutant phenotype suggests that precise oscillations of Notch pathway transcripts are not essential for establishing segmental pattern in the presomitic mesoderm.

A hyperactive transcriptional state marks genome reactivation at the mitosis–G1 transition

PubMed Central

Hsiung, Chris C.-S.; Bartman, Caroline R.; Huang, Peng; Ginart, Paul; Stonestrom, Aaron J.; Keller, Cheryl A.; Face, Carolyne; Jahn, Kristen S.; Evans, Perry; Sankaranarayanan, Laavanya; Giardine, Belinda; Hardison, Ross C.; Raj, Arjun; Blobel, Gerd A.

2016-01-01

During mitosis, RNA polymerase II (Pol II) and many transcription factors dissociate from chromatin, and transcription ceases globally. Transcription is known to restart in bulk by telophase, but whether de novo transcription at the mitosis–G1 transition is in any way distinct from later in interphase remains unknown. We tracked Pol II occupancy genome-wide in mammalian cells progressing from mitosis through late G1. Unexpectedly, during the earliest rounds of transcription at the mitosis–G1 transition, ∼50% of active genes and distal enhancers exhibit a spike in transcription, exceeding levels observed later in G1 phase. Enhancer–promoter chromatin contacts are depleted during mitosis and restored rapidly upon G1 entry but do not spike. Of the chromatin-associated features examined, histone H3 Lys27 acetylation levels at individual loci in mitosis best predict the mitosis–G1 transcriptional spike. Single-molecule RNA imaging supports that the mitosis–G1 transcriptional spike can constitute the maximum transcriptional activity per DNA copy throughout the cell division cycle. The transcriptional spike occurs heterogeneously and propagates to cell-to-cell differences in mature mRNA expression. Our results raise the possibility that passage through the mitosis–G1 transition might predispose cells to diverge in gene expression states. PMID:27340175

GA-DELLA pathway is involved in regulation of nitrogen deficiency-induced anthocyanin accumulation.

PubMed

Zhang, Yongqiang; Liu, Zhongjuan; Liu, Jianping; Lin, Sheng; Wang, Jianfeng; Lin, Wenxiong; Xu, Weifeng

2017-04-01

DELLA proteins positively regulate nitrogen deficiency-induced anthocyanin accumulation through directly interaction with PAP1 to enhance its transcriptional activity on anthocyanin biosynthetic gene expressions. Plants can survive a limiting nitrogen supply by undergoing adaptive responses, including induction of anthocyanin production. However, the detailed mechanism is still unclear. In this study, we found that this process was impaired and enhanced, respectively, by exogenous GA 3 (an active form of GAs) and paclobutrazol (PAC, a specific GA biosynthesis inhibitor) in Arabidopsis seedlings. Consistently, the nitrogen deficiency-induced transcript levels of several key genes involved in anthocyanin biosynthesis, including F3'H, DFR, LDOX, and UF3GT, were decreased and enhanced by exogenous GA 3 and PAC, respectively. Moreover, the nitrogen deficiency-induced anthocyanin accumulation and biosynthesis gene expressions were impaired in the loss-of-function mutant gai-t6/rga-t2/rgl1-1/rgl2-1/rgl3-1 (della) but enhanced in the GA-insensitive mutant gai, suggesting that DELLA proteins, known as repressors of GA signaling, are necessary for fully induction of nitrogen deficiency-driven anthocyanin biosynthesis. Using yeast two-hybrid (Y2H) assay, pull-down assay, and luciferase complementation assay, it was found that RGA, a DELLA of Arabidopsis, could strongly interact with PAP1, a known regulatory transcription factor positively involved in anthocyanin biosynthesis. Furthermore, transient expression assays indicated that RGA and GAI could enhance the transcriptional activities of PAP1 on its downstream genes, including F3'H and DFR. Taken together, this study suggests that DELLAs are necessary regulators for nitrogen deficiency-induced anthocyanin accumulation through interaction with PAP1 and enhancement of PAP1's transcriptional activity on its target genes. GA-DELLA-involved anthocyanin accumulation is important for plant adaptation to nitrogen deficiency.

Targeting the UPR transcription factor XBP1 protects against Huntington's disease through the regulation of FoxO1 and autophagy

PubMed Central

Vidal, Rene L.; Figueroa, Alicia; Court, Felipe A.; Thielen, Peter; Molina, Claudia; Wirth, Craig; Caballero, Benjamin; Kiffin, Roberta; Segura-Aguilar, Juan; Cuervo, Ana Maria; Glimcher, Laurie H.; Hetz, Claudio

2012-01-01

Mutations leading to expansion of a poly-glutamine track in Huntingtin (Htt) cause Huntington's disease (HD). Signs of endoplasmic reticulum (ER) stress have been recently reported in animal models of HD, associated with the activation of the unfolded protein response (UPR). Here we have investigated the functional contribution of ER stress to HD by targeting the expression of two main UPR transcription factors, XBP1 and ATF4 (activating transcription factor 4), in full-length mutant Huntingtin (mHtt) transgenic mice. XBP1-deficient mice were more resistant to developing disease features, associated with improved neuronal survival and motor performance, and a drastic decrease in mHtt levels. The protective effects of XBP1 deficiency were associated with enhanced macroautophagy in both cellular and animal models of HD. In contrast, ATF4 deficiency did not alter mHtt levels. Although, XBP1 mRNA splicing was observed in the striatum of HD transgenic brains, no changes in the levels of classical ER stress markers were detected in symptomatic animals. At the mechanistic level, we observed that XBP1 deficiency led to augmented expression of Forkhead box O1 (FoxO1), a key transcription factor regulating autophagy in neurons. In agreement with this finding, ectopic expression of FoxO1 enhanced autophagy and mHtt clearance in vitro. Our results provide strong evidence supporting an involvement of XBP1 in HD pathogenesis probably due to an ER stress-independent mechanism involving the control of FoxO1 and autophagy levels. PMID:22337954

Differential Effects of Methyl Jasmonate on the Expression of the Early Light-Inducible Proteins and Other Light-Regulated Genes in Barley1

PubMed Central

Wierstra, Inken; Kloppstech, Klaus

2000-01-01

The effects of methyl jasmonate (JA-Me) on early light-inducible protein (ELIP) expression in barley (Hordeum vulgare L. cv Apex) have been studied. Treatment of leaf segments with JA-Me induces the same symptoms as those exhibited by norflurazon bleaching, including a loss of pigments and enhanced light stress that results in increased ELIP expression under both high- and low-light conditions. The expression of both low- and high-molecular-mass ELIP families is considerably down-regulated by JA-Me at the transcript and protein levels. This repression occurs despite increased photoinhibition measurable as a massive degradation of D1 protein and a delayed recovery of photosystem II activity. In JA-Me-treated leaf segments, the decrease of the photochemical efficiency of photosystem II under high light is substantially more pronounced as compared to controls in water. The repression of ELIP expression by JA-Me is superimposed on the effect of the increased light stress that leads to enhanced ELIP expression. The fact that the reduction of ELIP transcript levels is less pronounced than those of light-harvesting complex II and small subunit of Rubisco transcripts indicates that light stress is still affecting gene expression in the presence of JA-Me. The jasmonate-induced protein transcript levels that are induced by JA-Me decline under light stress conditions. PMID:11027731

The Molecular Mechanism of Ethylene-Mediated Root Hair Development Induced by Phosphate Starvation

PubMed Central

Song, Li; Yu, Haopeng; Dong, Jinsong; Liu, Dong

2016-01-01

Enhanced root hair production, which increases the root surface area for nutrient uptake, is a typical adaptive response of plants to phosphate (Pi) starvation. Although previous studies have shown that ethylene plays an important role in root hair development induced by Pi starvation, the underlying molecular mechanism is not understood. In this work, we characterized an Arabidopsis mutant, hps5, that displays constitutive ethylene responses and increased sensitivity to Pi starvation due to a mutation in the ethylene receptor ERS1. hps5 accumulates high levels of EIN3 protein, a key transcription factor involved in the ethylene signaling pathway, under both Pi sufficiency and deficiency. Pi starvation also increases the accumulation of EIN3 protein. Combined molecular, genetic, and genomic analyses identified a group of genes that affect root hair development by regulating cell wall modifications. The expression of these genes is induced by Pi starvation and is enhanced in the EIN3-overexpressing line. In contrast, the induction of these genes by Pi starvation is suppressed in ein3 and ein3eil1 mutants. EIN3 protein can directly bind to the promoter of these genes, some of which are also the immediate targets of RSL4, a key transcription factor that regulates root hair development. Based on these results, we propose that under normal growth conditions, the level of ethylene is low in root cells; a group of key transcription factors, including RSL4 and its homologs, trigger the transcription of their target genes to promote root hair development; Pi starvation increases the levels of the protein EIN3, which directly binds to the promoters of the genes targeted by RSL4 and its homologs and further increase their transcription, resulting in the enhanced production of root hairs. This model not only explains how ethylene mediates root hair responses to Pi starvation, but may provide a general mechanism for how ethylene regulates root hair development under both stress and non-stress conditions. PMID:27427911

7SK-BAF axis controls pervasive transcription at enhancers

PubMed Central

Flynn, Ryan A.; Do, Brian T.; Rubin, Adam J.; Calo, Eliezer; Lee, Byron; Kuchelmeister, Hannes; Rale, Michael; Chu, Ci; Kool, Eric T.; Wysocka, Joanna; Khavari, Paul A.

2016-01-01

RNA functions at enhancers remain mysterious. Here we show that the 7SK small nuclear RNA (snRNA) inhibits enhancer transcription by modulating nucleosome position. 7SK occupies enhancers and super enhancers genome-wide in mouse and human cells, and 7SK is required to limit eRNA initiation and synthesis in a manner distinct from promoter pausing. Clustered elements at super enhancers uniquely require 7SK to prevent convergent transcription and DNA damage signaling. 7SK physically interacts with the BAF chromatin remodeling complex, recruit BAF to enhancers, and inhibits enhancer transcription by modulating chromatin structure. In turn, 7SK occupancy at enhancers coincides with Brd4 and is exquisitely sensitive to the bromodomain inhibitor JQ1. Thus, 7SK employs distinct mechanisms to counteract diverse consequences of pervasive transcription that distinguish super enhancers, enhancers, and promoters. PMID:26878240

Upregulation of CREB-mediated transcription enhances both short- and long-term memory.

PubMed

Suzuki, Akinobu; Fukushima, Hotaka; Mukawa, Takuya; Toyoda, Hiroki; Wu, Long-Jun; Zhao, Ming-Gao; Xu, Hui; Shang, Yuze; Endoh, Kengo; Iwamoto, Taku; Mamiya, Nori; Okano, Emiko; Hasegawa, Shunsuke; Mercaldo, Valentina; Zhang, Yue; Maeda, Ryouta; Ohta, Miho; Josselyn, Sheena A; Zhuo, Min; Kida, Satoshi

2011-06-15

Unraveling the mechanisms by which the molecular manipulation of genes of interest enhances cognitive function is important to establish genetic therapies for cognitive disorders. Although CREB is thought to positively regulate formation of long-term memory (LTM), gain-of-function effects of CREB remain poorly understood, especially at the behavioral level. To address this, we generated four lines of transgenic mice expressing dominant active CREB mutants (CREB-Y134F or CREB-DIEDML) in the forebrain that exhibited moderate upregulation of CREB activity. These transgenic lines improved not only LTM but also long-lasting long-term potentiation in the CA1 area in the hippocampus. However, we also observed enhanced short-term memory (STM) in contextual fear-conditioning and social recognition tasks. Enhanced LTM and STM could be dissociated behaviorally in these four lines of transgenic mice, suggesting that the underlying mechanism for enhanced STM and LTM are distinct. LTM enhancement seems to be attributable to the improvement of memory consolidation by the upregulation of CREB transcriptional activity, whereas higher basal levels of BDNF, a CREB target gene, predicted enhanced shorter-term memory. The importance of BDNF in STM was verified by microinfusing BDNF or BDNF inhibitors into the hippocampus of wild-type or transgenic mice. Additionally, increasing BDNF further enhanced LTM in one of the lines of transgenic mice that displayed a normal BDNF level but enhanced LTM, suggesting that upregulation of BDNF and CREB activity cooperatively enhances LTM formation. Our findings suggest that CREB positively regulates memory consolidation and affects memory performance by regulating BDNF expression.

Functional cooperativity between two TPA responsive elements in undifferentiated F9 embryonic stem cells.

PubMed Central

Okuda, A; Imagawa, M; Sakai, M; Muramatsu, M

1990-01-01

We have recently identified an enhancer, termed GPEI, in the 5'-flanking region of the rat glutathione transferase P gene, that is composed of two imperfect TPA (phorbol 12-O-tetradecanoate 13-acetate) responsive elements (TREs). Unlike other TRE-containing enhancers, GPEI exhibits a strong transcriptional enhancing activity in F9 embryonic stem cells. Mutational analyses have revealed that the high activity of GPEI is mediated by two imperfect TREs. Each TRE-like sequence has no activity by itself but acts synergistically to form a strong enhancer which is active even in the very low level of AP-1 activity in F9 cells. Furthermore, we show that synthetic DNAs containing two perfect TREs in certain arrangements have strong transcriptional enhancing activities in F9 cells and the activity is greatly influenced by the relative orientation and the distance of two TREs. Images Fig. 1. Fig. 2. Fig. 3. PMID:2323334

TALE-mediated modulation of transcriptional enhancers in vivo.

PubMed

Crocker, Justin; Stern, David L

2013-08-01

We tested whether transcription activator-like effectors (TALEs) could mediate repression and activation of endogenous enhancers in the Drosophila genome. TALE repressors (TALERs) targeting each of the five even-skipped (eve) stripe enhancers generated repression specifically of the focal stripes. TALE activators (TALEAs) targeting the eve promoter or enhancers caused increased expression primarily in cells normally activated by the promoter or targeted enhancer, respectively. This effect supports the view that repression acts in a dominant fashion on transcriptional activators and that the activity state of an enhancer influences TALE binding or the ability of the VP16 domain to enhance transcription. In these assays, the Hairy repression domain did not exhibit previously described long-range transcriptional repression activity. The phenotypic effects of TALER and TALEA expression in larvae and adults are consistent with the observed modulations of eve expression. TALEs thus provide a novel tool for detection and functional modulation of transcriptional enhancers in their native genomic context.

GmSBH1, a homeobox transcription factor gene, relates to growth and development and involves in response to high temperature and humidity stress in soybean.

PubMed

Shu, Yingjie; Tao, Yuan; Wang, Shuang; Huang, Liyan; Yu, Xingwang; Wang, Zhankui; Chen, Ming; Gu, Weihong; Ma, Hao

2015-11-01

GmSBH1 involves in response to high temperature and humidity stress. Homeobox transcription factors are key switches that control plant development processes. Glycine max H1 Sbh1 (GmSBH1) was the first homeobox gene isolated from soybean. In the present study, the full ORF of GmSBH1 was isolated, and the encoded protein was found to be a typical class I KNOX homeobox transcription factor. Subcellular localization and transcriptional activation assays showed that GmSBH1 is a nuclear protein and possesses transcriptional activation activity in the homeodomain. The KNOX1 domain was found to play a clear role in suppressing the transcriptional activation activity of GmSBH1. GmSBH1 showed different expression levels among different soybean tissues and was involved in response to high temperature and humidity (HTH) stress in developing soybean seeds. The overexpression of GmSBH1 in Arabidopsis altered leaf and stoma phenotypes and enhanced seed tolerance to HTH stress. Overall, our results indicated that GmSBH1 is involved in growth, development, and enhances tolerance to pre-harvest seed deterioration caused by HTH stress in soybean.

Characterization of docosahexaenoic acid (DHA)-induced heme oxygenase-1 (HO-1) expression in human cancer cells: the importance of enhanced BTB and CNC homology 1 (Bach1) degradation.

PubMed

Wang, Shuai; Hannafon, Bethany N; Wolf, Roman F; Zhou, Jundong; Avery, Jori E; Wu, Jinchang; Lind, Stuart E; Ding, Wei-Qun

2014-05-01

The effect of docosahexaenoic acid (DHA) on heme oxygenase-1 (HO-1) expression in cancer cells has never been characterized. This study examines DHA-induced HO-1 expression in human cancer cell model systems. DHA enhanced HO-1 gene expression in a time- and concentration-dependent manner, with maximal induction at 21 h of treatment. This induction of HO-1 expression was confirmed in vivo using a xenograft nude mouse model fed a fish-oil-enriched diet. The increase in HO-1 gene transcription induced by DHA was significantly attenuated by the antioxidant N-acetyl cysteine, suggesting the involvement of oxidative stress. This was supported by direct measurement of lipid peroxide levels after DHA treatment. Using a human HO-1 gene promoter reporter construct, we identified two antioxidant response elements (AREs) that mediate the DHA-induced increase in HO-1 gene transcription. Knockdown of nuclear factor (erythroid-derived 2)-like 2 (Nrf2) expression compromised the DHA-induced increase in HO-1 gene transcription, indicating the importance of the Nrf2 pathway in this event. However, the nuclear protein levels of Nrf2 remained unchanged upon DHA treatment. Further studies demonstrated that DHA reduces nuclear Bach1 protein expression by promoting its degradation and attenuates Bach1 binding to the AREs in the HO-1 gene promoter. In contrast, DHA enhanced Nrf2 binding to the AREs without affecting nuclear Nrf2 expression levels, indicating a new cellular mechanism that mediates DHA's induction of HO-1 gene transcription. To our knowledge, this is the first characterization of DHA-induced HO-1 expression in human malignant cells. Copyright © 2014 Elsevier Inc. All rights reserved.

Mammalian Sterile 20-like Kinase 1 (Mst1) Enhances the Stability of Forkhead Box P3 (Foxp3) and the Function of Regulatory T Cells by Modulating Foxp3 Acetylation.

PubMed

Li, Jiang; Du, Xingrong; Shi, Hao; Deng, Kejing; Chi, Hongbo; Tao, Wufan

2015-12-25

Regulatory T cells (Tregs) play crucial roles in maintaining immune tolerance. The transcription factor Foxp3 is a critical regulator of Treg development and function, and its expression is regulated at both transcriptional and post-translational levels. Acetylation by lysine acetyl transferases/lysine deacetylases is one of the main post-translational modifications of Foxp3, which regulate Foxp3's stability and transcriptional activity. However, the mechanism(s) by which the activities of these lysine acetyl transferases/lysine deacetylases are regulated to preserve proper Foxp3 acetylation during Treg development and maintenance of Treg function remains to be determined. Here we report that Mst1 can enhance Foxp3 stability, its transcriptional activity, and Treg function by modulating the Foxp3 protein at the post-translational level. We discovered that Mst1 could increase the acetylation of Foxp3 by inhibiting Sirt1 activity, which requires the Mst1 kinase activity. We also found that Mst1 could attenuate Sirt1-mediated deacetylation of Foxp3 through directly interacting with Foxp3 to prevent or interfere the interaction between Sirt1 and Foxp3. Therefore, Mst1 can regulate Foxp3 stability in kinase-dependent and kinase-independent manners. Finally, we showed that treatment of Mst1(-/-) Tregs with Ex-527, a Sirt1-specific inhibitor, partially restored the suppressive function of Mst1(-/-) Tregs. Our studies reveal a novel mechanism by which Mst1 enhances Foxp3 expression and Treg function at the post-translational level. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Enhanced stability and polyadenylation of select mRNAs support rapid thermogenesis in the brown fat of a hibernator

PubMed Central

Grabek, Katharine R; Diniz Behn, Cecilia; Barsh, Gregory S; Hesselberth, Jay R; Martin, Sandra L

2015-01-01

During hibernation, animals cycle between torpor and arousal. These cycles involve dramatic but poorly understood mechanisms of dynamic physiological regulation at the level of gene expression. Each cycle, Brown Adipose Tissue (BAT) drives periodic arousal from torpor by generating essential heat. We applied digital transcriptome analysis to precisely timed samples to identify molecular pathways that underlie the intense activity cycles of hibernator BAT. A cohort of transcripts increased during torpor, paradoxical because transcription effectively ceases at these low temperatures. We show that this increase occurs not by elevated transcription but rather by enhanced stabilization associated with maintenance and/or extension of long poly(A) tails. Mathematical modeling further supports a temperature-sensitive mechanism to protect a subset of transcripts from ongoing bulk degradation instead of increased transcription. This subset was enriched in a C-rich motif and genes required for BAT activation, suggesting a model and mechanism to prioritize translation of key proteins for thermogenesis. DOI: http://dx.doi.org/10.7554/eLife.04517.001 PMID:25626169

A unique enhancer boundary complex on the mouse ribosomal RNA genes persists after loss of Rrn3 or UBF and the inactivation of RNA polymerase I transcription

PubMed Central

Stefanovsky, Victor Y.; Tremblay, Michel G.; Lindsay, Helen; Robinson, Mark D.

2017-01-01

Transcription of the several hundred of mouse and human Ribosomal RNA (rRNA) genes accounts for the majority of RNA synthesis in the cell nucleus and is the determinant of cytoplasmic ribosome abundance, a key factor in regulating gene expression. The rRNA genes, referred to globally as the rDNA, are clustered as direct repeats at the Nucleolar Organiser Regions, NORs, of several chromosomes, and in many cells the active repeats are transcribed at near saturation levels. The rDNA is also a hotspot of recombination and chromosome breakage, and hence understanding its control has broad importance. Despite the need for a high level of rDNA transcription, typically only a fraction of the rDNA is transcriptionally active, and some NORs are permanently silenced by CpG methylation. Various chromatin-remodelling complexes have been implicated in counteracting silencing to maintain rDNA activity. However, the chromatin structure of the active rDNA fraction is still far from clear. Here we have combined a high-resolution ChIP-Seq protocol with conditional inactivation of key basal factors to better understand what determines active rDNA chromatin. The data resolve questions concerning the interdependence of the basal transcription factors, show that preinitiation complex formation is driven by the architectural factor UBF (UBTF) independently of transcription, and that RPI termination and release corresponds with the site of TTF1 binding. They further reveal the existence of an asymmetric Enhancer Boundary Complex formed by CTCF and Cohesin and flanked upstream by phased nucleosomes and downstream by an arrested RNA Polymerase I complex. We find that the Enhancer Boundary Complex is the only site of active histone modification in the 45kbp rDNA repeat. Strikingly, it not only delimits each functional rRNA gene, but also is stably maintained after gene inactivation and the re-establishment of surrounding repressive chromatin. Our data define a poised state of rDNA chromatin and place the Enhancer Boundary Complex as the likely entry point for chromatin remodelling complexes. PMID:28715449

A unique enhancer boundary complex on the mouse ribosomal RNA genes persists after loss of Rrn3 or UBF and the inactivation of RNA polymerase I transcription.

PubMed

Herdman, Chelsea; Mars, Jean-Clement; Stefanovsky, Victor Y; Tremblay, Michel G; Sabourin-Felix, Marianne; Lindsay, Helen; Robinson, Mark D; Moss, Tom

2017-07-01

Transcription of the several hundred of mouse and human Ribosomal RNA (rRNA) genes accounts for the majority of RNA synthesis in the cell nucleus and is the determinant of cytoplasmic ribosome abundance, a key factor in regulating gene expression. The rRNA genes, referred to globally as the rDNA, are clustered as direct repeats at the Nucleolar Organiser Regions, NORs, of several chromosomes, and in many cells the active repeats are transcribed at near saturation levels. The rDNA is also a hotspot of recombination and chromosome breakage, and hence understanding its control has broad importance. Despite the need for a high level of rDNA transcription, typically only a fraction of the rDNA is transcriptionally active, and some NORs are permanently silenced by CpG methylation. Various chromatin-remodelling complexes have been implicated in counteracting silencing to maintain rDNA activity. However, the chromatin structure of the active rDNA fraction is still far from clear. Here we have combined a high-resolution ChIP-Seq protocol with conditional inactivation of key basal factors to better understand what determines active rDNA chromatin. The data resolve questions concerning the interdependence of the basal transcription factors, show that preinitiation complex formation is driven by the architectural factor UBF (UBTF) independently of transcription, and that RPI termination and release corresponds with the site of TTF1 binding. They further reveal the existence of an asymmetric Enhancer Boundary Complex formed by CTCF and Cohesin and flanked upstream by phased nucleosomes and downstream by an arrested RNA Polymerase I complex. We find that the Enhancer Boundary Complex is the only site of active histone modification in the 45kbp rDNA repeat. Strikingly, it not only delimits each functional rRNA gene, but also is stably maintained after gene inactivation and the re-establishment of surrounding repressive chromatin. Our data define a poised state of rDNA chromatin and place the Enhancer Boundary Complex as the likely entry point for chromatin remodelling complexes.

An Enhancer Composed of Interlocking Sub-modules Controls Transcriptional Autoregulation of Suppressor of Hairless

PubMed Central

Liu, Feng; Posakony, James W.

2014-01-01

SUMMARY Positive autoregulation is an effective mechanism for the long-term maintenance of a transcription factor’s expression. This strategy is widely deployed in cell lineages, where the autoregulatory factor controls the activity of a battery of genes that constitute the differentiation program of a post-mitotic cell type. In Drosophila, the Notch pathway transcription factor Suppressor of Hairless activates its own expression specifically in the socket cell of external sensory organs, via an autoregulatory enhancer called the ASE. Here we show that the ASE is composed of several enhancer sub-modules, each of which can independently initiate weak Su(H) autoregulation. Cross-activation by these sub-modules is critical to ensuring that Su(H) rises above a threshold level necessary to activate a maintenance sub-module, which then sustains long-term Su(H) autoregulation. Our study reveals the use of interlinked positive feedback loops to control autoregulation dynamically, and provides mechanistic insight into initiation, establishment, and maintenance of the autoregulatory state. PMID:24735880

Survivin expression promotes VEGF-induced tumor angiogenesis via PI3K/Akt enhanced β-catenin/Tcf-Lef dependent transcription.

PubMed

Fernández, Jaime G; Rodríguez, Diego A; Valenzuela, Manuel; Calderon, Claudia; Urzúa, Ulises; Munroe, David; Rosas, Carlos; Lemus, David; Díaz, Natalia; Wright, Mathew C; Leyton, Lisette; Tapia, Julio C; Quest, Andrew Fg

2014-09-09

Early in cancer development, tumour cells express vascular endothelial growth factor (VEGF), a secreted molecule that is important in all stages of angiogenesis, an essential process that provides nutrients and oxygen to the nascent tumor and thereby enhances tumor-cell survival and facilitates growth. Survivin, another protein involved in angiogenesis, is strongly expressed in most human cancers, where it promotes tumor survival by reducing apoptosis as well as favoring endothelial cell proliferation and migration. The mechanisms by which cancer cells induce VEGF expression and angiogenesis upon survivin up-regulation remain to be fully established. Since the PI3K/Akt signalling and β-catenin-Tcf/Lef dependent transcription have been implicated in the expression of many cancer-related genes, including survivin and VEGF, we evaluated whether survivin may favor VEGF expression, release from tumor cells and induction of angiogenesis in a PI3K/Akt-β-catenin-Tcf/Lef-dependent manner. Here, we provide evidence linking survivin expression in tumor cells to increased β-catenin protein levels, β-catenin-Tcf/Lef transcriptional activity and expression of several target genes of this pathway, including survivin and VEGF, which accumulates in the culture medium. Alternatively, survivin downregulation reduced β-catenin protein levels and β-catenin-Tcf/Lef transcriptional activity. Also, using inhibitors of PI3K and the expression of dominant negative Akt, we show that survivin acts upstream in an amplification loop to promote VEGF expression. Moreover, survivin knock-down in B16F10 murine melanoma cells diminished the number of blood vessels and reduced VEGF expression in tumors formed in C57BL/6 mice. Finally, in the chick chorioallantoid membrane assay, survivin expression in tumor cells enhanced VEGF liberation and blood vessel formation. Importantly, the presence of neutralizing anti-VEGF antibodies precluded survivin-enhanced angiogenesis in this assay. These findings provide evidence for the existance of a posititve feedback loop connecting survivin expression in tumor cells to PI3K/Akt enhanced β-catenin-Tcf/Lef-dependent transcription followed by secretion of VEGF and angiogenesis.

Stronger activation of SREBP-1a by nucleus-localized HBx

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

Wu, Qi; Qiao, Ling; Yang, Jian

2015-05-08

We previously showed that hepatitis B virus (HBV) X protein activates the sterol regulatory element-binding protein-1a (SREBP-1a). Here we examined the role of nuclear localization of HBx in this process. In comparison to the wild-type and cytoplasmic HBx, nuclear HBx had stronger effects on SREBP-1a and fatty acid synthase transcription activation, intracellular lipid accumulation and cell proliferation. Furthermore, nuclear HBx could activate HBV enhancer I/X promoter and was more effective on up-regulating HBV mRNA level in the context of HBV replication than the wild-type HBx, while the cytoplasmic HBx had no effect. Our results demonstrate the functional significance of themore » nucleus-localized HBx in regulating host lipogenic pathway and HBV replication. - Highlights: • Nuclear HBx is more effective on activating SREBP-1a and FASN transcription. • Nuclear HBx is more effective on enhancing intracellular lipid accumulation. • Nuclear HBx is more effective on enhancing cell proliferation. • Nuclear HBx up-regulates HBV enhancer I/X promoter activity. • Nuclear HBx increases HBV mRNA level in the context of HBV replication.« less

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.

Enhancing the laccase production and laccase gene expression in the white-rot fungus Trametes velutina 5930 with great potential for biotechnological applications by different metal ions and aromatic compounds.

PubMed

Yang, Yang; Wei, Fuxiang; Zhuo, Rui; Fan, Fangfang; Liu, Huahua; Zhang, Chen; Ma, Li; Jiang, Mulan; Zhang, Xiaoyu

2013-01-01

Laccase is useful for various biotechnological and industrial applications. The white-rot fungus Trametes velutina 5930 and its laccase, isolated from the Shennongjia Nature Reserve in China by our laboratory, has great potential for practical application in environmental biotechnology. However, the original level of laccase produced by Trametes velutina 5930 was relatively low in the absence of any inducer. Therefore, in order to enhance the laccase production by Trametes velutina 5930 and make better use of this fungus in the field of environmental biotechnology, the regulation of laccase production and laccase gene expression in Trametes velutina 5930 were investigated in this study. Different metal ions such as Cu(2+) and Fe(2+) could stimulate the laccase synthesis and laccase gene transcription in Trametes velutina 5930. Some aromatic compounds structurally related to lignin, such as tannic acid, syringic acid, cinnamic acid, gallic acid and guaiacol, could also enhance the level of laccase activity and laccase gene transcription. We also found that there existed a positive synergistic effect of aromatic compound and metal ion on the laccase production and laccase gene transcription in Trametes velutina 5930. Taken together, our study may contribute to the improvement of laccase productivity by Trametes velutina 5930.

Drug-activated multiple pathways of defensin mRNA regulation in HL-60 cells are defined by reversed roles of participating protein kinases.

PubMed

Herwig, S; Su, Q; Tempst, P

1998-10-01

Defensin transcription in HL-60 promyelocytic leukemia cells is greatly enhanced during retinoic acid (RA)-induced differentiation. We have probed this regulatory pathway by selective modulation of various kinase activities. Induction was potentiated by elevated cAMP and attenuated by protein kinase C inhibition, entirely correlated to enhanced or blocked morphological differentiation, respectively. Yet, defensin mRNA was also induced in undifferentiated HL-60 cells, but not in others, by cAMP alone. By contrast, modulators that cooperated with RA had adverse effects on the normal capacity of dimethyl sulfoxide to up regulate these transcripts as well. Thus, defensin mRNA accumulation can be selectively uncoupled from maturation stage; and transcript levels may be regulated by multiple pathways, each independently acted upon by different chemical inducers.

The MNS glycophorin variant GP.Mur affects differential erythroid expression of Rh/RhAG transcripts.

PubMed

Hsu, K; Kuo, M-S; Yao, C-C; Cheng, H-C; Lin, H-J; Chan, Y-S; Lin, M

2017-10-01

The band 3 macrocomplex (also known as the ankyrin-associated complex) on the red cell membrane comprises two interacting subcomplexes: a band 3/glycophorin A subcomplex, and a Rh/RhAG subcomplex. Glycophorin B (GPB) is a component of the Rh/RhAG subcomplex that is also structurally associated with glycophorin A (GPA). Expression of glycophorin B-A-B hybrid GP.Mur enhances band 3 expression and is associated with lower levels of Rh-associated glycoprotein (RhAG) and Rh polypeptides. The goal of this study was to determine whether GP.Mur influenced erythroid Rh/RhAG expression at the transcript level. GP.Mur was serologically determined in healthy participants from Taitung County, Taiwan. RNA was extracted from the reticulocyte-enriched fraction of peripheral blood, followed by reverse transcription and quantitative PCR for RhAG, RhD and RhCcEe. Quantification by real-time PCR revealed significantly fewer RhAG and RhCcEe transcripts in the reticulocytes from subjects with homozygous GYP*Mur. Independent from GYP.Mur, both RhAG and RhD transcript levels were threefold or higher than that of RhCcEe. Also, in GYP.Mur and the control samples alike, direct quantitative associations were observed between the transcript levels of RhAG and RhD, but not between that of RhAG and RhCcEe. Erythroid RhD and RhCcEe were differentially expressed at the transcript levels, which could be related to their different degrees of interaction or sensitivity to RhAG. Further, the reduction or absence of glycophorin B in GYP.Mur erythroid cells affected transcript expressions of RhAG and RhCcEe. Thus, GPB and GP.Mur differentially influenced Rh/RhAG expressions prior to protein translation. © 2017 International Society of Blood Transfusion.

Transcriptional regulation of the novel monoamine oxidase renalase: Crucial roles of transcription factors Sp1, STAT3, and ZBP89.

PubMed

Sonawane, Parshuram J; Gupta, Vinayak; Sasi, Binu K; Kalyani, Ananthamohan; Natarajan, Bhargavi; Khan, Abrar A; Sahu, Bhavani S; Mahapatra, Nitish R

2014-11-11

Renalase, a novel monoamine oxidase, is emerging as an important regulator of cardiovascular, metabolic, and renal diseases. However, the mechanism of transcriptional regulation of this enzyme remains largely unknown. We undertook a systematic analysis of the renalase gene to identify regulatory promoter elements and transcription factors. Computational analysis coupled with transfection of human renalase promoter/luciferase reporter plasmids (5'-promoter-deletion constructs) into various cell types (HEK-293, IMR32, and HepG2) identified two crucial promoter domains at base pairs -485 to -399 and -252 to -150. Electrophoretic mobility shift assays using renalase promoter oligonucleotides with and without potential binding sites for transcription factors Sp1, STAT3, and ZBP89 displayed formation of specific complexes with HEK-293 nuclear proteins. Consistently, overexpression of Sp1, STAT3, and ZBP89 augmented renalase promoter activity; additionally, siRNA-mediated downregulation of Sp1, STAT3, and ZBP89 reduced the level of endogenous renalase transcription as well as the transfected renalase promoter activity. In addition, chromatin immunoprecipitation assays showed in vivo interactions of these transcription factors with renalase promoter. Interestingly, renalase promoter activity was augmented by nicotine and catecholamines; while Sp1 and STAT3 synergistically activated the nicotine-induced effect, Sp1 appeared to enhance epinephrine-evoked renalase transcription. Moreover, renalase transcript levels in mouse models of human essential hypertension were concomitantly associated with endogenous STAT3 and ZBP89 levels, suggesting crucial roles for these transcription factors in regulating renalase gene expression in cardiovascular pathological conditions.

Understanding Transcriptional Enhancement in Monoclonal Antibody-Producing Chinese Hamster Ovary Cells

NASA Astrophysics Data System (ADS)

Nicoletti, Sarah E.

With the demand for monoclonal antibody (mAB) therapeutics continually increasing, the need to better understand what makes a high productivity clone has gained substantial interest. Monoclonal antibody producing Chinese hamster ovary (CHO) cells with different productivities were provided by a biopharmaceutical company for investigation. Gene copy numbers, mRNA levels, and mAb productivities were previously determined for two low producing clones and their amplified progeny. These results showed an increase in mRNA copy number in amplified clones, which correlated to the observed increases in specific productivity of these clones. The presence of multiple copies of mRNA per one copy of DNA in the higher productivity clones has been coined as transcriptional enhancement. The methylation status of the CMV promoter as well as transcription factor/promoter interactions were evaluated to determine the cause of transcriptional enhancement. Methylation analysis via bisulfite sequencing revealed no significant difference in overall methylation status of the CMV promoter. These data did, however, reveal the possibility of differential interactions of transcription factors between the high and low productivity cell clones. This finding was further supported by chromatin immunoprecipitations previously performed in the lab, as well as literature studies. Transcription activator-like effector (TALE) binding proteins were constructed and utilized to selectively immunoprecipitate the CMV promoter along with its associated transcription factors in the different CHO cell clones. Cells were transfected with the TALE proteins, harvested and subjected to a ChIP-like procedure. Results obtained from the TALE ChIP demonstrated the lack of binding of the protein to the promoter and the need to redesign the TALE. Overall, results obtained from this study were unable to give a clear indication as to the causes of transcriptional enhancement in the amplified CHO cell clones. Further investigations into both epigenetic modifications and transcription factor interactions will help gain a better understanding of what characteristics make a higher producing CHO cell line. Discovery of these characteristics will aid in better genetic engineering strategies for future recombinant cell lines, improving productivity and shortening the clonal selection process.

A class of circadian long non-coding RNAs mark enhancers modulating long-range circadian gene regulation

PubMed Central

Fan, Zenghua; Zhao, Meng; Joshi, Parth D.; Li, Ping; Zhang, Yan; Guo, Weimin; Xu, Yichi; Wang, Haifang; Zhao, Zhihu

2017-01-01

Abstract Circadian rhythm exerts its influence on animal physiology and behavior by regulating gene expression at various levels. Here we systematically explored circadian long non-coding RNAs (lncRNAs) in mouse liver and examined their circadian regulation. We found that a significant proportion of circadian lncRNAs are expressed at enhancer regions, mostly bound by two key circadian transcription factors, BMAL1 and REV-ERBα. These circadian lncRNAs showed similar circadian phases with their nearby genes. The extent of their nuclear localization is higher than protein coding genes but less than enhancer RNAs. The association between enhancer and circadian lncRNAs is also observed in tissues other than liver. Comparative analysis between mouse and rat circadian liver transcriptomes showed that circadian transcription at lncRNA loci tends to be conserved despite of low sequence conservation of lncRNAs. One such circadian lncRNA termed lnc-Crot led us to identify a super-enhancer region interacting with a cluster of genes involved in circadian regulation of metabolism through long-range interactions. Further experiments showed that lnc-Crot locus has enhancer function independent of lnc-Crot's transcription. Our results suggest that the enhancer-associated circadian lncRNAs mark the genomic loci modulating long-range circadian gene regulation and shed new lights on the evolutionary origin of lncRNAs. PMID:28335007

Pim kinases are upregulated during Epstein-Barr virus infection and enhance EBNA2 activity

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

Rainio, Eeva-Marja; Turku Graduate School of Biomedical Sciences, 20520 Turku; Ahlfors, Helena

Latent Epstein-Barr virus (EBV) infection is strongly associated with B-cell proliferative diseases such as Burkitt's lymphoma. Here we show that the oncogenic serine/threonine kinases Pim-1 and Pim-2 enhance the activity of the viral transcriptional activator EBNA2. During EBV infection of primary B-lymphocytes, the mRNA expression levels of pim genes, especially of pim-2, are upregulated and remain elevated in latently infected B-cell lines. Thus, EBV-induced upregulation of Pim kinases and Pim-stimulated EBNA2 transcriptional activity may contribute to the ability of EBV to immortalize B-cells and predispose them to malignant growth.

Dom34 Links Translation to Protein O-mannosylation

PubMed Central

van Wijlick, Lasse; Geissen, René; Hilbig, Jessica S.; Lagadec, Quentin; Cantero, Pilar D.; Juchimiuk, Mateusz; Kluge, Sven; Wickert, Stephan; Alepuz, Paula; Ernst, Joachim F.

2016-01-01

In eukaryotes, Dom34 upregulates translation by securing levels of activatable ribosomal subunits. We found that in the yeast Saccharomyces cerevisiae and the human fungal pathogen Candida albicans, Dom34 interacts genetically with Pmt1, a major isoform of protein O-mannosyltransferase. In C. albicans, lack of Dom34 exacerbated defective phenotypes of pmt1 mutants, while they were ameliorated by Dom34 overproduction that enhanced Pmt1 protein but not PMT1 transcript levels. Translational effects of Dom34 required the 5′-UTR of the PMT1 transcript, which bound recombinant Dom34 directly at a CA/AC-rich sequence and regulated in vitro translation. Polysomal profiling revealed that Dom34 stimulates general translation moderately, but that it is especially required for translation of transcripts encoding Pmt isoforms 1, 4 and 6. Because defective protein N- or O-glycosylation upregulates transcription of PMT genes, it appears that Dom34-mediated specific translational upregulation of the PMT transcripts optimizes cellular responses to glycostress. Its translational function as an RNA binding protein acting at the 5′-UTR of specific transcripts adds another facet to the known ribosome-releasing functions of Dom34 at the 3′-UTR of transcripts. PMID:27768707

Dom34 Links Translation to Protein O-mannosylation.

PubMed

van Wijlick, Lasse; Geissen, René; Hilbig, Jessica S; Lagadec, Quentin; Cantero, Pilar D; Pfeifer, Eugen; Juchimiuk, Mateusz; Kluge, Sven; Wickert, Stephan; Alepuz, Paula; Ernst, Joachim F

2016-10-01

In eukaryotes, Dom34 upregulates translation by securing levels of activatable ribosomal subunits. We found that in the yeast Saccharomyces cerevisiae and the human fungal pathogen Candida albicans, Dom34 interacts genetically with Pmt1, a major isoform of protein O-mannosyltransferase. In C. albicans, lack of Dom34 exacerbated defective phenotypes of pmt1 mutants, while they were ameliorated by Dom34 overproduction that enhanced Pmt1 protein but not PMT1 transcript levels. Translational effects of Dom34 required the 5'-UTR of the PMT1 transcript, which bound recombinant Dom34 directly at a CA/AC-rich sequence and regulated in vitro translation. Polysomal profiling revealed that Dom34 stimulates general translation moderately, but that it is especially required for translation of transcripts encoding Pmt isoforms 1, 4 and 6. Because defective protein N- or O-glycosylation upregulates transcription of PMT genes, it appears that Dom34-mediated specific translational upregulation of the PMT transcripts optimizes cellular responses to glycostress. Its translational function as an RNA binding protein acting at the 5'-UTR of specific transcripts adds another facet to the known ribosome-releasing functions of Dom34 at the 3'-UTR of transcripts.

CD86 and beta2-adrenergic receptor signaling pathways, respectively, increase Oct-2 and OCA-B Expression and binding to the 3'-IgH enhancer in B cells.

PubMed

Podojil, Joseph R; Kin, Nicholas W; Sanders, Virginia M

2004-05-28

Stimulation of CD86 (formerly known as B7-2) and/or the beta2-adrenergic receptor on a CD40 ligand/interleukin-4-activated B cell increased the rate of mature IgG1 transcription. To identify the mechanism responsible for this effect, we determined whether CD86 and/or beta2-adrenergic receptor stimulation regulated transcription factor expression and binding to the 3'-IgH enhancer in vitro and in vivo. We showed that CD86 stimulation increased the nuclear localization of NF-kappaB1 (p50) and phosphorylated RelA (p65) and increased Oct-2 expression and binding to the 3'-IgH enhancer, in a protein kinase C-dependent manner. These effects were lost when CD86-deficient or NF-kappaB1-deficient B cells were used. CD86 stimulation also increased the level of IkappaB-alpha phosphorylation but in a protein kinase C-independent manner. Beta2-adrenergic receptor stimulation increased CREB phosphorylation, OCA-B expression, and OCA-B binding to the 3'-IgH enhancer in a protein kinase A-dependent manner, an effect lost when beta2-adrenergic receptor-deficient B cells were used. Also, the beta2-adrenergic receptor-induced increase in the level of mature IgG1 transcript was lost when OCA-B-deficient B cells were used. These data are the first to show that CD86 stimulation up-regulates the expression of the transcription factor Oct-2 in a protein kinase C- and NF-kappaB1-dependent manner, and that beta2-adrenergic receptor stimulation up-regulates the expression of the coactivator OCA-B in a protein kinase A-dependent manner to cooperate with Oct-2 binding to the 3'-IgH enhancer.

Nitric Oxide Exerts Basal and Insulin-Dependent Anorexigenic Actions in POMC Hypothalamic Neurons

PubMed Central

Wellhauser, Leigh; Chalmers, Jennifer A.

2016-01-01

The arcuate nucleus of the hypothalamus represents a key center for the control of appetite and feeding through the regulation of 2 key neuronal populations, notably agouti-related peptide/neuropeptide Y and proopimelanocortin (POMC)/cocaine- and amphetamine-regulated transcript neurons. Altered regulation of these neuronal networks, in particular the dysfunction of POMC neurons upon high-fat consumption, is a major pathogenic mechanism involved in the development of obesity and type 2 diabetes mellitus. Efforts are underway to preserve the integrity or enhance the functionality of POMC neurons in order to prevent or treat these metabolic diseases. Here, we report for the first time that the nitric oxide (NO−) donor, sodium nitroprusside (SNP) mediates anorexigenic actions in both hypothalamic tissue and hypothalamic-derived cell models by mediating the up-regulation of POMC levels. SNP increased POMC mRNA in a dose-dependent manner and enhanced α-melanocortin-secreting hormone production and secretion in mHypoA-POMC/GFP-2 cells. SNP also enhanced insulin-driven POMC expression likely by inhibiting the deacetylase activity of sirtuin 1. Furthermore, SNP enhanced insulin-dependent POMC expression, likely by reducing the transcriptional repression of Foxo1 on the POMC gene. Prolonged SNP exposure prevented the development of insulin resistance. Taken together, the NO− donor SNP enhances the anorexigenic potential of POMC neurons by promoting its transcriptional expression independent and in cooperation with insulin. Thus, increasing cellular NO− levels represents a hormone-independent method of promoting anorexigenic output from the existing POMC neuronal populations and may be advantageous in the fight against these prevalent disorders. PMID:26930171

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

DNA breaks and chromatin structural changes enhance the transcription of autoimmune regulator target genes.

PubMed

Guha, Mithu; Saare, Mario; Maslovskaja, Julia; Kisand, Kai; Liiv, Ingrid; Haljasorg, Uku; Tasa, Tõnis; Metspalu, Andres; Milani, Lili; Peterson, Pärt

2017-04-21

The autoimmune regulator (AIRE) protein is the key factor in thymic negative selection of autoreactive T cells by promoting the ectopic expression of tissue-specific genes in the thymic medullary epithelium. Mutations in AIRE cause a monogenic autoimmune disease called autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy. AIRE has been shown to promote DNA breaks via its interaction with topoisomerase 2 (TOP2). In this study, we investigated topoisomerase-induced DNA breaks and chromatin structural alterations in conjunction with AIRE-dependent gene expression. Using RNA sequencing, we found that inhibition of TOP2 religation activity by etoposide in AIRE-expressing cells had a synergistic effect on genes with low expression levels. AIRE-mediated transcription was not only enhanced by TOP2 inhibition but also by the TOP1 inhibitor camptothecin. The transcriptional activation was associated with structural rearrangements in chromatin, notably the accumulation of γH2AX and the exchange of histone H1 with HMGB1 at AIRE target gene promoters. In addition, we found the transcriptional up-regulation to co-occur with the chromatin structural changes within the genomic cluster of carcinoembryonic antigen-like cellular adhesion molecule genes. Overall, our results suggest that the presence of AIRE can trigger molecular events leading to an altered chromatin landscape and the enhanced transcription of low-expressed genes. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

DNA breaks and chromatin structural changes enhance the transcription of autoimmune regulator target genes

PubMed Central

Guha, Mithu; Saare, Mario; Maslovskaja, Julia; Kisand, Kai; Liiv, Ingrid; Haljasorg, Uku; Tasa, Tõnis; Metspalu, Andres; Milani, Lili; Peterson, Pärt

2017-01-01

The autoimmune regulator (AIRE) protein is the key factor in thymic negative selection of autoreactive T cells by promoting the ectopic expression of tissue-specific genes in the thymic medullary epithelium. Mutations in AIRE cause a monogenic autoimmune disease called autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy. AIRE has been shown to promote DNA breaks via its interaction with topoisomerase 2 (TOP2). In this study, we investigated topoisomerase-induced DNA breaks and chromatin structural alterations in conjunction with AIRE-dependent gene expression. Using RNA sequencing, we found that inhibition of TOP2 religation activity by etoposide in AIRE-expressing cells had a synergistic effect on genes with low expression levels. AIRE-mediated transcription was not only enhanced by TOP2 inhibition but also by the TOP1 inhibitor camptothecin. The transcriptional activation was associated with structural rearrangements in chromatin, notably the accumulation of γH2AX and the exchange of histone H1 with HMGB1 at AIRE target gene promoters. In addition, we found the transcriptional up-regulation to co-occur with the chromatin structural changes within the genomic cluster of carcinoembryonic antigen-like cellular adhesion molecule genes. Overall, our results suggest that the presence of AIRE can trigger molecular events leading to an altered chromatin landscape and the enhanced transcription of low-expressed genes. PMID:28242760

Hes1 promotes the IL-22-mediated antimicrobial response by enhancing STAT3-dependent transcription in human intestinal epithelial cells

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

Murano, Tatsuro; Okamoto, Ryuichi, E-mail: rokamoto.gast@tmd.ac.jp; Department of Advanced GI Therapeutics, Graduate School, Tokyo Medical and Dental University, Tokyo

Highlights: •Hes1 enhances IL-22-STAT3 signaling in human intestinal epithelial cells. •Hes1 enhances REG family gene induction by IL-22-STAT3 signaling. •Protein level of Hes1 restricts the response to IL-22. •Present regulation of a cytokine signal represents a new mode of Hes1 function. -- Abstract: Notch signaling plays an essential role in the proliferation and differentiation of intestinal epithelial cells (IECs). We have previously shown that Notch signaling is up-regulated in the inflamed mucosa of ulcerative colitis (UC) and thereby plays an indispensable role in tissue regeneration. Here we show that in addition to Notch signaling, STAT3 signaling is highly activated inmore » the inflamed mucosa of UC. Forced expression of the Notch target gene Hes1 dramatically enhanced the IL-22-mediated STAT3-dependent transcription in human IECs. This enhancement of STAT3-dependent transcription was achieved by the extended phosphorylation of STAT3 by Hes1. Microarray analysis revealed that Hes1-mediated enhancement of IL-22-STAT3 signaling significantly increased the induction of genes encoding antimicrobial peptides, such as REG1A, REG3A and REG3G, in human IECs. Conversely, the reduction of Hes1 protein levels with a γ-secretase inhibitor significantly down-regulated the induction of those genes in IECs, resulting in a markedly poor response to IL-22. Our present findings identify a new role for the molecular function of Hes1 in which the protein can interact with cytokine signals and regulate the immune response of IECs.« less

Stk1-mediated phosphorylation stimulates the DNA-binding properties of the Staphylococcus aureus SpoVG transcriptional factor.

PubMed

Bischoff, Markus; Brelle, Solène; Minatelli, Sabrina; Molle, Virginie

2016-05-13

The stage V sporulation protein G (SpoVG) homolog of Staphylococcus aureus is a modulator of virulence factor synthesis and antibiotic resistance in this clinically important gram-positive pathogen. Here we demonstrate that SpoVG can be phosphorylated by the staphylococcal Ser/Thr protein kinase Stk1 and that phosphorylation positively affects its DNA-binding properties. Mass spectrometric analyses and site directed mutagenesis identified Thr4, Thr13, Thr24 and Ser41 as phospho-acceptors. Stk1-mediated phosphorylation markedly enhanced the DNA binding activity of SpoVG towards the promoter regions of target genes such as capA, lip, and nuc1. Similarly, trans-complementation of the S. aureus ΔyabJ-spoVG mutant SM148 with a SpoVG derivative that mimics constitutive phosphorylation, SpoVG_Asp, exhibited capA, lip, and nuc1 transcript levels that were comparable to the levels seen with the wild-type, whereas trans-complementation with a phosphoablative variant of SpoVG (SpoVG_Ala) produced transcript levels similar to the ones seen in SM148. Our data suggest that the expression/activity of this transcription factor is tightly controlled in S. aureus by transcriptional, post-transcriptional and post-translational mechanisms. Copyright © 2016 Elsevier Inc. All rights reserved.

Up-Regulation of Bcl-xl by Hepatocyte Growth Factor in Human Mesothelioma Cells Involves ETS Transcription Factors

PubMed Central

Cao, Xiaobo; Littlejohn, James; Rodarte, Charles; Zhang, Lidong; Martino, Benjamin; Rascoe, Philip; Hamid, Kamran; Jupiter, Daniel; Smythe, W. Roy

2009-01-01

Bcl-xl and the hepatocyte growth factor (HGF) receptor c-Met are both highly expressed in mesotheliomas, where they protect cells from apoptosis and can confer resistance to conventional therapeutic agents. In our current study, we investigate a model for the transcriptional control of Bcl-xl that involves ETS transcription factors and the HGF/Met axis. In addition, the effects of activated c-Met on the phosphorylation of the ETS family transcriptional factors were examined. The transient expression of ETS-2 and PU.1 cDNAs in mesothelioma cell lines resulted in an increase in the promoter activity of Bcl-xl and consequently in its mRNA and protein expression levels, whereas the transcriptional repressor Tel suppressed Bcl-xl transcription. The activation of the HGF/Met axis led to rapid phosphorylation of ETS family transcription factors in mesothelioma cells through the mitogen-activated protein kinase pathway and via nuclear accumulation of ETS-2 and PU.1. A chromatin immunoprecipitation assay further demonstrated that the activation of c-Met enhanced the binding of ETS transcriptional factors to the Bcl-x promoter. Finally, we determined the Bcl-xl and phosphorylated c-Met expression levels in mesothelioma patient samples; these data suggest a strong correlation between Bcl-xl and phosphorylated c-Met levels. Taken together, these findings support a role for c-Met as an inhibitor of apoptosis and an activator of Bcl-xl. PMID:19834061

Functional Variants at the 11q13 Risk Locus for Breast Cancer Regulate Cyclin D1 Expression through Long-Range Enhancers

PubMed Central

French, Juliet D.; Ghoussaini, Maya; Edwards, Stacey L.; Meyer, Kerstin B.; Michailidou, Kyriaki; Ahmed, Shahana; Khan, Sofia; Maranian, Mel J.; O’Reilly, Martin; Hillman, Kristine M.; Betts, Joshua A.; Carroll, Thomas; Bailey, Peter J.; Dicks, Ed; Beesley, Jonathan; Tyrer, Jonathan; Maia, Ana-Teresa; Beck, Andrew; Knoblauch, Nicholas W.; Chen, Constance; Kraft, Peter; Barnes, Daniel; González-Neira, Anna; Alonso, M. Rosario; Herrero, Daniel; Tessier, Daniel C.; Vincent, Daniel; Bacot, Francois; Luccarini, Craig; Baynes, Caroline; Conroy, Don; Dennis, Joe; Bolla, Manjeet K.; Wang, Qin; Hopper, John L.; Southey, Melissa C.; Schmidt, Marjanka K.; Broeks, Annegien; Verhoef, Senno; Cornelissen, Sten; Muir, Kenneth; Lophatananon, Artitaya; Stewart-Brown, Sarah; Siriwanarangsan, Pornthep; Fasching, Peter A.; Loehberg, Christian R.; Ekici, Arif B.; Beckmann, Matthias W.; Peto, Julian; dos Santos Silva, Isabel; Johnson, Nichola; Aitken, Zoe; Sawyer, Elinor J.; Tomlinson, Ian; Kerin, Michael J.; Miller, Nicola; Marme, Frederik; Schneeweiss, Andreas; Sohn, Christof; Burwinkel, Barbara; Guénel, Pascal; Truong, Thérèse; Laurent-Puig, Pierre; Menegaux, Florence; Bojesen, Stig E.; Nordestgaard, Børge G.; Nielsen, Sune F.; Flyger, Henrik; Milne, Roger L.; Zamora, M. Pilar; Arias Perez, Jose Ignacio; Benitez, Javier; Anton-Culver, Hoda; Brenner, Hermann; Müller, Heiko; Arndt, Volker; Stegmaier, Christa; Meindl, Alfons; Lichtner, Peter; Schmutzler, Rita K.; Engel, Christoph; Brauch, Hiltrud; Hamann, Ute; Justenhoven, Christina; Aaltonen, Kirsimari; Heikkilä, Päivi; Aittomäki, Kristiina; Blomqvist, Carl; Matsuo, Keitaro; Ito, Hidemi; Iwata, Hiroji; Sueta, Aiko; Bogdanova, Natalia V.; Antonenkova, Natalia N.; Dörk, Thilo; Lindblom, Annika; Margolin, Sara; Mannermaa, Arto; Kataja, Vesa; Kosma, Veli-Matti; Hartikainen, Jaana M.; Wu, Anna H.; Tseng, Chiu-chen; Van Den Berg, David; Stram, Daniel O.; Lambrechts, Diether; Peeters, Stephanie; Smeets, Ann; Floris, Giuseppe; Chang-Claude, Jenny; Rudolph, Anja; Nickels, Stefan; Flesch-Janys, Dieter; Radice, Paolo; Peterlongo, Paolo; Bonanni, Bernardo; Sardella, Domenico; Couch, Fergus J.; Wang, Xianshu; Pankratz, Vernon S.; Lee, Adam; Giles, Graham G.; Severi, Gianluca; Baglietto, Laura; Haiman, Christopher A.; Henderson, Brian E.; Schumacher, Fredrick; Le Marchand, Loic; Simard, Jacques; Goldberg, Mark S.; Labrèche, France; Dumont, Martine; Teo, Soo Hwang; Yip, Cheng Har; Ng, Char-Hong; Vithana, Eranga Nishanthie; Kristensen, Vessela; Zheng, Wei; Deming-Halverson, Sandra; Shrubsole, Martha; Long, Jirong; Winqvist, Robert; Pylkäs, Katri; Jukkola-Vuorinen, Arja; Grip, Mervi; Andrulis, Irene L.; Knight, Julia A.; Glendon, Gord; Mulligan, Anna Marie; Devilee, Peter; Seynaeve, Caroline; García-Closas, Montserrat; Figueroa, Jonine; Chanock, Stephen J.; Lissowska, Jolanta; Czene, Kamila; Klevebring, Daniel; Schoof, Nils; Hooning, Maartje J.; Martens, John W.M.; Collée, J. Margriet; Tilanus-Linthorst, Madeleine; Hall, Per; Li, Jingmei; Liu, Jianjun; Humphreys, Keith; Shu, Xiao-Ou; Lu, Wei; Gao, Yu-Tang; Cai, Hui; Cox, Angela; Balasubramanian, Sabapathy P.; Blot, William; Signorello, Lisa B.; Cai, Qiuyin; Pharoah, Paul D.P.; Healey, Catherine S.; Shah, Mitul; Pooley, Karen A.; Kang, Daehee; Yoo, Keun-Young; Noh, Dong-Young; Hartman, Mikael; Miao, Hui; Sng, Jen-Hwei; Sim, Xueling; Jakubowska, Anna; Lubinski, Jan; Jaworska-Bieniek, Katarzyna; Durda, Katarzyna; Sangrajrang, Suleeporn; Gaborieau, Valerie; McKay, James; Toland, Amanda E.; Ambrosone, Christine B.; Yannoukakos, Drakoulis; Godwin, Andrew K.; Shen, Chen-Yang; Hsiung, Chia-Ni; Wu, Pei-Ei; Chen, Shou-Tung; Swerdlow, Anthony; Ashworth, Alan; Orr, Nick; Schoemaker, Minouk J.; Ponder, Bruce A.J.; Nevanlinna, Heli; Brown, Melissa A.; Chenevix-Trench, Georgia; Easton, Douglas F.; Dunning, Alison M.

2013-01-01

Analysis of 4,405 variants in 89,050 European subjects from 41 case-control studies identified three independent association signals for estrogen-receptor-positive tumors at 11q13. The strongest signal maps to a transcriptional enhancer element in which the G allele of the best candidate causative variant rs554219 increases risk of breast cancer, reduces both binding of ELK4 transcription factor and luciferase activity in reporter assays, and may be associated with low cyclin D1 protein levels in tumors. Another candidate variant, rs78540526, lies in the same enhancer element. Risk association signal 2, rs75915166, creates a GATA3 binding site within a silencer element. Chromatin conformation studies demonstrate that these enhancer and silencer elements interact with each other and with their likely target gene, CCND1. PMID:23540573

Regulation of expression of transgenes in developing fish.

PubMed

Moav, B; Liu, Z; Caldovic, L D; Gross, M L; Faras, A J; Hackett, P B

1993-05-01

The transcriptional regulatory elements of the beta-actin gene of carp (Cyprinus carpio) have been examined in zebrafish and goldfish harbouring transgenes. The high sequence conservation of the putative regulatory elements in the beta-actin genes of animals suggested that their function would be conserved, so that transgenic constructs with the same transcriptional control elements would promote similar levels of transgene expression in different species of transgenic animals. To test this assumption, we analysed the temporal expression of a reporter gene under the control of transcriptional control sequences from the carp beta-actin gene in zebrafish (Brachydanio rerio) and goldfish (Carrasius auratus). Our results indicated that, contrary to expectations, combinations of different transcriptional control elements affected the level, duration, and onset of gene expression differently in developing zebrafish and goldfish. The major differences in expression of beta-actin/CAT (chloramphenicol acetyltransferase) constructs in zebrafish and goldfish were: (1) overall expression was almost 100-fold higher in goldfish than in zebrafish embryos, (2) the first intron had an enhancing effect on gene expression in zebrafish but not in goldfish, and (3) the serum-responsive/CArG-containing regulatory element in the proximal promoter was not always required for maximal CAT activity in goldfish, but was required in zebrafish. These results suggest that in the zebrafish, but not in the goldfish, there may be interactions between motifs in the proximal promoter and the first intron which appear to be required for maximal enhancement of transcription.

General statistics of stochastic process of gene expression in eukaryotic cells.

PubMed Central

Kuznetsov, V A; Knott, G D; Bonner, R F

2002-01-01

Thousands of genes are expressed at such very low levels (< or =1 copy per cell) that global gene expression analysis of rarer transcripts remains problematic. Ambiguity in identification of rarer transcripts creates considerable uncertainty in fundamental questions such as the total number of genes expressed in an organism and the biological significance of rarer transcripts. Knowing the distribution of the true number of genes expressed at each level and the corresponding gene expression level probability function (GELPF) could help resolve these uncertainties. We found that all observed large-scale gene expression data sets in yeast, mouse, and human cells follow a Pareto-like distribution model skewed by many low-abundance transcripts. A novel stochastic model of the gene expression process predicts the universality of the GELPF both across different cell types within a multicellular organism and across different organisms. This model allows us to predict the frequency distribution of all gene expression levels within a single cell and to estimate the number of expressed genes in a single cell and in a population of cells. A random "basal" transcription mechanism for protein-coding genes in all or almost all eukaryotic cell types is predicted. This fundamental mechanism might enhance the expression of rarely expressed genes and, thus, provide a basic level of phenotypic diversity, adaptability, and random monoallelic expression in cell populations. PMID:12136033

CaWRKY58, encoding a group I WRKY transcription factor of Capsicum annuum, negatively regulates resistance to Ralstonia solanacearum infection.

PubMed

Wang, Yuna; Dang, Fengfeng; Liu, Zhiqin; Wang, Xu; Eulgem, Thomas; Lai, Yan; Yu, Lu; She, Jianju; Shi, Youliang; Lin, Jinhui; Chen, Chengcong; Guan, Deyi; Qiu, Ailian; He, Shuilin

2013-02-01

WRKY transcription factors are encoded by large gene families across the plant kingdom. So far, their biological and molecular functions in nonmodel plants, including pepper (Capsicum annuum) and other Solanaceae, remain poorly understood. Here, we report on the functional characterization of a new group I WRKY protein from pepper, termed CaWRKY58. Our data indicate that CaWRKY58 can be localized to the nucleus and can activate the transcription of the reporter β-glucuronidase (GUS) gene driven by the 35S core promoter with two copies of the W-box in its proximal upstream region. In pepper plants infected with the bacterial pathogen Ralstonia solanacearum, CaWRKY58 transcript levels showed a biphasic response, manifested in an early/transient down-regulation and late up-regulation. CaWRKY58 transcripts were suppressed by treatment with methyl jasmonate and abscisic acid. Tobacco plants overexpressing CaWRKY58 did not show any obvious morphological phenotypes, but exhibited disease symptoms of greater severity than did wild-type plants. The enhanced susceptibility of CaWRKY58-overexpressing tobacco plants correlated with the decreased expression of hypersensitive response marker genes, as well as various defence-associated genes. Consistently, CaWRKY58 pepper plants silenced by virus-induced gene silencing (VIGS) displayed enhanced resistance to the highly virulent R. solanacearum strain FJC100301, and this was correlated with enhanced transcripts of defence-related pepper genes. Our results suggest that CaWRKY58 acts as a transcriptional activator of negative regulators in the resistance of pepper to R. solanacearum infection. © 2012 THE AUTHORS. MOLECULAR PLANT PATHOLOGY © 2012 BSPP AND BLACKWELL PUBLISHING LTD.

The mediator subunit Med23 contributes to controlling T-cell activation and prevents autoimmunity.

PubMed

Sun, Yang; Zhu, Xiaoyan; Chen, Xufeng; Liu, Haifeng; Xu, Yu; Chu, Yajing; Wang, Gang; Liu, Xiaolong

2014-10-10

T-cell activation is critical for successful immune responses and is controlled at multiple levels. Although many changes of T-cell receptor-associated signalling molecules affect T-cell activation, the transcriptional mechanisms that control this process remain largely unknown. Here we find that T cell-specific deletion of the mediator subunit Med23 leads to hyperactivation of T cells and aged Med23-deficient mice exhibit an autoimmune syndrome. Med23 specifically and consistently promotes the transcription of multiple negative regulators of T-cell activation. In the absence of Med23, the T-cell activation threshold is lower, which results in enhanced antitumour T-cell function. Cumulatively, our data suggest that Med23 contributes to controlling T-cell activation at the transcriptional level and prevents the development of autoimmunity.

Persistent α-Fetoprotein Elevation in Healthy Adults and Mutational Analysis of α-Fetoprotein Promoter, Enhancer, and Silencer Regions.

PubMed

Jeon, Yejoo; Choi, Yun Suk; Jang, Eun Sun; Kim, Jin Wook; Jeong, Sook-Hyang

2017-01-15

α-Fetoprotein (AFP) is normally <10 ng/mL in adults without malignancy or liver regeneration. However, hereditary or nonhereditary persistence of AFP in healthy adults may be encountered in clinical practice. This study describes four cases of persistent AFP elevation in healthy adults and investigates mutations in key transcription regulatory regions of the AFP gene as potential drivers of AFP overexpression. Four healthy adults with persistently elevated AFP levels (12.1 to 186.1 ng/mL) for >1 year, and 20 controls with low AFP levels (<0.61 to 2.9 ng/mL) were included in the study. AFP levels were collected from the families of two of the patients. We sequenced five regions that are critical for AFP expression: a promoter, two enhancers, and two silencers. One of the two cases in which family information was represented is the first case of hereditary persistence of AFP in South Korea. Mutations related to AFP overexpression were not found in the transcription regulatory regions among the four patients. Persistent AFP elevation is a heterogeneous condition with or without a hereditary pattern and may be caused by factors outside of transcription regulatory region changes. Further research on the mechanism of AFP elevation is needed.

Persistent α-Fetoprotein Elevation in Healthy Adults and Mutational Analysis of α-Fetoprotein Promoter, Enhancer, and Silencer Regions

PubMed Central

Jeon, Yejoo; Choi, Yun Suk; Jang, Eun Sun; Kim, Jin Wook; Jeong, Sook-Hyang

2017-01-01

Background/Aims α-Fetoprotein (AFP) is normally <10 ng/mL in adults without malignancy or liver regeneration. However, hereditary or nonhereditary persistence of AFP in healthy adults may be encountered in clinical practice. This study describes four cases of persistent AFP elevation in healthy adults and investigates mutations in key transcription regulatory regions of the AFP gene as potential drivers of AFP overexpression. Methods Four healthy adults with persistently elevated AFP levels (12.1 to 186.1 ng/mL) for >1 year, and 20 controls with low AFP levels (<0.61 to 2.9 ng/mL) were included in the study. AFP levels were collected from the families of two of the patients. We sequenced five regions that are critical for AFP expression: a promoter, two enhancers, and two silencers. Results One of the two cases in which family information was represented is the first case of hereditary persistence of AFP in South Korea. Mutations related to AFP overexpression were not found in the transcription regulatory regions among the four patients. Conclusions Persistent AFP elevation is a heterogeneous condition with or without a hereditary pattern and may be caused by factors outside of transcription regulatory region changes. Further research on the mechanism of AFP elevation is needed. PMID:27609486

An in vitro bioassay for xenobiotics using the SXR-driven human CYP3A4/lacZ reporter gene.

PubMed

Lee, Mi R; Kim, Yeon J; Hwang, Dae Y; Kang, Tae S; Hwang, Jin H; Lim, Chae H; Kang, Hyung K; Goo, Jun S; Lim, Hwa J; Ahn, Kwang S; Cho, Jung S; Chae, Kap R; Kim, Yong K

2003-01-01

The dose and time effect of nine xenobiotics, including 17beta-estradiol, corticosterone, dexamethasone, progesterone, nifedipine, bisphenol A, rifampicin, methamphetamine, and nicotine were investigated, in vitro, using human steroid and xenobiotics receptor (SXR)-binding sites on the human CYP3A4 promoter, which can enhance the linked lacZ reporter gene transcription. To test this, liver-specific SAP (human serum amyloid P component)-SXR (SAP/SXR) and human CYP3A4 promoter-regulated lacZ (hCYP3A4/lacZ) constructs were transiently transfected into HepG2 and NIH3T3 cells to compare the xenobiotic responsiveness between human and nonhuman cell lines. In the HepG2 cells, rifampicin, followed by corticosterone, nicotine, methamphetamine, and dexamethasone, exhibited enhanced levels of the lacZ transcript, whereas those of bisphenol A and nifedipine were found to be reduced. No significant responses were observed with 17beta-estradiol or progesterone. In addition, 17beta-estradiol and progesterone did not change the levels of the lacZ transcripts in the HepG2 cells, but did induce significant increases in the transcripts of the NIH3T3 cells. Treatment with corticosterone and dexamethasone, which were highly expressed in the HepG2 cells, did not affect the levels of the lacZ transcript in NIH3T3 cells. These results show that lacZ transcripts can be measured, rapidly and reproducibly, using reverse transcriptase-polymerase chain reaction (RT-PCR) based on the expression of the hCYP3A4/lacZ reporter gene, and was mediated by the SXR. Thus, this in vitro reporter gene bioassay is useful for measuring xenobiotic activities, and is a means to a better relevant bioassay, using human cells, human genes and human promoters, in order to get a closer look at actual human exposure.

The muscle creatine kinase gene is regulated by multiple upstream elements, including a muscle-specific enhancer

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

Jaynes, J.B.; Johnson, J.E.; Buskin, J.N.

1988-01-01

Muscle creatine kinase (MCK) is induced to high levels during skeletal muscle differentiation. The authors examined the upstream regulatory elements of the mouse MCK gene which specify its activation during myogenesis in culture. Fusion genes containing up to 3,300 nucleotides (nt) of MCK 5' flanking DNA in various positions and orientations relative to the bacterial chloramphenicol acetyltransferase (CAT) structural gene were transfected into cultured cells. Transient expression of CAT was compared between proliferating and differentiated MM14 mouse myoblasts and with nonmyogenic mouse L cells. The major effector of high-level expression was found to have the properties of a transcriptional enhancer.more » This element, located between 1,050 and 1,256 nt upstream of the transcription start site, was also found to have a major influence on the tissue and differentiation specificity of MCK expression; it activated either the MCK promoter or heterologous promoters only in differentiated muscle cells. Comparisons of viral and cellular enhancer sequences with the MCK enhancer revealed some similarities to essential regions of the simian virus 40 enhancer as well as to a region of the immunoglobulin heavy-chain enhancer, which has been implicated in tissue-specific protein binding. Even in the absence of the enhancer, low-level expression from a 776-nt MCK promoter retained differentiation specificity. In addition to positive regulatory elements, our data provide some evidence for negative regulatory elements with activity in myoblasts. These may contribute to the cell type and differentiation specificity of MCK expression.« less

Brain transcriptome perturbations in the Hfe(-/-) mouse model of genetic iron loading.

PubMed

Johnstone, Daniel; Graham, Ross M; Trinder, Debbie; Delima, Roheeth D; Riveros, Carlos; Olynyk, John K; Scott, Rodney J; Moscato, Pablo; Milward, Elizabeth A

2012-04-11

Severe disruption of brain iron homeostasis can cause fatal neurodegenerative disease, however debate surrounds the neurologic effects of milder, more common iron loading disorders such as hereditary hemochromatosis, which is usually caused by loss-of-function polymorphisms in the HFE gene. There is evidence from both human and animal studies that HFE gene variants may affect brain function and modify risks of brain disease. To investigate how disruption of HFE influences brain transcript levels, we used microarray and real-time reverse transcription polymerase chain reaction to assess the brain transcriptome in Hfe(-/-) mice relative to wildtype AKR controls (age 10 weeks, n≥4/group). The Hfe(-/-) mouse brain showed numerous significant changes in transcript levels (p<0.05) although few of these related to proteins directly involved in iron homeostasis. There were robust changes of at least 2-fold in levels of transcripts for prominent genes relating to transcriptional regulation (FBJ osteosarcoma oncogene Fos, early growth response genes), neurotransmission (glutamate NMDA receptor Grin1, GABA receptor Gabbr1) and synaptic plasticity and memory (calcium/calmodulin-dependent protein kinase IIα Camk2a). As previously reported for dietary iron-supplemented mice, there were altered levels of transcripts for genes linked to neuronal ceroid lipofuscinosis, a disease characterized by excessive lipofuscin deposition. Labile iron is known to enhance lipofuscin generation which may accelerate brain aging. The findings provide evidence that iron loading disorders can considerably perturb levels of transcripts for genes essential for normal brain function and may help explain some of the neurologic signs and symptoms reported in hemochromatosis patients. Copyright © 2012 Elsevier B.V. All rights reserved.

MiR-590-3p suppresses hepatocellular carcinoma growth by targeting TEAD1.

PubMed

Ge, Xin; Gong, Liansheng

2017-03-01

MicroRNA signature is altered in different disease states including cancer, and some microRNAs act as oncogenes or tumor suppressors. MiR-590-3p has been shown to be involved in human cancer progression. However, its role in hepatocellular carcinoma remains unknown. In this study, miR-590-3p level was measured, and clinicopathological features were determined in hepatocellular carcinoma tissues. The function of miR-590-3p was examined in vitro and in vivo. Real-time reverse transcription polymerase chain reaction analysis demonstrated downregulation of miR-590-3p in hepatocellular carcinoma tissues, and its downregulation was associated with a poor overall survival of hepatocellular carcinoma patients. Ectopic expression of miR-590-3p promoted growth of hepatocellular carcinoma cells, whereas its depletion inhibited cell growth. Transcriptional enhancer activator domain 1 was identified as a validated miR-590-3p target. Upregulation of transcriptional enhancer activator domain 1 was found in hepatocellular carcinoma tissues and inversely correlated with miR-590-3p. Our results indicate a tumor suppressor role of miR-590-3p in hepatocellular carcinoma through targeting transcriptional enhancer activator domain 1 and suggest its use in the diagnosis and prognosis of liver cancer.

[Construction and characterization of a gspL mutant of avian pathogenic Escherichia coli].

PubMed

Fan, Guobo; Han, Yue; Zhang, Yuxi; Han, Xiangan; Wang, Shaohui; Bai, Hao; Meng, Qingmei; Qi, Kezong; Ding, Chan; Yu, Shengqing

2015-01-04

To study the role of gspL gene in avian pathogenic Escherichia coli. The gspL mutant of Avian pathogenic Escherichia coli (APEC) was constructed by homologous recombination assay. The growth characteristics, the ability of adhesion and invasion to DF1 cells, the virulence genes transcription level and median lethal dose (LD50) were analyzed between the gspL mutant strain and the wild strain. Compared with the wild strain, the mutant strain had no significant difference in the growth status. However, its ability of adhesion and invasion was significantly lower. The transcription of genes pfs, fyuA, iss and vat increased obviously, the tsh decreased and the transcription level of luxS, ibeA, stx2f and ompA had no significant change. LD50 showed that the gspL mutant strain had 12-fold increase in virulence. The deletion of gspL gene could abate the ability of adhesion and invasion, regulate and control some virulence gene transcription level, enhance the virulence of APEC. The results show that the gspL gene play roles in pathogenicity of APEC.

Unraveling uranium induced oxidative stress related responses in Arabidopsis thaliana seedlings. Part I: responses in the roots.

PubMed

Vanhoudt, Nathalie; Vandenhove, Hildegarde; Horemans, Nele; Remans, Tony; Opdenakker, Kelly; Smeets, Karen; Bello, Daniel Martinez; Wannijn, Jean; Van Hees, May; Vangronsveld, Jaco; Cuypers, Ann

2011-06-01

When aiming to evaluate the environmental impact of uranium contamination, it is important to unravel the mechanisms by which plants respond to uranium stress. As oxidative stress seems an important modulator under other heavy metal stress, this study aimed to investigate oxidative stress related responses in Arabidopsis thaliana exposed to uranium concentrations ranging from 0.1 to 100 μM for 1, 3 and 7 days. Besides analyzing relevant reactive oxygen species-producing and -scavenging enzymes at protein and transcriptional level, the importance of the ascorbate-glutathione cycle under uranium stress was investigated. These results are reported separately for roots and leaves in two papers: Part I dealing with responses in the roots and Part II unraveling responses in the leaves and presenting general conclusions. Results of Part I indicate that oxidative stress related responses in the roots were only triggered following exposure to the highest uranium concentration of 100 μM. A fast oxidative burst was suggested based on the observed enhancement of lipoxygenase (LOX1) and respiratory burst oxydase homolog (RBOHD) transcript levels already after 1 day. The first line of defense was attributed to superoxide dismutase (SOD), also triggered from the first day. The enhanced SOD-capacity observed at protein level corresponded with an enhanced expression of iron SOD (FSD1) located in the plastids. For the detoxification of H(2)O(2), an early increase in catalase (CAT1) transcript levels was observed while peroxidase capacities were enhanced at the later stage of 3 days. Although the ascorbate peroxidase capacity and gene expression (APX1) increased, the ascorbate/dehydroascorbate redox balance was completely disrupted and shifted toward the oxidized form. This disrupted balance could not be inverted by the glutathione part of the cycle although the glutathione redox balance could be maintained. Copyright © 2011 Elsevier Ltd. All rights reserved.

CCAR1/CoCoA pair-mediated recruitment of the Mediator defines a novel pathway for GATA1 function.

PubMed

Mizuta, Shumpei; Minami, Tomoya; Fujita, Haruka; Kaminaga, Chihiro; Matsui, Keiji; Ishino, Ruri; Fujita, Azusa; Oda, Kasumi; Kawai, Asami; Hasegawa, Natsumi; Urahama, Norinaga; Roeder, Robert G; Ito, Mitsuhiro

2014-01-01

The MED1 subunit of the Mediator transcriptional coregulator complex coactivates GATA1 and induces erythropoiesis. Here, we show the dual mechanism of GATA1- and MED1-mediated transcription. MED1 expression levels in K562 erythroleukemia cells paralleled the levels of GATA1-targeted gene transcription and erythroid differentiation. An N-terminal fragment of MED1, MED1(1-602), which is incapable of interacting with GATA1, enhanced GATA1-targeted gene transcription and erythroid differentiation, and introduction of MED1(1-602) into Med1(-/-) mouse embryonic fibroblasts (MEFs) partially rescued GATA1-mediated transcription. The C-terminal zinc-finger domain of GATA1 interacts with the MED1(1-602)-interacting coactivator CCAR1, CoCoA and MED1(681-715). CCAR1 and CoCoA synergistically enhanced GATA1-mediated transcription from the γ-globin promoter in MEFs. Recombinant GATA1, CCAR1, CoCoA and MED1(1-602) formed a complex in vitro, and GATA1, CCAR1, CoCoA and MED1 were recruited to the γ-globin promoter in K562 cells during erythroid differentiation. Therefore, in addition to the direct interaction between GATA1 and MED1, CoCoA and CCAR1 appear to relay the GATA1 signal to MED1, and multiple modes of the GATA1-MED1 axis may help to fine-tune GATA1 function during GATA1-mediated homeostasis events. © 2013 The Authors Genes to Cells © 2013 by the Molecular Biology Society of Japan and Wiley Publishing Asia Pty Ltd.

Bean Metal-Responsive Element-Binding Transcription Factor Confers Cadmium Resistance in Tobacco1

PubMed Central

Sun, Na; Liu, Meng; Zhang, Wentao; Yang, Wanning; Bei, Xiujuan; Ma, Hui; Qiao, Fan; Qi, Xiaoting

2015-01-01

Cadmium (Cd) is highly toxic to plants. Modulation of Cd-responsive transcription is an important way for Cd detoxification in plants. Metal-responsive element (MRE) is originally described in animal metallothionein genes. Although functional MREs also exist in Cd-regulated plant genes, specific transcription factors that bind MRE to regulate Cd tolerance have not been identified. Previously, we showed that Cd-inducible bean (Phaseolus vulgaris) stress-related gene2 (PvSR2) produces a short (S) PvSR2 transcript (S-PvSR2) driven by an intronic promoter. Here, we demonstrate that S-PvSR2 encodes a bean MRE-binding transcription factor1 (PvMTF-1) that confers Cd tolerance in tobacco (Nicotiana tabacum). PvMTF-1 expression was up-regulated by Cd at the levels of RNA and protein. Importantly, expression of PvMTF-1 in tobacco enhanced Cd tolerance, indicating its role in regulating Cd resistance in planta. This was achieved through direct regulation of a feedback-insensitive Anthranilate Synthase α-2 chain gene (ASA2), which catalyzes the first step for tryptophan biosynthesis. In vitro and in vivo DNA-protein interaction studies further revealed that PvMTF-1 directly binds to the MRE in the ASA2 promoter, and this binding depends on the zinc finger-like motif of PvMTF-1. Through modulating ASA2 up-regulation by Cd, PvMTF-1 increased free tryptophan level and subsequently reduced Cd accumulation, thereby enhancing Cd tolerance of transgenic tobacco plants. Consistent with this observation, tobacco transiently overexpressing ASA2 also exhibited increased tolerance to Cd. We conclude that PvMTF-1 is a zinc finger-like transcription factor that links MRE to Cd resistance in transgenic tobacco through activation of tryptophan biosynthesis. PMID:25624396

Enhanced cellulase production from Trichoderma reesei Rut-C30 by engineering with an artificial zinc finger protein library.

PubMed

Zhang, Fei; Bai, Fengwu; Zhao, Xinqing

2016-10-01

Trichoderma reesei Rut-C30 is a well-known cellulase producer, and improvement of its cellulase production is of great interest. An artificial zinc finger protein (AZFP) library is constructed for expression in T. reesei Rut-C30, and a mutant strain T. reesei U3 is selected based on its enhanced cellulase production. The U3 mutant shows a 55% rise in filter paper activity and 8.1-fold increased β-glucosidase activity, when compared to the native strain T. reesei Rut-C30. It is demonstrated that enhanced β-glucosidase activity was due to elevated transcription level of β-glucosidase gene in the U3 mutant. Moreover, significant elevation in transcription levels of several putative Azfp-U3 target genes is detected in the U3 mutant, including genes encoding hypothetical transcription factors and a putative glycoside hydrolase. Furthermore, U3 cellulase shows 115% higher glucose yield from pretreated corn stover, when compared to the cellulase of T. reesei Rut-C30. These results demonstrate that AZFP can be used to improve cellulase production in T. reesei Rut-C30. Our current work offers the establishment of an alternative strategy to develop fungal cell factories for improved production of high value industrial products. Copyright © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Transcription factor FoxA (HNF3) on a nucleosome at an enhancer complex in liver chromatin.

PubMed

Chaya, D; Hayamizu, T; Bustin, M; Zaret, K S

2001-11-30

Nucleosome-like particles and acetylated histones occur near active promoters and enhancers, and certain transcription factors can recognize their target sites on the surface of a nucleosome in vitro; yet it has been unclear whether transcription factors can occupy target sites on nucleosomes in native chromatin. We developed a method for sequential chromatin immunoprecipitation of distinct nuclear proteins that are simultaneously cross-linked to nucleosome-sized genomic DNA segments. We find that core histone H2A co-occupies, along with the FoxA (hepatocyte nuclear factor-3) transcription factor, DNA for the albumin transcriptional enhancer in native liver chromatin, where the enhancer is active. Because histone H2A on nuclear DNA is only known to exist in nucleosomes, we conclude that transcription factors can form a stable complex on nucleosomes at an active enhancer element in vivo.

Spatiotemporal coupling and decoupling of gene transcription with DNA replication origins during embryogenesis in C. elegans

PubMed Central

Pourkarimi, Ehsan; Bellush, James M; Whitehouse, Iestyn

2016-01-01

The primary task of developing embryos is genome replication, yet how DNA replication is integrated with the profound cellular changes that occur through development is largely unknown. Using an approach to map DNA replication at high resolution in C. elegans, we show that replication origins are marked with specific histone modifications that define gene enhancers. We demonstrate that the level of enhancer associated modifications scale with the efficiency at which the origin is utilized. By mapping replication origins at different developmental stages, we show that the positions and activity of origins is largely invariant through embryogenesis. Contrary to expectation, we find that replication origins are specified prior to the broad onset of zygotic transcription, yet when transcription initiates it does so in close proximity to the pre-defined replication origins. Transcription and DNA replication origins are correlated, but the association breaks down when embryonic cell division ceases. Collectively, our data indicate that replication origins are fundamental organizers and regulators of gene activity through embryonic development. DOI: http://dx.doi.org/10.7554/eLife.21728.001 PMID:28009254

ANKRD1 Acts as a Transcriptional Repressor of MMP13 via the AP-1 Site

PubMed Central

Almodóvar-García, Karinna; Kwon, Minjae; Samaras, Susan E.

2014-01-01

The transcriptional cofactor ANKRD1 is sharply induced during wound repair, and its overexpression enhances healing. We recently found that global deletion of murine Ankrd1 impairs wound contraction and enhances necrosis of ischemic wounds. A quantitative PCR array of Ankrd1−/− (KO) fibroblasts indicated that ANKRD1 regulates MMP genes. Yeast two-hybrid and coimmunoprecipitation analyses associated ANKRD1 with nucleolin, which represses AP-1 activation of MMP13. Ankrd1 deletion enhanced both basal and phorbol 12-myristate 13-acetate (PMA)-induced MMP13 promoter activity; conversely, Ankrd1 overexpression in control cells decreased PMA-induced MMP13 promoter activity. Ankrd1 reconstitution in KO fibroblasts decreased MMP13 mRNA, while Ankrd1 knockdown increased these levels. MMP13 mRNA and protein were elevated in intact skin and wounds of KO versus Ankrd1fl/fl (FLOX) mice. Electrophoretic mobility shift assay gel shift patterns suggested that additional transcription factors bind to the MMP13 AP-1 site in the absence of Ankrd1, and this concept was reinforced by chromatin immunoprecipitation analysis as greater binding of c-Jun to the AP-1 site in extracts from FLOX versus KO fibroblasts. We propose that ANKRD1, in association with factors such as nucleolin, represses MMP13 transcription. Ankrd1 deletion additionally relieved MMP10 transcriptional repression. Nuclear ANKRD1 appears to modulate extracellular matrix remodeling by MMPs. PMID:24515436

Transcription regulation by distal enhancers

PubMed Central

Stadhouders, Ralph; van den Heuvel, Anita; Kolovos, Petros; Jorna, Ruud; Leslie, Kris; Grosveld, Frank; Soler, Eric

2012-01-01

Genome-wide chromatin profiling efforts have shown that enhancers are often located at large distances from gene promoters within the noncoding genome. Whereas enhancers can stimulate transcription initiation by communicating with promoters via chromatin looping mechanisms, we propose that enhancers may also stimulate transcription elongation by physical interactions with intronic elements. We review here recent findings derived from the study of the hematopoietic system. PMID:22771987

Apolipoprotein E Enhances microRNA-146a in Monocytes and Macrophages to Suppress Nuclear Factor-κB–Driven Inflammation and Atherosclerosis

PubMed Central

Li, Kang; Ching, Daniel; Luk, Fu Sang; Raffai, Robert L.

2015-01-01

Rationale Apolipoprotein E (apoE) exerts anti-inflammatory properties that protect against atherosclerosis and other inflammatory diseases. However, mechanisms by which apoE suppresses the cellular activation of leukocytes commonly associated with atherosclerosis remain incompletely understood. Objective To test the hypothesis that apoE suppresses inflammation and atherosclerosis by regulating cellular microRNA levels in these leukocytes. Methods and Results An assessment of apoE expression among such leukocyte subsets in wild-type mice revealed that only macrophages and monocytes express apoE abundantly. An absence of apoE expression in macrophages and monocytes resulted in enhanced nuclear factor-κB (NF-κB) signaling and an exaggerated inflammatory response upon stimulation with lipopolysaccharide. This correlated with reduced levels of microRNA-146a, a critical negative regulator of NF-κB signaling. Ectopic apoE expression in Apoe−/− macrophages and monocytes raised miR-146a levels, while its silencing in wild-type cells had an opposite effect. Mechanistically, apoE increased the expression of transcription factor PU.1, which raised levels of pri-miR-146 transcripts, demonstrating that apoE exerts transcriptional control over miR-146a. In vivo, even a small amount of apoE expression in macrophages and monocytes of hypomorphic apoE mice led to increased miR-146a levels, and inhibited macrophage pro-inflammatory responses, Ly-6Chigh monocytosis, and atherosclerosis in the settings of hyperlipidemia. Accordingly, cellular enrichment of miR-146a through the systemic delivery of miR-146a mimetics in Apoe−/−Ldlr−/− and Ldlr−/− mice attenuated monocyte/macrophage activation and atherosclerosis in the absence of plasma lipid reduction. Conclusions Our data demonstrate that cellular apoE expression suppresses NF-κB–mediated inflammation and atherosclerosis by enhancing miR-146a levels in monocytes and macrophages. PMID:25904598

Tryptophan derivatives regulate the transcription of Oct4 in stem-like cancer cells.

PubMed

Cheng, Jie; Li, Wenxin; Kang, Bo; Zhou, Yanwen; Song, Jiasheng; Dan, Songsong; Yang, Ying; Zhang, Xiaoqian; Li, Jingchao; Yin, Shengyong; Cao, Hongcui; Yao, Hangping; Zhu, Chenggang; Yi, Wen; Zhao, Qingwei; Xu, Xiaowei; Zheng, Min; Zheng, Shusen; Li, Lanjuan; Shen, Binghui; Wang, Ying-Jie

2015-06-10

The aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor that responds to environmental toxicants, is increasingly recognized as a key player in embryogenesis and tumorigenesis. Here we show that a variety of tryptophan derivatives that act as endogenous AhR ligands can affect the transcription level of the master pluripotency factor Oct4. Among them, ITE enhances the binding of the AhR to the promoter of Oct4 and suppresses its transcription. Reduction of endogenous ITE levels in cancer cells by tryptophan deprivation or hypoxia leads to Oct4 elevation, which can be reverted by administration with synthetic ITE. Consequently, synthetic ITE induces the differentiation of stem-like cancer cells and reduces their tumorigenic potential in both subcutaneous and orthotopic xenograft tumour models. Thus, our results reveal a role of tryptophan derivatives and the AhR signalling pathway in regulating cancer cell stemness and open a new therapeutic avenue to target stem-like cancer cells.

Tryptophan derivatives regulate the transcription of Oct4 in stem-like cancer cells

PubMed Central

Cheng, Jie; Li, Wenxin; Kang, Bo; Zhou, Yanwen; Song, Jiasheng; Dan, Songsong; Yang, Ying; Zhang, Xiaoqian; Li, Jingchao; Yin, Shengyong; Cao, Hongcui; Yao, Hangping; Zhu, Chenggang; Yi, Wen; Zhao, Qingwei; Xu, Xiaowei; Zheng, Min; Zheng, Shusen; Li, Lanjuan; Shen, Binghui; Wang, Ying-Jie

2015-01-01

The aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor that responds to environmental toxicants, is increasingly recognized as a key player in embryogenesis and tumorigenesis. Here we show that a variety of tryptophan derivatives that act as endogenous AhR ligands can affect the transcription level of the master pluripotency factor Oct4. Among them, ITE enhances the binding of the AhR to the promoter of Oct4 and suppresses its transcription. Reduction of endogenous ITE levels in cancer cells by tryptophan deprivation or hypoxia leads to Oct4 elevation, which can be reverted by administration with synthetic ITE. Consequently, synthetic ITE induces the differentiation of stem-like cancer cells and reduces their tumorigenic potential in both subcutaneous and orthotopic xenograft tumour models. Thus, our results reveal a role of tryptophan derivatives and the AhR signalling pathway in regulating cancer cell stemness and open a new therapeutic avenue to target stem-like cancer cells. PMID:26059097

Hedgehog signaling is synergistically enhanced by nutritional deprivation and ligand stimulation in human fibroblasts of Gorlin syndrome.

PubMed

Mizuochi, Hiromi; Fujii, Katsunori; Shiohama, Tadashi; Uchikawa, Hideki; Shimojo, Naoki

2015-02-13

Hedgehog signaling is a pivotal developmental pathway that comprises hedgehog, PTCH1, SMO, and GLI proteins. Mutations in PTCH1 are responsible for Gorlin syndrome, which is characterized by developmental defects and tumorigenicity. Although the hedgehog pathway has been investigated extensively in Drosophila and mice, its functional roles have not yet been determined in human cells. In order to elucidate the mechanism by which transduction of the hedgehog signal is regulated in human tissues, we employed human fibroblasts derived from three Gorlin syndrome patients and normal controls. We investigated GLI1 transcription, downstream of hedgehog signaling, to assess native signal transduction, and then treated fibroblasts with a recombinant human hedgehog protein with or without serum deprivation. We also examined the transcriptional levels of hedgehog-related genes under these conditions. The expression of GLI1 mRNA was significantly higher in Gorlin syndrome-derived fibroblasts than in control cells. Hedgehog stimulation and nutritional deprivation synergistically enhanced GLI1 transcription levels, and this was blocked more efficiently by vismodegib, a SMO inhibitor, than by the natural compound, cyclopamine. Messenger RNA profiling revealed the increased expression of Wnt signaling and morphogenetic molecules in these fibroblasts. These results indicated that the hedgehog stimulation and nutritional deprivation synergistically activated the hedgehog signaling pathway in Gorlin syndrome fibroblasts, and this was associated with increments in the transcription levels of hedgehog-related genes such as those involved in Wnt signaling. These fibroblasts may become a significant tool for predicting the efficacies of hedgehog molecular-targeted therapies such as vismodegib. Copyright © 2015 Elsevier Inc. All rights reserved.

The cysteine2/histidine2-type transcription factor ZINC FINGER OF ARABIDOPSIS THALIANA 6-activated C-REPEAT-BINDING FACTOR pathway is essential for melatonin-mediated freezing stress resistance in Arabidopsis.

PubMed

Shi, Haitao; Chan, Zhulong

2014-09-01

Melatonin (N-acetyl-5-methoxytryptamine) is not only a widely known animal hormone, but also an important regulator in plant development and multiple abiotic stress responses. Recently, it has been revealed that melatonin alleviated cold stress through mediating several cold-related genes, including C-REPEAT-BINDING FACTORs (CBFs)/Drought Response Element Binding factors (DREBs), COR15a, and three transcription factors (CAMTA1, ZINC FINGER OF ARABIDOPSIS THALIANA 10 (ZAT10), and ZAT12). In this study, we quantified the endogenous melatonin level in Arabidopsis plant leaves and found the endogenous melatonin levels were significantly induced by cold stress (4 °C) treatment. In addition, we found one cysteine2/histidine2-type zinc finger transcription factor, ZAT6, was involved in melatonin-mediated freezing stress response in Arabidopsis. Interestingly, exogenous melatonin enhanced freezing stress resistance was largely alleviated in AtZAT6 knockdown plants, but was enhanced in AtZAT6 overexpressing plants. Moreover, the expression levels of AtZAT6 and AtCBFs were commonly upregulated by cold stress (4 °C) and exogenous melatonin treatments, and modulation of AtZAT6 expression significantly affected the induction AtCBFs transcripts by cold stress (4 °C) and exogenous melatonin treatments. Taken together, AtZAT6-activated CBF pathway might be essential for melatonin-mediated freezing stress response in Arabidopsis. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Glucocorticoids facilitate the transcription from the human cytomegalovirus major immediate early promoter in glucocorticoid receptor- and nuclear factor-I-like protein-dependent manner

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

Inoue-Toyoda, Maki; Kato, Kohsuke; Faculty of Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba 305-8575

Human cytomegalovirus (HCMV) is a common and usually asymptomatic virus agent in healthy individuals. Initiation of HCMV productive infection depends on expression of the major immediate early (MIE) genes. The transcription of HCMV MIE genes is regulated by a diverse set of transcription factors. It was previously reported that productive HCMV infection is triggered probably by elevation of the plasma hydroxycorticoid level. However, it is poorly understood whether the transcription of MIE genes is directly regulated by glucocorticoid. Here, we found that the dexamethasone (DEX), a synthetic glucocorticoid, facilitates the transcription of HCMV MIE genes through the MIE promoter andmore » enhancer in a glucocorticoid receptor (GR)-dependent manner. By competitive EMSA and reporter assays, we revealed that an NF-I like protein is involved in DEX-mediated transcriptional activation of the MIE promoter. Thus, this study supports a notion that the increased level of hydroxycorticoid in the third trimester of pregnancy reactivates HCMV virus production from the latent state. - Highlights: • DEX facilitates the transcription from the HCMV MIE promoter. • GR is involved in DEX-dependent transcription from the HCMV MIE promoter. • A 17 bp repeat is responsible for the HCMV MIE promoter activation by DEX. • An NF-I-like protein is involved in the HCMV MIE promoter activation by DEX.« less

Transcriptional regulation of PNPLA3 and its impact on susceptibility to nonalcoholic fatty liver Disease (NAFLD) in humans

PubMed Central

Wang, Xiaoliang; Gawrieh, Samer; Gamazon, Eric R.; Athinarayanan, Shaminie; Liu, Yang-Lin; Darlay, Rebecca; Cordell, Heather J; Daly, Ann K

2017-01-01

The increased expression of PNPLA3148M leads to hepatosteatosis in mice. This study aims to investigate the genetic control of hepatic PNPLA3 transcription and to explore its impact on NAFLD risk in humans. Through a locus-wide expression quantitative trait loci (eQTL) mapping in two human liver sample sets, a PNPLA3 intronic SNP, rs139051 A>G was identified as a significant eQTL (p = 6.6×10−8) influencing PNPLA3 transcription, with the A allele significantly associated with increased PNPLA3 mRNA. An electrophoresis mobility shift assay further demonstrated that the A allele has enhanced affinity to nuclear proteins than the G allele. The impact of this eQTL on NAFLD risk was further tested in three independent populations. We found that rs139051 did not independently affect the NAFLD risk, whilst rs738409 did not significantly modulate PNPLA3 transcription but was associated with NAFLD risk. The A-G haplotype associated with higher transcription of the disease-risk rs738409 G allele conferred similar risk for NAFLD compared to the G-G haplotype that possesses a lower transcription level. Our study suggests that the pathogenic role of PNPLA3148M in NAFLD is independent of the gene transcription in humans, which may be attributed to the high endogenous transcription level of PNPLA3 gene in human livers. PMID:27744419

Transcriptional regulation of PNPLA3 and its impact on susceptibility to nonalcoholic fatty liver Disease (NAFLD) in humans.

PubMed

Liu, Wanqing; Anstee, Quentin M; Wang, Xiaoliang; Gawrieh, Samer; Gamazon, Eric R; Athinarayanan, Shaminie; Liu, Yang-Lin; Darlay, Rebecca; Cordell, Heather J; Daly, Ann K; Day, Chris P; Chalasani, Naga

2016-10-13

The increased expression of PNPLA3 148M leads to hepatosteatosis in mice. This study aims to investigate the genetic control of hepatic PNPLA3 transcription and to explore its impact on NAFLD risk in humans. Through a locus-wide expression quantitative trait loci (eQTL) mapping in two human liver sample sets, a PNPLA3 intronic SNP, rs139051 A>G was identified as a significant eQTL ( p = 6.6×10 -8 ) influencing PNPLA3 transcription, with the A allele significantly associated with increased PNPLA3 mRNA. An electrophoresis mobility shift assay further demonstrated that the A allele has enhanced affinity to nuclear proteins than the G allele. The impact of this eQTL on NAFLD risk was further tested in three independent populations. We found that rs139051 did not independently affect the NAFLD risk, whilst rs738409 did not significantly modulate PNPLA3 transcription but was associated with NAFLD risk. The A-G haplotype associated with higher transcription of the disease-risk rs738409 G allele conferred similar risk for NAFLD compared to the G-G haplotype that possesses a lower transcription level. Our study suggests that the pathogenic role of PNPLA3 148M in NAFLD is independent of the gene transcription in humans, which may be attributed to the high endogenous transcription level of PNPLA3 gene in human livers.

Control of Flagellar Gene Regulation in Legionella pneumophila and Its Relation to Growth Phase▿ †

PubMed Central

Albert-Weissenberger, Christiane; Sahr, Tobias; Sismeiro, Odile; Hacker, Jörg; Heuner, Klaus; Buchrieser, Carmen

2010-01-01

The bacterial pathogen Legionella pneumophila responds to environmental changes by differentiation. At least two forms are well described: replicative bacteria are avirulent; in contrast, transmissive bacteria express virulence traits and flagella. Phenotypic analysis, Western blotting, and electron microscopy of mutants of the regulatory genes encoding RpoN, FleQ, FleR, and FliA demonstrated that flagellin expression is strongly repressed and that the mutants are nonflagellated in the transmissive phase. Transcriptome analyses elucidated that RpoN, together with FleQ, enhances transcription of 14 out of 31 flagellar class II genes, which code for the basal body, hook, and regulatory proteins. Unexpectedly, FleQ independent of RpoN enhances the transcription of fliA encoding sigma 28. Expression analysis of a fliA mutant showed that FliA activates three out of the five remaining flagellar class III genes and the flagellar class IV genes. Surprisingly, FleR does not induce but inhibits expression of at least 14 flagellar class III genes on the transcriptional level. Thus, we propose that flagellar class II genes are controlled by FleQ and RpoN, whereas the transcription of the class III gene fliA is controlled in a FleQ-dependent but RpoN-independent manner. However, RpoN and FleR might influence flagellin synthesis on a posttranscriptional level. In contrast to the commonly accepted view that enhancer-binding proteins such as FleQ always interact with RpoN to fullfill their regulatory functions, our results strongly indicate that FleQ regulates gene expression that is RpoN dependent and RpoN independent. Finally, FliA induces expression of flagellar class III and IV genes leading to the complete synthesis of the flagellum. PMID:19915024

Downregulation of RND3/RhoE in glioblastoma patients promotes tumorigenesis through augmentation of notch transcriptional complex activity.

PubMed

Liu, Baohui; Lin, Xi; Yang, Xiangsheng; Dong, Huimin; Yue, Xiaojing; Andrade, Kelsey C; Guo, Zhentao; Yang, Jian; Wu, Liquan; Zhu, Xiaonan; Zhang, Shenqi; Tian, Daofeng; Wang, Junmin; Cai, Qiang; Chen, Qizuan; Mao, Shanping; Chen, Qianxue; Chang, Jiang

2015-09-01

Activation of Notch signaling contributes to glioblastoma multiform (GBM) tumorigenesis. However, the molecular mechanism that promotes the Notch signaling augmentation during GBM genesis remains largely unknown. Identification of new factors that regulate Notch signaling is critical for tumor treatment. The expression levels of RND3 and its clinical implication were analyzed in GBM patients. Identification of RND3 as a novel factor in GBM genesis was demonstrated in vitro by cell experiments and in vivo by a GBM xenograft model. We found that RND3 expression was significantly decreased in human glioblastoma. The levels of RND3 expression were inversely correlated with Notch activity, tumor size, and tumor cell proliferation, and positively correlated with patient survival time. We demonstrated that RND3 functioned as an endogenous repressor of the Notch transcriptional complex. RND3 physically interacted with NICD, CSL, and MAML1, the Notch transcriptional complex factors, promoted NICD ubiquitination, and facilitated the degradation of these cofactor proteins. We further revealed that RND3 facilitated the binding of NICD to FBW7, a ubiquitin ligase, and consequently enhanced NICD protein degradation. Therefore, Notch transcriptional activity was inhibited. Forced expression of RND3 repressed Notch signaling, which led to the inhibition of glioblastoma cell proliferation in vitro and tumor growth in the xenograft mice in vivo. Downregulation of RND3, however, enhanced Notch signaling activity, and subsequently promoted glioma cell proliferation. Inhibition of Notch activity abolished RND3 deficiency-mediated GBM cell proliferation. We conclude that downregulation of RND3 is responsible for the enhancement of Notch activity that promotes glioblastoma genesis. © 2015 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

Downregulation of RND3/RhoE in glioblastoma patients promotes tumorigenesis through augmentation of notch transcriptional complex activity

PubMed Central

Liu, Baohui; Lin, Xi; Yang, Xiangsheng; Dong, Huimin; Yue, Xiaojing; Andrade, Kelsey C; Guo, Zhentao; Yang, Jian; Wu, Liquan; Zhu, Xiaonan; Zhang, Shenqi; Tian, Daofeng; Wang, Junmin; Cai, Qiang; Chen, Qizuan; Mao, Shanping; Chen, Qianxue; Chang, Jiang

2015-01-01

Activation of Notch signaling contributes to glioblastoma multiform (GBM) tumorigenesis. However, the molecular mechanism that promotes the Notch signaling augmentation during GBM genesis remains largely unknown. Identification of new factors that regulate Notch signaling is critical for tumor treatment. The expression levels of RND3 and its clinical implication were analyzed in GBM patients. Identification of RND3 as a novel factor in GBM genesis was demonstrated in vitro by cell experiments and in vivo by a GBM xenograft model. We found that RND3 expression was significantly decreased in human glioblastoma. The levels of RND3 expression were inversely correlated with Notch activity, tumor size, and tumor cell proliferation, and positively correlated with patient survival time. We demonstrated that RND3 functioned as an endogenous repressor of the Notch transcriptional complex. RND3 physically interacted with NICD, CSL, and MAML1, the Notch transcriptional complex factors, promoted NICD ubiquitination, and facilitated the degradation of these cofactor proteins. We further revealed that RND3 facilitated the binding of NICD to FBW7, a ubiquitin ligase, and consequently enhanced NICD protein degradation. Therefore, Notch transcriptional activity was inhibited. Forced expression of RND3 repressed Notch signaling, which led to the inhibition of glioblastoma cell proliferation in vitro and tumor growth in the xenograft mice in vivo. Downregulation of RND3, however, enhanced Notch signaling activity, and subsequently promoted glioma cell proliferation. Inhibition of Notch activity abolished RND3 deficiency-mediated GBM cell proliferation. We conclude that downregulation of RND3 is responsible for the enhancement of Notch activity that promotes glioblastoma genesis. PMID:26108681

Constitutive expression of a salinity-induced wheat WRKY transcription factor enhances salinity and ionic stress tolerance in transgenic Arabidopsis thaliana

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

Qin, Yuxiang, E-mail: yuxiangqin@126.com; Tian, Yanchen; Han, Lu

Highlights: •A class II WRKY transcription factor, TaWRKY79 was isolated and characterized. •TaWRKY79 was induced by NaCl or abscisic acid. •843 bp regulatory segment was sufficient to respond to ABA or NaCl treatment. •TaWRKY79 enhanced salinity and ionic tolerance while reduced sensitivity to ABA. •TaWRKY79 increased salinity and ionic tolerance in an ABA-dependent pathway. -- Abstract: The isolation and characterization of TaWRKY79, a wheat class II WRKY transcription factor, is described. Its 1297 bp coding region includes a 987 bp long open reading frame. TaWRKY79 was induced by stressing seedlings with either NaCl or abscisic acid (ABA). When a fusionmore » between an 843 bp segment upstream of the TaWRKY79 coding sequence and GUS was introduced into Arabidopsis thaliana, GUS staining indicated that this upstream segment captured the sequence(s) required to respond to ABA or NaCl treatment. When TaWRKY79 was constitutively expressed as a transgene in A. thaliana, the transgenic plants showed an improved capacity to extend their primary root in the presence of either 100 mM NaCl, 10 mM LiCl or 2 μM ABA. The inference was that TaWRKY79 enhanced the level of tolerance to both salinity and ionic stress, while reducing the level of sensitivity to ABA. The ABA-related genes ABA1, ABA2 ABI1 and ABI5 were all up-regulated in the TaWRKY79 transgenic plants, suggesting that the transcription factor operates in an ABA-dependent pathway.« less

Mechanism of enhanced responses after combination photodynamic therapy (cPDT) in carcinoma cells involves C/EBP-mediated transcriptional upregulation of the coproporphyrinogen oxidase (CPO) gene

NASA Astrophysics Data System (ADS)

Anand, Sanjay; Hasan, Tayyaba; Maytin, Edward V.

2013-03-01

Photodynamic therapy (PDT) with aminolevulinate (ALA) is widely accepted as an effective treatment for superficial carcinomas and pre-cancers. However, PDT is still suboptimal for deeper tumors, mainly due to inadequate ALA penetration and subsequent conversion to PpIX. We are interested in improving the effectiveness of photodynamic therapy (PDT) for deep tumors, using a combination approach (cPDT) in which target protoporphyrin (PpIX) levels are significantly enhanced by differentiation caused by giving Vitamin D or methotrexate (MTX) for 3 days prior to ALAPDT. In LNCaP and MEL cells, a strong correlation between inducible differentiation and expression of C/EBP transcription factors, as well as between differentiation and mRNA levels of CPO (a key heme-synthetic enzyme), indicates the possibility of CPO transcriptional regulation by the C/EBPs. Sequence analysis of the first 1300 base pairs of the murine CPO upstream region revealed 15 consensus C/EBP binding sites. Electrophoretic Mobility Shift Assays (EMSA) proved that these sites form specific complexes that have strong, moderate or weak affinities for C/EBPs. However, in the context of the full-length CPO promoter, inactivation of any type of site (strong or weak) reduced CPO promoter activity (luciferase assay) to nearly the same extent, suggesting cooperative interactions. A comparative analysis of murine and human CPO promoters revealed possible protein-protein interactions between C/EBPs and several neighboring transcription factors such as NFkB, Sp1, AP-1, CBP/p300 and CREB (an enhanceosome complex). Overall, these results confirm that C/EBP's are important for CPO expression via complex mechanisms which upregulate PpIX and enhance the outcome of cPDT.

Identifying Novel Transcriptional and Epigenetic Features of Nuclear Lamina-associated Genes.

PubMed

Wu, Feinan; Yao, Jie

2017-03-07

Because a large portion of the mammalian genome is associated with the nuclear lamina (NL), it is interesting to study how native genes resided there are transcribed and regulated. In this study, we report unique transcriptional and epigenetic features of nearly 3,500 NL-associated genes (NL genes). Promoter regions of active NL genes are often excluded from NL-association, suggesting that NL-promoter interactions may repress transcription. Active NL genes with higher RNA polymerase II (Pol II) recruitment levels tend to display Pol II promoter-proximal pausing, while Pol II recruitment and Pol II pausing are not correlated among non-NL genes. At the genome-wide scale, NL-association and H3K27me3 distinguishes two large gene classes with low transcriptional activities. Notably, NL-association is anti-correlated with both transcription and active histone mark levels among genes not significantly enriched with H3K9me3 or H3K27me3, suggesting that NL-association may represent a novel gene repression pathway. Interestingly, an NL gene subgroup is not significantly enriched with H3K9me3 or H3K27me3 and is transcribed at higher levels than the rest of NL genes. Furthermore, we identified distal enhancers associated with active NL genes and reported their epigenetic features.

Transcription regulation by distal enhancers: who's in the loop?

PubMed

Stadhouders, Ralph; van den Heuvel, Anita; Kolovos, Petros; Jorna, Ruud; Leslie, Kris; Grosveld, Frank; Soler, Eric

2012-01-01

Genome-wide chromatin profiling efforts have shown that enhancers are often located at large distances from gene promoters within the noncoding genome. Whereas enhancers can stimulate transcription initiation by communicating with promoters via chromatin looping mechanisms, we propose that enhancers may also stimulate transcription elongation by physical interactions with intronic elements. We review here recent findings derived from the study of the hematopoietic system.

Therapeutic doses of irradiation activate viral transcription and induce apoptosis in HIV-1 infected cells

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

Iordanskiy, Sergey; Van Duyne, Rachel; Center for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, MD 21702

The highly active antiretroviral therapy reduces HIV-1 RNA in plasma to undetectable levels. However, the virus continues to persist in the long-lived resting CD4{sup +} T cells, macrophages and astrocytes which form a viral reservoir in infected individuals. Reactivation of viral transcription is critical since the host immune response in combination with antiretroviral therapy may eradicate the virus. Using the chronically HIV-1 infected T lymphoblastoid and monocytic cell lines, primary quiescent CD4{sup +} T cells and humanized mice infected with dual-tropic HIV-1 89.6, we examined the effect of various X-ray irradiation (IR) doses (used for HIV-related lymphoma treatment and lowermore » doses) on HIV-1 transcription and viability of infected cells. Treatment of both T cells and monocytes with IR, a well-defined stress signal, led to increase of HIV-1 transcription, as evidenced by the presence of RNA polymerase II and reduction of HDAC1 and methyl transferase SUV39H1 on the HIV-1 promoter. This correlated with the increased GFP signal and elevated level of intracellular HIV-1 RNA in the IR-treated quiescent CD4{sup +} T cells infected with GFP-encoding HIV-1. Exposition of latently HIV-1infected monocytes treated with PKC agonist bryostatin 1 to IR enhanced transcription activation effect of this latency-reversing agent. Increased HIV-1 replication after IR correlated with higher cell death: the level of phosphorylated Ser46 in p53, responsible for apoptosis induction, was markedly higher in the HIV-1 infected cells following IR treatment. Exposure of HIV-1 infected humanized mice with undetectable viral RNA level to IR resulted in a significant increase of HIV-1 RNA in plasma, lung and brain tissues. Collectively, these data point to the use of low to moderate dose of IR alone or in combination with HIV-1 transcription activators as a potential application for the “Shock and Kill” strategy for latently HIV-1 infected cells. - Highlights: • X-ray irradiation (IR) increases HIV-1 transcription in latently-infected cells. • IR enhances activating effect of bryostatin 1 on HIV-1 transcription in monocytes. • IR induces apoptosis in HIV-1 infected cells via phosphorylation of p53 Ser46. • IR of HIV-1 infected humanized mice increases HIV-1 RNA in plasma, lung and brain.« less

Promoter-dependent and -independent activation of insulin-like growth factor binding protein-5 gene expression by prostaglandin E2 in primary rat osteoblasts

NASA Technical Reports Server (NTRS)

McCarthy, T. L.; Casinghino, S.; Mittanck, D. W.; Ji, C. H.; Centrella, M.; Rotwein, P.

1996-01-01

Insulin-like growth factor (IGF) action is mediated by high affinity cell surface IGF receptors and modulated by a family of secreted IGF binding proteins (IGFBPs). IGFBP-5, the most conserved of six IGFBPs characterized to date, uniquely potentiates the anabolic actions of IGF-I for skeletal cells. In osteoblasts, IGFBP-5 production is stimulated by prostaglandin E2 (PGE2), a local factor that mediates certain effects induced by parathyroid hormone, cytokines such as interleukin-1 and transforming growth factor-beta, and mechanical strain. In this study, we show that transcriptional and post-transcriptional events initiated by PGE2 collaborate to enhance IGFBP-5 gene expression in primary fetal rat osteoblast cultures. PGE2 treatment stimulated up to a 7-fold rise in steady-state levels of IGFBP-5 mRNA throughout 32 h of incubation. Analysis of nascent IGFBP-5 mRNA suggested that PGE2 had only a modest stimulatory effect on IGFBP-5 gene transcription, and transient transfection studies with IGFBP-5 promoter-reporter genes confirmed that PGE2 enhanced promoter activity by approximately 2-fold. Similar stimulatory effects were seen with forskolin. A DNA fragment with only 51 base pairs of the 5'-flanking sequence retained hormonal responsiveness, which may be mediated by a binding site for transcription factor AP-2 located at positions -44 to -36 in the proximal IGFBP-5 promoter. Incubation of osteoblasts with the mRNA transcriptional inhibitor 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole demonstrated that PGE2 enhanced IGFBP-5 mRNA stability by 2-fold, increasing the t1/2 from 9 to 18 h. The effects of PGE2 on steady-state IGFBP-5 transcripts were abrogated by preincubating cells with cycloheximide, indicating that the effects of PGE2 on both gene transcription and mRNA stability required ongoing protein synthesis. Therefore, both promoter-dependent and -independent pathways converge to enhance IGFBP-5 gene expression in response to PGE2 in osteoblasts.

ABP9, a maize bZIP transcription factor, enhances tolerance to salt and drought in transgenic cotton.

PubMed

Wang, Chunling; Lu, Guoqing; Hao, Yuqiong; Guo, Huiming; Guo, Yan; Zhao, Jun; Cheng, Hongmei

2017-09-01

ABP9 , encoding a bZIP transcription factor from maize, enhances tolerance to multiple stresses and may participate in the ABA signaling pathway in transgenic cotton by altering physiological and biochemical processes and stress-related gene expression. Abiotic stresses, such as soil salinity and drought, negatively affect growth, development, and yield in cotton. Gene ABP9, which encodes a bZIP transcription factor, binds to the abscisic acid (ABA)-responsive-element (ABRE2) motif of the maize catalase1 gene. Its expression significantly improves tolerance in Arabidopsis to multiple abiotic stresses, but little is known about its role in cotton. In the present study, the ABP9 gene was introduced into upland cotton (Gossypium hirsutum L.) cultivar R15 by Agrobacterium tumefaciens-mediated transformation, and 12 independent transgenic cotton lines were obtained. Cotton plants over-expressing ABP9 have enhanced tolerance to salt and osmotic stress. Under stress, they developed better root systems in a greenhouse and higher germination, reduced stomatal aperture, and stomatal density in a growth chamber. Under drought conditions, survival rate and relative water content (RWC) of transgenic cotton were higher than those of R15 plants. Under salt and osmotic stresses, chlorophyll, proline, and soluble sugar contents significantly increased in transgenic cotton leaves and the malondialdehyde (MDA) content was lower than in R15. Overexpression of ABP9 also enhanced oxidative stress tolerance, reduced cellular levels of reactive oxygen species (ROS) through increased activities of antioxidative enzymes, and alleviated oxidative damage to cell. Interestingly, ABP9 over-expressing cotton was more sensitive to exogenous ABA than R15 at seed germination, root growth, stomatal aperture, and stomatal density. Moreover, ABP9 overexpression upregulated significantly the transcription levels of stress-related genes such as GhDBP2, GhNCED2, GhZFP1, GhERF1, GhHB1, and GhSAP1 under salt treatment. Conjointly, these results showed that overexpression of ABP9 conferred enhanced tolerance to multiple abiotic stresses in cotton. The stress-tolerant transgenic lines provide valuable resources for cotton breeding.

Dual Roles for Ikaros in Regulation of Macrophage Chromatin State and Inflammatory Gene Expression.

PubMed

Oh, Kyu-Seon; Gottschalk, Rachel A; Lounsbury, Nicolas W; Sun, Jing; Dorrington, Michael G; Baek, Songjoon; Sun, Guangping; Wang, Ze; Krauss, Kathleen S; Milner, Joshua D; Dutta, Bhaskar; Hager, Gordon L; Sung, Myong-Hee; Fraser, Iain D C

2018-06-13

Macrophage activation by bacterial LPS leads to induction of a complex inflammatory gene program dependent on numerous transcription factor families. The transcription factor Ikaros has been shown to play a critical role in lymphoid cell development and differentiation; however, its function in myeloid cells and innate immune responses is less appreciated. Using comprehensive genomic analysis of Ikaros-dependent transcription, DNA binding, and chromatin accessibility, we describe unexpected dual repressor and activator functions for Ikaros in the LPS response of murine macrophages. Consistent with the described function of Ikaros as transcriptional repressor, Ikzf1 -/- macrophages showed enhanced induction for select responses. In contrast, we observed a dramatic defect in expression of many delayed LPS response genes, and chromatin immunoprecipitation sequencing analyses support a key role for Ikaros in sustained NF-κB chromatin binding. Decreased Ikaros expression in Ikzf1 +/- mice and human cells dampens these Ikaros-enhanced inflammatory responses, highlighting the importance of quantitative control of Ikaros protein level for its activator function. In the absence of Ikaros, a constitutively open chromatin state was coincident with dysregulation of LPS-induced chromatin remodeling, gene expression, and cytokine responses. Together, our data suggest a central role for Ikaros in coordinating the complex macrophage transcriptional program in response to pathogen challenge.

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

A coherent transcriptional feed-forward motif model for mediating auxin-sensitive PIN3 expression during lateral root development

PubMed Central

Chen, Qian; Liu, Yang; Maere, Steven; Lee, Eunkyoung; Van Isterdael, Gert; Xie, Zidian; Xuan, Wei; Lucas, Jessica; Vassileva, Valya; Kitakura, Saeko; Marhavý, Peter; Wabnik, Krzysztof; Geldner, Niko; Benková, Eva; Le, Jie; Fukaki, Hidehiro; Grotewold, Erich; Li, Chuanyou; Friml, Jiří; Sack, Fred; Beeckman, Tom; Vanneste, Steffen

2015-01-01

Multiple plant developmental processes, such as lateral root development, depend on auxin distribution patterns that are in part generated by the PIN-formed family of auxin-efflux transporters. Here we propose that AUXIN RESPONSE FACTOR7 (ARF7) and the ARF7-regulated FOUR LIPS/MYB124 (FLP) transcription factors jointly form a coherent feed-forward motif that mediates the auxin-responsive PIN3 transcription in planta to steer the early steps of lateral root formation. This regulatory mechanism might endow the PIN3 circuitry with a temporal ‘memory' of auxin stimuli, potentially maintaining and enhancing the robustness of the auxin flux directionality during lateral root development. The cooperative action between canonical auxin signalling and other transcription factors might constitute a general mechanism by which transcriptional auxin-sensitivity can be regulated at a tissue-specific level. PMID:26578065

Nuclear Lamin-A Scales with Tissue Stiffness and Enhances Matrix-Directed Differentiation

PubMed Central

Swift, Joe; Ivanovska, Irena L.; Buxboim, Amnon; Harada, Takamasa; Dingal, P. C. Dave P.; Pinter, Joel; Pajerowski, J. David; Spinler, Kyle R.; Shin, Jae-Won; Tewari, Manorama; Rehfeldt, Florian; Speicher, David W.; Discher, Dennis E.

2014-01-01

Tissues can be soft like fat, which bears little stress, or stiff like bone, which sustains high stress, but whether there is a systematic relationship between tissue mechanics and differentiation is unknown. Here, proteomics analyses revealed that levels of the nucleoskeletal protein lamin-A scaled with tissue elasticity, E, as did levels of collagens in the extracellular matrix that determine E. Stem cell differentiation into fat on soft matrix was enhanced by low lamin-A levels, whereas differentiation into bone on stiff matrix was enhanced by high lamin-A levels. Matrix stiffness directly influenced lamin-A protein levels, and, although lamin-A transcription was regulated by the vitamin A/retinoic acid (RA) pathway with broad roles in development, nuclear entry of RA receptors was modulated by lamin-A protein. Tissue stiffness and stress thus increase lamin-A levels, which stabilize the nucleus while also contributing to lineage determination. PMID:23990565

E2F mediates enhanced alternative polyadenylation in proliferation.

PubMed

Elkon, Ran; Drost, Jarno; van Haaften, Gijs; Jenal, Mathias; Schrier, Mariette; Oude Vrielink, Joachim A F; Agami, Reuven

2012-07-02

The majority of mammalian genes contain multiple poly(A) sites in their 3' UTRs. Alternative cleavage and polyadenylation are emerging as an important layer of gene regulation as they generate transcript isoforms that differ in their 3' UTRs, thereby modulating genes' response to 3' UTR-mediated regulation. Enhanced cleavage at 3' UTR proximal poly(A) sites resulting in global 3' UTR shortening was recently linked to proliferation and cancer. However, mechanisms that regulate this enhanced alternative polyadenylation are unknown. Here, we explored, on a transcriptome-wide scale, alternative polyadenylation events associated with cellular proliferation and neoplastic transformation. We applied a deep-sequencing technique for identification and quantification of poly(A) sites to two human cellular models, each examined under proliferative, arrested and transformed states. In both cell systems we observed global 3' UTR shortening associated with proliferation, a link that was markedly stronger than the association with transformation. Furthermore, we found that proliferation is also associated with enhanced cleavage at intronic poly(A) sites. Last, we found that the expression level of the set of genes that encode for 3'-end processing proteins is globally elevated in proliferation, and that E2F transcription factors contribute to this regulation. Our results comprehensively identify alternative polyadenylation events associated with cellular proliferation and transformation, and demonstrate that the enhanced alternative polyadenylation in proliferative conditions results not only in global 3' UTR shortening but also in enhanced premature cleavage in introns. Our results also indicate that E2F-mediated co-transcriptional regulation of 3'-end processing genes is one of the mechanisms that links enhanced alternative polyadenylation to proliferation.

Cloning and characterization of rat density-enhanced phosphatase-1, a protein tyrosine phosphatase expressed by vascular cells.

PubMed

Borges, L G; Seifert, R A; Grant, F J; Hart, C E; Disteche, C M; Edelhoff, S; Solca, F F; Lieberman, M A; Lindner, V; Fischer, E H; Lok, S; Bowen-Pope, D F

1996-09-01

We have cloned from cultured vascular smooth muscle cells a protein tyrosine phosphatase, rat density-enhanced phosphatase-1 (rDEP-1), which is a probable rat homologue of DEP-1/HPTP eta. rDEP-1 is encoded by an 8.7-kb transcript and is expressed as a 180- to 220-kD protein. The rDEP-1 gene is located on human chromosome 11 (region p11.2) and on mouse chromosome 2 (region 2E). The cDNA sequence predicts a transmembrane protein consisting of a single phosphatase catalytic domain in the intracellular region, a single transmembrane domain, and eight fibronectin type III repeats in the extracellular region (GenBank accession number U40790). In situ hybridization analysis demonstrates that rDEP-1 is widely expressed in vivo but that expression is highest in cells that form epithelioid monolayers. In cultured cells with epitheliod morphology, including endothelial cells and newborn smooth muscle cells, but not in fibroblast-like cells, rDEP-1 transcript levels are dramatically upregulated as population density increases. In vivo, quiescent endothelial cells in normal arteries express relatively high levels of rDEP-1. During repair of vascular injury, expression of rDEP-1 is downregulated in migrating and proliferating endothelial cells. In vivo, rDEP-1 transcript levels are present in very high levels in megakaryocytes, and circulating plates have high levels of the rDEP-1 protein. In vitro, initiation of differentiation of the human megakaryoblastic cell line CHRF-288-11 with phorbol 12-myristate 13-acetate leads to a very strong upregulation of rDEP-1 transcripts. The deduced structure and the regulation of expression of rDEP-1 suggest that it may play a role in adhesion and/or signaling events involving cell-cell and cell-matrix contact.

Glucocorticoid Regulation of the Vitamin D Receptor

PubMed Central

Hidalgo, Alejandro A.; Trump, Donald L.; Johnson, Candace S.

2010-01-01

Many studies indicate calcitriol has potent anti-tumor activity in different types of cancers. However, high levels of vitamin D can produce hypercalcemia in some patients. Glucocorticoids are used to ameliorate hypercalcemia and to enhance calcitriol anti-tumor activity. Calcitriol in combination with the glucocorticoid dexamethasone (Dex) increased vitamin D receptor (VDR) protein levels and ligand binding in squamous cell carcinoma VII (SCC). In this study we found that both calcitriol and Dex induce VDR- and glucocorticoid receptor (GR)-mediated transcription respectively, indicating both hormone receptors are active in SCC. Pre-treatment with Dex increases VDR-mediated transcription at the human CYP24A1 promoter. Whereas, pre-treatment with other steroid hormones, including dihydrotestosterone and R1881, has no effect on VDR-mediated transcription. Real-time PCR indicates treatment with Dex increases Vdr transcripts in a time-dependent manner, suggesting Dex may directly regulate expression of Vdr. Numerous putative glucocorticoid response elements (GREs) were found in the Vdr gene. Chromatin immunoprecipitation (ChIP) assay demonstrated GR binding at several putative GREs located within the mouse Vdr gene. However, none of the putative GREs studied increase GR-mediated transcription in luciferase reporter assays. In an attempt to identify the response element responsible for Vdr transcript regulation, future studies will continue to analyze newly identified GREs more distal from the Vdr gene promoter. PMID:20398752

Transcription co-activator Arabidopsis ANGUSTIFOLIA3 (AN3) regulates water-use efficiency and drought tolerance by modulating stomatal density and improving root architecture by the transrepression of YODA (YDA).

PubMed

Meng, Lai-Sheng; Yao, Shun-Qiao

2015-09-01

One goal of modern agriculture is the improvement of plant drought tolerance and water-use efficiency (WUE). Although stomatal density has been linked to WUE, the causal molecular mechanisms and engineered alternations of this relationship are not yet fully understood. Moreover, YODA (YDA), which is a MAPKK kinase gene, negatively regulates stomatal development. BR-INSENSITIVE 2 interacts with phosphorylates and inhibits YDA. However, whether YDA is modulated in the transcriptional level is still unclear. Plants lacking ANGUSTIFOLIA3 (AN3) activity have high drought stress tolerance because of low stomatal densities and improved root architecture. Such plants also exhibit enhanced WUE through declining transpiration without a demonstrable reduction in biomass accumulation. AN3 negatively regulated YDA expression at the transcriptional level by target-gene analysis. Chromatin immunoprecipitation analysis indicated that AN3 was associated with a region of the YDA promoter in vivo. YDA mutation significantly decreased the stomatal density and root length of an3 mutant, thus proving the participation of YDA in an3 drought tolerance and WUE enhancement. These components form an AN3-YDA complex, which allows the integration of water deficit stress signalling into the production or spacing of stomata and cell proliferation, thus leading to drought tolerance and enhanced WUE. © 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

Cell cycle-dependent transcription factors control the expression of yeast telomerase RNA.

PubMed

Dionne, Isabelle; Larose, Stéphanie; Dandjinou, Alain T; Abou Elela, Sherif; Wellinger, Raymund J

2013-07-01

Telomerase is a specialized ribonucleoprotein that adds repeated DNA sequences to the ends of eukaryotic chromosomes to preserve genome integrity. Some secondary structure features of the telomerase RNA are very well conserved, and it serves as a central scaffold for the binding of associated proteins. The Saccharomyces cerevisiae telomerase RNA, TLC1, is found in very low copy number in the cell and is the limiting component of the known telomerase holoenzyme constituents. The reasons for this low abundance are unclear, but given that the RNA is very stable, transcriptional control mechanisms must be extremely important. Here we define the sequences forming the TLC1 promoter and identify the elements required for its low expression level, including enhancer and repressor elements. Within an enhancer element, we found consensus sites for Mbp1/Swi4 association, and chromatin immunoprecipitation (ChIP) assays confirmed the binding of Mbp1 and Swi4 to these sites of the TLC1 promoter. Furthermore, the enhancer element conferred cell cycle-dependent regulation to a reporter gene, and mutations in the Mbp1/Swi4 binding sites affected the levels of telomerase RNA and telomere length. Finally, ChIP experiments using a TLC1 RNA-binding protein as target showed cell cycle-dependent transcription of the TLC1 gene. These results indicate that the budding yeast TLC1 RNA is transcribed in a cell cycle-dependent fashion late in G1 and may be part of the S phase-regulated group of genes involved in DNA replication.

Epigenetic regulation of TTF-I-mediated promoter–terminator interactions of rRNA genes

PubMed Central

Németh, Attila; Guibert, Sylvain; Tiwari, Vijay Kumar; Ohlsson, Rolf; Längst, Gernot

2008-01-01

Ribosomal RNA synthesis is the eukaryotic cell's main transcriptional activity, but little is known about the chromatin domain organization and epigenetics of actively transcribed rRNA genes. Here, we show epigenetic and spatial organization of mouse rRNA genes at the molecular level. TTF-I-binding sites subdivide the rRNA transcription unit into functional chromatin domains and sharply delimit transcription factor occupancy. H2A.Z-containing nucleosomes occupy the spacer promoter next to a newly characterized TTF-I-binding site. The spacer and the promoter proximal TTF-I-binding sites demarcate the enhancer. DNA from both the enhancer and the coding region is hypomethylated in actively transcribed repeats. 3C analysis revealed an interaction between promoter and terminator regions, which brings the beginning and end of active rRNA genes into close contact. Reporter assays show that TTF-I mediates this interaction, thereby linking topology and epigenetic regulation of the rRNA genes. PMID:18354495

ZEB2 drives immature T-cell lymphoblastic leukaemia development via enhanced tumour-initiating potential and IL-7 receptor signalling

PubMed Central

Goossens, Steven; Radaelli, Enrico; Blanchet, Odile; Durinck, Kaat; Van der Meulen, Joni; Peirs, Sofie; Taghon, Tom; Tremblay, Cedric S.; Costa, Magdaline; Ghahremani, Morvarid Farhang; De Medts, Jelle; Bartunkova, Sonia; Haigh, Katharina; Schwab, Claire; Farla, Natalie; Pieters, Tim; Matthijssens, Filip; Van Roy, Nadine; Best, J. Adam; Deswarte, Kim; Bogaert, Pieter; Carmichael, Catherine; Rickard, Adam; Suryani, Santi; Bracken, Lauryn S.; Alserihi, Raed; Canté-Barrett, Kirsten; Haenebalcke, Lieven; Clappier, Emmanuelle; Rondou, Pieter; Slowicka, Karolina; Huylebroeck, Danny; Goldrath, Ananda W.; Janzen, Viktor; McCormack, Matthew P.; Lock, Richard B.; Curtis, David J.; Harrison, Christine; Berx, Geert; Speleman, Frank; Meijerink, Jules P. P.; Soulier, Jean; Van Vlierberghe, Pieter; Haigh, Jody J.

2015-01-01

Early T-cell precursor leukaemia (ETP-ALL) is a high-risk subtype of human leukaemia that is poorly understood at the molecular level. Here we report translocations targeting the zinc finger E-box-binding transcription factor ZEB2 as a recurrent genetic lesion in immature/ETP-ALL. Using a conditional gain-of-function mouse model, we demonstrate that sustained Zeb2 expression initiates T-cell leukaemia. Moreover, Zeb2-driven mouse leukaemia exhibit some features of the human immature/ETP-ALL gene expression signature, as well as an enhanced leukaemia-initiation potential and activated Janus kinase (JAK)/signal transducers and activators of transcription (STAT) signalling through transcriptional activation of IL7R. This study reveals ZEB2 as an oncogene in the biology of immature/ETP-ALL and paves the way towards pre-clinical studies of novel compounds for the treatment of this aggressive subtype of human T-ALL using our Zeb2-driven mouse model. PMID:25565005

Chromatin looping and eRNA transcription precede the transcriptional activation of gene in the β-globin locus

PubMed Central

Kim, Yea Woon; Lee, Sungkung; Yun, Jangmi; Kim, AeRi

2015-01-01

Enhancers are closely positioned with actively transcribed target genes by chromatin looping. Non-coding RNAs are often transcribed on active enhancers, referred to as eRNAs (enhancer RNAs). To explore the kinetics of enhancer–promoter looping and eRNA transcription during transcriptional activation, we induced the β-globin locus by chemical treatment and analysed cross-linking frequency between the β-globin gene and locus control region (LCR) and the amount of eRNAs transcribed on the LCR in a time course manner. The cross-linking frequency was increased after chemical induction but before the transcriptional activation of gene in the β-globin locus. Transcription of eRNAs was increased in concomitant with the increase in cross-linking frequency. These results show that chromatin looping and eRNA transcription precedes the transcriptional activation of gene. Concomitant occurrence of the two events suggests functional relationship between them. PMID:25588787

SMN transcript levels in leukocytes of SMA patients determined by absolute real-time PCR

PubMed Central

Tiziano, Francesco Danilo; Pinto, Anna Maria; Fiori, Stefania; Lomastro, Rosa; Messina, Sonia; Bruno, Claudio; Pini, Antonella; Pane, Marika; D'Amico, Adele; Ghezzo, Alessandro; Bertini, Enrico; Mercuri, Eugenio; Neri, Giovanni; Brahe, Christina

2010-01-01

Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder caused by homozygous mutations of the SMN1 gene. Three forms of SMA are recognized (type I–III) on the basis of clinical severity. All patients have at least one or more (usually 2–4) copies of a highly homologous gene (SMN2), which produces insufficient levels of functional SMN protein, because of alternative splicing of exon 7. Recently, evidence has been provided that SMN2 expression can be enhanced by pharmacological treatment. However, no reliable biomarkers are available to test the molecular efficacy of the treatments. At present, the only potential biomarker is the dosage of SMN products in peripheral blood. However, the demonstration that SMN full-length (SMN-fl) transcript levels are reduced in leukocytes of patients compared with controls remains elusive (except for type I). We have developed a novel assay based on absolute real-time PCR, which allows the quantification of SMN1-fl/SMN2-fl transcripts. For the first time, we have shown that SMN-fl levels are reduced in leukocytes of type II–III patients compared with controls. We also found that transcript levels are related to clinical severity as in type III patients SMN2-fl levels are significantly higher compared with type II and directly correlated with functional ability in type II patients and with age of onset in type III patients. Moreover, in haploidentical siblings with discordant phenotype, the less severely affected individuals showed significantly higher transcript levels. Our study shows that SMN2-fl dosage in leukocytes can be considered a reliable biomarker and can provide the rationale for SMN dosage in clinical trials. PMID:19603064

Characterization of regulatory elements within the coat protein (CP) coding region of Tobacco mosaic virus affecting subgenomic transcription and green fluorescent protein expression from the CP subgenomic RNA promoter.

PubMed

Man, Michal; Epel, Bernard L

2004-06-01

A replicon based on Tobacco mosaic virus that was engineered to express the open reading frame (ORF) of the green fluorescent protein (GFP) gene in place of the native coat protein (CP) gene from a minimal CP subgenomic (sg) RNA promoter was found to accumulate very low levels of GFP. Regulatory regions within the CP ORF were identified that, when presented as untranslated regions flanking the GFP ORF, enhanced or inhibited sg transcription and GFP expression. Full GFP expression from the CP sgRNA promoter required more than the first 20 nt of the CP ORF but not beyond the first 56 nt. Further analysis indicated the presence of an enhancer element between nt +25 and +55 with respect to the CP translation start site. The inclusion of this enhancer sequence upstream of the GFP ORF led to elevated sg transcription and to a 50-fold increase in GFP accumulation in comparison with a minimal CP promoter in which the entire CP ORF was displaced by the GFP ORF. Inclusion of the 3'-terminal 22 nt had a minor positive effect on GFP accumulation, but the addition of extended untranslated sequences from the 3' terminus of the CP ORF downstream of the GFP ORF was basically found to inhibit sg transcription. Secondary structure analysis programs predicted the CP sgRNA promoter to reside within two stable stem-loop structures, which are followed by an enhancer region.

Kub5-Hera, the human Rtt103 homolog, plays dual functional roles in transcription termination and DNA repair

PubMed Central

Morales, Julio C.; Richard, Patricia; Rommel, Amy; Fattah, Farjana J.; Motea, Edward A.; Patidar, Praveen L.; Xiao, Ling; Leskov, Konstantin; Wu, Shwu-Yuan; Hittelman, Walter N.; Chiang, Cheng-Ming; Manley, James L.; Boothman, David A.

2014-01-01

Functions of Kub5-Hera (In Greek Mythology Hera controlled Artemis) (K-H), the human homolog of the yeast transcription termination factor Rtt103, remain undefined. Here, we show that K-H has functions in both transcription termination and DNA double-strand break (DSB) repair. K-H forms distinct protein complexes with factors that repair DSBs (e.g. Ku70, Ku86, Artemis) and terminate transcription (e.g. RNA polymerase II). K-H loss resulted in increased basal R-loop levels, DSBs, activated DNA-damage responses and enhanced genomic instability. Significantly lowered Artemis protein levels were detected in K-H knockdown cells, which were restored with specific K-H cDNA re-expression. K-H deficient cells were hypersensitive to cytotoxic agents that induce DSBs, unable to reseal complex DSB ends, and showed significantly delayed γ-H2AX and 53BP1 repair-related foci regression. Artemis re-expression in K-H-deficient cells restored DNA-repair function and resistance to DSB-inducing agents. However, R loops persisted consistent with dual roles of K-H in transcription termination and DSB repair. PMID:24589584

Kub5-Hera, the human Rtt103 homolog, plays dual functional roles in transcription termination and DNA repair.

PubMed

Morales, Julio C; Richard, Patricia; Rommel, Amy; Fattah, Farjana J; Motea, Edward A; Patidar, Praveen L; Xiao, Ling; Leskov, Konstantin; Wu, Shwu-Yuan; Hittelman, Walter N; Chiang, Cheng-Ming; Manley, James L; Boothman, David A

2014-04-01

Functions of Kub5-Hera (In Greek Mythology Hera controlled Artemis) (K-H), the human homolog of the yeast transcription termination factor Rtt103, remain undefined. Here, we show that K-H has functions in both transcription termination and DNA double-strand break (DSB) repair. K-H forms distinct protein complexes with factors that repair DSBs (e.g. Ku70, Ku86, Artemis) and terminate transcription (e.g. RNA polymerase II). K-H loss resulted in increased basal R-loop levels, DSBs, activated DNA-damage responses and enhanced genomic instability. Significantly lowered Artemis protein levels were detected in K-H knockdown cells, which were restored with specific K-H cDNA re-expression. K-H deficient cells were hypersensitive to cytotoxic agents that induce DSBs, unable to reseal complex DSB ends, and showed significantly delayed γ-H2AX and 53BP1 repair-related foci regression. Artemis re-expression in K-H-deficient cells restored DNA-repair function and resistance to DSB-inducing agents. However, R loops persisted consistent with dual roles of K-H in transcription termination and DSB repair.

Quantitative proteomic and transcriptomic analyses of molecular mechanisms associated with low silk production in silkworm Bombyx mori.

PubMed

Wang, Shao-Hua; You, Zheng-Ying; Ye, Lu-Peng; Che, Jiaqian; Qian, Qiujie; Nanjo, Yohei; Komatsu, Setsuko; Zhong, Bo-Xiong

2014-02-07

To investigate the molecular mechanisms underlying the low fibroin production of the ZB silkworm strain, we used both SDS-PAGE-based and gel-free-based proteomic techniques and transcriptomic sequencing technique. Combining the data from two different proteomic techniques was preferable in the characterization of the differences between the ZB silkworm strain and the original Lan10 silkworm strain. The correlation analysis showed that the individual protein and transcript were not corresponded well, however, the differentially changed proteins and transcripts showed similar regulated direction in function at the pathway level. In the ZB strain, numerous ribosomal proteins and transcripts were down-regulated, along with the transcripts of translational related elongation factors and genes of important components of fibroin. The proteasome pathway was significantly enhanced in the ZB strain, indicating that protein degradation began on the third day of fifth instar when fibroin would have been produced in the Lan10 strain normally and plentifully. From proteome and transcriptome levels of the ZB strain, the energy-metabolism-related pathways, oxidative phosphorylation, glycolysis/gluconeogenesis, and citrate cycle were enhanced, suggesting that the energy metabolism was vigorous in the ZB strain, while the silk production was low. This may due to the inefficient energy employment in fibroin synthesis in the ZB strain. These results suggest that the reason for the decreasing of the silk production might be related to the decreased ability of fibroin synthesis, the degradation of proteins, and the inefficiency of the energy exploiting.

Gene Expression Signature in Adipose Tissue of Acromegaly Patients

PubMed Central

Hochberg, Irit; Tran, Quynh T.; Barkan, Ariel L.; Saltiel, Alan R.; Chandler, William F.; Bridges, Dave

2015-01-01

To study the effect of chronic excess growth hormone on adipose tissue, we performed RNA sequencing in adipose tissue biopsies from patients with acromegaly (n = 7) or non-functioning pituitary adenomas (n = 11). The patients underwent clinical and metabolic profiling including assessment of HOMA-IR. Explants of adipose tissue were assayed ex vivo for lipolysis and ceramide levels. Patients with acromegaly had higher glucose, higher insulin levels and higher HOMA-IR score. We observed several previously reported transcriptional changes (IGF1, IGFBP3, CISH, SOCS2) that are known to be induced by GH/IGF-1 in liver but are also induced in adipose tissue. We also identified several novel transcriptional changes, some of which may be important for GH/IGF responses (PTPN3 and PTPN4) and the effects of acromegaly on growth and proliferation. Several differentially expressed transcripts may be important in GH/IGF-1-induced metabolic changes. Specifically, induction of LPL, ABHD5, and NRIP1 can contribute to enhanced lipolysis and may explain the elevated adipose tissue lipolysis in acromegalic patients. Higher expression of TCF7L2 and the fatty acid desaturases FADS1, FADS2 and SCD could contribute to insulin resistance. Ceramides were not different between the two groups. In summary, we have identified the acromegaly gene expression signature in human adipose tissue. The significance of altered expression of specific transcripts will enhance our understanding of the metabolic and proliferative changes associated with acromegaly. PMID:26087292

Isolation and Characterization of a Lycopene ε-Cyclase Gene of Chlorella (Chromochloris) zofingiensis. Regulation of the Carotenogenic Pathway by Nitrogen and Light

PubMed Central

Cordero, Baldo F.; Couso, Inmaculada; Leon, Rosa; Rodriguez, Herminia; Vargas, Maria Angeles

2012-01-01

The isolation and characterization of the lycopene ε-cyclase gene from the green microalga Chlorella (Chromochloris) zofingiensis (Czlcy-e) was performed. This gene is involved in the formation of the carotenoids α-carotene and lutein. Czlcy-e gene encoded a polypeptide of 654 amino acids. A single copy of Czlcy-e was found in C. zofingiensis. Functional analysis by heterologous complementation in Escherichia coli showed the ability of this protein to convert lycopene to δ-carotene. In addition, the regulation of the carotenogenic pathway by light and nitrogen was also studied in C. zofingiensis. High irradiance stress did not increase mRNA levels of neither lycopene β-cyclase gene (lcy-b) nor lycopene ε-cyclase gene (lcy-e) as compared with low irradiance conditions, whereas the transcript levels of psy, pds, chyB and bkt genes were enhanced, nevertheless triggering the synthesis of the secondary carotenoids astaxanthin, canthaxanthin and zeaxanthin and decreasing the levels of the primary carotenoids α-carotene, lutein, violaxanthin and β-carotene. Nitrogen starvation per se enhanced mRNA levels of all genes considered, except lcy-e and pds, but did not trigger the synthesis of astaxanthin, canthaxanthin nor zeaxanthin. The combined effect of both high light and nitrogen starvation stresses enhanced significantly the accumulation of these carotenoids as well as the transcript levels of bkt gene, as compared with the effect of only high irradiance stress. PMID:23118722

Analysis of Class I Major Histocompatibility Complex Gene Transcription in Human Tumors Caused by Human Papillomavirus Infection

PubMed Central

Gameiro, Steven F.; Zhang, Ali; Ghasemi, Farhad; Barrett, John W.; Mymryk, Joe S.

2017-01-01

Oncoproteins from high-risk human papillomaviruses (HPV) downregulate the transcription of the class I major histocompatibility complex (MHC-I) antigen presentation apparatus in tissue culture model systems. This could allow infected or transformed cells to evade the adaptive immune response. Using data from over 800 human cervical and head & neck tumors from The Cancer Genome Atlas (TCGA), we determined the impact of HPV status on the mRNA expression of all six MHC-I heavy chain genes, and the β2 microglobulin light chain. Unexpectedly, these genes were all expressed at high levels in HPV positive (HPV+) cancers compared with normal control tissues. Indeed, many of these genes were expressed at significantly enhanced levels in HPV+ tumors. Similarly, the transcript levels of several other components of the MHC-I peptide-loading complex were also high in HPV+ cancers. The coordinated expression of high mRNA levels of the MHC-I antigen presentation apparatus could be a consequence of the higher intratumoral levels of interferon γ in HPV+ carcinomas, which correlate with signatures of increased infiltration by T- and NK-cells. These data, which were obtained from both cervical and oral tumors in large human cohorts, indicates that HPV oncoproteins do not efficiently suppress the transcription of the antigen presentation apparatus in human tumors. PMID:28891951

Enhancers and super-enhancers have an equivalent regulatory role in embryonic stem cells through regulation of single or multiple genes

PubMed Central

Moorthy, Sakthi D.; Davidson, Scott; Shchuka, Virlana M.; Singh, Gurdeep; Malek-Gilani, Nakisa; Langroudi, Lida; Martchenko, Alexandre; So, Vincent; Macpherson, Neil N.; Mitchell, Jennifer A.

2017-01-01

Transcriptional enhancers are critical for maintaining cell-type–specific gene expression and driving cell fate changes during development. Highly transcribed genes are often associated with a cluster of individual enhancers such as those found in locus control regions. Recently, these have been termed stretch enhancers or super-enhancers, which have been predicted to regulate critical cell identity genes. We employed a CRISPR/Cas9-mediated deletion approach to study the function of several enhancer clusters (ECs) and isolated enhancers in mouse embryonic stem (ES) cells. Our results reveal that the effect of deleting ECs, also classified as ES cell super-enhancers, is highly variable, resulting in target gene expression reductions ranging from 12% to as much as 92%. Partial deletions of these ECs which removed only one enhancer or a subcluster of enhancers revealed partially redundant control of the regulated gene by multiple enhancers within the larger cluster. Many highly transcribed genes in ES cells are not associated with a super-enhancer; furthermore, super-enhancer predictions ignore 81% of the potentially active regulatory elements predicted by cobinding of five or more pluripotency-associated transcription factors. Deletion of these additional enhancer regions revealed their robust regulatory role in gene transcription. In addition, select super-enhancers and enhancers were identified that regulated clusters of paralogous genes. We conclude that, whereas robust transcriptional output can be achieved by an isolated enhancer, clusters of enhancers acting on a common target gene act in a partially redundant manner to fine tune transcriptional output of their target genes. PMID:27895109

Cocaine Administration and Its Withdrawal Enhance the Expression of Genes Encoding Histone-Modifying Enzymes and Histone Acetylation in the Rat Prefrontal Cortex.

PubMed

Sadakierska-Chudy, Anna; Frankowska, Małgorzata; Jastrzębska, Joanna; Wydra, Karolina; Miszkiel, Joanna; Sanak, Marek; Filip, Małgorzata

2017-07-01

Chronic exposure to cocaine, craving, and relapse are attributed to long-lasting changes in gene expression arising through epigenetic and transcriptional mechanisms. Although several brain regions are involved in these processes, the prefrontal cortex seems to play a crucial role not only in motivation and decision-making but also in extinction and seeking behavior. In this study, we applied cocaine self-administration and extinction training procedures in rats with a yoked triad to determine differentially expressed genes in prefrontal cortex. Microarray analysis showed significant upregulation of several genes encoding histone modification enzymes during early extinction training. Subsequent real-time PCR testing of these genes following cocaine self-administration or early (third day) and late (tenth day) extinction revealed elevated levels of their transcripts. Interestingly, we found the enrichment of Brd1 messenger RNA in rats self-administering cocaine that lasted until extinction training during cocaine withdrawal with concomitant increased acetylation of H3K9 and H4K8. However, despite elevated levels of methyl- and demethyltransferase-encoded transcripts, no changes in global di- and tri-methylation of histone H3 at lysine 4, 9, 27, and 79 were observed. Surprisingly, at the end of extinction training (10 days of cocaine withdrawal), most of the analyzed genes in the rats actively and passively administering cocaine returned to the control level. Together, the alterations identified in the rat prefrontal cortex may suggest enhanced chromatin remodeling and transcriptional activity induced by early cocaine abstinence; however, to know whether they are beneficial or not for the extinction of drug-seeking behavior, further in vivo evaluation is required.

Triptolide inhibits transcription of hTERT through down-regulation of transcription factor specificity protein 1 in primary effusion lymphoma cells

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

Long, Cong; Wang, Jingchao; Guo, Wei

Primary effusion lymphoma (PEL) is a rare and aggressive non-Hodgkin's lymphoma. Human telomerase reverse transcriptase (hTERT), a key component responsible for the regulation of telomerase activity, plays important roles in cellular immortalization and cancer development. Triptolide purified from Tripterygium extracts displays a broad-spectrum bioactivity profile, including immunosuppressive, anti-inflammatory, and anti-tumor. In this study, it is investigated whether triptolide reduces hTERT expression and suppresses its activity in PEL cells. The mRNA and protein levels of hTERT were examined by real time-PCR and Western blotting, respectively. The activity of hTERT promoter was determined by Dual luciferase reporter assay. Our results demonstrated thatmore » triptolide decreased expression of hTERT at both mRNA and protein levels. Further gene sequence analysis indicated that the activity of hTERT promoter was suppressed by triptolide. Triptolide also reduced the half-time of hTERT. Additionally, triptolide inhibited the expression of transcription factor specificity protein 1(Sp1) in PEL cells. Furthermore, knock-down of Sp1 by using specific shRNAs resulted in down-regulation of hTERT transcription and protein expression levels. Inhibition of Sp1 by specific shRNAs enhanced triptolide-induced cell growth inhibition and apoptosis. Collectively, our results demonstrate that the inhibitory effect of triptolide on hTERT transcription is possibly mediated by inhibition of transcription factor Sp1 in PEL cells. - Highlights: • Triptolide reduces expression of hTERT by decreasing its transcription level. • Triptolide reduces promoter activity and stability of hTERT. • Triptolide down-regulates expression of Sp1. • Special Sp1 shRNAs inhibit transcription and protein expression of hTERT. • Triptolide and Sp1 shRNA2 induce cell proliferation inhibition and apoptosis.« less

CITED2 modulates estrogen receptor transcriptional activity in breast cancer cells

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

Lau, Wen Min; Doucet, Michele; Huang, David

2013-07-26

Highlights: •The effects of elevated CITED2 on ER function in breast cancer cells are examined. •CITED2 enhances cell growth in the absence of estrogen and presence of tamoxifen. •CITED2 functions as a transcriptional co-activator of ER in breast cancer cells. -- Abstract: Cbp/p300-interacting transactivator with Glu/Asp-rich carboxy-terminal domain 2 (CITED2) is a member of the CITED family of non-DNA binding transcriptional co-activators of the p300/CBP-mediated transcription complex. Previously, we identified CITED2 as being overexpressed in human breast tumors relative to normal mammary epithelium. Upon further investigation within the estrogen receptor (ER)-positive subset of these breast tumor samples, we found thatmore » CITED2 mRNA expression was elevated in those associated with poor survival. In light of this observation, we investigated the effect of elevated CITED2 levels on ER function. While ectopic overexpression of CITED2 in three ER-positive breast cancer cell lines (MCF-7, T47D, and CAMA-1) did not alter cell proliferation in complete media, growth was markedly enhanced in the absence of exogenous estrogen. Correspondingly, cells overexpressing CITED2 demonstrated reduced sensitivity to the growth inhibitory effects of the selective estrogen receptor modulator, 4-hydroxytamoxifen. Subsequent studies revealed that basal ER transcriptional activity was elevated in CITED2-overexpressing cells and was further increased upon the addition of estrogen. Similarly, basal and estrogen-induced expression of the ER-regulated genes trefoil factor 1 (TFF1) and progesterone receptor (PGR) was higher in cells overexpressing CITED2. Concordant with this observation, ChIP analysis revealed higher basal levels of CITED2 localized to the TFF-1 and PGR promoters in cells with ectopic overexpression of CITED2, and these levels were elevated further in response to estrogen stimulation. Taken together, these data indicate that CITED2 functions as a transcriptional co-activator of ER in breast cancer cells and that its increased expression in tumors may result in estrogen-independent ER activation, thereby reducing estrogen dependence and response to anti-estrogen therapy.« less

CRTC2 Is a Coactivator of GR and Couples GR and CREB in the Regulation of Hepatic Gluconeogenesis.

PubMed

Hill, Micah J; Suzuki, Shigeru; Segars, James H; Kino, Tomoshige

2016-01-01

Glucocorticoid hormones play essential roles in the regulation of gluconeogenesis in the liver, an adaptive response that is required for the maintenance of circulating glucose levels during fasting. Glucocorticoids do this by cooperating with glucagon, which is secreted from pancreatic islets to activate the cAMP-signaling pathway in hepatocytes. The cAMP-response element-binding protein (CREB)-regulated transcription coactivator 2 (CRTC2) is a coactivator known to be specific to CREB and plays a central role in the glucagon-mediated activation of gluconeogenesis in the early phase of fasting. We show here that CRTC2 also functions as a coactivator for the glucocorticoid receptor (GR). CRTC2 strongly enhances GR-induced transcriptional activity of glucocorticoid-responsive genes. CRTC2 physically interacts with the ligand-binding domain of the GR through a region spanning amino acids 561-693. Further, CRTC2 is required for the glucocorticoid-associated cooperative mRNA expression of the glucose-6-phosphatase, a rate-limiting enzyme for hepatic gluconeogenesis, by facilitating the attraction of GR and itself to its promoter region already occupied by CREB. CRTC2 is required for the maintenance of blood glucose levels during fasting in mice by enhancing the GR transcriptional activity on both the G6p and phosphoenolpyruvate carboxykinase (Pepck) genes. Finally, CRTC2 modulates the transcriptional activity of the progesterone receptor, indicating that it may influence the transcriptional activity of other steroid/nuclear receptors. Taken together, these results reveal that CRTC2 plays an essential role in the regulation of hepatic gluconeogenesis through coordinated regulation of the glucocorticoid/GR- and glucagon/CREB-signaling pathways on the key genes G6P and PEPCK.

A NAP-AAO3 Regulatory Module Promotes Chlorophyll Degradation via ABA Biosynthesis in Arabidopsis Leaves[W][OPEN

PubMed Central

Yang, Jiading; Worley, Eric

2014-01-01

Chlorophyll degradation is an important part of leaf senescence, but the underlying regulatory mechanisms are largely unknown. Excised leaves of an Arabidopsis thaliana NAC-LIKE, ACTIVATED BY AP3/PI (NAP) transcription factor mutant (nap) exhibited lower transcript levels of known chlorophyll degradation genes, STAY-GREEN1 (SGR1), NON-YELLOW COLORING1 (NYC1), PHEOPHYTINASE (PPH), and PHEIDE a OXYGENASE (PaO), and higher chlorophyll retention than the wild type during dark-induced senescence. Transcriptome coexpression analysis revealed that abscisic acid (ABA) metabolism/signaling genes were disproportionately represented among those positively correlated with NAP expression. ABA levels were abnormally low in nap leaves during extended darkness. The ABA biosynthetic genes 9-CIS-EPOXYCAROTENOID DIOXYGENASE2, ABA DEFICIENT3, and ABSCISIC ALDEHYDE OXIDASE3 (AAO3) exhibited abnormally low transcript levels in dark-treated nap leaves. NAP transactivated the promoter of AAO3 in mesophyll cell protoplasts, and electrophoretic mobility shift assays showed that NAP can bind directly to a segment (−196 to −162 relative to the ATG start codon) of the AAO3 promoter. Exogenous application of ABA increased the transcript levels of SGR1, NYC1, PPH, and PaO and suppressed the stay-green phenotype of nap leaves during extended darkness. Overexpression of AAO3 in nap leaves also suppressed the stay-green phenotype under extended darkness. Collectively, the results show that NAP promotes chlorophyll degradation by enhancing transcription of AAO3, which leads to increased levels of the senescence-inducing hormone ABA. PMID:25516602

Interference of transcription across H-NS binding sites and repression by H-NS.

PubMed

Rangarajan, Aathmaja Anandhi; Schnetz, Karin

2018-05-01

Nucleoid-associated protein H-NS represses transcription by forming extended DNA-H-NS complexes. Repression by H-NS operates mostly at the level of transcription initiation. Less is known about how DNA-H-NS complexes interfere with transcription elongation. In vitro H-NS has been shown to enhance RNA polymerase pausing and to promote Rho-dependent termination, while in vivo inhibition of Rho resulted in a decrease of the genome occupancy by H-NS. Here we show that transcription directed across H-NS binding regions relieves H-NS (and H-NS/StpA) mediated repression of promoters in these regions. Further, we observed a correlation of transcription across the H-NS-bound region and de-repression. The data suggest that the transcribing RNA polymerase is able to remodel the H-NS complex and/or dislodge H-NS from the DNA and thus relieve repression. Such an interference of transcription and H-NS mediated repression may imply that poorly transcribed AT-rich loci are prone to be repressed by H-NS, while efficiently transcribed loci escape repression. © 2018 John Wiley & Sons Ltd.

Expression of a maize Myb transcription factor driven by a putative silk-specific promoter significantly enhances resistance to Helicoverpa zea in transgenic maize.

PubMed

Johnson, Eric T; Berhow, Mark A; Dowd, Patrick F

2007-04-18

Hi II maize (Zea mays) plants were engineered to express maize p1 cDNA, a Myb transcription factor, controlled by a putative silk specific promoter, for secondary metabolite production and corn earworm resistance. Transgene expression did not enhance silk color, but about half of the transformed plant silks displayed browning when cut, which indicated the presence of p1-produced secondary metabolites. Levels of maysin, a secondary metabolite with insect toxicity, were highest in newly emerged browning silks. The insect resistance of transgenic silks was also highest at emergence, regardless of maysin levels, which suggests that other unidentified p1-induced molecules likely contributed to larval mortality. Mean survivor weights of corn earworm larvae fed mature browning transgenic silks were significantly lower than weights of those fed mature nonbrowning transgenic silks. Some transgenic pericarps browned with drying and contained similar molecules found in pericarps expressing a dominant p1 allele, suggesting that the promoter may not be silk-specific.

Insulin/IGF-1 signaling mutants reprogram ER stress response regulators to promote longevity.

PubMed

Henis-Korenblit, Sivan; Zhang, Peichuan; Hansen, Malene; McCormick, Mark; Lee, Seung-Jae; Cary, Michael; Kenyon, Cynthia

2010-05-25

When unfolded proteins accumulate in the endoplasmic reticulum (ER), the unfolded protein response is activated. This ER stress response restores ER homeostasis by coordinating processes that decrease translation, degrade misfolded proteins, and increase the levels of ER-resident chaperones. Ribonuclease inositol-requiring protein-1 (IRE-1), an endoribonuclease that mediates unconventional splicing, and its target, the XBP-1 transcription factor, are key mediators of the unfolded protein response. In this study, we show that in Caenorhabditis elegans insulin/IGF-1 pathway mutants, IRE-1 and XBP-1 promote lifespan extension and enhance resistance to ER stress. We show that these effects are not achieved simply by increasing the level of spliced xbp-1 mRNA and expression of XBP-1's normal target genes. Instead, in insulin/IGF-1 pathway mutants, XBP-1 collaborates with DAF-16, a FOXO-transcription factor that is activated in these mutants, to enhance ER stress resistance and to activate new genes that promote longevity.

Bacillus subtilis δ Factor Functions as a Transcriptional Regulator by Facilitating the Open Complex Formation.

PubMed

Prajapati, Ranjit Kumar; Sengupta, Shreya; Rudra, Paulami; Mukhopadhyay, Jayanta

2016-01-15

Most bacterial RNA polymerases (RNAP) contain five conserved subunits, viz. 2α, β, β', and ω. However, in many Gram-positive bacteria, especially in fermicutes, RNAP is associated with an additional factor, called δ. For over three decades since its identification, it had been thought that δ functioned as a subunit of RNAP to enhance the level of transcripts by recycling RNAP. In support of the previous observations, we also find that δ is involved in recycling of RNAP by releasing the RNA from the ternary complex. We further show that δ binds to RNA and is able to recycle RNAP when the length of the nascent RNA reaches a critical length. However, in this work we decipher a new function of δ. Performing biochemical and mutational analysis, we show that Bacillus subtilis δ binds to DNA immediately upstream of the promoter element at A-rich sequences on the abrB and rrnB1 promoters and facilitates open complex formation. As a result, δ facilitates RNAP to initiate transcription in the second scale, compared with minute scale in the absence of δ. Using transcription assay, we show that δ-mediated recycling of RNAP cannot be the sole reason for the enhancement of transcript yield. Our observation that δ does not bind to RNAP holo enzyme but is required to bind to DNA upstream of the -35 promoter element for transcription activation suggests that δ functions as a transcriptional regulator. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Constitutive expression of a salinity-induced wheat WRKY transcription factor enhances salinity and ionic stress tolerance in transgenic Arabidopsis thaliana.

PubMed

Qin, Yuxiang; Tian, Yanchen; Han, Lu; Yang, Xinchao

2013-10-25

The isolation and characterization of TaWRKY79, a wheat class II WRKY transcription factor, is described. Its 1297 bp coding region includes a 987 bp long open reading frame. TaWRKY79 was induced by stressing seedlings with either NaCl or abscisic acid (ABA). When a fusion between an 843 bp segment upstream of the TaWRKY79 coding sequence and GUS was introduced into Arabidopsis thaliana, GUS staining indicated that this upstream segment captured the sequence(s) required to respond to ABA or NaCl treatment. When TaWRKY79 was constitutively expressed as a transgene in A. thaliana, the transgenic plants showed an improved capacity to extend their primary root in the presence of either 100 mM NaCl, 10 mM LiCl or 2 μM ABA. The inference was that TaWRKY79 enhanced the level of tolerance to both salinity and ionic stress, while reducing the level of sensitivity to ABA. The ABA-related genes ABA1, ABA2 ABI1 and ABI5 were all up-regulated in the TaWRKY79 transgenic plants, suggesting that the transcription factor operates in an ABA-dependent pathway. Copyright © 2013. Published by Elsevier Inc.

Exaptation of Transposable Elements into Novel Cis-Regulatory Elements: Is the Evidence Always Strong?

PubMed Central

de Souza, Flávio S.J.; Franchini, Lucía F.; Rubinstein, Marcelo

2013-01-01

Transposable elements (TEs) are mobile genetic sequences that can jump around the genome from one location to another, behaving as genomic parasites. TEs have been particularly effective in colonizing mammalian genomes, and such heavy TE load is expected to have conditioned genome evolution. Indeed, studies conducted both at the gene and genome levels have uncovered TE insertions that seem to have been co-opted—or exapted—by providing transcription factor binding sites (TFBSs) that serve as promoters and enhancers, leading to the hypothesis that TE exaptation is a major factor in the evolution of gene regulation. Here, we critically review the evidence for exaptation of TE-derived sequences as TFBSs, promoters, enhancers, and silencers/insulators both at the gene and genome levels. We classify the functional impact attributed to TE insertions into four categories of increasing complexity and argue that so far very few studies have conclusively demonstrated exaptation of TEs as transcriptional regulatory regions. We also contend that many genome-wide studies dealing with TE exaptation in recent lineages of mammals are still inconclusive and that the hypothesis of rapid transcriptional regulatory rewiring mediated by TE mobilization must be taken with caution. Finally, we suggest experimental approaches that may help attributing higher-order functions to candidate exapted TEs. PMID:23486611

Enhancement of cordyceps polysaccharide production via biosynthetic pathway analysis in Hirsutella sinensis.

PubMed

Lin, Shan; Liu, Zhi-Qiang; Baker, Peter James; Yi, Ming; Wu, Hui; Xu, Feng; Teng, Yi; Zheng, Yu-Guo

2016-11-01

The addition of various sulfates for enhanced cordyceps polysaccharide (CP) production in submerged cultivation of H. sinensis was investigated, and manganese sulfate was found the most effective. 2mM of manganese sulfate on 0day (d) was investigated as the optimal adding condition, and the CP production reached optimum with 5.33%, increasing by 93.3% compared with the control. Furthermore, the consumption of three main precursors of CP was studied over cultivation under two conditions. Intracellular mannose content decreased by 43.1% throughout 6days cultivation, which corresponded to CP accumulation rate sharply increased from 0 d to 6 d, and mannose was considered as the most preferred precursor for generating CP. Subsequently, mannose biosynthetic pathway was constructed and verified for the first time in H. sinensis, which constituted the important part of CP biosynthesis, and transcriptional levels of the biosynthetic genes were studied. Transcriptional level of gene cpsA was significantly up-regulated 5.35-fold and it was a key gene involved both in mannose and CP biosynthesis. This study demonstrated that manganese sulfate addition is an efficient and simple way to improve CP production. Transcriptional analysis based on biosynthetic pathway was helpful to find key genes and better understand CP biosynthesis. Copyright © 2016 Elsevier B.V. All rights reserved.

A Novel Method for Gene-Specific Enhancement of Protein Translation by Targeting 5’UTRs of Selected Tumor Suppressors

PubMed Central

Master, Adam; Wójcicka, Anna; Giżewska, Kamilla; Popławski, Piotr; Williams, Graham R.; Nauman, Alicja

2016-01-01

Background Translational control is a mechanism of protein synthesis regulation emerging as an important target for new therapeutics. Naturally occurring microRNAs and synthetic small inhibitory RNAs (siRNAs) are the most recognized regulatory molecules acting via RNA interference. Surprisingly, recent studies have shown that interfering RNAs may also activate gene transcription via the newly discovered phenomenon of small RNA-induced gene activation (RNAa). Thus far, the small activating RNAs (saRNAs) have only been demonstrated as promoter-specific transcriptional activators. Findings We demonstrate that oligonucleotide-based trans-acting factors can also specifically enhance gene expression at the level of protein translation by acting at sequence-specific targets within the messenger RNA 5’-untranslated region (5’UTR). We designed a set of short synthetic oligonucleotides (dGoligos), specifically targeting alternatively spliced 5’UTRs in transcripts expressed from the THRB and CDKN2A suppressor genes. The in vitro translation efficiency of reporter constructs containing alternative TRβ1 5’UTRs was increased by up to more than 55-fold following exposure to specific dGoligos. Moreover, we found that the most folded 5’UTR has higher translational regulatory potential when compared to the weakly folded TRβ1 variant. This suggests such a strategy may be especially applied to enhance translation from relatively inactive transcripts containing long 5’UTRs of complex structure. Significance This report represents the first method for gene-specific translation enhancement using selective trans-acting factors designed to target specific 5’UTR cis-acting elements. This simple strategy may be developed further to complement other available methods for gene expression regulation including gene silencing. The dGoligo-mediated translation-enhancing approach has the potential to be transferred to increase the translation efficiency of any suitable target gene and may have future application in gene therapy strategies to enhance expression of proteins including tumor suppressors. PMID:27171412

Resveratrol enhances exercise training responses in rats selectively bred for high running performance.

PubMed

Hart, Nikolett; Sarga, Linda; Csende, Zsolt; Koltai, Erika; Koch, Lauren G; Britton, Steven L; Davies, Kelvin J A; Kouretas, Dimitris; Wessner, Barbara; Radak, Zsolt

2013-11-01

High Capacity Runner (HCR) rats have been developed by divergent artificial selection for treadmill endurance running capacity to explore an aerobic biology-disease connection. The beneficial effects of resveratrol supplementation have been demonstrated in endurance running and the antioxidant capacity of resveratrol is also demonstrated. In this study we examine whether 12 weeks of treadmill exercise training and/or resveratrol can enhance performance in HCR. Indeed, resveratrol increased aerobic performance and strength of upper limbs of these rats. Moreover, we have found that resveratrol activated the AMP-activated protein kinase, SIRT1, and mitochondrial transcription factor A (p<0.05). The changes in mitochondrial fission/fusion and Lon protease/HSP78 levels suggest that exercise training does not significantly induce damage of proteins. Moreover, neither exercise training nor resveratrol supplementation altered the content of protein carbonyls. Changes in the levels of forkhead transcription factor 1 and SIRT4 could suggest increased fat utilization and improved insulin sensitivity. These data indicate, that resveratrol supplementation enhances aerobic performance due to the activation of the AMPK-SIRT1-PGC-1α pathway. Copyright © 2013 Elsevier Ltd. All rights reserved.

Super-lncRNAs: identification of lncRNAs that target super-enhancers via RNA:DNA:DNA triplex formation.

PubMed

Soibam, Benjamin

2017-11-01

Super-enhancers are characterized by high levels of Mediator binding and are major contributors to the expression of their associated genes. They exhibit high levels of local chromatin interactions and a higher order of local chromatin organization. On the other hand, lncRNAs can localize to specific DNA sites by forming a RNA:DNA:DNA triplex, which in turn can contribute to local chromatin organization. In this paper, we characterize a new class of lncRNAs called super-lncRNAs that target super-enhancers and which can contribute to the local chromatin organization of the super-enhancers. Using a logistic regression model based on the number of RNA:DNA:DNA triplex sites a lncRNA forms within the super-enhancer, we identify 442 unique super-lncRNA transcripts in 27 different human cell and tissue types; 70% of these super-lncRNAs were tissue restricted. They primarily harbor a single triplex-forming repeat domain, which forms an RNA:DNA:DNA triplex with multiple anchor DNA sites (originating from transposable elements) within the super-enhancers. Super-lncRNAs can be grouped into 17 different clusters based on the tissue or cell lines they target. Super-lncRNAs in a particular cluster share common short structural motifs and their corresponding super-enhancer targets are associated with gene ontology terms pertaining to the tissue or cell line. Super-lncRNAs may use these structural motifs to recruit and transport necessary regulators (such as transcription factors and Mediator complexes) to super-enhancers, influence chromatin organization, and act as spatial amplifiers for key tissue-specific genes associated with super-enhancers. © 2017 Soibam; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

The Bphi008a gene interacts with the ethylene pathway and transcriptionally regulates MAPK genes in the response of rice to brown planthopper feeding.

PubMed

Hu, Jing; Zhou, Jiangbo; Peng, Xinxin; Xu, Henghao; Liu, Caixiang; Du, Bo; Yuan, Hongyu; Zhu, Lili; He, Guangcun

2011-06-01

We examined ways in which the Brown planthopper induced008a (Bphi008a; AY256682) gene of rice (Oryza sativa) enhances the plant's resistance to a specialist herbivore, the brown planthopper (BPH; Nilaparvata lugens). Measurement of the expression levels of ethylene synthases and of ethylene emissions showed that BPH feeding rapidly initiated the ethylene signaling pathway and up-regulated Bphi008a transcript levels after 6 to 96 h of feeding. In contrast, blocking ethylene transduction (using 1-methylcyclopropene) reduced Bphi008a transcript levels in wild-type plants fed upon by BPH. In vitro kinase assays showed that Bphi008a can be phosphorylated by rice Mitogen-activated Protein Kinase5 (OsMPK5), and yeast two-hybrid assays demonstrated that the carboxyl-terminal proline-rich region of Bphi008a interacts directly with this kinase. Furthermore, bimolecular fluorescence complementation assays showed that this interaction occurs in the nucleus. Subsequently, we found that Bphi008a up-regulation and down-regulation were accompanied by different changes in transcription levels of OsMPK5, OsMPK12, OsMPK13, and OsMPK17 in transgenic plants. Immunoblot analysis also showed that the OsMPK5 protein level increased in overexpressing plants and decreased in RNA interference plants after BPH feeding. In transgenic lines, changes in the expression levels of several enzymes that are important components of the defenses against the BPH were also observed. Finally, yeast two-hybrid screening results showed that Bphi008a is able to interact with a b-ZIP transcription factor (OsbZIP60) and a RNA polymerase polypeptide (SDRP).

The Bphi008a Gene Interacts with the Ethylene Pathway and Transcriptionally Regulates MAPK Genes in the Response of Rice to Brown Planthopper Feeding1[C][W][OA

PubMed Central

Hu, Jing; Zhou, Jiangbo; Peng, Xinxin; Xu, Henghao; Liu, Caixiang; Du, Bo; Yuan, Hongyu; Zhu, Lili; He, Guangcun

2011-01-01

We examined ways in which the Brown planthopper induced008a (Bphi008a; AY256682) gene of rice (Oryza sativa) enhances the plant’s resistance to a specialist herbivore, the brown planthopper (BPH; Nilaparvata lugens). Measurement of the expression levels of ethylene synthases and of ethylene emissions showed that BPH feeding rapidly initiated the ethylene signaling pathway and up-regulated Bphi008a transcript levels after 6 to 96 h of feeding. In contrast, blocking ethylene transduction (using 1-methylcyclopropene) reduced Bphi008a transcript levels in wild-type plants fed upon by BPH. In vitro kinase assays showed that Bphi008a can be phosphorylated by rice Mitogen-activated Protein Kinase5 (OsMPK5), and yeast two-hybrid assays demonstrated that the carboxyl-terminal proline-rich region of Bphi008a interacts directly with this kinase. Furthermore, bimolecular fluorescence complementation assays showed that this interaction occurs in the nucleus. Subsequently, we found that Bphi008a up-regulation and down-regulation were accompanied by different changes in transcription levels of OsMPK5, OsMPK12, OsMPK13, and OsMPK17 in transgenic plants. Immunoblot analysis also showed that the OsMPK5 protein level increased in overexpressing plants and decreased in RNA interference plants after BPH feeding. In transgenic lines, changes in the expression levels of several enzymes that are important components of the defenses against the BPH were also observed. Finally, yeast two-hybrid screening results showed that Bphi008a is able to interact with a b-ZIP transcription factor (OsbZIP60) and a RNA polymerase polypeptide (SDRP). PMID:21487048

Transcriptional enhancer from milk protein genes

DOEpatents

Casperson, Gerald F.; Schmidhauser, Christian T.; Bissell, Mina J.

1999-01-01

The invention relates to novel enhancer nucleotide sequences which stimulate transcription of heterologous DNA in cells in culture. The enhancers are derived from major milk protein genes by the process of deletion mapping and functional analysis. The invention also relates to expression vectors containing the novel enhancers.

Integrating and mining the chromatin landscape of cell-type specificity using self-organizing maps.

PubMed

Mortazavi, Ali; Pepke, Shirley; Jansen, Camden; Marinov, Georgi K; Ernst, Jason; Kellis, Manolis; Hardison, Ross C; Myers, Richard M; Wold, Barbara J

2013-12-01

We tested whether self-organizing maps (SOMs) could be used to effectively integrate, visualize, and mine diverse genomics data types, including complex chromatin signatures. A fine-grained SOM was trained on 72 ChIP-seq histone modifications and DNase-seq data sets from six biologically diverse cell lines studied by The ENCODE Project Consortium. We mined the resulting SOM to identify chromatin signatures related to sequence-specific transcription factor occupancy, sequence motif enrichment, and biological functions. To highlight clusters enriched for specific functions such as transcriptional promoters or enhancers, we overlaid onto the map additional data sets not used during training, such as ChIP-seq, RNA-seq, CAGE, and information on cis-acting regulatory modules from the literature. We used the SOM to parse known transcriptional enhancers according to the cell-type-specific chromatin signature, and we further corroborated this pattern on the map by EP300 (also known as p300) occupancy. New candidate cell-type-specific enhancers were identified for multiple ENCODE cell types in this way, along with new candidates for ubiquitous enhancer activity. An interactive web interface was developed to allow users to visualize and custom-mine the ENCODE SOM. We conclude that large SOMs trained on chromatin data from multiple cell types provide a powerful way to identify complex relationships in genomic data at user-selected levels of granularity.

Integrating and mining the chromatin landscape of cell-type specificity using self-organizing maps

PubMed Central

Mortazavi, Ali; Pepke, Shirley; Jansen, Camden; Marinov, Georgi K.; Ernst, Jason; Kellis, Manolis; Hardison, Ross C.; Myers, Richard M.; Wold, Barbara J.

2013-01-01

We tested whether self-organizing maps (SOMs) could be used to effectively integrate, visualize, and mine diverse genomics data types, including complex chromatin signatures. A fine-grained SOM was trained on 72 ChIP-seq histone modifications and DNase-seq data sets from six biologically diverse cell lines studied by The ENCODE Project Consortium. We mined the resulting SOM to identify chromatin signatures related to sequence-specific transcription factor occupancy, sequence motif enrichment, and biological functions. To highlight clusters enriched for specific functions such as transcriptional promoters or enhancers, we overlaid onto the map additional data sets not used during training, such as ChIP-seq, RNA-seq, CAGE, and information on cis-acting regulatory modules from the literature. We used the SOM to parse known transcriptional enhancers according to the cell-type-specific chromatin signature, and we further corroborated this pattern on the map by EP300 (also known as p300) occupancy. New candidate cell-type-specific enhancers were identified for multiple ENCODE cell types in this way, along with new candidates for ubiquitous enhancer activity. An interactive web interface was developed to allow users to visualize and custom-mine the ENCODE SOM. We conclude that large SOMs trained on chromatin data from multiple cell types provide a powerful way to identify complex relationships in genomic data at user-selected levels of granularity. PMID:24170599

Gateways to the FANTOM5 promoter level mammalian expression atlas

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

Lizio, Marina; Harshbarger, Jayson; Shimoji, Hisashi

The FANTOM5 project investigates transcription initiation activities in more than 1,000 human and mouse primary cells, cell lines and tissues using CAGE. Based on manual curation of sample information and development of an ontology for sample classification, we assemble the resulting data into a centralized data resource (http://fantom.gsc.riken.jp/5/). In conclusion, this resource contains web-based tools and data-access points for the research community to search and extract data related to samples, genes, promoter activities, transcription factors and enhancers across the FANTOM5 atlas.

Gateways to the FANTOM5 promoter level mammalian expression atlas

DOE PAGES

Lizio, Marina; Harshbarger, Jayson; Shimoji, Hisashi; ...

2015-01-05

The FANTOM5 project investigates transcription initiation activities in more than 1,000 human and mouse primary cells, cell lines and tissues using CAGE. Based on manual curation of sample information and development of an ontology for sample classification, we assemble the resulting data into a centralized data resource (http://fantom.gsc.riken.jp/5/). In conclusion, this resource contains web-based tools and data-access points for the research community to search and extract data related to samples, genes, promoter activities, transcription factors and enhancers across the FANTOM5 atlas.

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

Zhao, Yu; Wang, Wenhui; Wang, Qi

Highlights: Black-Right-Pointing-Pointer 5-LOX is able to upregulate expression of NF-{kappa}B p65. Black-Right-Pointing-Pointer 5-LOX enhances nuclear translocation of NF-{kappa}B p65 via increasing p-I{kappa}B-{alpha} level. Black-Right-Pointing-Pointer 5-LOX stimulates transcriptional activity of NF-{kappa}B in hepatoma cells. Black-Right-Pointing-Pointer LTB4 activates transcriptional activity of NF-{kappa}B in hepatoma cells. -- Abstract: The issue that lipid metabolism enzyme and its metabolites regulate transcription factors in cancer cell is not fully understood. In this study, we first report that the lipid metabolism enzyme 5-Lipoxygenase (5-LOX) and its metabolite leukotriene B4 (LTB4) are capable of activating nuclear factor-{kappa}B (NF-{kappa}B) in hepatoma cells. We found that the treatment of MK886more » (an inhibitor of 5-LOX) or knockdown of 5-LOX was able to downregulate the expression of NF-{kappa}B p65 at the mRNA level and decreased the phosphorylation level of inhibitor {kappa}B{alpha} (I{kappa}B{alpha}) in the cytoplasm of hepatoma HepG2 or H7402 cells, which resulted in the decrease of the level of nuclear NF-{kappa}B p65. These were confirmed by immunofluorescence staining in HepG2 cell. Moreover, the above treatments were able to decrease the transcriptional activity of NF-{kappa}B in the cells. The LTB4, one of metabolites of 5-LOX, is responsible for 5-LOX-activated NF-{kappa}B in a dose-dependent manner. Thus, we conclude that the lipid metabolism enzyme 5-LOX and its metabolite LTB4 are capable of activating transcription factor NF-{kappa}B in hepatoma cells. Our finding provides new insight into the significance of lipid metabolism in activation of transcription factors in cancer.« less

Transcriptional profile of genes involved in ascorbate glutathione cycle in senescing leaves for an early senescence leaf (esl) rice mutant.

PubMed

Li, Zhaowei; Su, Da; Lei, Bingting; Wang, Fubiao; Geng, Wei; Pan, Gang; Cheng, Fangmin

2015-03-15

To clarify the complex relationship between ascorbate-glutathione (AsA-GSH) cycle and H2O2-induced leaf senescence, the genotype-dependent difference in some senescence-related physiological parameters and the transcript levels and the temporal patterns of genes involved in the AsA-GSH cycle during leaf senescence were investigated using two rice genotypes, namely, the early senescence leaf (esl) mutant and its wild type. Meanwhile, the triggering effect of exogenous H2O2 on the expression of OsAPX genes was examined using detached leaves. The results showed that the esl mutant had higher H2O2 level than its wild type at the initial stage of leaf senescence. At transcriptional level, the association of expression of various genes involved in the AsA-GSH cycle with leaf senescence was isoform dependent. For OsAPXs, the transcripts of two cytosolic OsAPX genes (OsAPX1 and OsAPX2), thylakoid-bound OsAPX8, chloroplastic OsAPX7 and peroxisomal OsAPX4 exhibited remarkable genotype-dependent variation in their expression levels and temporal patterns during leaf senescence, there were significantly increasing transcripts of OsAXP1 and OsAPX7, severely repressed transcripts of OsAPX4 and OsAPX8 for the esl rice at the initial leaf senescence. In contrast, the repressing transcript of OsAPX8 was highly sensitive to the increasing H2O2 level in the senescing rice leaves, while higher H2O2 concentration resulted in the enhancing transcripts of two cytosolic OsAPX genes, OsAPX7 transcript was greatly variable with different H2O2 concentrations and incubating duration, suggesting that the different OsAPXs isoforms played a complementary role in perceiving and scavenging H2O2 accumulation at various H2O2 concentrations during leaf senescence. Higher H2O2 level, increased AsA level, higher activities of APX and glutathione reductase (GR), and relatively stable GSH content during the entire sampling period in the leaves of esl mutant implied that a close interrelationship existed between AsA level and APX activity in the ongoing senescence of rice leaves. The GSH supply in rice leaves was not the limiting factor for the efficient maintenance of AsA-GSH cycle, despite the senescence-related change in GR activity between the two rice genotypes. Copyright © 2014 Elsevier GmbH. All rights reserved.

Enhanced levels of scrapie responsive gene mRNA in BSE-infected mouse brain.

PubMed

Dandoy-Dron, F; Benboudjema, L; Guillo, F; Jaegly, A; Jasmin, C; Dormont, D; Tovey, M G; Dron, M

2000-03-10

The expression of the mRNA of nine scrapie responsive genes was analyzed in the brains of FVB/N mice infected with bovine spongiform encephalopathy (BSE). The RNA transcripts of eight genes were overexpressed to a comparable extent in both BSE-infected and scrapie-infected mice, indicating a common series of pathogenic events in the two transmissible spongiform encephalopathies (TSEs). In contrast, the serine proteinase inhibitor spi 2, an analogue of the human alpha-1 antichymotrypsin gene, was overexpressed to a greater extent in the brains of scrapie-infected animals than in animals infected with BSE, reflecting either an agent specific or a mouse strain specific response. The levels of spi 2 mRNA were increased during the course of scrapie prior to the onset of clinical signs of the disease and the increase reached 11 to 45 fold relative to uninfected controls in terminally ill mice. Spi 2, in common with four of the other scrapie responsive genes studied, is known to be associated with pro-inflammatory processes. These observations underline the importance of cell reactivity in TSE. In addition, scrg2 mRNA the level of which is enhanced in TSE-infected mouse brain, was identified as a previously unrecognized long transcript of the murine aldolase C gene. However, the level of the principal aldolase C mRNA is unaffected in TSE. The increased representation of the longer transcript in the late stage of the disease may reflect changes in mRNA processing and/or stability in reactive astrocytes or in damaged Purkinje cells.

Stimulation of d- and l-lactate dehydrogenases transcriptional levels in presence of diammonium hydrogen phosphate resulting to enhanced lactic acid production by Lactobacillus strain.

PubMed

Singhvi, Mamata; Zendo, Takeshi; Iida, Hiroshi; Gokhale, Digambar; Sonomoto, Kenji

2017-12-01

The present study revealed the effect of nitrogen sources on lactic acid production and stimulation of d- and l-lactate dehydrogenases (LDH) of parent Lactobacillus lactis NCIM 2368 and its mutant RM2-24 generated after UV mutagenesis. Both the parent and mutant strains were evaluated for d-lactic acid production in control and modified media. The modified media did not show remarkable effect on lactic acid production in case of parent whereas mutant exhibited significant enhancement in d-lactic acid production along with the appearance of l-lactic acid in the broth. Both LDH activities and specific activities were found to be higher in mutant than the parent strain. These results suggested that the diammonium hydrogen phosphate in modified media triggered the expression of LDH genes leading to enhanced lactic acid production. This observation has been proved by studying the expression levels of d- and l-LDH genes of parent and mutant in control and modified media using quantitative RT-PCR technique. In case of mutant, the transcriptional levels of d-LDH and l-LDH increased ∼17 fold and ∼1.38 fold respectively in modified medium compared to the values obtained with control medium. In case of parent, no significant change in transcriptional levels of d- and l-LDH was found when the cells were grown in either control medium or modified medium. This study suggested that the mutant, RM2-24 has l-LDH gene which is expressed in presence of (NH 4 ) 2 HPO 4 resulting in l-lactic acid production. Co-production of l-lactic acid in d-lactic acid fermentation may be detrimental in the PLA production. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Drosophila mini-white model system: new insights into positive position effects and the role of transcriptional terminators and gypsy insulator in transgene shielding

PubMed Central

Silicheva, Margarita; Golovnin, Anton; Pomerantseva, Ekaterina; Parshikov, Aleksander; Georgiev, Pavel; Maksimenko, Oksana

2010-01-01

The white gene, which is responsible for eye pigmentation, is widely used to study position effects in Drosophila. As a result of insertion of P-element vectors containing mini-white without enhancers into random chromosomal sites, flies with different eye color phenotypes appear, which is usually explained by the influence of positive/negative regulatory elements located around the insertion site. We found that, in more than 70% of cases when mini-white expression was subject to positive position effects, deletion of the white promoter had no effect on eye pigmentation; in these cases, the transposon was inserted into the transcribed regions of genes. Therefore, transcription through the mini-white gene could be responsible for high levels of its expression in most of chromosomal sites. Consistently with this conclusion, transcriptional terminators proved to be efficient in protecting mini-white expression from positive position effects. On the other hand, the best characterized Drosophila gypsy insulator was poorly effective in terminating transcription and, as a consequence, only partially protected mini-white expression from these effects. Thus, to ensure maximum protection of a transgene from position effects, a perfect boundary/insulator element should combine three activities: to block enhancers, to provide a barrier between active and repressed chromatin, and to terminate transcription. PMID:19854952

E2F mediates enhanced alternative polyadenylation in proliferation

PubMed Central

2012-01-01

Background The majority of mammalian genes contain multiple poly(A) sites in their 3' UTRs. Alternative cleavage and polyadenylation are emerging as an important layer of gene regulation as they generate transcript isoforms that differ in their 3' UTRs, thereby modulating genes' response to 3' UTR-mediated regulation. Enhanced cleavage at 3' UTR proximal poly(A) sites resulting in global 3' UTR shortening was recently linked to proliferation and cancer. However, mechanisms that regulate this enhanced alternative polyadenylation are unknown. Results Here, we explored, on a transcriptome-wide scale, alternative polyadenylation events associated with cellular proliferation and neoplastic transformation. We applied a deep-sequencing technique for identification and quantification of poly(A) sites to two human cellular models, each examined under proliferative, arrested and transformed states. In both cell systems we observed global 3' UTR shortening associated with proliferation, a link that was markedly stronger than the association with transformation. Furthermore, we found that proliferation is also associated with enhanced cleavage at intronic poly(A) sites. Last, we found that the expression level of the set of genes that encode for 3'-end processing proteins is globally elevated in proliferation, and that E2F transcription factors contribute to this regulation. Conclusions Our results comprehensively identify alternative polyadenylation events associated with cellular proliferation and transformation, and demonstrate that the enhanced alternative polyadenylation in proliferative conditions results not only in global 3' UTR shortening but also in enhanced premature cleavage in introns. Our results also indicate that E2F-mediated co-transcriptional regulation of 3'-end processing genes is one of the mechanisms that links enhanced alternative polyadenylation to proliferation. PMID:22747694

Onset and organ specificity of Tk2 deficiency depends on Tk1 down-regulation and transcriptional compensation.

PubMed

Dorado, Beatriz; Area, Estela; Akman, Hasan O; Hirano, Michio

2011-01-01

Deficiency of thymidine kinase 2 (TK2) is a frequent cause of isolated myopathy or encephalomyopathy in children with mitochondrial DNA (mtDNA) depletion. To determine the bases of disease onset, organ specificity and severity of TK2 deficiency, we have carefully characterized Tk2 H126N knockin mice (Tk2-/-). Although normal until postnatal day 8, Tk2-/- mice rapidly develop fatal encephalomyopathy between postnatal days 10 and 13. We have observed that wild-type Tk2 activity is constant in the second week of life, while Tk1 activity decreases significantly between postnatal days 8 and 13. The down-regulation of Tk1 activity unmasks Tk2 deficiency in Tk2-/- mice and correlates with the onset of mtDNA depletion in the brain and the heart. Resistance to pathology in Tk2 mutant organs depends on compensatory mechanisms to the reduced mtDNA level. Our analyses at postnatal day 13 have revealed that Tk2-/- heart significantly increases mitochondrial transcript levels relative to the mtDNA content. This transcriptional compensation allows the heart to maintain normal levels of mtDNA-encoded proteins. The up-regulation in mitochondrial transcripts is not due to increased expression of the master mitochondrial biogenesis regulators peroxisome proliferator-activated receptor-gamma coactivator 1 alpha and nuclear respiratory factors 1 and 2, or to enhanced expression of the mitochondrial transcription factors A, B1 or B2. Instead, Tk2-/- heart compensates for mtDNA depletion by down-regulating the expression of the mitochondrial transcriptional terminator transcription factor 3 (MTERF3). Understanding the molecular mechanisms that allow Tk2 mutant organs to be spared may help design therapies for Tk2 deficiency.

Onset and organ specificity of Tk2 deficiency depends on Tk1 down-regulation and transcriptional compensation

PubMed Central

Dorado, Beatriz; Area, Estela; Akman, Hasan O.; Hirano, Michio

2011-01-01

Deficiency of thymidine kinase 2 (TK2) is a frequent cause of isolated myopathy or encephalomyopathy in children with mitochondrial DNA (mtDNA) depletion. To determine the bases of disease onset, organ specificity and severity of TK2 deficiency, we have carefully characterized Tk2 H126N knockin mice (Tk2−/−). Although normal until postnatal day 8, Tk2−/− mice rapidly develop fatal encephalomyopathy between postnatal days 10 and 13. We have observed that wild-type Tk2 activity is constant in the second week of life, while Tk1 activity decreases significantly between postnatal days 8 and 13. The down-regulation of Tk1 activity unmasks Tk2 deficiency in Tk2−/− mice and correlates with the onset of mtDNA depletion in the brain and the heart. Resistance to pathology in Tk2 mutant organs depends on compensatory mechanisms to the reduced mtDNA level. Our analyses at postnatal day 13 have revealed that Tk2−/− heart significantly increases mitochondrial transcript levels relative to the mtDNA content. This transcriptional compensation allows the heart to maintain normal levels of mtDNA-encoded proteins. The up-regulation in mitochondrial transcripts is not due to increased expression of the master mitochondrial biogenesis regulators peroxisome proliferator-activated receptor-gamma coactivator 1 alpha and nuclear respiratory factors 1 and 2, or to enhanced expression of the mitochondrial transcription factors A, B1 or B2. Instead, Tk2−/− heart compensates for mtDNA depletion by down-regulating the expression of the mitochondrial transcriptional terminator transcription factor 3 (MTERF3). Understanding the molecular mechanisms that allow Tk2 mutant organs to be spared may help design therapies for Tk2 deficiency. PMID:20940150

Cloning, tissue distribution and effects of fasting on pituitary adenylate cyclase-activating polypeptide in largemouth bass

NASA Astrophysics Data System (ADS)

Li, Shengjie; Han, Linqiang; Bai, Junjie; Ma, Dongmei; Quan, Yingchun; Fan, Jiajia; Jiang, Peng; Yu, Lingyun

2015-03-01

Pituitary adenylate cyclase activating polypeptide (PACAP) has a wide range of biological functions. We cloned the full-length cDNAs encoding PACAP and PACAP-related peptide (PRP) from the brain of largemouth bass ( Micropterus salmoides) and used real-time quantitative PCR to detect PRP-PACAP mRNA expression. The PRP-PACAP cDNA has two variants expressed via alternative splicing: a long form, which encodes both PRP and PACAP, and a short form, which encodes only PACAP. Sequence analysis results are consistent with a higher conservation of PACAP than PRP peptide sequences. The expression of PACAP-long and PACAP-short transcripts was highest in the forebrain, followed by the medulla, midbrain, pituitary, stomach, cerebellum, intestine, and kidney; however, these transcripts were either absent or were weakly expressed in the muscle, spleen, gill, heart, fatty tissue, and liver. The level of PACAP-short transcript expression was significantly higher than expression of the long transcript in the forebrain, cerebella, pituitary and intestine, but lower than that of the long transcript in the stomach. PACAP-long and PACAP-short transcripts were first detected at the blastula stage of embryogenesis, and the level of expression increased markedly between the muscular contraction stage and 3 d post hatch (dph). The expression of PACAP-long and PACAP-short transcripts decreased significantly in the brain following 4 d fasting compared with the control diet group. The down-regulation effect was enhanced as fasting continued. Conversely, expression levels increased significantly after 3 d of re-feeding. Our results suggest that PRP-PACAP acts as an important factor in appetite regulation in largemouth bass.

C3 exoenzyme impairs cell proliferation and apoptosis by altering the activity of transcription factors.

PubMed

von Elsner, Leonie; Hagemann, Sandra; Just, Ingo; Rohrbeck, Astrid

2016-09-01

C3 exoenzyme from C. botulinum is an ADP-ribosyltransferase that inactivates selectively RhoA, B, and C by coupling an ADP-ribose moiety. Rho-GTPases are involved in various cellular processes, such as regulation of actin cytoskeleton, cell proliferation, and apoptosis. Previous studies of our group with the murine hippocampal cell line HT22 revealed a C3-mediated inhibition of cell proliferation after 48 h and a prevention of serum-starved cells from apoptosis. For both effects, alterations of various signaling pathways are already known, including also changes on the transcriptional level. Investigations on the transcriptional activity in HT22 cells treated with C3 for 48 h identified five out of 48 transcription factors namely Sp1, ATF2, E2F-1, CBF, and Stat6 with a significantly regulated activity. For validation of identified transcription factors, studies on the protein level of certain target genes were performed. Western blot analyses exhibited an enhanced abundance of Sp1 target genes p21 and COX-2 as well as an increase in phosphorylation of c-Jun. In contrast, the level of p53 and apoptosis-inducing GADD153, a target gene of ATF2, was decreased. Our results reveal that C3 regulates the transcriptional activity of Sp1 and ATF2 resulting downstream in an altered protein abundance of various target genes. As the affected proteins are involved in the regulation of cell proliferation and apoptosis, thus the C3-mediated anti-proliferative and anti-apoptotic effects are consequences of the Rho-dependent alterations of the activity of certain transcriptional factors.

Student Transcript-Enhanced Placement Study (STEPS). Technical Report

ERIC Educational Resources Information Center

Willett, Terrence

2013-01-01

Prior research suggests that using high school transcripts may improve English and math placement accuracy at colleges. Between 2012 and 2013, the Research and Planning Group for California Community Colleges (RP Group) conducted the Student Transcript Enhanced Placement Study (STEPS) in partnership with the California Community Colleges…

Small Cell Lung Cancer Exhibits Frequent Inactivating Mutations in the Histone Methyltransferase KMT2D/MLL2: CALGB 151111 (Alliance)

PubMed Central

Augert, Arnaud; Zhang, Qing; Bates, Breanna; Cui, Min; Wang, Xiaofei; Wildey, Gary; Dowlati, Afshin; MacPherson, David

2017-01-01

Introduction SCLC is a lethal neuroendocrine tumor type that is highly prone to metastasis. There is an urgency to understand the mutated genes that promote SCLC, as there are no approved targeted therapies yet available. SCLC is rarely resected, limiting the number of samples available for genomic analyses of somatic mutations. Methods To identify potential driver mutations in human SCLC we sequenced the whole exomes of 18 primary SCLCs and seven cell lines along with matched normal controls. We extended these data by resequencing a panel of genes across 40 primary SCLCs and 48 cell lines. Results We report frequent mutations in the lysine methyltransferase 2D gene (KMT2D) (also known as MLL2), a key regulator of transcriptional enhancer function. KMT2D exhibited truncating nonsense/frameshift/splice site mutations in 8% of SCLC tumors and 17% of SCLC cell lines. We found that KMT2D mutation in human SCLC cell lines was associated with reduced lysine methyltransferase 2D protein levels and reduced monomethylation of histone H3 lysine 4, a mark associated with transcriptional enhancers. We also found mutations in other genes associated with transcriptional enhancer control, including CREB binding protein gene (CREBBP), E1A binding protein p300 gene (EP300), and chromodomain helicase DNA binding protein 7 gene (CHD7), and we report mutations in additional chromatin remodeling genes such as polybromo 1 gene (PBRM1). Conclusions These data indicate that KMT2D is one of the major mutated genes in SCLC, and they point to perturbation of transcriptional enhancer control as potentially contributing to SCLC. PMID:28007623

The transcription factor Grainy head primes epithelial enhancers for spatiotemporal activation by displacing nucleosomes.

PubMed

Jacobs, Jelle; Atkins, Mardelle; Davie, Kristofer; Imrichova, Hana; Romanelli, Lucia; Christiaens, Valerie; Hulselmans, Gert; Potier, Delphine; Wouters, Jasper; Taskiran, Ibrahim I; Paciello, Giulia; González-Blas, Carmen B; Koldere, Duygu; Aibar, Sara; Halder, Georg; Aerts, Stein

2018-06-04

Transcriptional enhancers function as docking platforms for combinations of transcription factors (TFs) to control gene expression. How enhancer sequences determine nucleosome occupancy, TF recruitment and transcriptional activation in vivo remains unclear. Using ATAC-seq across a panel of Drosophila inbred strains, we found that SNPs affecting binding sites of the TF Grainy head (Grh) causally determine the accessibility of epithelial enhancers. We show that deletion and ectopic expression of Grh cause loss and gain of DNA accessibility, respectively. However, although Grh binding is necessary for enhancer accessibility, it is insufficient to activate enhancers. Finally, we show that human Grh homologs-GRHL1, GRHL2 and GRHL3-function similarly. We conclude that Grh binding is necessary and sufficient for the opening of epithelial enhancers but not for their activation. Our data support a model positing that complex spatiotemporal expression patterns are controlled by regulatory hierarchies in which pioneer factors, such as Grh, establish tissue-specific accessible chromatin landscapes upon which other factors can act.

Epigenomics of macrophages

PubMed Central

Gosselin, David; Glass, Christopher K

2014-01-01

Summary Macrophages play essential roles in tissue homeostasis, pathogen elimination, and tissue repair. A defining characteristic of these cells is their ability to efficiently adapt to a variety of abruptly changing and complex environments. This ability is intrinsically linked to a capacity to quickly alter their transcriptome, and this is tightly associated with the epigenomic organization of these cells and, in particular, their enhancer repertoire. Indeed, enhancers are genomic sites that serve as platforms for the integration of signaling pathways with the mechanisms that regulate mRNA transcription. Notably, transcription is pervasive at active enhancers and enhancer RNAs (eRNAs) are tightly coupled to regulated transcription of protein-coding genes. Furthermore, given that each cell type possesses a defining enhancer repertoire, studies on enhancers provide a powerful method to study how specialization of functions among the diverse macrophage subtypes may arise. Here, we review recent studies providing insights into the distinct mechanisms that contribute to the establishment of enhancers and their role in the regulation of transcription in macrophages. PMID:25319330

E2F1 transcription is induced by genotoxic stress through ATM/ATR activation.

PubMed

Carcagno, Abel L; Ogara, María F; Sonzogni, Silvina V; Marazita, Mariela C; Sirkin, Pablo F; Ceruti, Julieta M; Cánepa, Eduardo T

2009-05-01

E2F1, a member of the E2F family of transcription factors, plays a critical role in controlling both cell cycle progression and apoptotic cell death in response to DNA damage and oncogene activation. Following genotoxic stresses, E2F1 protein is stabilized by phosphorylation and acetylation driven to its accumulation. The aim of the present work was to examine whether the increase in E2F1 protein levels observed after DNA damage is only a reflection of an increase in E2F1 protein stability or is also the consequence of enhanced transcription of the E2F1 gene. The data presented here demonstrates that UV light and other genotoxics induce the transcription of E2F1 gene in an ATM/ATR dependent manner, which results in increasing E2F1 mRNA and protein levels. After genotoxic stress, transcription of cyclin E, an E2F1 target gene, was significantly induced. This induction was the result of two well-differentiated effects, one of them dependent on de novo protein synthesis and the other on the protein stabilization. Our results strongly support a transcriptional effect of DNA damaging agents on E2F1 expression. The results presented herein uncover a new mechanism involving E2F1 in response to genotoxic stress.

Functional analysis of a WRKY transcription factor involved in transcriptional activation of the DBAT gene in Taxus chinensis.

PubMed

Li, S; Zhang, P; Zhang, M; Fu, C; Yu, L

2013-01-01

Although the regulation of taxol biosynthesis at the transcriptional level remains unclear, 10-deacetylbaccatin III-10 β-O-acetyl transferase (DBAT) is a critical enzyme in the biosynthesis of taxol. The 1740 bp fragment 5'-flanking sequence of the dbat gene was cloned from Taxus chinensis cells. Important regulatory elements needed for activity of the dbat promoter were located by deletion analyses in T. chinensis cells. A novel WRKY transcription factor, TcWRKY1, was isolated with the yeast one-hybrid system from a T. chinensis cell cDNA library using the important regulatory elements as bait. The gene expression of TcWRKY1 in T. chinensis suspension cells was specifically induced by methyl jasmonate (MeJA). Biochemical analysis indicated that TcWRKY1 protein specifically interacts with the two W-box (TGAC) cis-elements among the important regulatory elements. Overexpression of TcWRKY1 enhanced dbat expression in T. chinensis suspension cells, and RNA interference (RNAi) reduced the level of transcripts of dbat. These results suggest that TcWRKY1 participates in regulation of taxol biosynthesis in T. chinensis cells, and that dbat is a target gene of this transcription factor. This research also provides a potential candidate gene for engineering increased taxol accumulation in Taxus cell cultures. © 2012 German Botanical Society and The Royal Botanical Society of the Netherlands.

Gene Repression in Haloarchaea Using the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas I-B System.

PubMed

Stachler, Aris-Edda; Marchfelder, Anita

2016-07-15

The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system is used by bacteria and archaea to fend off foreign genetic elements. Since its discovery it has been developed into numerous applications like genome editing and regulation of transcription in eukaryotes and bacteria. For archaea currently no tools for transcriptional repression exist. Because molecular biology analyses in archaea become more and more widespread such a tool is vital for investigating the biological function of essential genes in archaea. Here we use the model archaeon Haloferax volcanii to demonstrate that its endogenous CRISPR-Cas system I-B can be harnessed to repress gene expression in archaea. Deletion of cas3 and cas6b genes results in efficient repression of transcription. crRNAs targeting the promoter region reduced transcript levels down to 8%. crRNAs targeting the reading frame have only slight impact on transcription. crRNAs that target the coding strand repress expression only down to 88%, whereas crRNAs targeting the template strand repress expression down to 8%. Repression of an essential gene results in reduction of transcription levels down to 22%. Targeting efficiencies can be enhanced by expressing a catalytically inactive Cas3 mutant. Genes can be targeted on plasmids or on the chromosome, they can be monocistronic or part of a polycistronic operon. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Gene Repression in Haloarchaea Using the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas I-B System*

PubMed Central

Stachler, Aris-Edda; Marchfelder, Anita

2016-01-01

The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system is used by bacteria and archaea to fend off foreign genetic elements. Since its discovery it has been developed into numerous applications like genome editing and regulation of transcription in eukaryotes and bacteria. For archaea currently no tools for transcriptional repression exist. Because molecular biology analyses in archaea become more and more widespread such a tool is vital for investigating the biological function of essential genes in archaea. Here we use the model archaeon Haloferax volcanii to demonstrate that its endogenous CRISPR-Cas system I-B can be harnessed to repress gene expression in archaea. Deletion of cas3 and cas6b genes results in efficient repression of transcription. crRNAs targeting the promoter region reduced transcript levels down to 8%. crRNAs targeting the reading frame have only slight impact on transcription. crRNAs that target the coding strand repress expression only down to 88%, whereas crRNAs targeting the template strand repress expression down to 8%. Repression of an essential gene results in reduction of transcription levels down to 22%. Targeting efficiencies can be enhanced by expressing a catalytically inactive Cas3 mutant. Genes can be targeted on plasmids or on the chromosome, they can be monocistronic or part of a polycistronic operon. PMID:27226589

The effect of centaurein on interferon-{gamma} expression and Listeria infection in mice

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

Chang, S.-L.; Department of Biological Science and Technology, National Chiao Tung University, 1001, Ta Hsueh Road, Hsinchu 300, Taiwan; Yeh, H.-H.

2007-02-15

We previously found that centaurein enhanced IFN-{gamma} transcription in T cells. Here, we demonstrate that centaurein increased the IFN-{gamma} expression in T and NK cells and the serum IFN-{gamma} level in mice. Centaurein elevated the transcription of T-bet but not GATA-3, which is consistent with its effect on that of IFN-{gamma} but not IL-4. Additionally, centaurein effectively protected mice against Listeria infection. Moreover, centaurein per se or in combination with antibiotics could treat Listeria infection. Our mechanistic studies suggest that centaurein augments IFN-{gamma} expression via a transcriptional up-regulation of T-bet and that centaurein protects against or treats Listeria infection viamore » a modulation of IFN-{gamma} expression.« less

Leptin upregulates telomerase activity and transcription of human telomerase reverse transcriptase in MCF-7 breast cancer cells

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

Ren, He, E-mail: herenrh@yahoo.com.cn; Zhao, Tiansuo; Wang, Xiuchao

2010-03-26

The aim was to analyze the mechanism of leptin-induced activity of telomerase in MCF-7 breast cancer cells. We found that leptin activated telomerase in a dose-dependent manner; leptin upregulated the expression of Human Telomerase Reverse Transcriptase (hTERT) at mRNA and protein levels; blockade of signal transducer and activator of transcription 3 (STAT3) phosphorylation significantly counteracted leptin-induced hTERT transcription and protein expression; chromatin immunoprecipitation analysis showed that leptin enhanced the binding of STAT3 to the hTERT promoter. This study uncovers a new mechanism of the proliferative effect of leptin on breast cancer cells and provides a new explanation of obesity-related breastmore » cancer.« less

Transcript level coordination of carbon pathways during silicon starvation-induced lipid accumulation in the diatom Thalassiosira pseudonana

DOE PAGES

Smith, Sarah R.; Gle, Corine; Abbriano, Raffaela M.; ...

2016-02-04

Diatoms are one of the most productive and successful photosynthetic taxa on Earth and possess attributes such as rapid growth rates and production of lipids, making them candidate sources of renewable fuels. Despite their significance, few details of the mechanisms used to regulate growth and carbon metabolism are currently known, hindering metabolic engineering approaches to enhance productivity. To characterize the transcript level component of metabolic regulation, genome-wide changes in transcript abundance were documented in the model diatom Thalassiosira pseudonana on a time-course of silicon starvation. Growth, cell cycle progression, chloroplast replication, fatty acid composition, pigmentation, and photosynthetic parameters were characterizedmore » alongside lipid accumulation. Extensive coordination of large suites of genes was observed, highlighting the existence of clusters of coregulated genes as a key feature of global gene regulation in T. pseudonana. The identity of key enzymes for carbon metabolic pathway inputs (photosynthesis) and outputs (growth and storage) reveals these clusters are organized to synchronize these processes. Coordinated transcript level responses to silicon starvation are probably driven by signals linked to cell cycle progression and shifts in photophysiology. A mechanistic understanding of how this is accomplished will aid efforts to engineer metabolism for development of algal-derived biofuels.« less

Pioneer factors govern super-enhancer dynamics in stem cell plasticity and lineage choice

PubMed Central

Adam, Rene C.; Yang, Hanseul; Rockowitz, Shira; Larsen, Samantha B.; Nikolova, Maria; Oristian, Daniel S.; Polak, Lisa; Kadaja, Meelis; Asare, Amma; Zheng, Deyou; Fuchs, Elaine

2015-01-01

Adult stem cells (SCs) reside in niches which balance self-renewal with lineage selection and progression during tissue homeostasis. Following injury, culture or transplantation, SCs outside their niche often display fate flexibility1-4. Here we show that super-enhancers5 underlie the identity, lineage commitment and plasticity of adult SCs in vivo. Using hair follicle (HF) as model, we map the global chromatin domains of HFSCs and their committed progenitors in their native microenvironments. We show that super-enhancers and their dense clusters (‘epicenters’) of transcription factor (TF) binding sites change upon lineage progression. New fate is acquired by decommissioning old and establishing new super-enhancers and/or epicenters, an auto-regulatory process that abates one master regulator subset while enhancing another. We further show that when outside their niche, either in vitro or in wound-repair, HFSCs dynamically remodel super-enhancers in response to changes in their microenvironment. Intriguingly, some key super-enhancers shift epicenters, enabling them to remain active and maintain a transitional state in an ever-changing transcriptional landscape. Finally, we identify SOX9 as a crucial chromatin rheostat of HFSC super-enhancers, and provide functional evidence that super-enhancers are dynamic, dense TF-binding platforms which are acutely sensitive to pioneer master regulators whose levels define not only spatial and temporal features of lineage-status, but also stemness, plasticity in transitional states and differentiation. PMID:25799994

Architectural and functional commonalities between enhancers and promoters

PubMed Central

Kim, Tae-Kyung; Shiekhattar, Ramin

2015-01-01

Summary With the explosion of genome-wide studies of regulated transcription, it has become clear that traditional definitions of enhancers and promoters need to be revisited. These control elements can now be characterized in terms of their local and regional architecture, their regulatory components including histone modifications and associated binding factors and their functional contribution to transcription. This review discusses unifying themes between promoters and enhancers in transcriptional regulatory mechanisms. PMID:26317464

Plant Enhancers: A Call for Discovery.

PubMed

Weber, Blaise; Zicola, Johan; Oka, Rurika; Stam, Maike

2016-11-01

Higher eukaryotes typically contain many different cell types, displaying different cellular functions that are influenced by biotic and abiotic cues. The different functions are characterized by specific gene expression patterns mediated by regulatory sequences such as transcriptional enhancers. Recent genome-wide approaches have identified thousands of enhancers in animals, reviving interest in enhancers in gene regulation. Although the regulatory roles of plant enhancers are as crucial as those in animals, genome-wide approaches have only very recently been applied to plants. Here we review characteristics of enhancers at the DNA and chromatin level in plants and other species, their similarities and differences, and techniques widely used for genome-wide discovery of enhancers in animal systems that can be implemented in plants. Copyright © 2016 Elsevier Ltd. All rights reserved.

Comparison of identical and functional Igh alleles reveals a nonessential role for Eμ in somatic hypermutation and class-switch recombination.

PubMed

Li, Fubin; Yan, Yi; Pieretti, Joyce; Feldman, Danielle A; Eckhardt, Laurel A

2010-11-15

Somatic hypermutation (SHM), coupled with Ag selection, provides a mechanism for generating Abs with high affinity for invading pathogens. Class-switch recombination (CSR) ensures that these Abs attain pathogen-appropriate effector functions. Although the enzyme critical to both processes, activation-induced cytidine deaminase, has been identified, it remains unclear which cis-elements within the Ig loci are responsible for recruiting activation-induced cytidine deaminase and promoting its activity. Studies showed that Ig gene-transcription levels are positively correlated with the frequency of SHM and CSR, making the intronic, transcriptional enhancer Eμ a likely contributor to both processes. Tests of this hypothesis yielded mixed results arising, in part, from the difficulty in studying B cell function in mice devoid of Eμ. In Eμ's absence, V(H) gene assembly is dramatically impaired, arresting B cell development. The current study circumvented this problem by modifying the murine Igh locus through simultaneous insertion of a fully assembled V(H) gene and deletion of Eμ. The behavior of this allele was compared with that of a matched allele carrying the same V(H) gene but with Eμ intact. Although IgH transcription was as great or greater on the Eμ-deficient allele, CSR and SHM were consistently, but modestly, reduced relative to the allele in which Eμ remained intact. We conclude that Eμ contributes to, but is not essential for, these complex processes and that its contribution is not as a transcriptional enhancer but, rather, is at the level of recruitment and/or activation of the SHM/CSR machinery.

RdreB1BI enhances drought tolerance by activating AQP-related genes in transgenic strawberry.

PubMed

Gu, Xianbin; Gao, Zhihong; Yan, Yichao; Wang, Xiuyun; Qiao, Yushan; Chen, Yahua

2017-10-01

The dehydration-responsive element binding protein (DREB) family of transcription factors is associated with abiotic stress responses during plant growth and development. This study focussed on the subfamily member DREB1B, which was initially described as highly and specifically responsive to low temperature. However, here it is shown that DREB1B is not only involved in cold tolerance but also other abiotic stress tolerances, such as that of drought. To further understand the genetic improvement effects of the drought tolerance provided by RdreB1BI in transgenic strawberry, drought stress responses of transgenic plants were evaluated at the morphological, physiological, and transcriptional levels. Transactivation assays revealed that RdreB1BI could activate the FvPIP2;1 like 1 promoter. RdreB1BI transgenic plants showed enhanced drought tolerance on the basis of lower rates of electrolyte leakage (EL), higher relative water content (RWC), and less stomatal aperture as well as increased peroxidase (POD) and superoxide dismutase (SOD) activities and less malondialdehyde (MDA) accumulation. The transgenic plants also accumulated higher levels of drought-related regulatory genes and functional gene transcripts, including those of PIP, NAC, RD22, ABI, and NCED. Together, these results demonstrate that RdreB1BI plays an essential role in the regulation of the drought stress response. DREB1B transcription constitutes a useful strategy to exploit in transgenic plants for coping with abiotic stresses, at least cold and drought stresses. The approach may be helpful for genetic engineering horticultural plants to have increased environmental adaptations. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Casein kinase 2 (CK2) increases survivin expression via enhanced β-catenin–T cell factor/lymphoid enhancer binding factor-dependent transcription

PubMed Central

Tapia, J. C.; Torres, V. A.; Rodriguez, D. A.; Leyton, L.; Quest, A. F. G.

2006-01-01

Increased expression of casein kinase 2 (CK2) is associated with hyperproliferation and suppression of apoptosis in cancer. Mutations in the tumor suppressor APC (adenomatous polyposis coli) are frequent in colon cancer and often augment β-catenin–T cell factor (Tcf)/lymphoid enhancer binding factor (Lef)-dependent transcription of genes such as c-myc and cyclin-D1. CK2 has also been implicated recently in the regulation of β-catenin stability. To identify mechanisms by which CK2 promotes survival, effects of the specific CK2 inhibitors 4,5,6,7-tetrabromobenzotriazole (TBB) and 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole were assessed. TBB and 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole significantly decreased proliferation and increased apoptosis of HT29(US) colon cancer cells. RT-PCR and immunoblot analysis revealed that both inhibitors decreased survivin mRNA and protein levels in HT29(US) cells. Similar effects were observed with TBB in human DLD-1 and SW-480 colorectal cells as well as ZR-75 breast cancer cells and HEK-293T embryonic kidney cells. Expression of GFP–CK2α in HEK-293T cells resulted in β-catenin–Tcf/Lef-dependent up-regulation of survivin and increased resistance to anticancer drugs. Augmented β-catenin–Tcf/Lef-dependent transcription and resistance to apoptosis observed upon GFP–CK2α expression were abolished by TBB. Alternatively, HEK-293T cells expressing GFP–survivin were resistant to TBB-induced apoptosis. Finally, siRNA-mediated down-regulation of CK2α in HEK-293T cells coincided with reduced β-catenin and survivin levels. Taken together, these results suggest that CK2 kinase activity promotes survival by increasing survivin expression via β-catenin–Tcf/Lef-mediated transcription. Hence, selective CK2 inhibition or down-regulation in tumors may provide an attractive opportunity for the development of novel cancer therapies. PMID:17005722

Osterix Controls Cementoblast Differentiation through Downregulation of Wnt-signaling via Enhancing DKK1 Expression

PubMed Central

Cao, Zhengguo; Liu, Rubing; Zhang, Hua; Liao, Haiqing; Zhang, Yufeng; Hinton, Robert J.; Feng, Jian Q.

2015-01-01

Osterix (Osx), a transcriptional factor essential for osteogenesis, is also critical for in vivo cellular cementum formation. However, the molecular mechanism by which Osx regulates cementoblasts is largely unknown. In this study, we initially demonstrated that overexpression of Osx in a cementoblast cell line upregulated the expression of markers vital to cementogenesis such as osteopontin (OPN), osteocalcin (OCN), and bone sialoprotein (BSP) at both mRNA and protein levels, and enhanced alkaline phosphatase (ALP) activity. Unexpectedly, we demonstrated a sharp increase in the expression of DKK1 (a potent canonical Wnt antagonist), and a great reduction in protein levels of β-catenin and its nuclear translocation by overexpression of Osx. Further, transient transfection of Osx reduced protein levels of TCF1 (a target transcription factor of β-catenin), which were partially reversed by an addition of DKK1. We also demonstrated that activation of canonical Wnt signaling by LiCl or Wnt3a significantly enhanced levels of TCF1 and suppressed the expression of OPN, OCN, and BSP, as well as ALP activity and formation of extracellular mineralized nodules. Importantly, we confirmed that there were a sharp reduction in DKK1 and a concurrent increase in β-catenin in Osx cKO mice (crossing between the Osx loxP and 2.3 Col 1-Cre lines), in agreement with the in vitro data. Thus, we conclude that the key role of Osx in control of cementoblast proliferation and differentiation is to maintain a low level of Wnt-β-catenin via direct up-regulation of DKK1. PMID:25678852

USF-related transcription factor, HIV-TF1, stimulates transcription of human immunodeficiency virus-1.

PubMed

Maekawa, T; Sudo, T; Kurimoto, M; Ishii, S

1991-09-11

The transcription factor HIV-TF1, which binds to a region about 60 bp upstream from the enhancer of the human immunodeficiency virus-1 (HIV-1), was purified from human B cells. HIV-TF1 had a molecular weight of 39,000. Binding of HIV-TF1 to the HIV long terminal repeat (LTR) activated transcription from the HIV promoter in vitro. The HIV-TF1-binding site in HIV LTR was similar to the site recognized by upstream stimulatory factor (USF) in the adenovirus major late promoter. DNA-binding properties of HIV-TF1 suggested that HIV-TF1 might be identical or related to USF. Interestingly, treatment of purified HIV-TF1 by phosphatase greatly reduced its DNA-binding activity, suggesting that phosphorylation of HIV-TF1 was essential for DNA binding. The disruption of HIV-TF1-binding site induced a 60% decrease in the level of transcription from the HIV promoter in vivo. These results suggest that HIV-TF1 is involved in transcriptional regulation of HIV-1.

Coordination of m6A mRNA methylation and gene transcription by ZFP217 regulates pluripotency and reprogramming

PubMed Central

Aguilo, Francesca; Zhang, Fan; Sancho, Ana; Fidalgo, Miguel; Di Cecilia, Serena; Vashisht, Ajay; Lee, Dung-Fang; Chen, Chih-Hung; Rengasamy, Madhumitha; Andino, Blanca; Jahouh, Farid; Roman, Angel; Krig, Sheryl R.; Wang, Rong; Zhang, Weijia; Wohlschlegel, James A.; Wang, Jianlong; Walsh, Martin J.

2015-01-01

SUMMARY Epigenetic and epitranscriptomic networks have important functions in maintaining pluripotency of embryonic stem cells (ESCs) and somatic cell reprogramming. However the mechanisms integrating the actions of these distinct networks are only partially understood. Here, we show that the chromatin-associated zinc finger protein 217 (ZFP217) coordinates epigenetic and epitranscriptomic regulation. ZFP217 interacts with several epigenetic regulators, activates transcription of key pluripotency genes, and modulates N6-methyladenosine (m6A) deposition on their transcripts by sequestering the enzyme m6A methyltransferase-like 3 (METTL3). Consistently, Zfp217 depletion compromises ESC self-renewal and somatic cell reprogramming, globally increases m6A RNA levels, and enhances m6A modification of Nanog, Sox2, Klf4, and c-Myc mRNAs, promoting their degradation. ZFP217 binds its own target gene mRNAs, which are also METTL3-associated, and is enriched at promoters of m6A-modified transcripts. Collectively, these findings shed light on how a transcription factor can tightly couple gene transcription to m6A RNA modification to insure ESC identity. PMID:26526723

Combinatorial influence of environmental parameters on transcription factor activity.

PubMed

Knijnenburg, T A; Wessels, L F A; Reinders, M J T

2008-07-01

Cells receive a wide variety of environmental signals, which are often processed combinatorially to generate specific genetic responses. Changes in transcript levels, as observed across different environmental conditions, can, to a large extent, be attributed to changes in the activity of transcription factors (TFs). However, in unraveling these transcription regulation networks, the actual environmental signals are often not incorporated into the model, simply because they have not been measured. The unquantified heterogeneity of the environmental parameters across microarray experiments frustrates regulatory network inference. We propose an inference algorithm that models the influence of environmental parameters on gene expression. The approach is based on a yeast microarray compendium of chemostat steady-state experiments. Chemostat cultivation enables the accurate control and measurement of many of the key cultivation parameters, such as nutrient concentrations, growth rate and temperature. The observed transcript levels are explained by inferring the activity of TFs in response to combinations of cultivation parameters. The interplay between activated enhancers and repressors that bind a gene promoter determine the possible up- or downregulation of the gene. The model is translated into a linear integer optimization problem. The resulting regulatory network identifies the combinatorial effects of environmental parameters on TF activity and gene expression. The Matlab code is available from the authors upon request. Supplementary data are available at Bioinformatics online.

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.

Archaeal TFEα/β is a hybrid of TFIIE and the RNA polymerase III subcomplex hRPC62/39

PubMed Central

Blombach, Fabian; Salvadori, Enrico; Fouqueau, Thomas; Yan, Jun; Reimann, Julia; Sheppard, Carol; Smollett, Katherine L; Albers, Sonja V; Kay, Christopher WM; Thalassinos, Konstantinos; Werner, Finn

2015-01-01

Transcription initiation of archaeal RNA polymerase (RNAP) and eukaryotic RNAPII is assisted by conserved basal transcription factors. The eukaryotic transcription factor TFIIE consists of α and β subunits. Here we have identified and characterised the function of the TFIIEβ homologue in archaea that on the primary sequence level is related to the RNAPIII subunit hRPC39. Both archaeal TFEβ and hRPC39 harbour a cubane 4Fe-4S cluster, which is crucial for heterodimerization of TFEα/β and its engagement with the RNAP clamp. TFEα/β stabilises the preinitiation complex, enhances DNA melting, and stimulates abortive and productive transcription. These activities are strictly dependent on the β subunit and the promoter sequence. Our results suggest that archaeal TFEα/β is likely to represent the evolutionary ancestor of TFIIE-like factors in extant eukaryotes. DOI: http://dx.doi.org/10.7554/eLife.08378.001 PMID:26067235

Regulation of transcriptional activators by DNA-binding domain ubiquitination

PubMed Central

Landré, Vivien; Revi, Bhindu; Mir, Maria Gil; Verma, Chandra; Hupp, Ted R; Gilbert, Nick; Ball, Kathryn L

2017-01-01

Ubiquitin is a key component of the regulatory network that maintains gene expression in eukaryotes, yet the molecular mechanism(s) by which non-degradative ubiquitination modulates transcriptional activator (TA) function is unknown. Here endogenous p53, a stress-activated transcription factor required to maintain health, is stably monoubiquitinated, following pathway activation by IR or Nutlin-3 and localized to the nucleus where it becomes tightly associated with chromatin. Comparative structure–function analysis and in silico modelling demonstrate a direct role for DNA-binding domain (DBD) monoubiquitination in TA activation. When attached to the DBD of either p53, or a second TA IRF-1, ubiquitin is orientated towards, and makes contact with, the DNA. The contact is made between a predominantly cationic surface on ubiquitin and the anionic DNA. Our data demonstrate an unexpected role for ubiquitin in the mechanism of TA-activity enhancement and provides insight into a new level of transcriptional regulation. PMID:28362432

Insulin Signaling Augments eIF4E-Dependent Nonsense-Mediated mRNA Decay in Mammalian Cells.

PubMed

Park, Jungyun; Ahn, Seyoung; Jayabalan, Aravinth K; Ohn, Takbum; Koh, Hyun Chul; Hwang, Jungwook

2016-07-01

Nonsense-mediated mRNA decay (NMD) modulates the level of mRNA harboring a premature termination codon (PTC) in a translation-dependent manner. Inhibition of translation is known to impair NMD; however, few studies have investigated the correlation between enhanced translation and increased NMD. Here, we demonstrate that insulin signaling events increase translation, leading to an increase in NMD of eIF4E-bound transcripts. We provide evidence that (i) insulin-mediated enhancement of translation augments NMD and rapamycin abrogates this enhancement; (ii) an increase in AKT phosphorylation due to inhibition of PTEN facilitates NMD; (iii) insulin stimulation increases the binding of up-frameshift factor 1 (UPF1), most likely to eIF4E-bound PTC-containing transcripts; and (iv) insulin stimulation induces the colocalization of UPF1 and eIF4E in processing bodies. These results illustrate how extracellular signaling promotes the removal of eIF4E-bound NMD targets. Copyright © 2015 Elsevier B.V. All rights reserved.

Epstein-Barr Virus oncoprotein super-enhancers control B cell growth

PubMed Central

Zhou, Hufeng; Schmidt, Stefanie CS; Jiang, Sizun; Willox, Bradford; Bernhardt, Katharina; Liang, Jun; Johannsen, Eric C; Kharchenko, Peter; Gewurz, Benjamin E; Kieff, Elliott; Zhao, Bo

2015-01-01

Summary Super-enhancers are clusters of gene-regulatory sites bound by multiple transcription factors that govern cell transcription, development, phenotype, and oncogenesis. By examining Epstein-Barr virus (EBV) transformed lymphoblastoid cell lines (LCLs), we identified four EBV oncoproteins and five EBV-activated NF-κB subunits co-occupying ~1800 enhancer sites. Of these, 187 had markedly higher and broader histone H3K27ac signals characteristic of super-enhancers, and were designated “EBV super-enhancers”. EBV super-enhancer-associated genes included the MYC and BCL2 oncogenes, enabling LCL proliferation and survival. EBV super-enhancers were enriched for B cell transcription factor motifs and had a high co-occupancy of the transcription factors STAT5 and NFAT. EBV super-enhancer-associated genes were more highly expressed than other LCL genes. Disrupting EBV super-enhancers by the bromodomain inhibitor, JQ1 or conditionally inactivating an EBV oncoprotein or NF-κB decreased MYC or BCL2 expression and arrested LCL growth. These findings provide insight into mechanisms of EBV-induced lymphoproliferation and identify potential therapeutic interventions. PMID:25639793

BFV activates the NF-kappaB pathway through its transactivator (BTas) to enhance viral transcription

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

Wang Jian; Tan Juan; Zhang Xihui

2010-05-10

Multiple families of viruses have evolved sophisticated strategies to regulate nuclear factor-kappaB (NF-kappaB) signaling, which plays a pivotal role in diverse cellular events, including virus-host interactions. In this study, we report that bovine foamy virus (BFV) is able to activate the NF-kappaB pathway through the action of its transactivator, BTas. Both cellular IKKbeta and IkappaBalpha also participate in this activation. In addition, we demonstrate that BTas induces the processing of p100, which implies that BTas can activate NF-kappaB through a noncanonical pathway as well. Co-immunoprecipitation analysis shows that BTas interacts with IKK catalytic subunits (IKKalpha and IKKbeta), which may bemore » responsible for regulation of IKK kinase activity and persistent NF-kappaB activation. Furthermore, our results indicate that the level of BTas-mediated LTR transcription correlates with the activity of cellular NF-kappaB. Together, this study suggests that BFV activates the NF-kappaB pathway through BTas to enhance viral transcription.« less

BFV activates the NF-kappaB pathway through its transactivator (BTas) to enhance viral transcription.

PubMed

Wang, Jian; Tan, Juan; Zhang, Xihui; Guo, Hongyan; Zhang, Qicheng; Guo, Tingting; Geng, Yunqi; Qiao, Wentao

2010-05-10

Multiple families of viruses have evolved sophisticated strategies to regulate nuclear factor-kappaB (NF-kappaB) signaling, which plays a pivotal role in diverse cellular events, including virus-host interactions. In this study, we report that bovine foamy virus (BFV) is able to activate the NF-kappaB pathway through the action of its transactivator, BTas. Both cellular IKKbeta and IkappaBalpha also participate in this activation. In addition, we demonstrate that BTas induces the processing of p100, which implies that BTas can activate NF-kappaB through a noncanonical pathway as well. Co-immunoprecipitation analysis shows that BTas interacts with IKK catalytic subunits (IKKalpha and IKKbeta), which may be responsible for regulation of IKK kinase activity and persistent NF-kappaB activation. Furthermore, our results indicate that the level of BTas-mediated LTR transcription correlates with the activity of cellular NF-kappaB. Together, this study suggests that BFV activates the NF-kappaB pathway through BTas to enhance viral transcription. Copyright 2010 Elsevier Inc. All rights reserved.

Sequestration of cAMP response element-binding proteins by transcription factor decoys causes collateral elaboration of regenerating Aplysia motor neuron axons.

PubMed

Dash, P K; Tian, L M; Moore, A N

1998-07-07

Axonal injury increases intracellular Ca2+ and cAMP and has been shown to induce gene expression, which is thought to be a key event for regeneration. Increases in intracellular Ca2+ and/or cAMP can alter gene expression via activation of a family of transcription factors that bind to and modulate the expression of CRE (Ca2+/cAMP response element) sequence-containing genes. We have used Aplysia motor neurons to examine the role of CRE-binding proteins in axonal regeneration after injury. We report that axonal injury increases the binding of proteins to a CRE sequence-containing probe. In addition, Western blot analysis revealed that the level of ApCREB2, a CRE sequence-binding repressor, was enhanced as a result of axonal injury. The sequestration of CRE-binding proteins by microinjection of CRE sequence-containing plasmids enhanced axon collateral formation (both number and length) as compared with control plasmid injections. These findings show that Ca2+/cAMP-mediated gene expression via CRE-binding transcription factors participates in the regeneration of motor neuron axons.

A novel de novo activating mutation in STAT3 identified in a patient with common variable immunodeficiency (CVID).

PubMed

Russell, Mark A; Pigors, Manuela; Houssen, Maha E; Manson, Ania; Kelsell, David; Longhurst, Hilary; Morgan, Noel G

2018-02-01

Common variable immunodeficiency (CVID) is characterised by repeated infection associated with primary acquired hypogammaglobulinemia. CVID frequently has a complex aetiology but, in certain cases, it has a monogenic cause. Recently, variants within the gene encoding the transcription factor STAT3 were implicated in monogenic CVID. Here, we describe a patient presenting with symptoms synonymous with CVID, who displayed reduced levels of IgG and IgA, repeated viral infections and multiple additional co-morbidities. Whole-exome sequencing revealed a de novo novel missense mutation in the coiled-coil domain of STAT3 (c.870A>T; p.K290N). Accordingly, the K290N variant of STAT3 was generated, and a STAT3 responsive dual-luciferase reporter assay revealed that the variant strongly enhances STAT3 transcriptional activity both under basal and stimulated (with IL-6) conditions. Overall, these data complement earlier studies in which CVID-associated STAT3 mutations are predicted to enhance transcriptional activity, suggesting that such patients may respond favourably to IL-6 receptor antagonists (e.g. tocilizumab). Copyright © 2017 Elsevier Inc. All rights reserved.

Translational regulation of inhibin βA by TGFβ via the RNA-binding protein hnRNP E1 enhances the invasiveness of epithelial-to-mesenchymal transitioned cells.

PubMed

Howley, B V; Hussey, G S; Link, L A; Howe, P H

2016-03-31

The epithelial-to-mesenchymal transition (EMT) is a cellular process that functions during embryonic development and tissue regeneration, thought to be aberrantly activated in epithelial-derived cancer and has an important role in the process of metastasis. The transforming growth factor (TGF)-β signaling pathway is a key inducer of EMT and we have elucidated a posttranscriptional mechanism by which TGFβ modulates expression of select transcripts via the RNA-binding protein hnRNP E1 during EMT. One such transcript inhibin βA is a member of the TGFβ superfamily. Here, we show by polysome profiling that inhibin βA is translationally regulated by TGFβ via hnRNP E1. TGFβ treatment or knockdown of hnRNP E1 relieves silencing of the inhibin βA transcript, resulting in increased protein expression and secreted levels of the inhibin βA homodimer, activin A. Our data indicate that the translational upregulation of inhibin βA enhances the migration and invasion of cells that have undergone an EMT and promotes cancer progression in vivo.

Cohesin regulates tissue-specific expression by stabilizing highly occupied cis-regulatory modules

PubMed Central

Faure, Andre J.; Schmidt, Dominic; Watt, Stephen; Schwalie, Petra C.; Wilson, Michael D.; Xu, Huiling; Ramsay, Robert G.; Odom, Duncan T.; Flicek, Paul

2012-01-01

The cohesin protein complex contributes to transcriptional regulation in a CTCF-independent manner by colocalizing with master regulators at tissue-specific loci. The regulation of transcription involves the concerted action of multiple transcription factors (TFs) and cohesin's role in this context of combinatorial TF binding remains unexplored. To investigate cohesin-non-CTCF (CNC) binding events in vivo we mapped cohesin and CTCF, as well as a collection of tissue-specific and ubiquitous transcriptional regulators using ChIP-seq in primary mouse liver. We observe a positive correlation between the number of distinct TFs bound and the presence of CNC sites. In contrast to regions of the genome where cohesin and CTCF colocalize, CNC sites coincide with the binding of master regulators and enhancer-markers and are significantly associated with liver-specific expressed genes. We also show that cohesin presence partially explains the commonly observed discrepancy between TF motif score and ChIP signal. Evidence from these statistical analyses in wild-type cells, and comparisons to maps of TF binding in Rad21-cohesin haploinsufficient mouse liver, suggests that cohesin helps to stabilize large protein–DNA complexes. Finally, we observe that the presence of mirrored CTCF binding events at promoters and their nearby cohesin-bound enhancers is associated with elevated expression levels. PMID:22780989

Deacetylases and NF-κB in Redox Regulation of Cigarette Smoke induced Lung Inflammation: Implications in Pathogenesis of COPD

PubMed Central

Rajendrasozhan, Saravanan; Yang, Se-Ran; Edirisinghe, Indika; Yao, Hongwei; Adenuga, David; Rahman, Irfan

2009-01-01

Oxidative stress has been implicated in the pathogenesis of several inflammatory lung disorders including chronic obstructive pulmonary disease (COPD) due to its effect on pro-inflammatory gene transcription. Cigarette smoke-mediated oxidative stress activates NF-κB-dependent transcription of pro-inflammatory mediators either through activation of inhibitor κB-α kinase (IKK) and/or the enhanced recruitment and activation of transcriptional co-activators. Enhanced NF-κB-co-activator complex formation results in targeted increase in chromatin modifications, such as histone acetylation leading to inflammatory gene transcription. NF-κB-dependent gene expression, at least in part, is regulated by changes in deacetylases such as histone deacetylases (HDACs) and sirtuins. Cigarette smoke and oxidants also alter the levels/activity of HDAC by post-translational modifications and in doing so further induces gene expression of pro-inflammatory mediators. In addition, cigarette smoke/oxidants can reduce glucocorticoid sensitivity by attenuating HDAC2 activity and expression, which may account for the glucocorticoid insensitivity in patients with COPD. Understanding the mechanisms of NF-κB regulation, and the balance between histone acetylation and deacetylation may lead to the development of novel therapies based on the pharmacological manipulation of IKK and deacetylases in lung inflammation and injury. PMID:18220485

A wheat salinity-induced WRKY transcription factor TaWRKY93 confers multiple abiotic stress tolerance in Arabidopsis thaliana.

PubMed

Qin, Yuxiang; Tian, Yanchen; Liu, Xiuzhi

2015-08-21

Wheat is an important crop in the world. But most of the cultivars are salt sensitive, and often adversely affected by salt stress. WRKY transcription factors play a major role in plant responses to salt stress, but the effective salinity regulatory WRKYs identified in bread wheat are limited and the mechanism of salt stress tolerance is also not well explored. Here, we identified a salt (NaCl) induced class II WRKY transcription factor TaWRKY93. Its transcript level was strongly induced by salt (NaCl) and exogenous abscisic acid (ABA). Over-expression of TaWRKY93 in Arabidopsis thaliana enhanced salt (NaCl), drought, low temperature and osmotic (mannitol) stress tolerance, mainly demonstrated by transgenic plants forming longer primary roots or more lateral roots on MS plates supplemented with NaCl and mannitol individually, higher survival rate under drought and low temperature stress. Further, transgenic plants maintained a more proline content, higher relative water content and less electrolyte leakage than the wild type plants. The transcript abundance of a series of abiotic stress-related genes was up-regulated in the TaWRKY93 transgenic plants. In summary, TaWRKY93 is a new positive regulator of abiotic stress, it may increase salinity, drought and low temperature stress tolerance through enhancing osmotic adjustment, maintaining membrane stability and increasing transcription of stress related genes, and contribute to the superior agricultural traits of SR3 through promoting root development. It can be used as a candidate gene for wheat transgenic engineering breeding against abiotic stress. Copyright © 2015 Elsevier Inc. All rights reserved.

Physiological plasticity related to zonation affects hsp70 expression in the reef-building coral Pocillopora verrucosa

PubMed Central

Poli, Davide; Fabbri, Elena; Goffredo, Stefano; Airi, Valentina

2017-01-01

This study investigates for the first time the transcriptional regulation of a stress-inducible 70-kDa heat shock protein (hsp70) in the scleractinian coral Pocillopora verrucosa sampled at three locations and two depths (3 m and 12 m) in Bangka Island waters (North Sulawesi, Indonesia). Percentage of coral cover indicated reduced habitat suitability with depth and at the Tanjung Husi (TA) site, which also displayed relatively higher seawater temperatures. Expression of the P. verrucosa hsp70 transcript evaluated under field conditions followed a depth-related profile, with relatively higher expression levels in 3-m collected nubbins compared to the 12-m ones. Expression levels of metabolism-related transcripts ATP synthase and NADH dehydrogenase indicated metabolic activation of nubbins to cope with habitat conditions of the TA site at 3 m. After a 14-day acclimatization to common and fixed temperature conditions in the laboratory, corals were subjected for 7 days to an altered thermal regime, where temperature was elevated at 31°C during the light phase and returned to 28°C during the dark phase. Nubbins collected at 12 m were relatively more sensitive to thermal stress, as they significantly over-expressed the selected transcripts. Corals collected at 3 m appeared more resilient, as they showed unaffected mRNA expressions. The results indicated that local habitat conditions may influence transcription of stress-related genes in P. verrucosa. Corals exhibiting higher basal hsp70 levels may display enhanced tolerance towards environmental stressors. PMID:28199351

Negatively supercoiled simian virus 40 DNA contains Z-DNA segments within transcriptional enhancer sequences

NASA Technical Reports Server (NTRS)

Nordheim, A.; Rich, A.

1983-01-01

Three 8-base pair (bp) segments of alternating purine-pyrimidine from the simian virus 40 enhancer region form Z-DNA on negative supercoiling; minichromosome DNase I-hypersensitive sites determined by others bracket these three segments. A survey of transcriptional enhancer sequences reveals a pattern of potential Z-DNA-forming regions which occur in pairs 50-80 bp apart. This may influence local chromatin structure and may be related to transcriptional activation.

Regulation of circadian clock transcriptional output by CLOCK:BMAL1

PubMed Central

Trott, Alexandra J.

2018-01-01

The mammalian circadian clock relies on the transcription factor CLOCK:BMAL1 to coordinate the rhythmic expression of 15% of the transcriptome and control the daily regulation of biological functions. The recent characterization of CLOCK:BMAL1 cistrome revealed that although CLOCK:BMAL1 binds synchronously to all of its target genes, its transcriptional output is highly heterogeneous. By performing a meta-analysis of several independent genome-wide datasets, we found that the binding of other transcription factors at CLOCK:BMAL1 enhancers likely contribute to the heterogeneity of CLOCK:BMAL1 transcriptional output. While CLOCK:BMAL1 rhythmic DNA binding promotes rhythmic nucleosome removal, it is not sufficient to generate transcriptionally active enhancers as assessed by H3K27ac signal, RNA Polymerase II recruitment, and eRNA expression. Instead, the transcriptional activity of CLOCK:BMAL1 enhancers appears to rely on the activity of ubiquitously expressed transcription factors, and not tissue-specific transcription factors, recruited at nearby binding sites. The contribution of other transcription factors is exemplified by how fasting, which effects several transcription factors but not CLOCK:BMAL1, either decreases or increases the amplitude of many rhythmically expressed CLOCK:BMAL1 target genes. Together, our analysis suggests that CLOCK:BMAL1 promotes a transcriptionally permissive chromatin landscape that primes its target genes for transcription activation rather than directly activating transcription, and provides a new framework to explain how environmental or pathological conditions can reprogram the rhythmic expression of clock-controlled genes. PMID:29300726

Sost, independent of the non-coding enhancer ECR5, is required for bone mechanoadaptation

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

Robling, Alexander G.; Kang, Kyung Shin; Bullock, Whitney A.

Here, sclerostin ( Sost) is a negative regulator of bone formation that acts upon the Wnt signaling pathway. Sost is mechanically regulated at both mRNA and protein level such that loading represses and unloading enhances Sost expression, in osteocytes and in circulation. The non-coding evolutionarily conserved enhancer ECR5 has been previously reported as a transcriptional regulatory element required for modulating Sost expression in osteocytes. Here we explored the mechanisms by which ECR5, or several other putative transcriptional enhancers regulate Sost expression, in response to mechanical stimulation. We found that in vivo ulna loading is equally osteoanabolic in wildtype and Sostmore » –/– mice, although Sost is required for proper distribution of load-induced bone formation to regions of high strain. Using Luciferase reporters carrying the ECR5 non-coding enhancer and heterologous or homologous h SOST promoters, we found that ECR5 is mechanosensitive in vitro and that ECR5-driven Luciferase activity decreases in osteoblasts exposed to oscillatory fluid flow. Yet, ECR5–/– mice showed similar magnitude of load-induced bone formation and similar periosteal distribution of bone formation to high-strain regions compared to wildtype mice. Further, we found that in contrast to Sost–/– mice, which are resistant to disuse-induced bone loss, ECR5–/– mice lose bone upon unloading to a degree similar to wildtype control mice. ECR5 deletion did not abrogate positive effects of unloading on Sost, suggesting that additional transcriptional regulators and regulatory elements contribute to load-induced regulation of Sost.« less

Sost, independent of the non-coding enhancer ECR5, is required for bone mechanoadaptation

DOE PAGES

Robling, Alexander G.; Kang, Kyung Shin; Bullock, Whitney A.; ...

2016-09-04

Here, sclerostin ( Sost) is a negative regulator of bone formation that acts upon the Wnt signaling pathway. Sost is mechanically regulated at both mRNA and protein level such that loading represses and unloading enhances Sost expression, in osteocytes and in circulation. The non-coding evolutionarily conserved enhancer ECR5 has been previously reported as a transcriptional regulatory element required for modulating Sost expression in osteocytes. Here we explored the mechanisms by which ECR5, or several other putative transcriptional enhancers regulate Sost expression, in response to mechanical stimulation. We found that in vivo ulna loading is equally osteoanabolic in wildtype and Sostmore » –/– mice, although Sost is required for proper distribution of load-induced bone formation to regions of high strain. Using Luciferase reporters carrying the ECR5 non-coding enhancer and heterologous or homologous h SOST promoters, we found that ECR5 is mechanosensitive in vitro and that ECR5-driven Luciferase activity decreases in osteoblasts exposed to oscillatory fluid flow. Yet, ECR5–/– mice showed similar magnitude of load-induced bone formation and similar periosteal distribution of bone formation to high-strain regions compared to wildtype mice. Further, we found that in contrast to Sost–/– mice, which are resistant to disuse-induced bone loss, ECR5–/– mice lose bone upon unloading to a degree similar to wildtype control mice. ECR5 deletion did not abrogate positive effects of unloading on Sost, suggesting that additional transcriptional regulators and regulatory elements contribute to load-induced regulation of Sost.« less

Identification of the first PAR1 deletion encompassing upstream SHOX enhancers in a family with idiopathic short stature.

PubMed

Benito-Sanz, Sara; Aza-Carmona, Miriam; Rodríguez-Estevez, Amaya; Rica-Etxebarria, Ixaso; Gracia, Ricardo; Campos-Barros, Angel; Heath, Karen E

2012-01-01

Short stature homeobox-containing gene, MIM 312865 (SHOX) is located within the pseudoautosomal region 1 (PAR1) of the sex chromosomes. Mutations in SHOX or its downstream transcriptional regulatory elements represent the underlying molecular defect in ~60% of Léri-Weill dyschondrosteosis (LWD) and ~5-15% of idiopathic short stature (ISS) patients. Recently, three novel enhancer elements have been identified upstream of SHOX but to date, no PAR1 deletions upstream of SHOX have been observed that only encompass these enhancers in LWD or ISS patients. We set out to search for genetic alterations of the upstream SHOX regulatory elements in 63 LWD and 100 ISS patients with no known alteration in SHOX or the downstream enhancer regions using a specifically designed MLPA assay, which covers the PAR1 upstream of SHOX. An upstream SHOX deletion was identified in an ISS proband and her affected father. The deletion was confirmed and delimited by array-CGH, to extend ~286 kb. The deletion included two of the upstream SHOX enhancers without affecting SHOX. The 13.3-year-old proband had proportionate short stature with normal GH and IGF-I levels. In conclusion, we have identified the first PAR1 deletion encompassing only the upstream SHOX transcription regulatory elements in a family with ISS. The loss of these elements may result in SHOX haploinsufficiency because of decreased SHOX transcription. Therefore, this upstream region should be included in the routine analysis of PAR1 in patients with LWD, LMD and ISS.

Identification of the first PAR1 deletion encompassing upstream SHOX enhancers in a family with idiopathic short stature

PubMed Central

Benito-Sanz, Sara; Aza-Carmona, Miriam; Rodríguez-Estevez, Amaya; Rica-Etxebarria, Ixaso; Gracia, Ricardo; Campos-Barros, Ángel; Heath, Karen E

2012-01-01

Short stature homeobox-containing gene, MIM 312865 (SHOX) is located within the pseudoautosomal region 1 (PAR1) of the sex chromosomes. Mutations in SHOX or its downstream transcriptional regulatory elements represent the underlying molecular defect in ∼60% of Léri-Weill dyschondrosteosis (LWD) and ∼5–15% of idiopathic short stature (ISS) patients. Recently, three novel enhancer elements have been identified upstream of SHOX but to date, no PAR1 deletions upstream of SHOX have been observed that only encompass these enhancers in LWD or ISS patients. We set out to search for genetic alterations of the upstream SHOX regulatory elements in 63 LWD and 100 ISS patients with no known alteration in SHOX or the downstream enhancer regions using a specifically designed MLPA assay, which covers the PAR1 upstream of SHOX. An upstream SHOX deletion was identified in an ISS proband and her affected father. The deletion was confirmed and delimited by array-CGH, to extend ∼286 kb. The deletion included two of the upstream SHOX enhancers without affecting SHOX. The 13.3-year-old proband had proportionate short stature with normal GH and IGF-I levels. In conclusion, we have identified the first PAR1 deletion encompassing only the upstream SHOX transcription regulatory elements in a family with ISS. The loss of these elements may result in SHOX haploinsufficiency because of decreased SHOX transcription. Therefore, this upstream region should be included in the routine analysis of PAR1 in patients with LWD, LMD and ISS. PMID:22071895

AML1/ETO trans-activates c-KIT expression through the long range interaction between promoter and intronic enhancer.

PubMed

Tian, Ying; Wang, Genjie; Hu, Qingzhu; Xiao, Xichun; Chen, Shuxia

2018-04-01

The AML1/ETO onco-fusion protein is crucial for the genesis of t(8;21) acute myeloid leukemia (AML) and is well documented as a transcriptional repressor through dominant-negative effect. However, little is known about the transactivation mechanism of AML1/ETO. Through large cohort of patient's expression level data analysis and a series of experimental validation, we report here that AML1/ETO transactivates c-KIT expression through directly binding to and mediating the long-range interaction between the promoter and intronic enhancer regions of c-KIT. Gene expression analyses verify that c-KIT expression is significantly high in t(8;21) AML. Further ChIP-seq analysis and motif scanning identify two regulatory regions located in the promoter and intronic enhancer region of c-KIT, respectively. Both regions are enriched by co-factors of AML1/ETO, such as AML1, CEBPe, c-Jun, and c-Fos. Further luciferase reporter assays show that AML1/ETO trans-activates c-KIT promoter activity through directly recognizing the AML1 motif and the co-existence of co-factors. The induction of c-KIT promoter activity is reinforced with the existence of intronic enhancer region. Furthermore, ChIP-3C-qPCR assays verify that AML1/ETO mediates the formation of DNA-looping between the c-KIT promoter and intronic enhancer region through the long-range interaction. Collectively, our data uncover a novel transcriptional activity mechanism of AML1/ETO and enrich our knowledge of the onco-fusion protein mediated transcription regulation. © 2017 Wiley Periodicals, Inc.

B-DNA to Z-DNA structural transitions in the SV40 enhancer: stabilization of Z-DNA in negatively supercoiled DNA minicircles

NASA Technical Reports Server (NTRS)

Gruskin, E. A.; Rich, A.

1993-01-01

During replication and transcription, the SV40 control region is subjected to significant levels of DNA unwinding. There are three, alternating purine-pyrimidine tracts within this region that can adopt the Z-DNA conformation in response to negative superhelix density: a single copy of ACACACAT and two copies of ATGCATGC. Since the control region is essential for both efficient transcription and replication, B-DNA to Z-DNA transitions in these vital sequence tracts may have significant biological consequences. We have synthesized DNA minicircles to detect B-DNA to Z-DNA transitions in the SV40 enhancer, and to determine the negative superhelix density required to stabilize the Z-DNA. A variety of DNA sequences, including the entire SV40 enhancer and the two segments of the enhancer with alternating purine-pyrimidine tracts, were incorporated into topologically relaxed minicircles. Negative supercoils were generated, and the resulting topoisomers were resolved by electrophoresis. Using an anti-Z-DNA Fab and an electrophoretic mobility shift assay, Z-DNA was detected in the enhancer-containing minicircles at a superhelix density of -0.05. Fab saturation binding experiments demonstrated that three, independent Z-DNA tracts were stabilized in the supercoiled minicircles. Two other minicircles, each with one of the two alternating purine-pyrimidine tracts, also contained single Z-DNA sites. These results confirm the identities of the Z-DNA-forming sequences within the control region. Moreover, the B-DNA to Z-DNA transitions were detected at superhelix densities observed during normal replication and transcription processes in the SV40 life cycle.

B29 Gene Silencing in Pituitary Cells is Regulated by Its 3′ Enhancer

PubMed Central

Malone, Cindy S.; Kuraishy, Ali I.; Fike, Francesca M.; Loya, Ruchika G.; Mikkili, Minil R.; Teitell, Michael A.; Wall, Randolph

2007-01-01

Summary B cell-specific B29 (Igβ, CD79b) genes in rat, mouse, and human are situated between the 5′ growth hormone (GH) locus control region (LCR) and the 3′ GH gene cluster. The entire GH genomic region is DNase1 hypersensitive in GH-expressing pituitary cells, which predicts an “open” chromatin configuration, and yet B29 is not expressed. The B29 promoter and enhancers exhibit histone deacetylation in pituitary cells, but histone deacetylase inhibition failed to activate B29 expression. The B29 promoter and a 3′ enhancer showed local dense DNA methylation in both pituitary and non-lymphoid cells consistent with gene silencing. However, DNA methyltransferase inhibition did not activate B29 expression either. B29 promoter constructs were minimally activated in transfected pituitary cells. Co-transfection of the B cell-specific octamer transcriptional co-activator Bob1 with the B29 promoter construct resulted in high level promoter activity in pituitary cells comparable to B29 promoter activity in transfected B cells. Unexpectedly, inclusion of the B29 3′ enhancer in B29 promoter constructs strongly inhibited B29 transcriptional activity even when pituitary cells were co-transfected with Bob1. Both Oct-1 and Pit-1 bind the B29 3′ enhancer in in vitro EMSA and in in vivo chromatin immunoprecipitation analyses. These data indicate that the GH locus-embedded, tissue-specific B29 gene is silenced in GH-expressing pituitary cells by epigenetic mechanisms, the lack of a B cell-specific transcription factor, and likely by the B29 3′ enhancer acting as a powerful silencer in a context and tissue-specific manner. PMID:16920149

Hydroquinone, a reactive metabolite of benzene, enhances interleukin-4 production in CD4+ T cells and increases immunoglobulin E levels in antigen-primed mice

PubMed Central

Lee, M H; Chung, S W; Kang, B Y; Kim, K-M; Kim, T S

2002-01-01

Exposure to cigarette smoke is known to increase the risk of the development of allergic disease. The mechanism is not well understood. In this study, we determined the effect of hydroquinone (HQ), a major metabolite of benzene present in large quantities in cigarette tar, on interleukin-4 (IL-4) production by CD4+ T cells. HQ significantly enhanced IL-4 production by keyhole limpet haemocyanin (KLH)-primed CD4+ T cells in a dose-dependent manner. The enhancing effect of HQ on IL-4 production was maximal at a concentration of 50 µm. It increased the level of IL-4 production approximately 10-fold. HQ enhanced IL-4 mRNA expression and also IL-4 gene promoter activity, suggesting that the enhancing effect of HQ on IL-4 production may occur at the transcriptional level. Furthermore, the injection of KLH-primed mice with HQ resulted in a significant increase in the levels of IL-4 and immunoglobulin E. These findings provide evidence that HQ, a major component of cigarette tar, may enhance allergic immune responses by inducing the production of IL-4 in CD4+ T cells. PMID:12153512

Increased linear bone growth by GH in the absence of SOCS2 is independent of IGF-1.

PubMed

Dobie, Ross; Ahmed, Syed F; Staines, Katherine A; Pass, Chloe; Jasim, Seema; MacRae, Vicky E; Farquharson, Colin

2015-11-01

Growth hormone (GH) signaling is essential for postnatal linear bone growth, but the relative importance of GHs actions on the liver and/or growth plate cartilage remains unclear. The importance of liver derived insulin like-growth factor-1 (IGF-1) for endochondral growth has recently been challenged. Here, we investigate linear growth in Suppressor of Cytokine Signaling-2 (SOCS2) knockout mice, which have enhanced growth despite normal systemic GH/IGF-1 levels. Wild-type embryonic ex vivo metatarsals failed to exhibit increased linear growth in response to GH, but displayed increased Socs2 transcript levels (P < 0.01). In the absence of SOCS2, GH treatment enhanced metatarsal linear growth over a 12 day period. Despite this increase, IGF-1 transcript and protein levels were not increased in response to GH. In accordance with these data, IGF-1 levels were unchanged in GH-challenged postnatal Socs2(-/-) conditioned medium despite metatarsals showing enhanced linear growth. Growth-plate Igf1 mRNA levels were not elevated in juvenile Socs2(-/-) mice. GH did however elevate IGF-binding protein 3 levels in conditioned medium from GH challenged metatarsals and this was more apparent in Socs2(-/-) metatarsals. GH did not enhance the growth of Socs2(-/-) metatarsals when the IGF receptor was inhibited, suggesting that IGF receptor mediated mechanisms are required. IGF-2 may be responsible as IGF-2 promoted metatarsal growth and Igf2 expression was elevated in Socs2(-/-) (but not WT) metatarsals in response to GH. These studies emphasise the critical importance of SOCS2 in regulating GHs ability to promote bone growth. Also, GH appears to act directly on the metatarsals of Socs2(-/-) mice, promoting growth via a mechanism that is independent of IGF-1. © 2014 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals, Inc.

Tissue-specific thyroid hormone regulation of gene transcripts encoding iodothyronine deiodinases and thyroid hormone receptors in striped parrotfish (Scarus iseri).

PubMed

Johnson, Kaitlin M; Lema, Sean C

2011-07-01

In fish as in other vertebrates, the diverse functions of thyroid hormones are mediated at the peripheral tissue level through iodothyronine deiodinase (dio) enzymes and thyroid hormone receptor (tr) proteins. In this study, we examined thyroid hormone regulation of mRNAs encoding the three deiodinases dio1, dio2 and dio3 - as well as three thyroid hormone receptors trαA, trαB and trβ - in initial phase striped parrotfish (Scarus iseri). Parrotfish were treated with dissolved phase T(3) (20 nM) or methimazole (3 mM) for 3 days. Treatment with exogenous T(3) elevated circulating T(3), while the methimazole treatment depressed plasma T(4). Experimentally-induced hyperthyroidism increased the relative abundance of transcripts encoding trαA and trβ in the liver and brain, but did not affect trαB mRNA levels in either tissue. In both sexes, methimazole-treated fish exhibited elevated dio2 transcripts in the liver and brain, suggesting enhanced outer-ring deiodination activity in these tissues. Accordingly, systemic hyperthyroidism elevated relative dio3 transcript levels in these same tissues. In the gonad, however, patterns of transcript regulation were distinctly different with elevated T(3) increasing mRNAs encoding dio2 in testicular and ovarian tissues and dio3, trαA and trαB in the testes only. Thyroid hormone status did not affect dio1 transcript abundance in the liver, brain or gonads. Taken as a whole, these results demonstrate that thyroidal status influences relative transcript abundance for dio2 and dio3 in the liver, provide new evidence for similar patterns of dio2 and dio3 mRNA regulation in the brain, and make evident that fish exhibit tr subtype-specific transcript abundance changes to altered thyroid status. Copyright © 2011 Elsevier Inc. All rights reserved.

Enhancer Sharing Promotes Neighborhoods of Transcriptional Regulation Across Eukaryotes

PubMed Central

Quintero-Cadena, Porfirio; Sternberg, Paul W.

2016-01-01

Enhancers physically interact with transcriptional promoters, looping over distances that can span multiple regulatory elements. Given that enhancer–promoter (EP) interactions generally occur via common protein complexes, it is unclear whether EP pairing is predominantly deterministic or proximity guided. Here, we present cross-organismic evidence suggesting that most EP pairs are compatible, largely determined by physical proximity rather than specific interactions. By reanalyzing transcriptome datasets, we find that the transcription of gene neighbors is correlated over distances that scale with genome size. We experimentally show that nonspecific EP interactions can explain such correlation, and that EP distance acts as a scaling factor for the transcriptional influence of an enhancer. We propose that enhancer sharing is commonplace among eukaryotes, and that EP distance is an important layer of information in gene regulation. PMID:27799341

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.

Cross-species approaches to seed dormancy and germination: conservation and biodiversity of ABA-regulated mechanisms and the Brassicaceae DOG1 genes.

PubMed

Graeber, Kai; Linkies, Ada; Müller, Kerstin; Wunchova, Andrea; Rott, Anita; Leubner-Metzger, Gerhard

2010-05-01

Seed dormancy is genetically determined with substantial environmental influence mediated, at least in part, by the plant hormone abscisic acid (ABA). The ABA-related transcription factor ABI3/VP1 (ABA INSENSITIVE3/VIVIPAROUS1) is widespread among green plants. Alternative splicing of its transcripts appears to be involved in regulating seed dormancy, but the role of ABI3/VP1 goes beyond seeds and dormancy. In contrast, DOG1 (DELAY OF GERMINATION 1), a major quantitative trait gene more specifically involved in seed dormancy, was so far only known from Arabidopsis thaliana (AtDOG1) and whether it also has roles during the germination of non-dormant seeds was not known. Seed germination of Lepidium sativum ('garden cress') is controlled by ABA and its antagonists gibberellins and ethylene and involves the production of apoplastic hydroxyl radicals. We found orthologs of AtDOG1 in the Brassicaceae relatives L. sativum (LesaDOG1) and Brassica rapa (BrDOG1) and compared their gene structure and the sequences of their transcripts expressed in seeds. Tissue-specific analysis of LesaDOG1 transcript levels in L. sativum seeds showed that they are degraded upon imbibition in the radicle and the micropylar endosperm. ABA inhibits germination in that it delays radicle protrusion and endosperm weakening and it increased LesaDOG1 transcript levels during early germination due to enhanced transcription and/or inhibited degradation. A reduced decrease in LesaDOG1 transcript levels upon ABA treatment is evident in the late germination phase in both tissues. This temporal and ABA-related transcript expression pattern suggests a role for LesaDOG1 in the control of germination timing of non-dormant L. sativum seeds. The possible involvement of the ABA-related transcription factors ABI3 and ABI5 in the regulation of DOG1 transcript expression is discussed. Other species of the monophyletic genus Lepidium showed coat or embryo dormancy and are therefore highly suited for comparative seed biology.

Loss of calreticulin function decreases NFκB activity by stabilizing IκB protein.

PubMed

Massaeli, Hamid; Jalali, Shahrzad; Viswanathan, Divya; Mesaeli, Nasrin

2014-11-01

Transcription factor NFκB is activated by several processes including inflammation, endoplasmic-reticulum (ER) stress, increase in Akt signaling and enhanced proteasomal degradation. Calreticulin (CRT) is an ER Ca(2+)-binding chaperone that regulates many cellular processes. Gene-targeted deletion of CRT has been shown to induce ER stress that is accompanied with a significant increase in the proteasome activity. Loss of CRT function increases the resistance of CRT-deficient (crt-/-) cells to UV- and drug-induced apoptosis. Based on these reports we hypothesized that loss of CRT will activate NFκB signaling thus contributing to enhanced resistance to apoptosis. In contrast to our hypothesis, we observed a significant decrease in the basal transcriptional activity of NFκB in CRT-deficient cells. Treatment with lipopolysaccharide failed to increase the transcriptional activity of NFκB in the crt-/- cells to the same level as in the wt cells. Our data illustrate that the mechanism of decreased NFκB activity in CRT-deficient cells is mediated by a significant increase in IκB protein expression. Furthermore, we showed a significant increase in protein phosphatase 2A activity inhibition which resulted in decreased IκBα protein level in CRT-deficient cells. Based on our data we concluded that loss of CRT increases the stability of IκB protein thus reducing NFκB activity. Copyright © 2014 Elsevier B.V. All rights reserved.

The immunomodulatory effect of vitamin D in chickens is dose-dependent and influenced by calcium and phosphorus levels.

PubMed

Rodriguez-Lecompte, J C; Yitbarek, A; Cuperus, T; Echeverry, H; van Dijk, A

2016-11-01

Vitamin D requirement is estimated to be higher than recommended values for the first two weeks of a broiler chicken's life, and is heavily dependent on the concentrations of Ca and P in the diet. There are data indicating the beneficial effect of higher vitamin D levels on performance and overall health of the chickens. However, data on the role of higher vitamin D levels on the innate immune response of chickens are limited. Therefore, in the current study, we examined the effect of higher doses of vitamin D supplementation on the innate immune response in broiler chickens receiving optimal or calcium (Ca) and phosphorus (P) deficient diets. Three hundred Ross-308 male broiler chicks were randomly allocated into 60 cages with 5 birds per cage in a 3 × 2 factorial design with three levels of vitamin D and two levels of Ca/P with each experimental diet fed to 10 cages (10 replicates). Quantitative reverse transcription PCR (n = 5) was used to assess Toll-like receptor (TLR2b and 4), cytokine/chemokine (IL-12, IFN-γ, IL-10, IL-4, IL-13, IL-18, CxCLi2) and cathelicidin (CATH1, CATHB1, CATH3) transcription levels in peripheral blood mononuclear cells (PBMCs), spleen, and bursa of Fabricius. Vitamin D supplementation of the Ca and P deficient diet considerably augmented transcription of TLR2b, TLR4, CATH1, and CATHB1 and predominantly Th2 cytokines in spleen. Supplementation of the control diet with vitamin D downregulated TLR4 transcription, and dose-dependently increased CATH1, CATHB1, Th1, and Th2 cytokine transcription (Th2>Th1). All diets downregulated CATH3 transcription. In conclusion, vitamin D or its derivative 25-OH-D 3 both have a robust immunomodulatory property with a more favorable Th2 response, while at the same time enhancing observed Th2 cytokine responses under both optimal and lower Ca and P inclusion levels in the diets of broiler chickens. © 2016 Poultry Science Association Inc.

Adenosine Deaminase Acting on RNA 1 (ADAR1) Suppresses the Induction of Interferon by Measles Virus

PubMed Central

Li, Zhiqun; Okonski, Kristina M.

2012-01-01

ADAR1, the interferon (IFN)-inducible adenosine deaminase acting on RNA, catalyzes the C-6 deamination of adenosine (A) to produce inosine (I) in RNA substrates with a double-stranded character. Because double-stranded RNA is a known inducer of IFN, we tested the role of ADAR1 in IFN induction following virus infection. HeLa cells made stably deficient in ADAR1 (ADAR1kd) were compared to vector control (CONkd) and protein kinase PKR-deficient (PKRkd) cells for IFN-β induction following infection with either parental (wild-type [WT]) recombinant Moraten vaccine strain measles virus (MV) or isogenic knockout mutants deficient for either V (Vko) or C (Cko) protein expression. We observed potent IFN-β transcript induction in ADAR1kd cells by all three viruses; in contrast, in ADAR1-sufficient CONkd cells, only the Cko mutant virus was an effective inducer and the IFN-β RNA induction was amplified by PKR. The enhanced IFN-β transcript-inducing capacity of the WT and Vko viruses seen in ADAR1-deficient cells correlated with the enhanced activation of PKR, IFN regulatory factor IRF3, and activator of transcription ATF2, reaching levels similar to those seen in Cko virus-infected cells. However, the level of IFN-β protein produced was not proportional to the level of IFN-β RNA but rather correlated inversely with the level of activated PKR. These results suggest that ADAR1 functions as an important suppressor of MV-mediated responses, including the activation of PKR and IRF3 and the induction of IFN-β RNA. Our findings further implicate a balanced interplay between PKR and ADAR1 in modulating IFN-β protein production following virus infection. PMID:22278222

Over-expression of 3-hydroxy-3- methylglutaryl-coenzyme A reductase 1 (hmgr1) gene under super-promoter for enhanced latex biosynthesis in rubber tree (Hevea brasiliensis Muell. Arg.).

PubMed

Jayashree, R; Nazeem, P A; Rekha, K; Sreelatha, S; Thulaseedharan, A; Krishnakumar, R; Kala, R G; Vineetha, M; Leda, P; Jinu, U; Venkatachalam, P

2018-06-01

Natural rubber (cis-1, 4-polyisoprene) is being produced from bark laticifer cells of Hevea brasiliensis and the popular high latex yielding Indian rubber clones are easily prone to onset of tapping panel dryness syndrome (TPD) which is considered as a physiological syndrome affecting latex production either partially or completely. This report describes an efficient protocol for development of transgenic rubber plants by over-expression of 3-hydroxy 3-methylglutaryl Co-enzyme A reductase 1 (hmgr1) gene which is considered as rate limiting factor for latex biosynthesis via Agrobacterium-mediated transformation. The pBIB plasmid vector containing hmgr1 gene cloned under the control of a super-promoter was used for genetic transformation using embryogenic callus. Putatively transgenic cell lines were obtained on selection medium and produced plantlets with 44% regeneration efficiency. Transgene integration was confirmed by PCR amplification of 1.8 kb hmgr1 and 0.6 kb hpt genes from all putatively transformed callus lines as well as transgenic plants. Southern blot analysis showed the stable integration and presence of transgene in the transgenic plants. Over expression of hmgr1 transgene was determined by Northern blot hybridization, semi-quantitative PCR and real-time PCR (qRT-PCR) analysis. Accumulation of hmgr1 mRNA transcripts was more abundant in transgenic plants than control. Increased level of photosynthetic pigments, protein contents and HMGR enzyme activity was also noticed in transgenic plants over control. Interestingly, the latex yield was significantly enhanced in all transgenic plants compared to the control. The qRT-PCR results exhibit that the hmgr1 mRNA transcript levels was 160-fold more abundance in transgenic plants over untransformed control. These results altogether suggest that there is a positive correlation between latex yield and accumulation of mRNA transcripts level as well as HMGR enzyme activity in transgenic rubber plants. It is presumed that there is a possibility for enhanced level of latex biosynthesis in transgenic plants as the level of mRNA transcripts and HMGR enzyme activity is directly correlated with latex yield in rubber tree. Further, the present results clearly suggest that the quantification of HMGR enzyme activity in young seedlings will be highly beneficial for early selection of high latex yielding plants in rubber breeding programs. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

HES6 enhances the motility of alveolar rhabdomyosarcoma cells

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

Wickramasinghe, Caroline M; MRC Laboratory of Molecular Biology, Addenbrooke's Hospital Cambridge, CB2 0QH; Domaschenz, Renae

Absract: HES6, a member of the hairy-enhancer-of-split family of transcription factors, plays multiple roles in myogenesis. It is a direct target of the myogenic transcription factor MyoD and has been shown to regulate the formation of the myotome in development, myoblast cell cycle exit and the organization of the actin cytoskeleton during terminal differentiation. Here we investigate the expression and function of HES6 in rhabdomyosarcoma, a soft tissue tumor which expresses myogenic genes but fails to differentiate into muscle. We show that HES6 is expressed at high levels in the subset of alveolar rhabdomyosarcomas expressing PAX/FOXO1 fusion genes (ARMSp). Knockdownmore » of HES6 mRNA in the ARMSp cell line RH30 reduces proliferation and cell motility. This phenotype is rescued by expression of mouse Hes6 which is insensitive to HES6 siRNA. Furthermore, expression microarray analysis indicates that the HES6 knockdown is associated with a decrease in the levels of Transgelin, (TAGLN), a regulator of the actin cytoskeleton. Knockdown of TAGLN decreases cell motility, whilst TAGLN overexpression rescues the motility defect resulting from HES6 knockdown. These findings indicate HES6 contributes to the pathogenesis of ARMSp by enhancing both proliferation and cell motility.« less

Selective Cooperation between Fatty Acid Binding Proteins and Peroxisome Proliferator-Activated Receptors in Regulating Transcription

PubMed Central

Tan, Nguan-Soon; Shaw, Natacha S.; Vinckenbosch, Nicolas; Liu, Peng; Yasmin, Rubina; Desvergne, Béatrice; Wahli, Walter; Noy, Noa

2002-01-01

Lipophilic compounds such as retinoic acid and long-chain fatty acids regulate gene transcription by activating nuclear receptors such as retinoic acid receptors (RARs) and peroxisome proliferator-activated receptors (PPARs). These compounds also bind in cells to members of the family of intracellular lipid binding proteins, which includes cellular retinoic acid-binding proteins (CRABPs) and fatty acid binding proteins (FABPs). We previously reported that CRABP-II enhances the transcriptional activity of RAR by directly targeting retinoic acid to the receptor. Here, potential functional cooperation between FABPs and PPARs in regulating the transcriptional activities of their common ligands was investigated. We show that adipocyte FABP and keratinocyte FABP (A-FABP and K-FABP, respectively) selectively enhance the activities of PPARγ and PPARβ, respectively, and that these FABPs massively relocate to the nucleus in response to selective ligands for the PPAR isotype which they activate. We show further that A-FABP and K-FABP interact directly with PPARγ and PPARβ and that they do so in a receptor- and ligand-selective manner. Finally, the data demonstrate that the presence of high levels of K-FABP in keratinocytes is essential for PPARβ-mediated induction of differentiation of these cells. Taken together, the data establish that A-FABP and K-FABP govern the transcriptional activities of their ligands by targeting them to cognate PPARs in the nucleus, thereby enabling PPARs to exert their biological functions. PMID:12077340

Identification and characterization of an esterase involved in malathion resistance in the head louse Pediculus humanus capitis.

PubMed

Kwon, Deok Ho; Kim, Ju Hyeon; Kim, Young Ho; Yoon, Kyong Sup; Clark, J Marshall; Lee, Si Hyeock

2014-06-01

Enhanced malathion carboxylesterase (MCE) activity was previously reported to be involved in malathion resistance in the head louse Pediculus humanus capitis (Gao et al., 2006 [8]). To identify MCE, the transcriptional profiles of all five esterases that had been annotated to be catalytically active were determined and compared between the malathion-resistant (BR-HL) and malathion-susceptible (KR-HL) strains of head lice. An esterase gene, designated HLCbE3, exhibited approximately 5.4-fold higher transcription levels, whereas remaining four esterases did not exhibit a significant increase in their transcription in BR-HL, indicating that HLCbE3 may be the putative MCE. Comparison of the entire cDNA sequences of HLCbE3 revealed no sequence differences between the BR-HL and KR-HL strains and suggested that no single nucleotide polymorphism is associated with enhanced MCE activity. Two copies of the HLCbE3 gene were observed in BR-HL, implying that the over-transcription of HLCbE3 is due to the combination of a gene duplication and up-regulated transcription. Knockdown of HLCbE3 expression by RNA interference in the BR-HL strain led to increases in malathion susceptibility, confirming the identity of HLCbE3 as a MCE responsible for malathion resistance in the head louse. Phylogenetic analysis suggested that HLCbE3 is a typical dietary esterase and belongs to a clade containing various MCEs involved in malathion resistance. Copyright © 2014 Elsevier Inc. All rights reserved.

Postnatal ablation of osteoblast Smad4 enhances proliferative responses to canonical Wnt signaling through interactions with β-catenin

PubMed Central

Salazar, Valerie S.; Zarkadis, Nicholas; Huang, Lisa; Watkins, Marcus; Kading, Jacqueline; Bonar, Sheri; Norris, Jin; Mbalaviele, Gabriel; Civitelli, Roberto

2013-01-01

Summary Canonical Wnt (cWnt) signaling through β-catenin regulates osteoblast proliferation and differentiation to enhance bone formation. We previously reported that osteogenic action of β-catenin is dependent on BMP signaling. Here, we further examined interactions between cWnt and BMP in bone. In osteoprogenitors stimulated with BMP2, β-catenin localizes to the nucleus, physically interacts with Smad4, and is recruited to DNA-binding transcription complexes containing Smad4, R-Smad1/5 and TCF4. Furthermore, Tcf/Lef-dependent transcription, Ccnd1 expression and proliferation all increase when Smad4, 1 or 5 levels are low, whereas TCF/Lef activities decrease when Smad4 expression is high. The ability of Smad4 to antagonize transcription of Ccnd1 is dependent on DNA-binding activity but Smad4-dependent transcription is not required. In mice, conditional deletion of Smad4 in osterix+ cells increases mitosis of cells on trabecular bone surfaces as well as in primary osteoblast cultures from adult bone marrow and neonatal calvaria. By contrast, ablation of Smad4 delays differentiation and matrix mineralization by primary osteoblasts in response to Wnt3a, indicating that loss of Smad4 perturbs the balance between proliferation and differentiation in osteoprogenitors. We propose that Smad4 and Tcf/Lef transcription complexes compete for β-catenin, thus restraining cWnt-dependent proliferative signals while favoring the matrix synthesizing activity of osteoblasts. PMID:24101723

Diverse patterns of genomic targeting by transcriptional regulators in Drosophila melanogaster.

PubMed

Slattery, Matthew; Ma, Lijia; Spokony, Rebecca F; Arthur, Robert K; Kheradpour, Pouya; Kundaje, Anshul; Nègre, Nicolas; Crofts, Alex; Ptashkin, Ryan; Zieba, Jennifer; Ostapenko, Alexander; Suchy, Sarah; Victorsen, Alec; Jameel, Nader; Grundstad, A Jason; Gao, Wenxuan; Moran, Jennifer R; Rehm, E Jay; Grossman, Robert L; Kellis, Manolis; White, Kevin P

2014-07-01

Annotation of regulatory elements and identification of the transcription-related factors (TRFs) targeting these elements are key steps in understanding how cells interpret their genetic blueprint and their environment during development, and how that process goes awry in the case of disease. One goal of the modENCODE (model organism ENCyclopedia of DNA Elements) Project is to survey a diverse sampling of TRFs, both DNA-binding and non-DNA-binding factors, to provide a framework for the subsequent study of the mechanisms by which transcriptional regulators target the genome. Here we provide an updated map of the Drosophila melanogaster regulatory genome based on the location of 84 TRFs at various stages of development. This regulatory map reveals a variety of genomic targeting patterns, including factors with strong preferences toward proximal promoter binding, factors that target intergenic and intronic DNA, and factors with distinct chromatin state preferences. The data also highlight the stringency of the Polycomb regulatory network, and show association of the Trithorax-like (Trl) protein with hotspots of DNA binding throughout development. Furthermore, the data identify more than 5800 instances in which TRFs target DNA regions with demonstrated enhancer activity. Regions of high TRF co-occupancy are more likely to be associated with open enhancers used across cell types, while lower TRF occupancy regions are associated with complex enhancers that are also regulated at the epigenetic level. Together these data serve as a resource for the research community in the continued effort to dissect transcriptional regulatory mechanisms directing Drosophila development. © 2014 Slattery et al.; Published by Cold Spring Harbor Laboratory Press.

Differential Regulation of Telomerase Reverse Transcriptase Promoter Activation and Protein Degradation by Histone Deacetylase Inhibition.

PubMed

Qing, Hua; Aono, Jun; Findeisen, Hannes M; Jones, Karrie L; Heywood, Elizabeth B; Bruemmer, Dennis

2016-06-01

Telomerase reverse transcriptase (TERT) maintains telomeres and is rate limiting for replicative life span. While most somatic tissues silence TERT transcription resulting in telomere shortening, cells derived from cancer or cardiovascular diseases express TERT and activate telomerase. In the present study, we demonstrate that histone deacetylase (HDAC) inhibition induces TERT transcription and promoter activation. At the protein level in contrast, HDAC inhibition decreases TERT protein abundance through enhanced degradation, which decreases telomerase activity and induces senescence. Finally, we demonstrate that HDAC inhibition decreases TERT expression during vascular remodeling in vivo. These data illustrate a differential regulation of TERT transcription and protein stability by HDAC inhibition and suggest that TERT may constitute an important target for the anti-proliferative efficacy of HDAC inhibitors. © 2015 Wiley Periodicals, Inc.

Transcriptional regulation of methionine synthase by homocysteine and choline in Aspergillus nidulans.

PubMed Central

Kacprzak, Magdalena M; Lewandowska, Irmina; Matthews, Rowena G; Paszewski, Andrzej

2003-01-01

Roles played by homocysteine and choline in the regulation of MS (methionine synthase) have been examined in fungi. The Aspergillus nidulans metH gene encoding MS was cloned and characterized. Its transcription was not regulated by methionine, but was enhanced by homocysteine and repressed by choline and betaine. MS activity levels were regulated in a similar way. The repression by betaine was due to its metabolic conversion to choline, which was found to be very efficient in A. nidulans. Betaine and choline supplementation stimulated growth of leaky metH mutants apparently by decreasing the demand for methyl groups and thus saving methionine and S -adenosylmethionine. We have also found that homocysteine stimulates transcription of MS-encoding genes in Saccharomyces cerevisiae and Schizosaccharomyces pombe. PMID:12954077

An ABA-responsive DRE-binding protein gene from Setaria italica, SiARDP, the target gene of SiAREB, plays a critical role under drought stress.

PubMed

Li, Cong; Yue, Jing; Wu, Xiaowei; Xu, Cong; Yu, Jingjuan

2014-10-01

The DREB (dehydration-responsive element binding)-type transcription factors regulate the expression of stress-inducible genes by binding the DRE/CRT cis-elements in promoter regions. The upstream transcription factors that regulate the transcription of DREB transcription factors have not been clearly defined, although the function of DREB transcription factors in abiotic stress is known. In this study, an abscisic acid (ABA)-responsive DREB-binding protein gene (SiARDP) was cloned from foxtail millet (Setaria italica). The transcript level of SiARDP increased not only after drought, high salt, and low temperature stresses, but also after an ABA treatment in foxtail millet seedlings. Two ABA-responsive elements (ABRE1: ACGTGTC; ABRE2: ACGTGGC) exist in the promoter of SiARDP. Further analyses showed that two ABA-responsive element binding (AREB)-type transcription factors, SiAREB1 and SiAREB2, could physically bind to the ABRE core element in vitro and in vivo. The constitutive expression of SiARDP in Arabidopsis thaliana enhanced drought and salt tolerance during seed germination and seedling development, and overexpression of SiARDP in foxtail millet improved drought tolerance. The expression levels of target genes of SiARDP were upregulated in transgenic Arabidopsis and foxtail millet. These results reveal that SiARDP, one of the target genes of SiAREB, is involved in ABA-dependent signal pathways and plays a critical role in the abiotic stress response in plants. © The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Expression patterns of ERVWE1/Syncytin-1 and other placentally expressed human endogenous retroviruses along the malignant transformation process of hydatidiform moles.

PubMed

Bolze, Pierre-Adrien; Patrier, Sophie; Cheynet, Valérie; Oriol, Guy; Massardier, Jérôme; Hajri, Touria; Guillotte, Michèle; Bossus, Marc; Sanlaville, Damien; Golfier, François; Mallet, François

2016-03-01

Up to 20% of hydatidiform moles are followed by malignant transformation in gestational trophoblastic neoplasia and require chemotherapy. Syncytin-1 is involved in human placental morphogenesis and is also expressed in various cancers. We assessed the predictive value of the expression of Syncytin-1 and its interactants in the malignant transformation process of hydatidiform moles. Syncytin-1 glycoprotein was localized by immunohistochemistry in hydatidiform moles, gestational trophoblastic neoplasia and control placentas. The transcription levels of its locus ERVWE1, its interaction partners (hASCT1, hASCT2, TLR4 and DC-SIGN) and two loci (ERVFRDE1 and ERV3) involved the expression of other placental envelopes were assessed by real-time PCR. Syncytin-1 glycoprotein was expressed in syncytiotrophoblast of hydatidiform moles with an apical enhancement when compared with normal placentas. Moles with further malignant transformation had a higher staining intensity of Syncytin-1 surface unit C-terminus but the transcription level of its locus ERVWE1 was not different from that of moles with further remission and normal placentas. hASCT1 and TLR4, showed lower transcription levels in complete moles when compared to normal placentas. ERVWE1, ERVFRDE1 and ERV3 transcription was down-regulated in hydatidiform moles and gestational trophoblastic neoplasia. Variations of Syncytin-1 protein localization and down-regulation of hASCT1 and TLR4 transcription are likely to reflect altered functions of Syncytin-1 in the premalignant context of complete moles. The reduced transcription in gestational trophoblastic diseases of ERVWE1, ERVFRDE1 and ERV3, which expression during normal pregnancy is differentially regulated by promoter region methylation, suggest a joint dysregulation mechanism in malignant context. Copyright © 2016 Elsevier Ltd. All rights reserved.

Requirement of multiple cis-acting elements in the human cytomegalovirus major immediate-early distal enhancer for viral gene expression and replication.

PubMed

Meier, Jeffery L; Keller, Michael J; McCoy, James J

2002-01-01

We have shown previously that the human cytomegalovirus (HCMV) major immediate-early (MIE) distal enhancer is needed for MIE promoter-dependent transcription and viral replication at low multiplicities of infection (MOI). To understand how this region works, we constructed and analyzed a series of HCMVs with various distal enhancer mutations. We show that the distal enhancer is composed of at least two parts that function independently to coordinately activate MIE promoter-dependent transcription and viral replication. One such part is contained in a 47-bp segment that has consensus binding sites for CREB/ATF, SP1, and YY1. At low MOI, these working parts likely function in cis to directly activate MIE gene expression, thus allowing viral replication to ensue. Three findings support the view that these working parts are likely cis-acting elements. (i) Deletion of either part of a bisegmented distal enhancer only slightly alters MIE gene transcription and viral replication. (ii) Reversing the distal enhancer's orientation largely preserves MIE gene transcription and viral replication. (iii) Placement of stop codons at -300 or -345 in all reading frames does not impair MIE gene transcription and viral replication. Lastly, we show that these working parts are dispensable at high MOI, partly because of compensatory stimulation of MIE promoter activity and viral replication that is induced by a virion-associated component(s) present at a high viral particle/cell ratio. We conclude that the distal enhancer is a complex multicomponent cis-acting region that is required to augment both MIE promoter-dependent transcription and HCMV replication.

Sinensetin enhances adipogenesis and lipolysis by increasing cyclic adenosine monophosphate levels in 3T3-L1 adipocytes.

PubMed

Kang, Seong-Il; Shin, Hye-Sun; Kim, Se-Jae

2015-01-01

Sinensetin is a rare polymethoxylated flavone (PMF) found in certain citrus fruits. In this study, we investigated the effects of sinensetin on lipid metabolism in 3T3-L1 cells. Sinensetin promoted adipogenesis in 3T3-L1 preadipocytes growing in incomplete differentiation medium, which did not contain 3-isobutyl-1-methylxanthine. Sinensetin up-regulated expression of the adipogenic transcription factors peroxisome proliferator-activated receptor γ, CCAAT/enhancer-binding protein (C/EBP) α, and sterol regulatory element-binding protein 1c. It also potentiated expression of C/EBPβ and activation of cAMP-responsive element-binding protein. Sinensetin enhanced activation of protein kinase A and increased intracellular cAMP levels in 3T3-L1 preadipocytes. In mature 3T3-L1 adipocytes, sinensetin stimulated lipolysis via a cAMP pathway. Taken together, these results suggest that sinensetin enhances adipogenesis and lipolysis by increasing cAMP levels in adipocytes.

Ectopic expression of different cytokinin-regulated transcription factor genes of Arabidopsis thaliana alters plant growth and development.

PubMed

Köllmer, Ireen; Werner, Tomáš; Schmülling, Thomas

2011-08-15

The plant hormone cytokinin rapidly alters the steady state transcript levels of a number of transcription factor genes suggesting that these might have a function in mediating cytokinin effects. Here we report the analysis of Arabidopsis thaliana plants with an altered expression level of four different cytokinin-regulated transcription factor genes. These include GATA22 (also known as CGA1/GNL), two genes coding for members of the homeodomain zip (HD zip) class II transcription factor family (HAT4, HAT22), and bHLH64. Ectopic expression of the GATA22 gene induced the development of chloroplasts in root tissue where it is normally suppressed and led to the formation of shorter and less branched roots. Overexpression of HAT22 lowered the seedlings chlorophyll content and caused an earlier onset of leaf senescence. Enhanced expression of the HAT4 gene led to severe defects in inflorescence stem development and to a decrease in root growth and branching, while hat4 insertional mutants developed a larger root system. 35S:bHLH64 transgenic plants showed a pleiotropic phenotype, consisting of larger rosettes, reduced chlorophyll content and an elongated and thickened hypocotyl. Flower development was strongly disturbed leading to sterile plants. The results are consistent with specific functions of these transcription factor genes in regulating part of the cytokinin activities and suggest their action as convergence point with other signalling pathways, particularly those of gibberellin and light. Copyright © 2011 Elsevier GmbH. All rights reserved.

Changes in metabolites, antioxidant system, and gene expression in Microcystis aeruginosa under sodium chloride stress.

PubMed

Chen, Lei; Mao, Feijian; Kirumba, George Chira; Jiang, Cheng; Manefield, Mike; He, Yiliang

2015-12-01

Microcystis (M.) aeruginosa, one of the most common bloom-forming cyanobacteria, occurs worldwide. The Qingcaosha (QCS) Reservoir is undergoing eutrophication and faces the problem of saltwater intrusion. The aim of this study was to investigate the effects of sudden salinity changes on physiological parameters and related gene transcription in M. aeruginosa under controlled laboratory conditions. The results showed that sodium chloride (50, 200 and 500 mg L(-1) NaCl) inhibited the algal growth and decreased pigment concentrations (chlorophyll a, carotenoid and phycocyanin). Sodium chloride increased both the intracellular and extracellular microcystin contents and elevated the mcyD transcript level in M. aeruginosa. It also increased the malondialdehyde (MDA) content and caused cytomembrane damage. This damage caused the release of intracellular toxins into the culture medium. In addition, NaCl decreased the maximum electron transport rate, increased the levels of reactive oxygen species (ROS) and changed the cellular redox status. Consequently, NaCl inhibited the expression of cpcB, psbA and rbcL. Furthermore, NaCl increased the activities of superoxide dismutases (SOD), catalase (CAT), glutathione reductase (GR), and total glutathione peroxidase (GPx). The transcript levels of sod and reduced glutathione (gsh) were also increased after exposure to NaCl. Our results indicate that a sudden increase in salinity increases the production and excretion of microcystin, changes the cellular redox status, enhances the activities of antioxidant enzymes, inhibits photosynthesis, and affects transcript levels of related genes in M. aeruginosa. Copyright © 2015 Elsevier Inc. All rights reserved.

Transcriptional profiling of Medicago truncatula under salt stress identified a novel CBF transcription factor MtCBF4 that plays an important role in abiotic stress responses

PubMed Central

2011-01-01

Background Salt stress hinders the growth of plants and reduces crop production worldwide. However, different plant species might possess different adaptive mechanisms to mitigate salt stress. We conducted a detailed pathway analysis of transcriptional dynamics in the roots of Medicago truncatula seedlings under salt stress and selected a transcription factor gene, MtCBF4, for experimental validation. Results A microarray experiment was conducted using root samples collected 6, 24, and 48 h after application of 180 mM NaCl. Analysis of 11 statistically significant expression profiles revealed different behaviors between primary and secondary metabolism pathways in response to external stress. Secondary metabolism that helps to maintain osmotic balance was induced. One of the highly induced transcription factor genes was successfully cloned, and was named MtCBF4. Phylogenetic analysis revealed that MtCBF4, which belongs to the AP2-EREBP transcription factor family, is a novel member of the CBF transcription factor in M. truncatula. MtCBF4 is shown to be a nuclear-localized protein. Expression of MtCBF4 in M. truncatula was induced by most of the abiotic stresses, including salt, drought, cold, and abscisic acid, suggesting crosstalk between these abiotic stresses. Transgenic Arabidopsis over-expressing MtCBF4 enhanced tolerance to drought and salt stress, and activated expression of downstream genes that contain DRE elements. Over-expression of MtCBF4 in M. truncatula also enhanced salt tolerance and induced expression level of corresponding downstream genes. Conclusion Comprehensive transcriptomic analysis revealed complex mechanisms exist in plants in response to salt stress. The novel transcription factor gene MtCBF4 identified here played an important role in response to abiotic stresses, indicating that it might be a good candidate gene for genetic improvement to produce stress-tolerant plants. PMID:21718548

Epigenetic programming of T cells impacts immune reconstitution in hematopoietic stem cell transplant recipients.

PubMed

Hardy, Kristine; Smith, Corey; Tu, Wen Juan; McCuaig, Robert; Panikkar, Archana; Dasari, Vijayendra; Wu, Fan; Tey, Siok-Keen; Hill, Geoffrey R; Khanna, Rajiv; Rao, Sudha

2018-03-27

Immune reconstitution following hematopoietic stem cell transplantation (HSCT) is critical in preventing harmful sequelae in recipients with cytomegalovirus (CMV) infection. To understand the molecular mechanisms underlying immune reconstitution kinetics, we profiled the transcriptome-chromatin accessibility landscape of CMV-specific CD8 + T cells from HCST recipients with different immune reconstitution efficiencies. CMV-specific T cells from HSCT recipients with stable antiviral immunity expressed higher levels of interferon/defense response and cell cycle genes in an interconnected network involving PI3KCG , STAT5B , NFAT , RBPJ , and lower HDAC6 , increasing chromatin accessibility at the enhancer regions of immune and T-cell receptor signaling pathway genes. By contrast, the transcriptional and epigenomic signatures of CMV-specific T cells from HSCT recipients with unstable immune reconstitution showed commonalities with T-cell responses in other nonresolving chronic infections. These signatures included higher levels of EGR and KLF factors that, along with lower JARID2 expression, maintained higher accessibility at promoter and CpG-rich regions of genes associated with apoptosis. Furthermore, epigenetic targeting via inhibition of HDAC6 or JARID2 enhanced the transcription of genes associated with differential responses, suggesting that drugs targeting epigenomic modifiers may have therapeutic potential for enhancing immune reconstitution in HSCT recipients. Taken together, these analyses demonstrate that transcription factors and chromatin modulators create different chromatin accessibility landscapes in T cells of HSCT recipients that not only affect immediate gene expression but also differentially prime cells for responses to additional signals. Epigenetic therapy may be a promising strategy to promote immune reconstitution in HSCT recipients. © 2018 by The American Society of Hematology.

1,25-DIHYDROXYVITAMIN D3 INDUCES MONOCYTIC DIFFERENTIATION OF HUMAN MYELOID LEUKEMIA CELLS BY REGULATING C/EBPβ EXPRESSION THROUGH MEF2C

PubMed Central

Zheng, Ruifang; Wang, Xuening; Studzinski, George P.

2015-01-01

Myogenic enhancer factor2 (Mef2) consists of a family of transcription factors involved in morphogenesis of skeletal, cardiac and smooth muscle cells. Among the four isoforms (Mef2A, 2B, 2C, and 2D), Mef2C was also found to play important roles in hematopoiesis. At myeloid progenitor level, Mef2C expression favors monocytic differentiation. Previous studies from our laboratory demonstrated that ERK5 was activated in 1,25-dihydroxyvitamin D3 (1,25D)-induced monocytic differentiation in AML cells and ERK5 activation was accompanied by increased Mef2C phosphorylation. We therefore examined the role of Mef2C in 1,25D-induced monocytic differentiation in AML cell lines (HL60, U937 and THP1) and found that knockdown of Mef2C with small interfering RNA (siRNA) significantly decreases the expression of the monocytic marker, CD14, without affecting the expression of the general myeloid marker, CD11b. CCAAT/Enhancer-binding protein (C/EBP) β, which can bind to CD14 promoter and increase its transcription, has been shown to be the downstream effector of 1,25D-induced monocytic differentiation in AML cells. When Mef2C was knocked down, expression of C/EBPβ was reduced at both mRNA and protein levels. The protein expression levels of cell cycle regulators, p27Kip1 and cyclin D1, were not affected by Mef2C knockdown, nor the monopoiesis related transcription factor, ATF2 (Activating Transcription Factor 2). Thus, we conclude that 1,25D-induced monocytic differentiation, and CD14 expression in particular, is mediated through activation of ERK5-Mef2C-C/EBPβ signaling pathway, and that Mef2C does not seem to modulate cell cycle progression. PMID:25448741

The transcription factor LEF-1 induces an epithelial–mesenchymal transition in MDCK cells independent of β-catenin

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

Kobayashi, Wakako; Ozawa, Masayuki, E-mail: mozawa@m.kufm.kagoshima-u.ac.jp

2013-12-06

Highlights: •The transcription factor LEF-1 induces an EMT in MDCK cells. •A mutant LEF-1 that cannot interact with β-catenin retained the ability. •The nuclear function of β-catenin was not necessary for the LEF-1-induced EMT. •The mRNA levels of Slug, ZEB1, and ZEB2 increased significantly in these cells. -- Abstract: The epithelial–mesenchymal transition (EMT), a key process in the tumor metastatic cascade, is characterized by the loss of cell–cell junctions and cell polarity, as well as the acquisition of migratory and invasive properties. LEF-1 is a member of the lymphoid enhancer-binding factor/T-cell factor (LEF/TCF) family of DNA-binding transcription factors, which interactmore » with nuclear β-catenin and act as central transcriptional mediators of Wnt signaling. To investigate the role of LEF-1 in EMT, we generated stable LEF-1 transfectants using MDCK cells. The transfectants had a spindle-shaped mesenchymal morphology, and enhanced migration and invasiveness relative to control cells. These EMT changes were accompanied by the downregulation of an epithelial marker protein, E-cadherin, and the upregulation of mesenchymal marker proteins, vimentin and N-cadherin. Consistent with these observations, the mRNA levels of Slug, ZEB1, and ZEB2—EMT-related transcription factors—increased significantly. Although the N-terminally deleted mutant LEF-1 cannot interact with β-catenin, it retained the ability to induce EMT. Consistent with these observations, neither the expression of a dominant negative β-catenin/engrailed chimera, nor the expression of a cytoplasmic domain of E-cadherin that sequesters β-catenin from binding to LEF/TCF, reversed LEF-1-induced EMT. Together, these data indicated that the nuclear function of β-catenin was not necessary for the induction of Slug, ZEB1, and ZEB2 expression leading to EMT.« less

Genetic variation in the MITF promoter affects skin colour and transcriptional activity in black-boned chickens.

PubMed

Wang, G; Liao, J; Tang, M; Yu, S

2018-02-01

1. Microphthalmia-associated transcription factor (MITF) plays a pivotal role in melanocyte development by regulating the transcription of major pigmentation enzymes (e.g. TYR, TYRP1 and DCT). A single-nucleotide polymorphism (SNP), c.-638T>C, was identified in the MITF promoter, and genotyping of a population (n = 426) revealed that SNP c.-638T>C was associated with skin colour in black-boned chickens. 2. Individuals with genotypes CC and TC exhibited greater MTIF expression than those with genotype TT. Luciferase assays also revealed that genotype CC and TC promoters had higher activity levels than genotype TT. Expression of melanogenesis-related gene (TYR) was higher in the skin of chickens with the CC and CT genotype compared to TT chickens (P < 0.05). 3. Transcription factor-binding site analyses showed that the c.-638C allele contains a putative binding site for transcription factor sterol regulatory element-binding transcription factor 2, aryl hydrocarbon receptor nuclear translocator, transcription factor binding to IGHM enhancer 3 and upstream transcription factor 2. In contrast, the c.-638T allele contains binding sites for Sp3 transcription factor and Krüppel-like factor 1. 4. It was concluded that MITF promoter polymorphisms affected chicken skin colour. SNP c.-638T>C could be used for the marker-assisted selection of skin colour in black-boned chicken breeding.

Dexamethasone Enhances 1α,25-Dihydroxyvitamin D3 Effects by Increasing Vitamin D Receptor Transcription*

PubMed Central

Hidalgo, Alejandro A.; Deeb, Kristin K.; Pike, J. Wesley; Johnson, Candace S.; Trump, Donald L.

2011-01-01

Calcitriol, the active form of vitamin D, in combination with the glucocorticoid dexamethasone (Dex) has been shown to increase the antitumor effects of calcitriol in squamous cell carcinoma. In this study we found that pretreatment with Dex potentiates calcitriol effects by inhibiting cell growth and increasing vitamin D receptor (VDR) and VDR-mediated transcription. Treatment with actinomycin D inhibits Vdr mRNA synthesis, indicating that Dex regulates VDR expression at transcriptional level. Real time PCR shows that treatment with Dex increases Vdr transcripts in a time- and a dose-dependent manner, indicating that Dex directly regulates expression of Vdr. RU486, an inhibitor of glucocorticoids, inhibits Dex-induced Vdr expression. In addition, the silencing of glucocorticoid receptor (GR) abolishes the induction of Vdr by Dex, indicating that Dex increases Vdr transcripts in a GR-dependent manner. A fragment located 5.2 kb upstream of Vdr transcription start site containing two putative glucocorticoid response elements (GREs) was evaluated using a luciferase-based reporter assay. Treatment with 100 nm Dex induces transcription of luciferase driven by the fragment. Deletion of the GRE distal to transcription start site was sufficient to abolish Dex induction of luciferase. Also, chromatin immunoprecipitation reveals recruitment of GR to distal GRE with Dex treatment. We conclude that Dex increases VDR and vitamin D effects by increasing Vdr de novo transcription in a GR-dependent manner. PMID:21868377

Intestinal Master Transcription Factor CDX2 Controls Chromatin Access for Partner Transcription Factor Binding

PubMed Central

Verzi, Michael P.; Shin, Hyunjin; San Roman, Adrianna K.

2013-01-01

Tissue-specific gene expression requires modulation of nucleosomes, allowing transcription factors to occupy cis elements that are accessible only in selected tissues. Master transcription factors control cell-specific genes and define cellular identities, but it is unclear if they possess special abilities to regulate cell-specific chromatin and if such abilities might underlie lineage determination and maintenance. One prevailing view is that several transcription factors enable chromatin access in combination. The homeodomain protein CDX2 specifies the embryonic intestinal epithelium, through unknown mechanisms, and partners with transcription factors such as HNF4A in the adult intestine. We examined enhancer chromatin and gene expression following Cdx2 or Hnf4a excision in mouse intestines. HNF4A loss did not affect CDX2 binding or chromatin, whereas CDX2 depletion modified chromatin significantly at CDX2-bound enhancers, disrupted HNF4A occupancy, and abrogated expression of neighboring genes. Thus, CDX2 maintains transcription-permissive chromatin, illustrating a powerful and dominant effect on enhancer configuration in an adult tissue. Similar, hierarchical control of cell-specific chromatin states is probably a general property of master transcription factors. PMID:23129810

Integrated Metabolomics and Transcriptomics Reveal Enhanced Specialized Metabolism in Medicago truncatula Root Border Cells1[OPEN

PubMed Central

Watson, Bonnie S.; Bedair, Mohamed F.; Urbanczyk-Wochniak, Ewa; Huhman, David V.; Yang, Dong Sik; Allen, Stacy N.; Li, Wensheng; Tang, Yuhong; Sumner, Lloyd W.

2015-01-01

Integrated metabolomics and transcriptomics of Medicago truncatula seedling border cells and root tips revealed substantial metabolic differences between these distinct and spatially segregated root regions. Large differential increases in oxylipin-pathway lipoxygenases and auxin-responsive transcript levels in border cells corresponded to differences in phytohormone and volatile levels compared with adjacent root tips. Morphological examinations of border cells revealed the presence of significant starch deposits that serve as critical energy and carbon reserves, as documented through increased β-amylase transcript levels and associated starch hydrolysis metabolites. A substantial proportion of primary metabolism transcripts were decreased in border cells, while many flavonoid- and triterpenoid-related metabolite and transcript levels were increased dramatically. The cumulative data provide compounding evidence that primary and secondary metabolism are differentially programmed in border cells relative to root tips. Metabolic resources normally destined for growth and development are redirected toward elevated accumulation of specialized metabolites in border cells, resulting in constitutively elevated defense and signaling compounds needed to protect the delicate root cap and signal motile rhizobia required for symbiotic nitrogen fixation. Elevated levels of 7,4′-dihydroxyflavone were further increased in border cells of roots exposed to cotton root rot (Phymatotrichopsis omnivora), and the value of 7,4′-dihydroxyflavone as an antimicrobial compound was demonstrated using in vitro growth inhibition assays. The cumulative and pathway-specific data provide key insights into the metabolic programming of border cells that strongly implicate a more prominent mechanistic role for border cells in plant-microbe signaling, defense, and interactions than envisioned previously. PMID:25667316

Combined protein- and nucleic acid-level effects of rs1143679 (R77H), a lupus-predisposing variant within ITGAM.

PubMed

Maiti, Amit K; Kim-Howard, Xana; Motghare, Prasenjeet; Pradhan, Vandana; Chua, Kek Heng; Sun, Celi; Arango-Guerrero, María Teresa; Ghosh, Kanjaksha; Niewold, Timothy B; Harley, John B; Anaya, Juan-Manual; Looger, Loren L; Nath, Swapan K

2014-08-01

Integrin alpha M (ITGAM; CD11b) is a component of the macrophage-1 antigen complex, which mediates leukocyte adhesion, migration and phagocytosis as part of the immune system. We previously identified a missense polymorphism, rs1143679 (R77H), strongly associated with systemic lupus erythematosus (SLE). However, the molecular mechanisms of this variant are incompletely understood. A meta-analysis of published and novel data on 28 439 individuals with European, African, Hispanic and Asian ancestries reinforces genetic association between rs1143679 and SLE [Pmeta = 3.60 × 10(-90), odds ratio (OR) = 1.76]. Since rs1143679 is in the most active region of chromatin regulation and transcription factor binding in ITGAM, we quantitated ITGAM RNA and surface protein levels in monocytes from patients with each rs1143679 genotype. We observed that transcript levels significantly decreased for the risk allele ('A') relative to the non-risk allele ('G'), in a dose-dependent fashion: ('AA' < 'AG' < 'GG'). CD11b protein levels in patients' monocytes were directly correlated with RNA levels. Strikingly, heterozygous individuals express much lower (average 10- to 15-fold reduction) amounts of the 'A' transcript than 'G' transcript. We found that the non-risk sequence surrounding rs1143679 exhibits transcriptional enhancer activity in vivo and binds to Ku70/80, NFKB1 and EBF1 in vitro, functions that are significantly reduced with the risk allele. Mutant CD11b protein shows significantly reduced binding to fibrinogen and vitronectin, relative to non-risk, both in purified protein and in cellular models. This two-pronged contribution (nucleic acid- and protein-level) of the rs1143679 risk allele to decreasing ITGAM activity provides insight into the molecular mechanisms of its potent association with SLE. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Inflammatory response to Escherichia coli urinary tract infection in the neurogenic bladder of the spinal cord injured host.

PubMed

Chaudhry, Rajeev; Madden-Fuentes, Ramiro J; Ortiz, Tara K; Balsara, Zarine; Tang, Yuping; Nseyo, Unwanaobong; Wiener, John S; Ross, Sherry S; Seed, Patrick C

2014-05-01

Urinary tract infections cause significant morbidity in patients with spinal cord injury. An in vivo spinal cord injured rat model of experimental Escherichia coli urinary tract infection mimics human disease with enhanced susceptibility to urinary tract infection compared to controls. We hypothesized that a dysregulated inflammatory response contributes to enhanced susceptibility to urinary tract infection. Spinal cord injured and sham injured rats were inoculated transurethrally with E. coli. Transcript levels of 84 inflammatory pathway genes were measured in bladder tissue of each group before infection, 24 hours after infection and after 5 days of antibiotic therapy. Before infection quantitative polymerase chain reaction array revealed greater than twofold up-regulation in the proinflammatory factor transcripts slc11a1, ccl4 and il1β, and down-regulation of the antimicrobial peptides lcn2 and mpo in spinal cord injured vs control bladders. At 24 hours after infection spinal cord injured bladders showed an attenuated innate immune response with decreased expression of il6, slc11a1, il1β and lcn2, and decreased il10 and slpi expression compared to controls. Despite clearance of bacteriuria with antibiotics spinal cord injured rats had delayed induction of il6 transcription and a delayed anti-inflammatory response with decreased il10 and slpi transcript levels relative to controls. Spinal cord injured bladders fail to mount a characteristic inflammatory response to E. coli infection and cannot suppress inflammation after infection is eliminated. This may lead to increased susceptibility to urinary tract infection and persistent chronic inflammation through neural mediated pathways, which to our knowledge remain to be defined. Copyright © 2014 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.

Requirement of the cyclic adenosine monophosphate response element-binding protein for hepatitis B virus replication.

PubMed

Kim, Bo Kyung; Lim, Seoung Ok; Park, Yun Gyu

2008-08-01

The cyclic adenosine monophosphate-response element (CRE)-transcription factor complex participates in the regulation of viral gene expression and pathologic processes caused by various viruses. The hepatitis B virus (HBV) enhancer I directs liver-specific transcription of viral genes and contains a CRE sequence (HBV-CRE); however, whether the HBV-CRE and CRE-binding protein (CREB) are required for the HBV life cycle remains to be determined. This study was designed to investigate the role of CREB in HBV replication and gene expression. Sequence-comparison analysis of 984 HBVs reported worldwide showed that the HBV-CRE sequence is highly conserved, indicating the possibility that it plays an important role in the HBV life cycle. The binding of CREB to the HBV-CRE site was markedly inhibited by oligonucleotides containing HBV-CRE and consensus CRE sequences in vitro and in vivo. The HBV promoter activity was demonstrated to be dependent upon the transactivation activity of CREB. Treatment with CRE decoy oligonucleotides reduced HBV promoter activity, and this was reversed by CREB overexpression. The levels of viral transcripts, DNA, and antigens were remarkably decreased in response to the overexpression of CREB mutants or treatment with the CRE decoy oligonucleotides, whereas enhancing CREB activity increased the levels of viral transcripts. In addition, introduction of a three-base mutation into the HBV-CRE led to a marked reduction in HBV messenger RNA synthesis. Taken together, our results demonstrate that both replication and gene expression of HBV require a functional CREB and HBV-CRE. We have also demonstrated that CRE decoy oligonucleotides and the overexpression of CREB mutants can effectively block the HBV life cycle, suggesting that interventions against CREB activity could provide a new avenue to treat HBV infection.

R-spondin1/Wnt-enhanced Ascl2 autoregulation controls the self-renewal of colorectal cancer progenitor cells.

PubMed

Ye, Jun; Liu, Shanxi; Shang, Yangyang; Chen, Haoyuan; Wang, Rongquan

2018-06-25

The Wnt signaling pathway controls stem cell identity in the intestinal epithelium and cancer stem cells (CSCs). The transcription factor Ascl2 (Wnt target gene) is fate decider of intestinal cryptic stem cells and colon cancer stem cells. It is unclear how Wnt signaling is translated into Ascl2 expression and keeping the self-renewal of CRC progenitor cells. We showed that the exogenous Ascl2 in colorectal cancer (CRC) cells activated the endogenous Ascl2 expression via a direct autoactivatory loop, including Ascl2 binding to its own promoter and further transcriptional activation. Higher Ascl2 expression in human CRC cancerous tissues led to greater enrichment in Ascl2 immunoprecipitated DNA within the Ascl2 promoter in the CRC cancerous sample than the peri-cancerous mucosa. Ascl2 binding to its own promoter and inducing further transcriptional activation of the Ascl2 gene was predominant in the CD133 + CD44 + CRC population. R-spondin1/Wnt activated Ascl2 expression dose-dependently in the CD133 + CD44 + CRC population, but not in the CD133 - CD44 - CRC population, which was caused by differences in Ascl2 autoregulation under R-spondin1/Wnt activation. R-spondin1/Wnt treatment in the CD133 + CD44 + or CRC CD133 - CD44 - populations exerted a different pattern of stemness maintenance, which was defined by alterations of the mRNA levels of stemness-associated genes, the protein expression levels (Bmi1, C-myc, Oct-4 and Nanog) and tumorsphere formation. The results indicated that Ascl2 autoregulation formed a transcriptional switch that was enhanced by Wnt signaling in the CD133 + CD44 + CRC population, thus conferring their self-renewal.

Disruption of Higher-Order Folding by Core Histone Acetylation Dramatically Enhances Transcription of Nucleosomal Arrays by RNA Polymerase III

PubMed Central

Tse, Christin; Sera, Takashi; Wolffe, Alan P.; Hansen, Jeffrey C.

1998-01-01

We have examined the effects of core histone acetylation on the transcriptional activity and higher-order folding of defined 12-mer nucleosomal arrays. Purified HeLa core histone octamers containing an average of 2, 6, or 12 acetates per octamer (8, 23, or 46% maximal site occupancy, respectively) were assembled onto a DNA template consisting of 12 tandem repeats of a 208-bp Lytechinus 5S rRNA gene fragment. Reconstituted nucleosomal arrays were transcribed in a Xenopus oocyte nuclear extract and analyzed by analytical hydrodynamic and electrophoretic approaches to determine the extent of array compaction. Results indicated that in buffer containing 5 mM free Mg2+ and 50 mM KCl, high levels of acetylation (12 acetates/octamer) completely inhibited higher-order folding and concurrently led to a 15-fold enhancement of transcription by RNA polymerase III. The molecular mechanisms underlying the acetylation effects on chromatin condensation were investigated by analyzing the ability of differentially acetylated nucleosomal arrays to fold and oligomerize. In MgCl2-containing buffer the folding of 12-mer nucleosomal arrays containing an average of two or six acetates per histone octamer was indistinguishable, while a level of 12 acetates per octamer completely disrupted the ability of nucleosomal arrays to form higher-order folded structures at all ionic conditions tested. In contrast, there was a linear relationship between the extent of histone octamer acetylation and the extent of disruption of Mg2+-dependent oligomerization. These results have yielded new insight into the molecular basis of acetylation effects on both transcription and higher-order compaction of nucleosomal arrays. PMID:9671473

REDUCED DORMANCY5 Encodes a Protein Phosphatase 2C That Is Required for Seed Dormancy in Arabidopsis[C][W][OPEN

PubMed Central

Xiang, Yong; Nakabayashi, Kazumi; Ding, Jia; He, Fei; Bentsink, Leónie; Soppe, Wim J.J.

2014-01-01

Seed dormancy determines germination timing and contributes to crop production and the adaptation of natural populations to their environment. Our knowledge about its regulation is limited. In a mutagenesis screen of a highly dormant Arabidopsis thaliana line, the reduced dormancy5 (rdo5) mutant was isolated based on its strongly reduced seed dormancy. Cloning of RDO5 showed that it encodes a PP2C phosphatase. Several PP2C phosphatases belonging to clade A are involved in abscisic acid signaling and control seed dormancy. However, RDO5 does not cluster with clade A phosphatases, and abscisic acid levels and sensitivity are unaltered in the rdo5 mutant. RDO5 transcript could only be detected in seeds and was most abundant in dry seeds. RDO5 was found in cells throughout the embryo and is located in the nucleus. A transcriptome analysis revealed that several genes belonging to the conserved PUF family of RNA binding proteins, in particular Arabidopsis PUMILIO9 (APUM9) and APUM11, showed strongly enhanced transcript levels in rdo5 during seed imbibition. Further transgenic analyses indicated that APUM9 reduces seed dormancy. Interestingly, reduction of APUM transcripts by RNA interference complemented the reduced dormancy phenotype of rdo5, indicating that RDO5 functions by suppressing APUM transcript levels. PMID:25415980

Bovine foamy virus transactivator BTas interacts with cellular RelB to enhance viral transcription.

PubMed

Wang, Jian; Tan, Juan; Guo, Hongyan; Zhang, Qicheng; Jia, Rui; Xu, Xuan; Geng, Yunqi; Qiao, Wentao

2010-11-01

Viruses are obligate intracellular parasites that depend on cellular machinery for their efficient transcription and replication. In a previous study we reported that bovine foamy virus (BFV) is able to activate the nuclear factor κB (NF-κB) pathway through the action of its transactivator BTas to enhance viral transcription. However, the mechanism used by NF-κB to enhance BFV transcription remains elusive. To address this question, we employed a yeast two-hybrid assay to screen for BTas-interacting proteins. We found that RelB, a member of NF-κB protein family, interacts with BTas. We confirmed the putative RelB-BTas interaction in vitro and in vivo and identified the protein regions responsible for the RelB-BTas interaction. Using a luciferase reporter assay, we next showed that RelB enhances BFV transcription (BTas-induced long terminal repeat [LTR] transactivation) and that this process requires both the localization of the RelB-BTas interaction in the nucleus and the Rel homology domain of RelB. The knockdown of the cellular endogenous RelB protein using small interfering RNA (siRNA) significantly attenuated BTas-induced LTR transcription. The results of chromatin immunoprecipitation (ChIP) analysis showed that endogenous RelB binds to the viral LTR in BFV-infected cells. Together, these results suggest that BFV engages the RelB protein as a cotransactivator of BTas to enhance viral transcription. In addition, our findings indicate that BFV infection upregulates cellular RelB expression through BTas-induced NF-κB activation. Thus, this study demonstrates the existence of a positive-feedback circuit in which BFV utilizes the host's NF-κB pathway through the RelB protein for efficient viral transcription.

Thyroid Receptor β Involvement in the Effects of Acute Nicotine on Hippocampus-Dependent Memory

PubMed Central

Leach, Prescott T.; Kenney, Justin W.; Connor, David; Gould, Thomas J.

2015-01-01

Cigarette smoking is common despite adverse health effects. Nicotine’s effects on learning may contribute to addiction by enhancing drug-context associations. Effects of nicotine on learning could be direct or could occur by altering systems that modulate cognition. Because thyroid signaling can alter cognition and nicotine/smoking may change thyroid function, nicotine could affect learning through changes in thyroid signaling. These studies investigate the functional contributions of thyroid receptor (TR) subtypes β and α1 to nicotine-enhanced learning and characterize the effects of acute nicotine and learning on thyroid hormone levels. We conducted a high throughput screen of transcription factor activity to identify novel targets that may contribute to the effects of nicotine on learning. Based on these results, which showed that combined nicotine and learning uniquely acted to increase TR activation, we identified TRs as potential targets of nicotine. Further analyses were conducted to determine the individual and combined effects of nicotine and learning on thyroid hormone levels, but no changes were seen. Next, to determine the role of TRβ and TRα1 in the effects of nicotine on learning, mice lacking the TRβ or TRα1 gene and wildtype littermates were administered acute nicotine prior to fear conditioning. Nicotine enhanced contextual fear conditioning in TRα1 knockout mice and wildtypes from both lines but TRβ knockout mice did not show nicotine-enhanced learning. This finding supports involvement of TRβ signaling in the effect of acute nicotine on hippocampus-dependent memory. Acute nicotine enhances learning and these effects may involve processes regulated by the transcription factor TRβ. PMID:25666034

Dynamic and Differential Regulation of Stem Cell Factor FoxD3 in the Neural Crest Is Encrypted in the Genome

PubMed Central

Tan-Cabugao, Joanne; Sauka-Spengler, Tatjana; Bronner, Marianne E.

2012-01-01

The critical stem cell transcription factor FoxD3 is expressed by the premigratory and migrating neural crest, an embryonic stem cell population that forms diverse derivatives. Despite its important role in development and stem cell biology, little is known about what mediates FoxD3 activity in these cells. We have uncovered two FoxD3 enhancers, NC1 and NC2, that drive reporter expression in spatially and temporally distinct manners. Whereas NC1 activity recapitulates initial FoxD3 expression in the cranial neural crest, NC2 activity recapitulates initial FoxD3 expression at vagal/trunk levels while appearing only later in migrating cranial crest. Detailed mutational analysis, in vivo chromatin immunoprecipitation, and morpholino knock-downs reveal that transcription factors Pax7 and Msx1/2 cooperate with the neural crest specifier gene, Ets1, to bind to the cranial NC1 regulatory element. However, at vagal/trunk levels, they function together with the neural plate border gene, Zic1, which directly binds to the NC2 enhancer. These results reveal dynamic and differential regulation of FoxD3 in distinct neural crest subpopulations, suggesting that heterogeneity is encrypted at the regulatory level. Isolation of neural crest enhancers not only allows establishment of direct regulatory connections underlying neural crest formation, but also provides valuable tools for tissue specific manipulation and investigation of neural crest cell identity in amniotes. PMID:23284303

Role of the POZ zinc finger transcription factor FBI-1 in human and murine adipogenesis.

PubMed

Laudes, Matthias; Christodoulides, Constantinos; Sewter, Ciaran; Rochford, Justin J; Considine, Robert V; Sethi, Jaswinder K; Vidal-Puig, Antonio; O'Rahilly, Stephen

2004-03-19

Poxvirus zinc finger (POZ) zinc finger domain transcription factors have been shown to play a role in the control of growth arrest and differentiation in several types of mesenchymal cells but not, as yet, adipocytes. We found that a POZ domain protein, factor that binds to inducer of short transcripts-1 (FBI-1), was induced during both murine and human preadipocyte differentiation with maximal expression levels seen at days 2-4. FBI-1 mRNA was expressed in human adipose tissue with the highest levels found in samples from morbidly obese subjects. Murine cell lines constitutively expressing FBI-1 showed evidence for accelerated adipogenesis with earlier induction of markers of differentiation and enhanced lipid accumulation, suggesting that FBI-1 may be an active participant in the differentiation process. Consistent with the properties of this family of proteins in other cell systems, 3T3L1 cells stably overexpressing FBI-1 showed reduced DNA synthesis and reduced expression of cyclin A, cyclin-dependent kinase 2, and p107, proteins known to be involved in the regulation of mitotic clonal expansion. In addition, FBI-1 reduced the transcriptional activity of the cyclin A promoter. Thus, FBI-1, a POZ zinc finger transcription factor, is induced during the early phases of human and murine preadipocyte differentiation where it may contribute to adipogenesis through influencing the switch from cellular proliferation to terminal differentiation.

Combinatorial influence of environmental parameters on transcription factor activity

PubMed Central

Knijnenburg, T.A.; Wessels, L.F.A.; Reinders, M.J.T.

2008-01-01

Motivation: Cells receive a wide variety of environmental signals, which are often processed combinatorially to generate specific genetic responses. Changes in transcript levels, as observed across different environmental conditions, can, to a large extent, be attributed to changes in the activity of transcription factors (TFs). However, in unraveling these transcription regulation networks, the actual environmental signals are often not incorporated into the model, simply because they have not been measured. The unquantified heterogeneity of the environmental parameters across microarray experiments frustrates regulatory network inference. Results: We propose an inference algorithm that models the influence of environmental parameters on gene expression. The approach is based on a yeast microarray compendium of chemostat steady-state experiments. Chemostat cultivation enables the accurate control and measurement of many of the key cultivation parameters, such as nutrient concentrations, growth rate and temperature. The observed transcript levels are explained by inferring the activity of TFs in response to combinations of cultivation parameters. The interplay between activated enhancers and repressors that bind a gene promoter determine the possible up- or downregulation of the gene. The model is translated into a linear integer optimization problem. The resulting regulatory network identifies the combinatorial effects of environmental parameters on TF activity and gene expression. Availability: The Matlab code is available from the authors upon request. Contact: t.a.knijnenburg@tudelft.nl Supplementary information: Supplementary data are available at Bioinformatics online. PMID:18586711

Scleraxis is a transcriptional activator that regulates the expression of Tenomodulin, a marker of mature tenocytes and ligamentocytes.

PubMed

Shukunami, Chisa; Takimoto, Aki; Nishizaki, Yuriko; Yoshimoto, Yuki; Tanaka, Seima; Miura, Shigenori; Watanabe, Hitomi; Sakuma, Tetsushi; Yamamoto, Takashi; Kondoh, Gen; Hiraki, Yuji

2018-02-16

Tenomodulin (Tnmd) is a type II transmembrane glycoprotein predominantly expressed in tendons and ligaments. We found that scleraxis (Scx), a member of the Twist-family of basic helix-loop-helix transcription factors, is a transcriptional activator of Tnmd expression in tenocytes. During embryonic development, Scx expression preceded that of Tnmd. Tnmd expression was nearly absent in tendons and ligaments of Scx-deficient mice generated by transcription activator-like effector nucleases-mediated gene disruption. Tnmd mRNA levels were dramatically decreased during serial passages of rat tenocytes. Scx silencing by small interfering RNA significantly suppressed endogenous Tnmd mRNA levels in tenocytes. Mouse Tnmd contains five E-box sites in the ~1-kb 5'-flanking region. A 174-base pair genomic fragment containing a TATA box drives transcription in tenocytes. Enhancer activity was increased in the upstream region (-1030 to -295) of Tnmd in tenocytes, but not in NIH3T3 and C3H10T1/2 cells. Preferential binding of both Scx and Twist1 as a heterodimer with E12 or E47 to CAGATG or CATCTG and transactivation of the 5'-flanking region were confirmed by electrophoresis mobility shift and dual luciferase assays, respectively. Scx directly transactivates Tnmd via these E-boxes to positively regulate tenocyte differentiation and maturation.

Genotype-dependent activation or repression of HBV enhancer II by transcription factor COUP-TF1

PubMed Central

Fischer, Silke F; Schmidt, Katja; Fiedler, Nicola; Glebe, Dieter; Schüttler, Christian; Sun, Jianguang; Gerlich, Wolfram H; Repp, Reinald; Schaefer, Stephan

2006-01-01

AIM: To study the expression of HBV enhancer II by transcription factor COUP-TF1. METHODS: In order to study the regulation of HBV variants in the vicinity of the NRRE we cloned luciferase constructs containing the HBV enhancer II from variants and from HBV genotypes A and D and cotransfected them together with expression vectors for COUP-TF1 into HepG2 cells. RESULTS: Our findings show that enhancer II of HBV genotype A is also repressed by COUP-TF1. In contrast, two different enhancer II constructs of HBV genotype D were activated by COUP-TF1. The activation was independent of the NRRE because a natural variant with a deletion of nt 1763-1770 was still activated by COUP-TF1. CONCLUSION: Regulation of transcription of the HBV genome seems to differ among HBV genomes derived from different genotypes. These differences in transcriptional control among HBV genotypes may be the molecular basis for differences in the clinical course among HBV genotypes. PMID:17009409

Genotype-dependent activation or repression of HBV enhancer II by transcription factor COUP-TF1.

PubMed

Fischer, Silke F; Schmidt, Katja; Fiedler, Nicola; Glebe, Dieter; Schüttler, Christian; Sun, Jianguang; Gerlich, Wolfram H; Repp, Reinald; Schaefer, Stephan

2006-10-07

To study the expression of HBV enhancer II by transcription factor COUP-TF1. In order to study the regulation of HBV variants in the vicinity of the NRRE we cloned luciferase constructs containing the HBV enhancer II from variants and from HBV genotypes A and D and cotransfected them together with expression vectors for COUP-TF1 into HepG2 cells. Our findings show that enhancer II of HBV genotype A is also repressed by COUP-TF1. In contrast, two different enhancer II constructs of HBV genotype D were activated by COUP-TF1. The activation was independent of the NRRE because a natural variant with a deletion of nt 1763-1770 was still activated by COUP-TF1. Regulation of transcription of the HBV genome seems to differ among HBV genomes derived from different genotypes. These differences in transcriptional control among HBV genotypes may be the molecular basis for differences in the clinical course among HBV genotypes.

Cooperative activation of cardiac transcription through myocardin bridging of paired MEF2 sites

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

Anderson, Courtney M.; Hu, Jianxin; Thomas, Reuben

2017-03-28

Enhancers frequently contain multiple binding sites for the same transcription factor. These homotypic binding sites often exhibit synergy, whereby the transcriptional output from two or more binding sites is greater than the sum of the contributions of the individual binding sites alone. Although this phenomenon is frequently observed, the mechanistic basis for homotypic binding site synergy is poorly understood. Here in this paper, we identify a bona fide cardiac-specific Prkaa2 enhancer that is synergistically activated by homotypic MEF2 binding sites. We show that two MEF2 sites in the enhancer function cooperatively due to bridging of the MEF2C-bound sites by themore » SAP domain-containing co-activator protein myocardin, and we show that paired sites buffer the enhancer from integration site-dependent effects on transcription in vivo. Paired MEF2 sites are prevalent in cardiac enhancers, suggesting that this might be a common mechanism underlying synergy in the control of cardiac gene expression in vivo.« less

Colonization by the arbuscular mycorrhizal fungus Glomus versiforme induces a defense response against the root-knot nematode Meloidogyne incognita in the grapevine (Vitis amurensis Rupr.), which includes transcriptional activation of the class III chitinase gene VCH3.

PubMed

Li, Hai-Yan; Yang, Guo-Dong; Shu, Huai-Rui; Yang, Yu-Tao; Ye, Bao-Xing; Nishida, Ikuo; Zheng, Cheng-Chao

2006-01-01

Inoculation of the grapevine (Vitis amurensis Rupr.) with the arbuscular mycorrhizal (AM) fungus Glomus versiforme significantly increased resistance against the root-knot nematode (RKN) Meloidogyne incognita. Studies using relative quantitative reverse transcription-PCR (RQRT-PCR) analysis of grapevine root inoculation with the AM fungus revealed an up-regulation of VCH3 transcripts. This increase was greater than that observed following infection with RKN. However, inoculation of the mycorrhizal grapevine roots with RKN was able to enhance VCH3 transcript expression further. Moreover, the increase in VCH3 transcripts appeared to result in a higher level of resistance against subsequent RKN infection. Constitutive expression of VCH3 cDNA in transgenic tobacco under the control of the cauliflower mosaic virus 35S promoter also conferred resistance against RKN, but had no significant effect on the growth of the AM fungus. We analyzed beta-glucuronidase (GUS) activity directed by a 1,216 bp VCH3 promoter in transgenic tobacco following inoculation with both the AM fungus and RKN. GUS activity was negligible in the root tissues before inoculation, and was more effectively induced after inoculation with the AM fungus than with RKN. Moreover, GUS staining in the mycorrhizal transgenic tobacco roots was enhanced by subsequent RKN infection, and was found ubiquitously throughout the whole root tissue. Together, these results suggest that AM fungus induced a defense response against RKN in the mycorrhizal grapevine roots, which appeared to involve transcriptional control of VCH3 expression throughout the whole root tissue.

CREB, NF-Y and MEIS1 conserved binding sites are essential to balance Myostatin promoter/enhancer activity during early myogenesis.

PubMed

Grade, Carla Vermeulen Carvalho; Mantovani, Carolina Stefano; Fontoura, Marina Alves; Yusuf, Faisal; Brand-Saberi, Beate; Alvares, Lúcia Elvira

2017-10-01

Myostatin (MSTN) is a strong inhibitor of skeletal muscle growth in human and other vertebrates. Its transcription is controlled by a proximal promoter/enhancer (Mstn P/E) containing a TATA box besides CREB, NF-Y, MEIS1 and FXR transcription factor binding sites (TFBSs), which are conserved throughout evolution. The aim of this work was to investigate the role of these TFBSs on Mstn P/E activity and evaluate the potential of their putative ligands as Mstn trans regulators. Mstn P/E mutant constructs were used to establish the role of conserved TFBSs using dual-luciferase assays. Expression analyses were performed by RT-PCR and in situ hybridization in C2C12 myoblasts and E10.5 mouse embryos, respectively. Our results revealed that CREB, NF-Y and MEIS1 sites are required to balance Mstn P/E activity, keeping Mstn transcription within basal levels during myoblast proliferation. Furthermore, our data showed that NF-Y site is essential, although not sufficient, to mediate Mstn P/E transcriptional activity. In turn, CREB and MEIS1 binding sites seem to depend on the presence of NF-Y site to induce Mstn P/E. FXR appears not to confer any effect on Mstn P/E activity, except in the absence of all other conserved TFBS. Accordingly, expression studies pointed to CREB, NF-Y and MEIS1 but not to FXR factors as possible regulators of Mstn transcription in the myogenic context. Altogether, our findings indicated that CREB, NF-Y and MEIS1 conserved sites are essential to control basal Mstn transcription during early myogenesis, possibly by interacting with these or other related factors.

Repression of ESR1 transcription by MYOD potentiates letrozole-resistance in ERα-positive breast cancer cells.

PubMed

Zhang, Qiang; Liu, Xiao-Yan; Li, Shuang; Zhao, Zhao; Li, Juan; Cui, Ming-Ke; Wang, En-Hua

2017-10-21

Transcriptional silencing of estrogen receptor α (ERα) expression is an important etiology contributing to the letrozole-resistance in ERα-positive breast cancer (BCa) cells, but the transcription factors responsible for this transcriptional repression remain largely unidentified. Here we report that the expression of the basic helix-loop-helix myogenic regulatory factor MYOD was abnormally up-regulated in letrozole-resistant BCa tissues and in experimentally-induced letrozole-resistant BCa cells. Overexpression of the exogenous MYOD impaired ERα expression and potentiated letrozole-resistance in letrozole-sensitive MCF7 cells, whereas MYOD knockdown could effectively restore ERα expression and thereby promote letrozole-sensitivity in letrozole-resistant MCF-7/LR cells. Mechanistically, MYOD was shown to be a potent corepressor of ESR1 transcription, and this transcriptional repression was significantly enhanced in the presence of letrozole treatment. Thus, targeted inhibition of MYOD may restore ERα level and lead to resensitization to letrozole-based hormone therapy, providing a novel therapeutic strategy for relapsed ERα-positive BCa patients. Our data also underscore an unexpected chemotherapeutic facet of MYOD, which may operate as a novel regulator of BCa biology. Copyright © 2017 Elsevier Inc. All rights reserved.

Expression of the nifH Gene of a Herbaspirillum Endophyte in Wild Rice Species: Daily Rhythm during the Light-Dark Cycle

PubMed Central

You, Mu; Nishiguchi, Tomohiro; Saito, Asami; Isawa, Tsuyoshi; Mitsui, Hisayuki; Minamisawa, Kiwamu

2005-01-01

The expression of nitrogenase genes of Herbaspirillum sp. B501 associated in shoot (leaf and stem) of wild rice, Oryza officinalis, was studied by means of reverse transcription-PCR (RT-PCR) targeted at the nifH gene. RT-PCR analyses indicate that nifH transcript was detected exclusively from nitrogen-fixing cells of gfp-tagged strain B501gfp1 in both free-living and endophytic states by using a constitutive gfp gene transcript as a positive control. Transcription of nifH and nitrogen fixation in free-living cells were induced maximally at a 2% O2 concentration and repressed in free air (21% O2). nifH transcription was monitored in the endophytic cells by using total RNA extracted from B501gfp1-inoculated wild rice plants during daily light-dark cycles. The level of nifH transcription in planta varied dramatically, with a maximum during the light period. Moreover, the light radiation enhanced nifH expression even in free-living cells grown in culture. These results suggest that in planta nitrogen fixation by the endophyte shows a daily rhythm determined by the plant's light environment. PMID:16332801

Acute transcriptional up-regulation specific to osteoblasts/osteoclasts in medaka fish immediately after exposure to microgravity

PubMed Central

Chatani, Masahiro; Morimoto, Hiroya; Takeyama, Kazuhiro; Mantoku, Akiko; Tanigawa, Naoki; Kubota, Koji; Suzuki, Hiromi; Uchida, Satoko; Tanigaki, Fumiaki; Shirakawa, Masaki; Gusev, Oleg; Sychev, Vladimir; Takano, Yoshiro; Itoh, Takehiko; Kudo, Akira

2016-01-01

Bone loss is a serious problem in spaceflight; however, the initial action of microgravity has not been identified. To examine this action, we performed live-imaging of animals during a space mission followed by transcriptome analysis using medaka transgenic lines expressing osteoblast and osteoclast-specific promoter-driven GFP and DsRed. In live-imaging for osteoblasts, the intensity of osterix- or osteocalcin-DsRed fluorescence in pharyngeal bones was significantly enhanced 1 day after launch; and this enhancement continued for 8 or 5 days. In osteoclasts, the signals of TRAP-GFP and MMP9-DsRed were highly increased at days 4 and 6 after launch in flight. HiSeq from pharyngeal bones of juvenile fish at day 2 after launch showed up-regulation of 2 osteoblast- and 3 osteoclast- related genes. Gene ontology analysis for the whole-body showed that transcription of genes in the category “nucleus” was significantly enhanced; particularly, transcription-regulators were more up-regulated at day 2 than at day 6. Lastly, we identified 5 genes, c-fos, jun-B-like, pai-1, ddit4 and tsc22d3, which were up-regulated commonly in the whole-body at days 2 and 6, and in the pharyngeal bone at day 2. Our results suggested that exposure to microgravity immediately induced dynamic alteration of gene expression levels in osteoblasts and osteoclasts. PMID:28004797

The modification of Gat1p in nitrogen catabolite repression to enhance non-preferred nitrogen utilization in Saccharomyces cerevisiae

PubMed Central

Zhao, Xinrui; Zou, Huijun; Chen, Jian; Du, Guocheng; Zhou, Jingwen

2016-01-01

In Saccharomyces cerevisiae, when preferred nitrogen sources are present, the metabolism of non-preferred nitrogen is repressed. Previous work showed that this metabolic regulation is primarily controlled by nitrogen catabolite repression (NCR) related regulators. Among these regulators, two positive regulators (Gln3p and Gat1p) could be phosphorylated and sequestered in the cytoplasm leading to the transcription of non-preferred nitrogen metabolic genes being repressed. The nuclear localization signals (NLSs) and nuclear localization regulatory signals (NLRSs) in Gln3p and Gat1p play essential roles in the regulation of their localization in cells. However, compared with Gln3p, the information of NLS and NLRS for Gat1p remains unknown. In this study, residues 348–375 and 366–510 were identified as the NLS and NLRS of Gat1p firstly. In addition, the modifications of Gat1p (mutations on the NLS and truncation on the NLRS) were attempted to enhance the transcription of non-preferred nitrogen metabolic genes. Quantitative real-time PCR showed that the transcriptional levels of 15 non-preferred nitrogen metabolic genes increased. Furthermore, during the shaking-flask culture tests, the utilization of urea, proline and allantoine was significantly increased. Based on these results, the genetic engineering on Gat1p has a great potential in enhancing non-preferred nitrogen metabolism in S. cerevisiae. PMID:26899143

Ikaros controls isotype selection during immunoglobulin class switch recombination.

PubMed

Sellars, MacLean; Reina-San-Martin, Bernardo; Kastner, Philippe; Chan, Susan

2009-05-11

Class switch recombination (CSR) allows the humoral immune response to exploit different effector pathways through specific secondary antibody isotypes. However, the molecular mechanisms and factors that control immunoglobulin (Ig) isotype choice for CSR are unclear. We report that deficiency for the Ikaros transcription factor results in increased and ectopic CSR to IgG(2b) and IgG(2a), and reduced CSR to all other isotypes, regardless of stimulation. Ikaros suppresses active chromatin marks, transcription, and activation-induced cytidine deaminase (AID) accessibility at the gamma2b and gamma2a genes to inhibit class switching to these isotypes. Further, Ikaros directly regulates isotype gene transcription as it directly binds the Igh 3' enhancer and interacts with isotype gene promoters. Finally, Ikaros-mediated repression of gamma2b and gamma2a transcription promotes switching to other isotype genes by allowing them to compete for AID-mediated recombination at the single-cell level. Thus, our results reveal transcriptional competition between constant region genes in individual cells to be a critical and general mechanism for isotype specification during CSR. We show that Ikaros is a master regulator of this competition.

Mutations in SNRPB, encoding components of the core splicing machinery, cause cerebro-costo-mandibular syndrome.

PubMed

Bacrot, Séverine; Doyard, Mathilde; Huber, Céline; Alibeu, Olivier; Feldhahn, Niklas; Lehalle, Daphné; Lacombe, Didier; Marlin, Sandrine; Nitschke, Patrick; Petit, Florence; Vazquez, Marie-Paule; Munnich, Arnold; Cormier-Daire, Valérie

2015-02-01

Cerebro-costo-mandibular syndrome (CCMS) is a developmental disorder characterized by the association of Pierre Robin sequence and posterior rib defects. Exome sequencing and Sanger sequencing in five unrelated CCMS patients revealed five heterozygous variants in the small nuclear ribonucleoprotein polypeptides B and B1 (SNRPB) gene. This gene includes three transcripts, namely transcripts 1 and 2, encoding components of the core spliceosomal machinery (SmB' and SmB) and transcript 3 undergoing nonsense-mediated mRNA decay. All variants were located in the premature termination codon (PTC)-introducing alternative exon of transcript 3. Quantitative RT-PCR analysis revealed a significant increase in transcript 3 levels in leukocytes of CCMS individuals compared to controls. We conclude that CCMS is due to heterozygous mutations in SNRPB, enhancing inclusion of a SNRPB PTC-introducing alternative exon, and show that this developmental disease is caused by defects in the splicing machinery. Our finding confirms the report of SNRPB mutations in CCMS patients by Lynch et al. (2014) and further extends the clinical and molecular observations. © 2014 WILEY PERIODICALS, INC.

Butyrate-Induced Transcriptional Changes in Human Colonic Mucosa

PubMed Central

Vanhoutvin, Steven A. L. W.; Troost, Freddy J.; Hamer, Henrike M.; Lindsey, Patrick J.; Koek, Ger H.; Jonkers, Daisy M. A. E.; Kodde, Andrea; Venema, Koen; Brummer, Robert J. M.

2009-01-01

Background Fermentation of dietary fiber in the colon results in the production of short chain fatty acids (mainly propionate, butyrate and acetate). Butyrate modulates a wide range of processes, but its mechanism of action is mostly unknown. This study aimed to determine the effects of butyrate on the transcriptional regulation of human colonic mucosa in vivo. Methodology/Principal Findings Five hundred genes were found to be differentially expressed after a two week daily butyrate administration with enemas. Pathway analysis showed that the butyrate intervention mainly resulted in an increased transcriptional regulation of the pathways representing fatty acid oxidation, electron transport chain and oxidative stress. In addition, several genes associated with epithelial integrity and apoptosis, were found to be differentially expressed after the butyrate intervention. Conclusions/Significance Colonic administration of butyrate in concentrations that can be achieved by consumption of a high-fiber diet enhances the maintenance of colonic homeostasis in healthy subjects, by regulating fatty acid metabolism, electron transport and oxidative stress pathways on the transcriptional level and provide for the first time, detailed molecular insight in the transcriptional response of gut mucosa to butyrate. PMID:19707587

Transcription elongation factors are involved in programming hormone production in pituitary neuroendocrine GH4C1 cells.

PubMed

Fujita, Toshitsugu; Piuz, Isabelle; Schlegel, Werner

2010-05-05

Transcription elongation of many eukaryotic genes is regulated. Two negative transcription elongation factors, 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) sensitivity-inducing factor (DSIF) and negative elongation factor (NELF) are known to stall collaboratively RNA polymerase II promoter proximally. We discovered that DSIF and NELF are linked to hormone expression in rat pituitary GH4C1 cells. When NELF-E, a subunit of NELF or Spt5, a subunit of DSIF was stably knocked-down, prolactin (PRL) expression was increased both at the mRNA and protein levels. In contrast, stable knock-down of only Spt5 abolished growth hormone (GH) expression. Transient NELF-E knock-down increased coincidentally PRL expression and enhanced transcription of a PRL-promoter reporter gene. However, no direct interaction of NELF with the PRL gene could be demonstrated by chromatin immuno-precipitation. Thus, NELF suppressed PRL promoter activity indirectly. In conclusion, transcription regulation by NELF and DSIF is continuously involved in the control of hormone production and may contribute to neuroendocrine cell differentiation. Copyright 2010 Elsevier Ireland Ltd. All rights reserved.

Enterococcus faecium WEFA23 from infant lessens high-fat-diet-induced hyperlipidemia via cholesterol 7-alpha-hydroxylase gene by altering the composition of gut microbiota in rats.

PubMed

Huang, Fuqing; Zhang, Fen; Xu, Di; Zhang, Zhihong; Xu, Feng; Tao, Xueying; Qiu, Liang; Wei, Hua

2018-06-20

Enterococcus faecium WEFA23 is a potential probiotic strain from Chinese infants with the ability to decrease cholesterol levels. Aiming to explore the mechanism of E. faecium WEFA23 in lowering cholesterol in vivo, we examined the gene transcriptions related to cholesterol metabolism, the composition of bile acids in feces, the synthesis of trimethylamine N-oxide (TMAO) in liver, and the composition of the gut microbiota of rats. We found that E. faecium WEFA23 enhanced the synthesis of bile acids by promoting cholesterol excretion, upregulating the genes transcript level relevant to cholesterol decomposition and transportation, and downregulating the genes involved in cholesterol synthesis. In addition, E. faecium WEFA23 not only downregulated the transcript levels of farnesoid X receptor and fibroblast growth factor 15 as well as flavin-containing monooxygenase 3, but also decreased the TMAO production followed by increasing the CYP7A1 transcript level. Furthermore, when orally administered to rats for 35 d, E. faecium WEFA23 improved the gut microbiota diversity of rats fed a high-fat diet. Therein, the ratio of Bacteroidetes to Firmicutes and the abundance of Rikenellaceae increased, whereas the number of Veillonellaceae decreased. These results suggest that reduction of cholesterol level by E. faecium WEFA23 might be related to the changes in the gut microbiota. Our finding provides important information on lowering cholesterol by E. faecium and reveals that Enterococcus spp. might have the potential to decrease the TMAO level. Copyright © 2018 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

A Mechanism to Enhance Cellular Responsivity to Hormone Action: Krüppel-Like Factor 9 Promotes Thyroid Hormone Receptor-β Autoinduction During Postembryonic Brain Development

PubMed Central

Hu, Fang; Knoedler, Joseph R.

2016-01-01

Thyroid hormone (TH) receptor (TR)-β (trb) is induced by TH (autoinduced) in Xenopus tadpoles during metamorphosis. We previously showed that Krüppel-like factor 9 (Klf9) is rapidly induced by TH in the tadpole brain, associates in chromatin with the trb upstream region in a developmental stage and TH-dependent manner, and forced expression of Klf9 in the Xenopus laevis cell line XTC-2 accelerates and enhances trb autoinduction. Here we investigated whether Klf9 can promote trb autoinduction in tadpole brain in vivo. Using electroporation-mediated gene transfer, we transfected plasmids into premetamorphic tadpole brain to express wild-type or mutant forms of Klf9. Forced expression of Klf9 increased baseline trb mRNA levels in thyroid-intact but not in goitrogen-treated tadpoles, supporting that Klf9 enhances liganded TR action. As in XTC-2 cells, forced expression of Klf9 enhanced trb autoinduction in tadpole brain in vivo and also increased TH-dependent induction of the TR target genes klf9 and thbzip. Consistent with our previous mutagenesis experiments conducted in XTC-2 cells, the actions of Klf9 in vivo required an intact N-terminal region but not a functional DNA binding domain. Forced expression of TRβ in tadpole brain by electroporation-mediated gene transfer increased baseline and TH-induced TR target gene transcription, supporting a role for trb autoinduction during metamorphosis. Our findings support that Klf9 acts as an accessory transcription factor for TR at the trb locus during tadpole metamorphosis, enhancing trb autoinduction and transcription of other TR target genes, which increases cellular responsivity to further TH action on developmental gene regulation programs. PMID:26886257

Evolution of transcriptional enhancers and animal diversity

PubMed Central

Rubinstein, Marcelo; de Souza, Flávio S. J.

2013-01-01

Deciphering the genetic bases that drive animal diversity is one of the major challenges of modern biology. Although four decades ago it was proposed that animal evolution was mainly driven by changes in cis-regulatory DNA elements controlling gene expression rather than in protein-coding sequences, only now are powerful bioinformatics and experimental approaches available to accelerate studies into how the evolution of transcriptional enhancers contributes to novel forms and functions. In the introduction to this Theme Issue, we start by defining the general properties of transcriptional enhancers, such as modularity and the coexistence of tight sequence conservation with transcription factor-binding site shuffling as different mechanisms that maintain the enhancer grammar over evolutionary time. We discuss past and current methods used to identify cell-type-specific enhancers and provide examples of how enhancers originate de novo, change and are lost in particular lineages. We then focus in the central part of this Theme Issue on analysing examples of how the molecular evolution of enhancers may change form and function. Throughout this introduction, we present the main findings of the articles, reviews and perspectives contributed to this Theme Issue that together illustrate some of the great advances and current frontiers in the field. PMID:24218630

An enhanceosome containing the Jun B/Fra-2 heterodimer and the HMG-I(Y) architectural protein controls HPV 18 transcription.

PubMed

Bouallaga, I; Massicard, S; Yaniv, M; Thierry, F

2000-11-01

Recent studies have reported new mechanisms that mediate the transcriptional synergy of strong tissue-specific enhancers, involving the cooperative assembly of higher-order nucleoprotein complexes called enhanceosomes. Here we show that the HPV18 enhancer, which controls the epithelial-specific transcription of the E6 and E7 transforming genes, exhibits characteristic features of these structures. We used deletion experiments to show that a core enhancer element cooperates, in a specific helical phasing, with distant essential factors binding to the ends of the enhancer. This core sequence, binding a Jun B/Fra-2 heterodimer, cooperatively recruits the architectural protein HMG-I(Y) in a nucleoprotein complex, where they interact with each other. Therefore, in HeLa cells, HPV18 transcription seems to depend upon the assembly of an enhanceosome containing multiple cellular factors recruited by a core sequence interacting with AP1 and HMG-I(Y).

Invited review: Regulation of skeletal muscle GLUT-4 expression by exercise.

PubMed

Dohm, G Lynis

2002-08-01

The amount of GLUT-4 protein is a primary factor in determining the maximal rate of glucose transport into skeletal muscle. Therefore, it is important that we understand how exercise regulates GLUT-4 expression so that therapeutic strategies can be designed to increase muscle glucose disposal as a treatment for diabetes. Muscle contraction increases the rates of GLUT-4 transcription and translation. Transcriptional control likely requires at least two DNA binding proteins, myocyte enhancer factor-2 and GLUT-4 enhancer factor, which bind to the promoter. Increased GLUT-4 expression may be mediated by the enzyme AMP-activated kinase, which is activated during exercise and has been demonstrated to increase GLUT-4 transcription. Further research needs to be done to investigate whether AMP-activated kinase activates myocyte enhancer factor-2 and GLUT-4 enhancer factor to increase transcription of the GLUT-4 gene.

Mediator and Cohesin Connect Gene Expression and Chromatin Architecture

PubMed Central

Kagey, Michael H.; Newman, Jamie J.; Bilodeau, Steve; Zhan, Ye; Orlando, David A.; van Berkum, Nynke L.; Ebmeier, Christopher C.; Goossens, Jesse; Rahl, Peter B.; Levine, Stuart S.; Taatjes, Dylan J.; Dekker, Job; Young, Richard A.

2010-01-01

Summary Transcription factors control cell specific gene expression programs through interactions with diverse coactivators and the transcription apparatus. Gene activation may involve DNA loop formation between enhancer-bound transcription factors and the transcription apparatus at the core promoter, but this process is not well understood. We report here that Mediator and Cohesin physically and functionally connect the enhancers and core promoters of active genes in embryonic stem cells. Mediator, a transcriptional coactivator, forms a complex with Cohesin, which can form rings that connect two DNA segments. The Cohesin loading factor Nipbl is associated with Mediator/Cohesin complexes, providing a means to load Cohesin at promoters. DNA looping is observed between the enhancers and promoters occupied by Mediator and Cohesin. Mediator and Cohesin occupy different promoters in different cells, thus generating cell-type specific DNA loops linked to the gene expression program of each cell. PMID:20720539

Control of early cardiac-specific transcription of Nkx2-5 by a GATA-dependent enhancer.

PubMed

Lien, C L; Wu, C; Mercer, B; Webb, R; Richardson, J A; Olson, E N

1999-01-01

The homeobox gene Nkx2-5 is the earliest known marker of the cardiac lineage in vertebrate embryos. Nkx2-5 expression is first detected in mesodermal cells specified to form heart at embryonic day 7.5 in the mouse and expression is maintained throughout the developing and adult heart. In addition to the heart, Nkx2-5 is transiently expressed in the developing pharynx, thyroid and stomach. To investigate the mechanisms that initiate cardiac transcription during embryogenesis, we analyzed the Nkx2-5 upstream region for regulatory elements sufficient to direct expression of a lacZ transgene in the developing heart of transgenic mice. We describe a cardiac enhancer, located about 9 kilobases upstream of the Nkx2-5 gene, that fully recapitulates the expression pattern of the endogenous gene in cardiogenic precursor cells from the onset of cardiac lineage specification and throughout the linear and looping heart tube. Thereafter, as the atrial and ventricular chambers become demarcated, enhancer activity becomes restricted to the developing right ventricle. Transcription of Nkx2-5 in pharynx, thyroid and stomach is controlled by regulatory elements separable from the cardiac enhancer. This distal cardiac enhancer contains a high-affinity binding site for the cardiac-restricted zinc finger transcription factor GATA4 that is essential for transcriptional activity. These results reveal a novel GATA-dependent mechanism for activation of Nkx2-5 transcription in the developing heart and indicate that regulation of Nkx2-5 is controlled in a modular manner, with multiple regulatory regions responding to distinct transcriptional networks in different compartments of the developing heart.

H3K4me3 breadth is linked to cell identity and transcriptional consistency.

PubMed

Benayoun, Bérénice A; Pollina, Elizabeth A; Ucar, Duygu; Mahmoudi, Salah; Karra, Kalpana; Wong, Edith D; Devarajan, Keerthana; Daugherty, Aaron C; Kundaje, Anshul B; Mancini, Elena; Hitz, Benjamin C; Gupta, Rakhi; Rando, Thomas A; Baker, Julie C; Snyder, Michael P; Cherry, J Michael; Brunet, Anne

2014-07-31

Trimethylation of histone H3 at lysine 4 (H3K4me3) is a chromatin modification known to mark the transcription start sites of active genes. Here, we show that H3K4me3 domains that spread more broadly over genes in a given cell type preferentially mark genes that are essential for the identity and function of that cell type. Using the broadest H3K4me3 domains as a discovery tool in neural progenitor cells, we identify novel regulators of these cells. Machine learning models reveal that the broadest H3K4me3 domains represent a distinct entity, characterized by increased marks of elongation. The broadest H3K4me3 domains also have more paused polymerase at their promoters, suggesting a unique transcriptional output. Indeed, genes marked by the broadest H3K4me3 domains exhibit enhanced transcriptional consistency and [corrected] increased transcriptional levels, and perturbation of H3K4me3 breadth leads to changes in transcriptional consistency. Thus, H3K4me3 breadth contains information that could ensure transcriptional precision at key cell identity/function genes. Copyright © 2014 Elsevier Inc. All rights reserved.

Human PIRH2 Enhances Androgen Receptor Signaling through Inhibition of Histone Deacetylase 1 and Is Overexpressed in Prostate Cancer

PubMed Central

Logan, Ian R.; Gaughan, Luke; McCracken, Stuart R. C.; Sapountzi, Vasileia; Leung, Hing Y.; Robson, Craig N.

2006-01-01

The androgen receptor (AR) is a hormone-dependent transcription factor critically involved in human prostate carcinogenesis. Optimal transcriptional control of androgen-responsive genes by AR may require complex interaction among multiple coregulatory proteins. We have previously shown that the AR coregulator TIP60 can interact with human PIRH2 (hPIRH2). In this study, we uncover important new functional role(s) for hPIRH2 in AR signaling: (i) hPIRH2 interacts with AR and enhances AR-mediated transcription with a dynamic pattern of recruitment to androgen response elements in the prostate-specific antigen (PSA) gene; (ii) hPIRH2 interacts with the AR corepressor HDAC1, leading to reduced HDAC1 protein levels and inhibition of transcriptional repression; (iii) hPIRH2 is required for optimal PSA expression; and (iv) hPIRH2 is involved in prostate cancer cell proliferation. In addition, overexpression of hPIRH2 protein was detected in 73 of 82 (89%) resected prostate cancers, with a strong correlation between increased hPIRH2 expression and aggressive disease, as signified by high Gleason sum scores and the presence of metastatic disease (P = <0.0001 and 0.0004, respectively). Collectively, our data establish hPIRH2 as a key modulator of AR function, opening a new direction for targeted therapy in aggressive human prostate cancer. PMID:16914734

Human PIRH2 enhances androgen receptor signaling through inhibition of histone deacetylase 1 and is overexpressed in prostate cancer.

PubMed

Logan, Ian R; Gaughan, Luke; McCracken, Stuart R C; Sapountzi, Vasileia; Leung, Hing Y; Robson, Craig N

2006-09-01

The androgen receptor (AR) is a hormone-dependent transcription factor critically involved in human prostate carcinogenesis. Optimal transcriptional control of androgen-responsive genes by AR may require complex interaction among multiple coregulatory proteins. We have previously shown that the AR coregulator TIP60 can interact with human PIRH2 (hPIRH2). In this study, we uncover important new functional role(s) for hPIRH2 in AR signaling: (i) hPIRH2 interacts with AR and enhances AR-mediated transcription with a dynamic pattern of recruitment to androgen response elements in the prostate-specific antigen (PSA) gene; (ii) hPIRH2 interacts with the AR corepressor HDAC1, leading to reduced HDAC1 protein levels and inhibition of transcriptional repression; (iii) hPIRH2 is required for optimal PSA expression; and (iv) hPIRH2 is involved in prostate cancer cell proliferation. In addition, overexpression of hPIRH2 protein was detected in 73 of 82 (89%) resected prostate cancers, with a strong correlation between increased hPIRH2 expression and aggressive disease, as signified by high Gleason sum scores and the presence of metastatic disease (P = <0.0001 and 0.0004, respectively). Collectively, our data establish hPIRH2 as a key modulator of AR function, opening a new direction for targeted therapy in aggressive human prostate cancer.

Integrative Transcript and Metabolite Analysis of Nutritionally Enhanced DE-ETIOLATED1 Downregulated Tomato Fruit[W

PubMed Central

Enfissi, Eugenia M.A.; Barneche, Fredy; Ahmed, Ikhlak; Lichtlé, Christiane; Gerrish, Christopher; McQuinn, Ryan P.; Giovannoni, James J.; Lopez-Juez, Enrique; Bowler, Chris; Bramley, Peter M.; Fraser, Paul D.

2010-01-01

Fruit-specific downregulation of the DE-ETIOLATED1 (DET1) gene product results in tomato fruits (Solanum lycopersicum) containing enhanced nutritional antioxidants, with no detrimental effects on yield. In an attempt to further our understanding of how modulation of this gene leads to improved quality traits, detailed targeted and multilevel omic characterization has been performed. Metabolite profiling revealed quantitative increases in carotenoid, tocopherol, phenylpropanoids, flavonoids, and anthocyanidins. Qualitative differences could also be identified within the phenolics, including unique formation in fruit pericarp tissues. These changes resulted in increased total antioxidant content both in the polar and nonpolar fractions. Increased transcription of key biosynthetic genes is a likely mechanism producing elevated phenolic-based metabolites. By contrast, high levels of isoprenoids do not appear to result from transcriptional regulation but are more likely related to plastid-based parameters, such as increased plastid volume per cell. Parallel metabolomic and transcriptomic analyses reveal the widespread effects of DET1 downregulation on diverse sectors of metabolism and sites of synthesis. Correlation analysis of transcripts and metabolites independently indicated strong coresponses within and between related pathways/processes. Interestingly, despite the fact that secondary metabolites were the most severely affected in ripe tomato fruit, our integrative analyses suggest that the coordinated activation of core metabolic processes in cell types amenable to plastid biogenesis is the main effect of DET1 loss of function. PMID:20435899

Granulin, a novel STAT3-interacting protein, enhances STAT3 transcriptional function and correlates with poorer prognosis in breast cancer

PubMed Central

Yeh, Jennifer E.; Kreimer, Simion; Walker, Sarah R.; Emori, Megan M.; Krystal, Hannah; Richardson, Andrea; Ivanov, Alexander R.; Frank, David A.

2015-01-01

Since the neoplastic phenotype of a cell is largely driven by aberrant gene expression patterns, increasing attention has been focused on transcription factors that regulate critical mediators of tumorigenesis such as signal transducer and activator of transcription 3 (STAT3). As proteins that interact with STAT3 may be key in addressing how STAT3 contributes to cancer pathogenesis, we took a proteomics approach to identify novel STAT3-interacting proteins. We performed mass spectrometry-based profiling of STAT3-containing complexes from breast cancer cells that have constitutively active STAT3 and are dependent on STAT3 function for survival. We identified granulin (GRN) as a novel STAT3-interacting protein that was necessary for both constitutive and maximal leukemia inhibitory factor (LIF)induced STAT3 transcriptional activity. GRN enhanced STAT3 DNA binding and also increased the time-integrated amount of LIF-induced STAT3 activation in breast cancer cells. Furthermore, silencing GRN neutralized STAT3-mediated tumorigenic phenotypes including viability, clonogenesis, and migratory capacity. In primary breast cancer samples, GRN mRNA levels were positively correlated with STAT3 gene expression signatures and with reduced patient survival. These studies identify GRN as a functionally important STAT3-interacting protein that may serve as an important prognostic biomarker and potential therapeutic target in breast cancer. PMID:26000098

Activation of ethylene signaling pathways enhances disease resistance by regulating ROS and phytoalexin production in rice.

PubMed

Yang, Chao; Li, Wen; Cao, Jidong; Meng, Fanwei; Yu, Yongqi; Huang, Junkai; Jiang, Lan; Liu, Muxing; Zhang, Zhengguang; Chen, Xuewei; Miyamoto, Koji; Yamane, Hisakazu; Zhang, Jinsong; Chen, Shouyi; Liu, Jun

2017-01-01

Ethylene plays diverse roles in plant growth, development and stress responses. However, the roles of ethylene signaling in immune responses remain largely unknown. In this study, we showed that the blast fungus Magnaporthe oryzae infection activated ethylene biosynthesis in rice. Resistant rice cultivars accumulated higher levels of ethylene than susceptible ones. Ethylene signaling components OsEIN2 and the downstream transcription factor OsEIL1 positively regulated disease resistance. Mutation of OsEIN2 led to enhanced disease susceptibility. Whole-genome transcription analysis revealed that responsive genes of ethylene, jasmonates (JAs) and reactive oxygen species (ROS) signaling as well as phytoalexin biosynthesis genes were remarkably induced. Transcription of OsrbohA/B, which encode NADPH oxidases, and OsOPRs, the JA biosynthesis genes, were induced by M. oryzae infection. Furthermore, we demonstrated that OsEIL1 binds to the promoters of OsrbohA/OsrbohB and OsOPR4 to activate their expression. These data suggest that OsEIN2-mediated OsrbohA/OsrbohB and OsOPR transcription may play essential roles in ROS generation, JA biosynthesis and the subsequent phytoalexin accumulation. Therefore, the involvement of ethylene signaling in disease resistance is probably by activation of ROS and phytoalexin production in rice during M. oryzae infection. © 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.

ChiTaRS-3.1-the enhanced chimeric transcripts and RNA-seq database matched with protein-protein interactions.

PubMed

Gorohovski, Alessandro; Tagore, Somnath; Palande, Vikrant; Malka, Assaf; Raviv-Shay, Dorith; Frenkel-Morgenstern, Milana

2017-01-04

Discovery of chimeric RNAs, which are produced by chromosomal translocations as well as the joining of exons from different genes by trans-splicing, has added a new level of complexity to our study and understanding of the transcriptome. The enhanced ChiTaRS-3.1 database (http://chitars.md.biu.ac.il) is designed to make widely accessible a wealth of mined data on chimeric RNAs, with easy-to-use analytical tools built-in. The database comprises 34 922: chimeric transcripts along with 11 714: cancer breakpoints. In this latest version, we have included multiple cross-references to GeneCards, iHop, PubMed, NCBI, Ensembl, OMIM, RefSeq and the Mitelman collection for every entry in the 'Full Collection'. In addition, for every chimera, we have added a predicted Chimeric Protein-Protein Interaction (ChiPPI) network, which allows for easy visualization of protein partners of both parental and fusion proteins for all human chimeras. The database contains a comprehensive annotation for 34 922: chimeric transcripts from eight organisms, and includes the manual annotation of 200 sense-antiSense (SaS) chimeras. The current improvements in the content and functionality to the ChiTaRS database make it a central resource for the study of chimeric transcripts and fusion proteins. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

The AP-1 transcription factor Fra1 inhibits follicular B cell differentiation into plasma cells

PubMed Central

Grötsch, Bettina; Brachs, Sebastian; Lang, Christiane; Luther, Julia; Derer, Anja; Schlötzer-Schrehardt, Ursula; Bozec, Aline; Fillatreau, Simon; Berberich, Ingolf; Hobeika, Elias; Reth, Michael; Wagner, Erwin F.; Schett, Georg

2014-01-01

The cornerstone of humoral immunity is the differentiation of B cells into antibody-secreting plasma cells. This process is tightly controlled by a regulatory gene network centered on the transcriptional repressor B lymphocyte–induced maturation protein 1 (Blimp1). Proliferation of activated B cells is required to foster Blimp1 expression but needs to be terminated to avoid overshooting immune reactions. Activator protein 1 (AP-1) transcription factors become quickly up-regulated upon B cell activation. We demonstrate that Fra1, a Fos member of AP-1, enhances activation-induced cell death upon induction in activated B cells. Moreover, mice with B cell–specific deletion of Fra1 show enhanced plasma cell differentiation and exacerbated antibody responses. In contrast, transgenic overexpression of Fra1 blocks plasma cell differentiation and immunoglobulin production, which cannot be rescued by Bcl2. On the molecular level, Fra1 represses Blimp1 expression and interferes with binding of the activating AP-1 member c-Fos to the Blimp1 promoter. Conversely, overexpression of c-Fos in Fra1 transgenic B cells releases Blimp1 repression. As Fra1 lacks transcriptional transactivation domains, we propose that Fra1 inhibits Blimp1 expression and negatively controls plasma cell differentiation through binding to the Blimp1 promoter. In summary, we demonstrate that Fra1 negatively controls plasma cell differentiation by repressing Blimp1 expression. PMID:25288397

CARTILAGE OLIGOMERIC MATRIX PROTEIN ENHANCES MATRIX ASSEMBLY DURING CHONDROGENESIS OF HUMAN MESENCHYMAL STEM CELLS

PubMed Central

Haleem-Smith, Hana; Calderon, Raul; Song, Yingjie; Tuan, Rocky S.; Chen, Faye H.

2011-01-01

Cartilage oligomeric matrix protein/thrombospondin-5 (COMP/TSP5) is an abundant cartilage extracellular matrix (ECM) protein that interacts with major cartilage ECM components, including aggrecan and collagens. To test our hypothesis that COMP/TSP5 functions in the assembly of the ECM during cartilage morphogenesis, we have employed mesenchymal stem cell (MSC) chondrogenesis in vitro as a model to examine the effects of COMP over-expression on neo-cartilage formation. Human bone marrow-derived MSCs were transfected with either full-length COMP cDNA or control plasmid, followed by chondrogenic induction in three-dimensional pellet or alginate-hydrogel culture. MSC chondrogenesis and ECM production was estimated based on quantitation of sulfated glycosaminoglycan (sGAG) accumulation, immunohistochemistry of the presence and distribution of cartilage ECM proteins, and real-time RT-PCR analyis of mRNA expression of cartilage markers. Our results showed that COMP over-expression resulted in increased total sGAG content during the early phase of MSC chondrogenesis, and increased immuno-detectable levels of aggrecan and collagen type II in the ECM of COMP-transfected pellet and alginate cultures, indicating more abundant cartilaginous matrix. COMP transfection did not significantly increase the transcript levels of the early chondrogenic marker, Sox9, or aggrecan, suggesting that enhancement of MSC cartilage ECM was effected at post-transcriptional levels. These findings strongly suggest that COMP functions in mesenchymal chondrogenesis by enhancing cartilage ECM organization and assembly. The action of COMP is most likely mediated not via direct changes in cartilage matrix gene expression but via interactions of COMP with other cartilage ECM proteins, such as aggrecan and collagens, that result in enhanced assembly and retention. PMID:22095699

Interleukin-1 Receptor Type 2 Acts with c-Fos to Enhance the Expression of Interleukin-6 and Vascular Endothelial Growth Factor A in Colon Cancer Cells and Induce Angiogenesis*

PubMed Central

Mar, Ai-Chung; Chu, Chun-Ho; Lee, Hui-Ju; Chien, Chia-Wen; Cheng, Jing-Jy; Yang, Shung-Haur; Jiang, Jeng-Kai; Lee, Te-Chang

2015-01-01

Interleukin-1 receptor type 2 (IL1R2) acts as a decoy receptor of exogenous IL-1; however, its intracellular activity is poorly understood. We previously demonstrated that IL1R2 intracellularly activates the expression of several proinflammatory cytokines and affects cell migration. In this study, we found that intracellular IL1R2 expression was increased in human colorectal cancer cells (CRCs) compared with normal colon cells. We also observed that the mRNA levels of IL1R2 were highly correlated with IL-6 in tumor tissues of CRC patients. By modulating its expression in CRC cells, we verified that enhanced IL1R2 expression transcriptionally activated the expression of IL-6 and VEGF-A. Conditioned medium harvested from IL1R2-overexpressing CRC cells contained higher levels of IL-6 and VEGF-A than that from vector control cells and significantly enhanced the proliferation, migration, and tube formation of cultured endothelial cells. We further demonstrated a positive association of intracellular IL1R2 levels with tumor growth and microvessel density in xenograft mouse models. These results revealed that IL1R2 activates the expression of angiogenic factors. Mechanistically, we revealed that IL1R2 complexes with c-Fos and binds to the AP-1 site at the IL-6 and VEGF-A promoters. Together, these results reveal a novel function of intracellular IL1R2 that acts with c-Fos to enhance the transcription of IL-6 and VEGF-A, which promotes angiogenesis in CRC. PMID:26209639

Hes6 is required for actin cytoskeletal organization in differentiating C2C12 myoblasts

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

Malone, Caroline M.P.; Domaschenz, Renae; Amagase, Yoko

Hes6 is a member of the hairy-enhancer-of-split family of transcription factors that regulate proliferating cell fate in development and is known to be expressed in developing muscle. Here we investigate its function in myogenesis in vitro. We show that Hes6 is a direct transcriptional target of the myogenic transcription factors MyoD and Myf5, indicating that it is integral to the myogenic transcriptional program. The localization of Hes6 protein changes during differentiation, becoming predominantly nuclear. Knockdown of Hes6 mRNA levels by siRNA has no effect on cell cycle exit or induction of myosin heavy chain expression in differentiating C2C12 myoblasts, butmore » F-actin filament formation is disrupted and both cell motility and myoblast fusion are reduced. The knockdown phenotype is rescued by expression of Hes6 cDNA resistant to siRNA. These results define a novel role for Hes6 in actin cytoskeletal dynamics in post mitotic myoblasts.« less

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

Molecular integration of HoxB4 and STAT3 for self-renewal of hematopoietic stem cells: a model of molecular convergence for stemness.

PubMed

Hong, Sung-Hyun; Yang, Seung-Jip; Kim, Tae-Min; Shim, Jae-Seung; Lee, Ho-Sun; Lee, Ga-Young; Park, Bo-Bae; Nam, Suk Woo; Ryoo, Zae Young; Oh, Il-Hoan

2014-05-01

The upregulation of HoxB4 promotes self-renewal of hematopoietic stem cells (HSCs) without overriding the normal stem cell pool size. A similar enhancement of HSC self-renewal occurs when signal transducer and activator of transcription 3 (STAT3) is activated in HSCs. In this study, to gain insight into the functional organization of individual transcription factors (TFs) that have similar effects on HSCs, we investigated the molecular interplay between HoxB4 and STAT3 in the regulation of HSC self-renewal. We found that while STAT3-C or HoxB4 similarly enhanced the in vitro self-renewal and in vivo repopulating activities of HSCs, simultaneous transduction of both TFs did not have additive effects, indicating their functional redundancy in HSCs. In addition, activation of STAT3 did not cause changes in the expression levels of HoxB4. In contrast, the inhibition of STAT3 activity in HoxB4-overexpressing hematopoietic cells significantly abrogated the enhancing effects of HoxB4, and the upregulation of HoxB4 caused a ligand-independent Tyr-phosphorylation of STAT3. Microarray analysis revealed a significant overlap of the transcriptomes regulated by STAT3 and HoxB4 in undifferentiated hematopoietic cells. Moreover, a gene set enrichment analysis showed significant overlap in the candidate TFs that can recapitulate the transcriptional changes induced by HoxB4 or STAT3. Interestingly, among these common TFs were the pluripotency-related genes Oct-4 and Nanog. These results indicate that tissue-specific TFs regulating HSC self-renewal are functionally organized to play an equivalent role in transcription and provide insights into the functional convergence of multiple entries of TFs toward a conserved transcription program for the stem cell state. © 2014 AlphaMed Press.

Methods to Monitor and Manipulate TFEB Activity During Autophagy.

PubMed

Medina, D L; Settembre, C; Ballabio, A

2017-01-01

Macroautophagy is a catabolic process deputed to the turnover of intracellular components. Recent studies have revealed that transcriptional regulation is a major mechanism controlling autophagy. Currently, more than 20 transcription factors have been shown to modulate cellular autophagy levels. Among them, the transcription factor EB (TFEB) appears to have the broadest proautophagy role, given its capacity to control the biogenesis of lysosomes and autophagosomes, the two main organelles required for the autophagy pathway. TFEB has attracted major attention owing to its ability to enhance cellular clearance of pathogenic substrates in a variety of animal models of disease, such as lysosomal storage disorders, Parkinson's, Alzheimer's, α1-antitrypsin, obesity as well as others, suggesting that the TFEB pathway represents an extraordinary possibility for future development of innovative therapies. Importantly, the subcellular localization and activity of TFEB are regulated by its phosphorylation status, suggesting that TFEB activity can be pharmacologically targeted. Given the growing list of common and rare diseases in which manipulation of autophagy may be beneficial, in this chapter we describe a set of validated protocols developed to modulate and analyze TFEB-mediated enhancement of autophagy both in vitro and in vivo conditions. © 2017 Elsevier Inc. All rights reserved.

Dynamic regulation of EZH2 from HPSc to hepatocyte-like cell fate

PubMed Central

Helsen, Nicky; Vanhove, Jolien; Boon, Ruben; Xu, Zhuofei; Ordovas, Laura; Verfaillie, Catherine M.

2017-01-01

Currently, drug metabolization and toxicity studies rely on the use of primary human hepatocytes and hepatoma cell lines, which both have conceivable limitations. Human pluripotent stem cell (hPSC)—derived hepatocyte-like cells (HLCs) are an alternative and valuable source of hepatocytes that can overcome these limitations. EZH2 (enhancer of zeste homolog 2), a transcriptional repressor of the polycomb repressive complex 2 (PRC2), may play an important role in hepatocyte development, but its role during in vitro hPSC-HLC differentiation has not yet been assessed. We here demonstrate dynamic regulation of EZH2 during hepatic differentiation of hPSC. To enhance EZH2 expression, we inducibly overexpressed EZH2 between d0 and d8, demonstrating a significant improvement in definitive endoderm formation, and improved generation of HLCs. Despite induction of EZH2 overexpression until d8, EZH2 transcript and protein levels decreased from d4 onwards, which might be caused by expression of microRNAs predicted to inhibit EZH2 expression. In conclusion, our studies demonstrate that EZH2 plays a role in endoderm formation and hepatocyte differentiation, but its expression is tightly post-transcriptionally regulated during this process. PMID:29091973

Regulation of MHC class I expression by Foxp3 and its effect on Treg cell function

PubMed Central

Mu, Jie; Tai, Xuguang; Iyer, Shankar S.; Weissman, Jocelyn D.; Singer, Alfred; Singer, Dinah S.

2014-01-01

Expression of MHC class I molecules, which provide immune surveillance against intracellular pathogens, is higher on lymphoid cells than on any other cell types. In T cells, this is a result of activation of class I transcription by the T cell enhanceosome consisting of Runx1, CBFβ and LEF1. We now report that MHC class I transcription in T cells also is enhanced by Foxp3, resulting in higher levels of class I in CD4+CD25+ T regulatory cells than in conventional CD4+CD25− T cells. Interestingly, the effect of Foxp3 regulation of MHC class I transcription is cell-type specific: Foxp3 increases MHC class I expression in T cells but represses it in epithelial tumor cells. In both cell types, Foxp3 targets the upstream IRE and downstream core promoter of the class I gene. Importantly, expression of MHC class I contributes to the function of CD4+CD25+ T regulatory cells by enhancing immune suppression, both in in vitro and in vivo. These findings identify MHC class I genes as direct targets of Foxp3 whose expression augments regulatory T cell function. PMID:24523508

The Campylobacter jejuni MarR-like transcriptional regulators RrpA and RrpB both influence bacterial responses to oxidative and aerobic stresses.

PubMed

Gundogdu, Ozan; da Silva, Daiani T; Mohammad, Banaz; Elmi, Abdi; Mills, Dominic C; Wren, Brendan W; Dorrell, Nick

2015-01-01

The ability of the human intestinal pathogen Campylobacter jejuni to respond to oxidative stress is central to bacterial survival both in vivo during infection and in the environment. Re-annotation of the C. jejuni NCTC11168 genome revealed the presence of two MarR-type transcriptional regulators Cj1546 and Cj1556, originally annotated as hypothetical proteins, which we have designated RrpA and RrpB (regulator of response to peroxide) respectively. Previously we demonstrated a role for RrpB in both oxidative and aerobic (O2) stress and that RrpB was a DNA binding protein with auto-regulatory activity, typical of MarR-type transcriptional regulators. In this study, we show that RrpA is also a DNA binding protein and that a rrpA mutant in strain 11168H exhibits increased sensitivity to hydrogen peroxide oxidative stress. Mutation of either rrpA or rrpB reduces catalase (KatA) expression. However, a rrpAB double mutant exhibits higher levels of resistance to hydrogen peroxide oxidative stress, with levels of KatA expression similar to the wild-type strain. Mutation of either rrpA or rrpB also results in a reduction in the level of katA expression, but this reduction was not observed in the rrpAB double mutant. Neither the rrpA nor rrpB mutant exhibits any significant difference in sensitivity to either cumene hydroperoxide or menadione oxidative stresses, but both mutants exhibit a reduced ability to survive aerobic (O2) stress, enhanced biofilm formation and reduced virulence in the Galleria mellonella infection model. The rrpAB double mutant exhibits wild-type levels of biofilm formation and wild-type levels of virulence in the G mellonella infection model. Together these data indicate a role for both RrpA and RrpB in the C. jejuni peroxide oxidative and aerobic (O2) stress responses, enhancing bacterial survival in vivo and in the environment.

The SlNAC8 gene of the halophyte Suaeda liaotungensis enhances drought and salt stress tolerance in transgenic Arabidopsis thaliana.

PubMed

Wu, Dandan; Sun, Yinghao; Wang, Hongfei; Shi, He; Su, Mingxing; Shan, Hongyan; Li, Tongtong; Li, Qiuli

2018-07-01

NAC (NAM, ATAF1/2 and CUC) transcription factors play an important role in resisting abiotic stress in plants. In this study, a novel NAC gene, designated SlNAC8 from Suaeda liaotungensis K. was characterized. SlNAC8 protein is localized in the nucleus, and the yeast one-hybrid screening showed that it contains an activation domain in its C-terminus and functions as a transcriptional activator. Gene expression analysis revealed that it is induced by drought and salt stress. Arabidopsis plants overexpressing SlNAC8 demonstrated enhanced tolerance to drought and salt stress, showing significant advantages in seed germination, root growth, shoot growth, and survival rate compared with controls. Moreover, transgenic plants had a significantly higher proline concentration, antioxidant enzyme activity (superoxide dismutase, peroxidase, and catalase), and level of chlorophyll fluorescence than wild-type, and a significantly lower malondialdehyde concentration and electrolyte leakage under drought and salt stress. The overexpression of SlNAC8 in transgenic plants also enhanced the expression of stress-responsive genes such as RD20, GSTF6, COR47, RD29A, RD29B, and NYC1. In summary, SlNAC8, as a transcription factor, may change the physiological-biochemical characteristic of plants by regulating the expression of stress-responsive genes and enhance the drought and salt stress tolerance of plants. SlNAC8 can be utilized for developing drought and salinity tolerance in crop plants through genetic engineering. Copyright © 2018 Elsevier B.V. All rights reserved.

Requirement of Hsp105 in CoCl{sub 2}-induced HIF-1α accumulation and transcriptional activation

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

Mikami, Hiroki; Saito, Youhei, E-mail: ysaito@mb.kyoto-phu.ac.jp; Okamoto, Namiko

The mammalian stress protein Hsp105α protects cells from stress conditions. Several studies have indicated that Hsp105α is overexpressed in many types of solid tumors, which contain hypoxic microenvironments. However, the role of Hsp105α in hypoxic tumors remains largely unknown. We herein demonstrated the involvement of Hsp105α in HIF-1 functions induced by the hypoxia-mimetic agent CoCl{sub 2}. While Hsp105α is mainly localized in the cytoplasm under normal conditions, a treatment with CoCl{sub 2} induces the nuclear localization of Hsp105α, which correlated with HIF-1α expression levels. The overexpression of degradation-resistant HIF-1α enhances the nuclear localization of Hsp105α without the CoCl{sub 2} treatment.more » The CoCl{sub 2}-dependent transcriptional activation of HIF-1, which is measured using a reporter gene containing a HIF-responsive element, is reduced by the knockdown of Hsp105α. Furthermore, the CoCl{sub 2}-induced accumulation of HIF-1α is enhanced by heat shock, which results in the nuclear localization of Hsp105, and is suppressed by the knockdown of Hsp105. Hsp105 associates with HIF-1α in CoCl{sub 2}-treated cells. These results suggest that Hsp105α plays an important role in the functions of HIF-1 under hypoxic conditions, in which Hsp105α enhances the accumulation and transcriptional activity of HIF-1 through the HIF-1α-mediated nuclear localization of Hsp105α. - Highlights: • Hsp105α is required for the CoCl{sub 2}-induced transcriptional activation and accumulation of HIF-1. • Hsp105α localizes to the nucleus and interacts with HIF-1α in CoCl{sub 2}-treated cells. • Hsp105 enhances the CoCl{sub 2}-induced accumulation of HIF-1α under heat shock conditions.« less

The rice ERF transcription factor OsERF922 negatively regulates resistance to Magnaporthe oryzae and salt tolerance

PubMed Central

Liu, Dongfeng; Chen, Xujun; Liu, Jiqin; Ye, Jianchun; Guo, Zejian

2012-01-01

Rice OsERF922, encoding an APETELA2/ethylene response factor (AP2/ERF) type transcription factor, is rapidly and strongly induced by abscisic acid (ABA) and salt treatments, as well as by both virulent and avirulent pathovars of Magnaporthe oryzae, the causal agent of rice blast disease. OsERF922 is localized to the nucleus, binds specifically to the GCC box sequence, and acts as a transcriptional activator in plant cells. Knockdown of OsERF922 by means of RNAi enhanced resistance against M. oryzae. The elevated disease resistance of the RNAi plants was associated with increased expression of PR, PAL, and the other genes encoding phytoalexin biosynthetic enzymes and without M. oryzae infection. In contrast, OsERF922-overexpressing plants showed reduced expression of these defence-related genes and enhanced susceptibility to M. oryzae. In addition, the OsERF922-overexpressing lines exhibited decreased tolerance to salt stress with an increased Na+/K+ ratio in the shoots. The ABA levels were found increased in the overexpressing lines and decreased in the RNAi plants. Expression of the ABA biosynthesis-related genes, 9-cis-epoxycarotenoid dioxygenase (NCED) 3 and 4, was upregulated in the OsERF922-overexpressing plants, and NCED4 was downregulated in the RNAi lines. These results suggest that OsERF922 is integrated into the cross-talk between biotic and abiotic stress-signalling networks perhaps through modulation of the ABA levels. PMID:22442415

Proteomic characterization of the acid tolerance response in Lactobacillus delbrueckii subsp. bulgaricus CAUH1 and functional identification of a novel acid stress-related transcriptional regulator Ldb0677.

PubMed

Zhai, Zhengyuan; Douillard, François P; An, Haoran; Wang, Guohong; Guo, Xinghua; Luo, Yunbo; Hao, Yanling

2014-06-01

To overcome the deleterious effects of acid stress, Lactobacillus delbrueckii subsp. bulgaricus (L. bulgaricus) elicits an adaptive response to acid stress. In this study, proteomics approach complemented by transcriptional analysis revealed some cellular changes in L. bulgaricus CAUH1 during acid adaptation. We observed an increase of glycolysis-associated proteins, promoting an optimal utilization of carbohydrates. Also, rerouting of the pyruvate metabolism to fatty acid biosynthesis was observed, indicating a possible modification of the cell membrane rigidity and impermeability. In addition, expression of ribosomal protein S1 (RpsA) was repressed; however, the expression of EF-Tu, EF-G and TypA was up-regulated at both protein and transcript levels. This suggests a reduction of protein synthesis in response to acid stress along with possible enhancement of the translational accuracy and protein folding. It is noteworthy that the putative transcriptional regulator Ldb0677 was 1.84-fold up-regulated. Heterologous expression of Ldb0677 was shown to significantly enhance acid resistance in host strain Lactococcus lactis. To clarify its role in transcriptional regulation network, the DNA-binding specificity of Ldb0677 was determined using bacterial one-hybrid and electrophoretic mobility shift assay. The identification of a binding motif (SSTAGACR) present in the promoter regions of 22 genes indicates that it might function as a major regulator in acid stress response in L. bulgaricus. © 2013 Society for Applied Microbiology and John Wiley & Sons Ltd.

Global regulator Anr represses PlcH phospholipase activity in Pseudomonas aeruginosa when oxygen is limiting.

PubMed

Jackson, Angelyca A; Daniels, Emily F; Hammond, John H; Willger, Sven D; Hogan, Deborah A

2014-10-01

Haemolytic phospholipase C (PlcH) is a potent virulence and colonization factor that is expressed at high levels by Pseudomonas aeruginosa within the mammalian host. The phosphorylcholine liberated from phosphatidylcholine and sphingomyelin by PlcH is further catabolized into molecules that both support growth and further induce plcH expression. We have shown previously that the catabolism of PlcH-released choline leads to increased activity of Anr, a global transcriptional regulator that promotes biofilm formation and virulence. Here, we demonstrated the presence of a negative feedback loop in which Anr repressed plcH transcription and we proposed that this regulation allowed for PlcH levels to be maintained in a way that promotes productive host-pathogen interactions. Evidence for Anr-mediated regulation of PlcH came from data showing that growth at low oxygen (1%) repressed PlcH abundance and plcH transcription in the WT, and that plcH transcription was enhanced in an Δanr mutant. The plcH promoter featured an Anr consensus sequence that was conserved across all P. aeruginosa genomes and mutation of conserved nucleotides within the Anr consensus sequence increased plcH expression under hypoxic conditions. The Anr-regulated transcription factor Dnr was not required for this effect. The loss of Anr was not sufficient to completely derepress plcH transcription as GbdR, a positive regulator of plcH, was required for expression. Overexpression of Anr was sufficient to repress plcH transcription even at 21 % oxygen. Anr repressed plcH expression and phospholipase C activity in a cell culture model for P. aeruginosa-epithelial cell interactions. The Authors.

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

Xiang, Zhonghua; Qiao, Ling; Zhou, Yan

Research highlights: {yields} A chimeric subgenomic HCV replicon expresses HCV-3a NS5A in an HCV-1b backbone. {yields} HCV-3a NS5A increases mature SREBP-1c protein level. {yields} HCV-3a NS5A activates SREBP-1c transcription. {yields} Domain II of HCV-3a NS5A is more effective in SREBP-1c promoter activation. {yields} Transcription factor Sp1 is required for SREBP-1c activation by HCV-3a NS5A. -- Abstract: Steatosis is an important clinical manifestation of hepatitis C virus (HCV) infection. The molecular mechanisms of HCV-associated steatosis are not well understood. Sterol regulatory element-binding protein-1c (SREBP-1c) is a key transcription factor which activates the transcription of lipogenic genes. Here we showed that themore » nuclear, mature SREBP-1c level increases in the nucleus of replicon cells expressing HCV-3a nonstructural protein-5A (NS5A). We further showed that HCV-3a NS5A up-regulates SREBP-1c transcription. Additional analysis showed that transcriptional factor Sp1 is involved in SREBP-1c activation by HCV-3a NS5A because inhibition of Sp1 activity by mithramycin A or a dominant-negative Sp1 construct abrogated SREBP-1c promoter activation by HCV-3a NS5A. In addition, chromatin immunoprecipitation (ChIP) assay demonstrated enhanced binding of Sp1 on the SREBP-1c promoter in HCV-3a NS5A replicon cells. These results showed that HCV-3a NS5A activates SREBP-1c transcription through Sp1. Taken together, our results suggest that HCV-3a NS5A is a contributing factor for steatosis caused by HCV-3a infection.« less

Targeting MYCN-Driven Transcription By BET-Bromodomain Inhibition.

PubMed

Henssen, Anton; Althoff, Kristina; Odersky, Andrea; Beckers, Anneleen; Koche, Richard; Speleman, Frank; Schäfers, Simon; Bell, Emma; Nortmeyer, Maike; Westermann, Frank; De Preter, Katleen; Florin, Alexandra; Heukamp, Lukas; Spruessel, Annika; Astrahanseff, Kathy; Lindner, Sven; Sadowski, Natalie; Schramm, Alexander; Astorgues-Xerri, Lucile; Riveiro, Maria E; Eggert, Angelika; Cvitkovic, Esteban; Schulte, Johannes H

2016-05-15

Targeting BET proteins was previously shown to have specific antitumoral efficacy against MYCN-amplified neuroblastoma. We here assess the therapeutic efficacy of the BET inhibitor, OTX015, in preclinical neuroblastoma models and extend the knowledge on the role of BRD4 in MYCN-driven neuroblastoma. The efficacy of OTX015 was assessed in in vitro and in vivo models of human and murine MYCN-driven neuroblastoma. To study the effects of BET inhibition in the context of high MYCN levels, MYCN was ectopically expressed in human and murine cells. The effect of OTX015 on BRD4-regulated transcriptional pause release was analyzed using BRD4 and H3K27Ac chromatin immunoprecipitation coupled with DNA sequencing (ChIP-Seq) and gene expression analysis in neuroblastoma cells treated with OTX015 compared with vehicle control. OTX015 showed therapeutic efficacy against preclinical MYCN-driven neuroblastoma models. Similar to previously described BET inhibitors, concurrent MYCN repression was observed in OTX015-treated samples. Ectopic MYCN expression, however, did not abrogate effects of OTX015, indicating that MYCN repression is not the only target of BET proteins in neuroblastoma. When MYCN was ectopically expressed, BET inhibition still disrupted MYCN target gene transcription without affecting MYCN expression. We found that BRD4 binds to super-enhancers and MYCN target genes, and that OTX015 specifically disrupts BRD4 binding and transcription of these genes. We show that OTX015 is effective against mouse and human MYCN-driven tumor models and that BRD4 not only targets MYCN, but specifically occupies MYCN target gene enhancers as well as other genes associated with super-enhancers. Clin Cancer Res; 22(10); 2470-81. ©2015 AACR. ©2015 American Association for Cancer Research.

Arabidopsis thaliana G2-LIKE FLAVONOID REGULATOR and BRASSINOSTEROID ENHANCED EXPRESSION1 are low-temperature regulators of flavonoid accumulation.

PubMed

Petridis, Antonios; Döll, Stefanie; Nichelmann, Lars; Bilger, Wolfgang; Mock, Hans-Peter

2016-08-01

Flavonoid synthesis is predominantly regulated at the transcriptional level through the MYB-basic helix-loop-helix (bHLH)-WD40 (MBW) (MYB: transcription factor of the myeloblastosis protein family, WD40: tanscription factor with a short structural motif of 40 amino acids which terminates in an aspartic acid-tryptophan dipeptide) complex, and responds to both environmental and developmental stimuli. Although the developmental regulation of flavonoid accumulation in Arabidopsis thaliana has been examined in great detail, the response of the flavonoid synthesis pathway to abiotic stress (particularly low temperature) remains unclear. A screen of a Dissociation element (Ds) transposon-induced mutation collection identified two lines which exhibited an altered profile of phenylpropanoid accumulation following exposure to low-temperature stress. One of the mutated genes (BRASSINOSTEROID ENHANCED EXPRESSION1 (BEE1)) encoded a brassinosteroid enhanced expression transcription factor, while the other (G2-LIKE FLAVONOID REGULATOR (GFR)) encoded a G2-like flavonoid regulator. Phenylpropanoid-targeted analysis was performed using high-performance LC-MS, and gene expression analysis using quantitative reverse transcription-PCR. In both mutants, the accumulation of quercetins and scopolin was reduced under low-temperature growing conditions, whereas that of anthocyanin was increased. BEE1 and GFR were both shown to negatively regulate anthocyanin accumulation by inhibiting anthocyanin synthesis genes via the suppression of the bHLH (TRANSPARENT TESTA8 (TT8) and GLABROUS3 (GL3)) and/or the MYB (PRODUCTION OF ANTHOCYANIN PIGMENTS2 (PAP2)) components of the MBW complex. Our results provide new insight into the regulatory control of phenylpropanoid metabolism at low temperatures, and reveal that BEE1 and GFR act as important components of the signal transduction chain. © 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.

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.

Sirt1 activation ameliorates renal fibrosis by inhibiting the TGF-β/Smad3 pathway.

PubMed

Huang, Xin-Zhong; Wen, Donghai; Zhang, Min; Xie, Qionghong; Ma, Leting; Guan, Yi; Ren, Yueheng; Chen, Jing; Hao, Chuan-Ming

2014-05-01

TGF-β signaling plays an important role in the pathogenesis and progression of chronic kidney disease (CKD). Smad3, a transcription factor, is a critical fibrogenic mediator of TGF-β. Sirt1 is a NAD(+) -dependent deacetylase that has been reported to modify a number of transcription factors to exert certain beneficial health effects. This study examined the effect of Sirt1 on Smad3 and its role in CKD. Resveratrol attenuated the expression of extracelluar matrix proteins in both the remnant kidney of 5/6th nephrectomized rats and cultured mesangial cells (MMCs) exposed to TGF-β1. The effect of resveratrol was substantially attenuated in cultured MMCs for which Sirt1 had been knocked down by an shRNA lentivirus. Overexpression of Sirt1 attenuated TGF-β1-induced extracelluar matrix expression in cultured cells. Co-immunoprecipitation studies suggested that Sirt1 could bind with Smad3. Resveratrol treatment enhanced this binding and reduced acetylation levels of Smad3. Resveratrol inhibited the transcription activity of Smad3. Knockdown of Sirt1 increased acetylated Smad3 and substantially enhanced the transcriptional activity following TGF-β1. Finally, Sirt1 deficiency aggravated renal function damage and markedly enhanced fibrosis in the remnant kidney of 5/6 nephrectomized mice. Taken together, these results identify Sirt1 as an important protective factor for renal fibrosis in a CKD rodent model, and the protective function of Sirt1 is attributable to its action on TGF-β/Smad3 signaling. Therefore, we suggest that Sirt1 may be a potential therapeutic target for the treatment of CKD. © 2013 Wiley Periodicals, Inc.

Hormone-dependent control of developmental timing through regulation of chromatin accessibility

PubMed Central

Uyehara, Christopher M.; Nystrom, Spencer L.; Niederhuber, Matthew J.; Leatham-Jensen, Mary; Ma, Yiqin; Buttitta, Laura A.

2017-01-01

Specification of tissue identity during development requires precise coordination of gene expression in both space and time. Spatially, master regulatory transcription factors are required to control tissue-specific gene expression programs. However, the mechanisms controlling how tissue-specific gene expression changes over time are less well understood. Here, we show that hormone-induced transcription factors control temporal gene expression by regulating the accessibility of DNA regulatory elements. Using the Drosophila wing, we demonstrate that temporal changes in gene expression are accompanied by genome-wide changes in chromatin accessibility at temporal-specific enhancers. We also uncover a temporal cascade of transcription factors following a pulse of the steroid hormone ecdysone such that different times in wing development can be defined by distinct combinations of hormone-induced transcription factors. Finally, we show that the ecdysone-induced transcription factor E93 controls temporal identity by directly regulating chromatin accessibility across the genome. Notably, we found that E93 controls enhancer activity through three different modalities, including promoting accessibility of late-acting enhancers and decreasing accessibility of early-acting enhancers. Together, this work supports a model in which an extrinsic signal triggers an intrinsic transcription factor cascade that drives development forward in time through regulation of chromatin accessibility. PMID:28536147

Mongolian Almond (Prunus mongolica Maxim): The Morpho-Physiological, Biochemical and Transcriptomic Response to Drought Stress

PubMed Central

Bai, Shulan; Gao, Xiaomin; Liu, Min; Yan, Wei

2015-01-01

Prunus mongolica Maxim, which is widely established in the Gobi Desert, shows extreme tolerance to drought. However, there is a lack of available transcriptomic resources for this species related to its response to water deficiency. To investigate the mechanisms that allow P. mongolica to maintain growth in extremely arid environments, the response of P. mongolica seedlings to drought stress was analyzed using morphological, physiological, biochemical and high-throughput sequencing approaches. We generated 28,713,735 and 26,650,133 raw reads from no-stress control and drought-stressed P. mongolica seedlings, respectively. In total, we obtained 67,352 transcripts with an average length of 874.44 bp. Compared with the no-stress control, 3,365 transcripts were differentially expressed in the drought-stressed seedlings, including 55.75% (1,876 transcripts) up-regulated and 44.25% (1,489 transcripts) down-regulated transcripts. The photosynthesis response showed a decreasing tendency under drought stress, but the changes in the levels of hormones (auxins, cytokinins and abscisic acid) resulted in the closing of stomata and decreased cell enlargement and division; these changes were effective for promoting P. mongolica survival in Gobi Desert. Next, we analyzed the aquaporin and superoxide dismutase gene families due to their importance in plant resistance to drought stress. We found that all of the plasma membrane intrinsic protein transcripts were down-regulated in the drought-stressed treatment, whereas drought did not affect the expression of nodulin intrinsic protein or small basic intrinsic protein transcripts in P. mongolica seedlings. In addition, activation of iron superoxide dismutase transcription and enhanced transcription of manganese superoxide dismutase were observed in P. mongolica to promote tolerance of drought stress. This study identified drought response genes in P. mongolica seedlings. Our results provide a significant contribution to the understanding of how P. mongolica responds to drought stress at the transcriptome level, which may help to elucidate molecular mechanisms associated with the drought response of almond plants. PMID:25893685

Transcriptional activation of the suppressor of cytokine signaling-3 (SOCS-3) gene via STAT3 is increased in F9 REX1 (ZFP-42) knockout teratocarcinoma stem cells relative to wild-type cells.

PubMed

Xu, Juliana; Sylvester, Renia; Tighe, Ann P; Chen, Siming; Gudas, Lorraine J

2008-03-14

Rex1 (Zfp42), first identified as a gene that is transcriptionally repressed by retinoic acid (RA), encodes a zinc finger transcription factor expressed at high levels in F9 teratocarcinoma stem cells, embryonic stem cells, and other stem cells. Loss of both alleles of Rex1 by homologous recombination alters the RA-induced differentiation of F9 cells, a model of pluripotent embryonic stem cells. We identified Suppressor of Cytokine Signaling-3 (SOCS-3) as a gene that exhibits greatly increased transcriptional activation in RA, cAMP, and theophylline (RACT)-treated F9 Rex1(-/-) cells (approximately 25-fold) as compared to wild-type (WT) cells ( approximately 2.5-fold). By promoter deletion, mutation, and transient transfection analyses, we have shown that this transcriptional increase is mediated by the STAT3 DNA-binding elements located between -99 to -60 in the SOCS-3 promoter. Overexpression of STAT3 dominant-negative mutants greatly diminishes this SOCS-3 transcriptional increase in F9 Rex1(-/-) cells. This increase in SOCS-3 transcription is associated with a four- to fivefold higher level of tyrosine-phosphorylated STAT3 in the RACT-treated F9 Rex1(-/-) cells as compared to WT. Dominant-negative Src tyrosine kinase, Jak2, and protein kinase A partially reduce the transcriptional activation of the SOCS 3 gene in RACT-treated F9 Rex1 null cells. In contrast, parathyroid hormone peptide enhances the effect of RA in F9 Rex1(-/-) cells, but not in F9 WT. Thus, Rex1, which is highly expressed in stem cells, inhibits signaling via the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway, thereby modulating the differentiation of F9 cells.

Interaction of the Transcription Start Site Core Region and Transcription Factor YY1 Determine Ascorbate Transporter SVCT2 Exon 1a Promoter Activity

PubMed Central

Qiao, Huan; May, James M.

2012-01-01

Transcription of the ascorbate transporter, SVCT2, is driven by two distinct promoters in exon 1 of the transporter sequence. The exon 1a promoter lacks a classical transcription start site and little is known about regulation of promoter activity in the transcription start site core (TSSC) region. Here we present evidence that the TSSC binds the multifunctional initiator-binding protein YY1. Electrophoresis shift assays using YY1 antibody showed that YY1 is present as one of two major complexes that specifically bind to the TSSC. The other complex contains the transcription factor NF-Y. Mutations in the TSSC that decreased YY1 binding also impaired the exon 1a promoter activity despite the presence of an upstream activating NF-Y/USF complex, suggesting that YY1 is involved in the regulation of the exon 1a transcription. Furthermore, YY1 interaction with NF-Y and/or USF synergistically enhanced the exon 1a promoter activity in transient transfections and co-activator p300 enhanced their synergistic activation. We propose that the TSSC plays a vital role in the exon 1a transcription and that this function is partially carried out by the transcription factor YY1. Moreover, co-activator p300 might be able to synergistically enhance the TSSC function via a “bridge” mechanism with upstream sequences. PMID:22532872

The role of MEF2 transcription factors in dehydration and anoxia survival in Rana sylvatica skeletal muscle

PubMed Central

Hoyeck, Myriam P.; Hadj-Moussa, Hanane

2017-01-01

The wood frog (Rana sylvatica) can endure freezing of up to 65% of total body water during winter. When frozen, wood frogs enter a dormant state characterized by a cessation of vital functions (i.e., no heartbeat, blood circulation, breathing, brain activity, or movement). Wood frogs utilize various behavioural and biochemical adaptations to survive extreme freezing and component anoxia and dehydration stresses, including a global suppression of metabolic functions and gene expression. The stress-responsive myocyte enhancer factor-2 (MEF2) transcription factor family regulates the selective expression of genes involved in glucose transport, protein quality control, and phosphagen homeostasis. This study examined the role of MEF2A and MEF2C proteins as well as select downstream targets (glucose transporter-4, calreticulin, and muscle and brain creatine kinase isozymes) in 40% dehydration and 24 h anoxia exposure at the transcriptional, translational, and post-translational levels using qRT-PCR, immunoblotting, and subcellular localization. Mef2a/c transcript levels remained constant during dehydration and anoxia. Total, cytoplasmic, and nuclear MEF2A/C and phospho-MEF2A/C protein levels remained constant during dehydration, whereas a decrease in total MEF2C levels was observed during rehydration. Total and phospho-MEF2A levels remained constant during anoxia, whereas total MEF2C levels decreased during 24 h anoxia and P-MEF2C levels increased during 4 h anoxia. In contrast, cytoplasmic MEF2A levels and nuclear phospho-MEF2A/C levels were upregulated during anoxia. MEF2 downstream targets remained constant during dehydration and anoxia, with the exception of glut4 which was upregulated during anoxia. These results suggest that the upregulated MEF2 response reported in wood frogs during freezing may in part stem from their cellular responses to surviving prolonged anoxia, rather than dehydration, leading to an increase in GLUT4 expression which may have an important role during anoxia survival. PMID:29134152

Enhancement of Chlorogenic Acid Production in Hairy Roots of Platycodon grandiflorum by Over-Expression of An Arabidopsis thaliana Transcription Factor AtPAP1

PubMed Central

Tuan, Pham Anh; Kwon, Do Yeon; Lee, Sanghyun; Arasu, Mariadhas Valan; Al-Dhabi, Naif Abdullah; Park, Nam Il; Park, Sang Un

2014-01-01

To improve the production of chlorogenic acid (CGA) in hairy roots of Platycodon grandiflorum, we induced over-expression of Arabidopsis thaliana transcription factor production of anthocyanin pigment (AtPAP1) using an Agrobacterium rhizogenes-mediated transformation system. Twelve hairy root lines showing over-expression of AtPAP1 were generated. In order to investigate the regulation of AtPAP1 on the activities of CGA biosynthetic genes, the expression levels of seven P. grandiflorum CGA biosynthetic genes were analyzed in the hairy root line that had the greatest accumulation of AtPAP1 transcript, OxPAP1-1. The introduction of AtPAP1 increased the mRNA levels of all examined CGA biosynthetic genes and resulted in a 900% up-regulation of CGA accumulation in OxPAP1-1 hairy roots relative to controls. This suggests that P. grandiflorum hairy roots that over-express the AtPAP1 gene are a potential alternative source of roots for the production of CGA. PMID:25153629

Integrative analysis with ChIP-seq advances the limits of transcript quantification from RNA-seq

PubMed Central

Liu, Peng; Sanalkumar, Rajendran; Bresnick, Emery H.; Keleş, Sündüz; Dewey, Colin N.

2016-01-01

RNA-seq is currently the technology of choice for global measurement of transcript abundances in cells. Despite its successes, isoform-level quantification remains difficult because short RNA-seq reads are often compatible with multiple alternatively spliced isoforms. Existing methods rely heavily on uniquely mapping reads, which are not available for numerous isoforms that lack regions of unique sequence. To improve quantification accuracy in such difficult cases, we developed a novel computational method, prior-enhanced RSEM (pRSEM), which uses a complementary data type in addition to RNA-seq data. We found that ChIP-seq data of RNA polymerase II and histone modifications were particularly informative in this approach. In qRT-PCR validations, pRSEM was shown to be superior than competing methods in estimating relative isoform abundances within or across conditions. Data-driven simulations suggested that pRSEM has a greatly decreased false-positive rate at the expense of a small increase in false-negative rate. In aggregate, our study demonstrates that pRSEM transforms existing capacity to precisely estimate transcript abundances, especially at the isoform level. PMID:27405803

Transcriptional Repression of ATF4 Gene by CCAAT/Enhancer-binding Protein β (C/EBPβ) Differentially Regulates Integrated Stress Response*

PubMed Central

Dey, Souvik; Savant, Sudha; Teske, Brian F.; Hatzoglou, Maria; Calkhoven, Cornelis F.; Wek, Ronald C.

2012-01-01

Different environmental stresses induce the phosphorylation of eIF2 (eIF2∼P), repressing global protein synthesis coincident with preferential translation of ATF4. ATF4 is a transcriptional activator of genes involved in metabolism and nutrient uptake, antioxidation, and regulation of apoptosis. Because ATF4 is a common downstream target that integrates signaling from different eIF2 kinases and their respective stress signals, the eIF2∼P/ATF4 pathway is collectively referred to as the integrated stress response. Although eIF2∼P elicits translational control in response to many different stresses, there are selected stresses, such as exposure to UV irradiation, that do not increase ATF4 expression despite robust eIF2∼P. The rationale for this discordant induction of ATF4 expression and eIF2∼P in response to UV irradiation is that transcription of ATF4 is repressed, and therefore ATF4 mRNA is not available for preferential translation. In this study, we show that C/EBPβ is a transcriptional repressor of ATF4 during UV stress. C/EBPβ binds to critical elements in the ATF4 promoter, resulting in its transcriptional repression. Expression of C/EBPβ increases in response to UV stress, and the liver-enriched inhibitory protein (LIP) isoform of C/EBPβ, but not the liver-enriched activating protein (LAP) version, represses ATF4 transcription. Loss of the liver-enriched inhibitory protein isoform results in increased ATF4 mRNA levels in response to UV irradiation and subsequent recovery of ATF4 translation, leading to enhanced expression of its target genes. Together these results illustrate how eIF2∼P and translational control combined with transcription factors regulated by alternative signaling pathways can direct programs of gene expression that are specifically tailored to each environmental stress. PMID:22556424

ERalpha and ERbeta expression and transcriptional activity are differentially regulated by HDAC inhibitors

PubMed Central

Duong, Vanessa; Licznar, Anne; Margueron, Raphaël; Boulle, Nathalie; Busson, Muriel; Lacroix, Matthieu; Katzenellenbogen, Benita S.; Cavaillès, Vincent; Lazennec, Gwendal

2006-01-01

The proliferative action of ERα largely accounts for the carcinogenic activity of estrogens. By contrast, recent data show that ERβ displays tumor-suppressor properties, thus supporting the interest to identify compounds which could increase its activity. Here, we show that histone deacetylase inhibitors (HDI) up-regulated ERβ protein levels, whereas it decreased ERα expression. Part of this regulation took place at the mRNA level through a mechanism independent of de novo protein synthesis. In addition, we found that, in various cancer cells, the treatment with different HDI enhanced the ligand-dependent activity of ERβ more strongly than that of ERα. On the other hand, in MDA-MB231 and HeLa cells, the expression of ERs modified the transcriptional response to HDI. The use of deletion mutants of both receptors demonstrated that AF1 domain of the receptors was required. Finally, we show that ERβ expression led to a dramatic increased in the antiproliferative activity of HDI, which correlated with a modification of the transcription of genes involved in cell cycle control by HDI. Altogether, these data demonstrate that the interference of ERβ and HDAC on the control of transcription and cell proliferation constitute a promising approach for cancer therapy. PMID:16158045

Green tea (Camellia sinensis) administration induces expression of immune relevant genes and biochemical parameters in rainbow trout (Oncorhynchus mykiss).

PubMed

Nootash, Shahab; Sheikhzadeh, Najmeh; Baradaran, Behzad; Oushani, Ali Khani; Maleki Moghadam, Mohammad Reza; Nofouzi, Katayoon; Monfaredan, Amir; Aghebati, Leili; Zare, Fatemeh; Shabanzadeh, Sadigheh

2013-12-01

Present study elucidates the efficacy of green tea (Camellia sinensis) on growth performance, immune and antioxidant systems and cytokine gene expression in rainbow trout tissues. Green tea was supplemented at 20, 100, and 500 mg kg(-1) diet and fed to fish (average weight: 23.5 g) for 35 days. No remarkable changes in growth performance were observed among all test groups. Lower lipid peroxidation product and higher superoxide dismutase activity were noted in fish received the medium dose of green tea. Significant increase in serum bactericidal activity and total protein were recorded in all treatment groups. All doses of green tea up-regulated Interleukin-1β transcription in the spleen, while Interleukin-1β mRNA level decreased significantly in the kidney of low dose of green tea. Interleukin-6 mRNA level was up-regulated in the spleen of high dose of green tea and liver of middle and high doses of green tea. High dose and medium dose of green tea up-regulated the interleukin-8 transcription in the kidney and liver, respectively. Meanwhile, green tea inhibited the production of interleukin-10 in all treatment groups compared with control group. Medium dose of green tea up-regulated tumor necrosis factor-α transcription in all fish tissues, while high dose and low dose of green tea enhanced tumor necrosis factor-α mRNA levels in the kidney and spleen, respectively. Present study suggests that green tea especially at 100 mg kg(-1) feed may effectively enhance the antioxidant system and immune system in rainbow trout. Copyright © 2013 Elsevier Ltd. All rights reserved.

Protective effects of 3-alkyl luteolin derivatives are mediated by Nrf2 transcriptional activity and decreased oxidative stress in Huntington's disease mouse striatal cells.

PubMed

Oliveira, Ana M; Cardoso, Susana M; Ribeiro, Márcio; Seixas, Raquel S G R; Silva, Artur M S; Rego, A Cristina

2015-12-01

Huntington's disease (HD) is a polyglutamine-expansion neurodegenerative disorder caused by increased number of CAG repeats in the HTT gene, encoding for the huntingtin protein. The mutation is linked to several intracellular mechanisms, including oxidative stress. Flavones are compounds with a protective role in neurodegenerative pathologies. In the present study we analyzed the protective effect of luteolin (Lut, 3',4',5,7-tetrahydroxyflavone) and four luteolin derivatives bearing 3-alkyl chains of 1, 4, 6 and 10 carbons (Lut-C1, Lut-C4, Lut-C6, Lut-C10) in striatal cells derived from HD knock-in mice expressing mutant Htt (STHdh(Q111/Q111)) versus wild-type striatal cells (STHdh(Q7/Q7)). HD cells showed increased caspase-3-like activity and intracellular reactive oxygen species (ROS), which were significantly decreased following treatment with Lut-C4 and Lut-C6 under concentrations that enhanced cell viability. Interestingly, Lut-C4 and Lut-C6 rose the nuclear levels of phospho(Ser40)-nuclear factor (erythroid-derived-2)-like 2 (Nrf2) and Nrf2/ARE transcriptional activity. Concordantly with increased Nrf2/ARE transcription, Lut-C6 enhanced superoxide dismutase 1 (SOD1) mRNA and SOD activity and glutamate-cysteine ligase catalytic subunit (GCLc) mRNA and protein levels, while Lut-C4 induced mRNA levels of GCLc only in mutant striatal cells. Data suggest that Lut-C6 luteolin derivative (in particular) might be relevant for the development of antioxidant strategies in HD. Copyright © 2015 Elsevier Ltd. All rights reserved.

A Basic Helix-Loop-Helix Transcription Factor, PtrbHLH, of Poncirus trifoliata Confers Cold Tolerance and Modulates Peroxidase-Mediated Scavenging of Hydrogen Peroxide1[C][W

PubMed Central

Huang, Xiao-San; Wang, Wei; Zhang, Qian; Liu, Ji-Hong

2013-01-01

The basic helix-loop-helix (bHLH) transcription factors are involved in a variety of physiological processes. However, plant bHLHs functioning in cold tolerance and the underlying mechanisms remain poorly understood. Here, we report the identification and functional characterization of PtrbHLH isolated from trifoliate orange (Poncirus trifoliata). The transcript levels of PtrbHLH were up-regulated under various abiotic stresses, particularly cold. PtrbHLH was localized in the nucleus with transactivation activity. Overexpression of PtrbHLH in tobacco (Nicotiana tabacum) or lemon (Citrus limon) conferred enhanced tolerance to cold under chilling or freezing temperatures, whereas down-regulation of PtrbHLH in trifoliate orange by RNA interference (RNAi) resulted in elevated cold sensitivity. A range of stress-responsive genes was up-regulated or down-regulated in the transgenic lemon. Of special note, several peroxidase (POD) genes were induced after cold treatment. Compared with the wild type, POD activity was increased in the overexpression plants but decreased in the RNAi plants, which was inversely correlated with the hydrogen peroxide (H2O2) levels in the tested lines. Treatment of the transgenic tobacco plants with POD inhibitors elevated the H2O2 levels and greatly compromised their cold tolerance, while exogenous replenishment of POD enhanced cold tolerance of the RNAi line. In addition, transgenic tobacco and lemon plants were more tolerant to oxidative stresses. Yeast one-hybrid assay and transient expression analysis demonstrated that PtrbHLH could bind to the E-box elements in the promoter region of a POD gene. Taken together, these results demonstrate that PtrbHLH plays an important role in cold tolerance, at least in part, by positively regulating POD-mediated reactive oxygen species removal. PMID:23624854

Spi-C has opposing effects to PU.1 on gene expression in progenitor B cells.

PubMed

Schweitzer, Brock L; Huang, Kelly J; Kamath, Meghana B; Emelyanov, Alexander V; Birshtein, Barbara K; DeKoter, Rodney P

2006-08-15

The Ets transcription factor Spi-C, expressed in B cells and macrophages, is closely related to PU.1 and has the ability to recognize the same DNA consensus sequence. However, the function of Spi-C has yet to be determined. The purpose of this study is to further examine Spi-C activity in B cell development. First, using retroviral vectors to infect PU.1(-/-) fetal liver progenitors, Spi-C was found to be inefficient at inducing cytokine-dependent proliferation and differentiation of progenitor B (pro-B) cells or macrophages relative to PU.1 or Spi-B. Next, Spi-C was ectopically expressed in fetal liver-derived, IL-7-dependent pro-B cell lines. Wild-type (WT) pro-B cells ectopically expressing Spi-C (WT-Spi-C) have several phenotypic characteristics of pre-B cells such as increased CD25 and decreased c-Kit surface expression. In addition, WT-Spi-C pro-B cells express increased levels of IgH sterile transcripts and reduced levels of expression and transcription of the FcgammaRIIb gene. Gel-shift analysis suggests that Spi-C, ectopically expressed in pro-B cells, can bind PU.1 consensus sites in the IgH intronic enhancer and FcgammaRIIb promoter. Transient transfection analysis demonstrated that PU.1 functions to repress the IgH intronic enhancer and activate the FcgammaRIIb promoter, while Spi-C opposes these activities. WT-Spi-C pro-B cells have reduced levels of dimethylation on lysine 9 of histone H3 within the IgH 3' regulatory region, indicating that Spi-C can contribute to removal of repressive features in the IgH locus. Overall, these studies suggest that Spi-C may promote B cell differentiation by modulating the activity of PU.1-dependent genes.

Induction of activation of the antioxidant response element and stabilization of Nrf2 by 3-(3-pyridylmethylidene)-2-indolinone (PMID) confers protection against oxidative stress-induced cell death

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

Yao, Jia-Wei; Beijing Institute of Radiation Medicine, Beijing 100850; Liu, Jing

2012-03-01

The antioxidant response elements (ARE) are a cis-acting enhancer sequence located in regulatory regions of antioxidant and detoxifying genes. Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is a member of the Cap ‘n’ Collar family of transcription factors that binds to the ARE and regulates the transcription of specific ARE-containing genes. Under oxidative stress, Nrf2/ARE induction is fundamental to defense against reactive oxygen species (ROS) and serves as a key factor in the protection against toxic xenobiotics. 3-(3-Pyridylmethylidene)-2-Indolinone (PMID) is a derivative of 2-indolinone compounds which act as protein kinase inhibitors and show anti-tumor activity. However, the role of PMID inmore » the oxidative stress remains unknown. In the present study, we showed that PMID induced the activation of ARE-mediated transcription, increased the DNA-binding activity of Nrf2 and then up-regulated the expression of antioxidant genes such as HO-1, SOD, and NQO1. The level of Nrf2 protein was increased in cells treated with PMID by a post-transcriptional mechanism. Under CHX treatment, the stability of Nrf2 protein was enhanced by PMID with decreased turnover rate. We showed that PMID reduced the ubiquitination of Nrf2 and disrupted the Cullin3 (Cul3)-Keap1 interaction. Furthermore, cells treated with PMID showed resistance to cytotoxicity by H{sub 2}O{sub 2} and pro-oxidant 6-OHDA. PMID also up-regulated the antioxidant level in BALB/c mice. Taken together, the compound PMID induces the ARE-mediated gene expression through stabilization of Nrf2 protein and activation of Nrf2/ARE pathway and protects against oxidative stress-mediated cell death. -- Highlights: ► PMID up-regulates ARE-mediated antioxidant gene expression in vitro and in vivo. ► PMID enhances the stabilization of Nrf2 protein, decreasing Nrf2 turnover rate. ► PMID disrupted the Cullin3 (Cul3)-Keap1 interaction. ► PMID protects against cell death induced by H{sub 2}O{sub 2} and pro-oxidant 6-OHDA.« less

Imidacloprid, a neonicotinoid insecticide, facilitates tyrosine hydroxylase transcription and phenylethanolamine N-methyltransferase mRNA expression to enhance catecholamine synthesis and its nicotine-evoked elevation in PC12D cells.

PubMed

Kawahata, Ichiro; Yamakuni, Tohru

2018-02-01

Imidacloprid is a neonicotinoid insecticide acting as an agonist of nicotinic acetylcholine receptors (nAChRs) in the target insects. However, questions about the safety to mammals, including human have emerged. Overactivation of mammalian peripheral catecholaminergic systems leads to onset of tachycardia, hypertension, vomiting, etc., which have been observed in acutely imidacloprid-poisoned patients as well. Physiological activation of the nAChRs is known to drive catecholamine biosynthesis and secretion in mammalian adrenal chromaffin cells. Yet, the impacts of imidacloprid on the catecholaminergic function of the chromaffin cells remain to be evaluated. In this study using PC12D cells, a catecholaminergic cell line derived from the medulla chromaffin-cell tumors of rat adrenal gland, we examined whether imidacloprid itself could impact the catecholamine-synthesizing ability. Imidacloprid alone did facilitate tyrosine hydroxylase (TH) transcription via activation of α3β4 nAChR and the α7 subunit-comprising receptor. The insecticide showed the TH transcription-facilitating ability at the concentrations of 3 and 30 μM, at which acetylcholine is known to produce physiological responses, including catecholamine secretion through the nAChRs in adrenal chromaffin cells. The insecticide-facilitated TH transcription was also dependent on PKA- and RhoA-mediated signaling pathways. The insecticide coincidentally raised levels of TH and phenylethanolamine N-methyltransferase (PNMT) mRNA, and as a consequence, increased catecholamine production, although the efficacy of the neonicotinoid was lesser than that of nicotine, indicating its partial agonist-like action. Intriguingly, in cultured rat adrenal chromaffin cells, imidacloprid did increase levels of TH and PNMT protein. When the chromaffin cells were treated with nicotine in the presence of the insecticide, nicotine-elevated adrenaline production was enhanced due to facilitation of nicotine-increased TH and PNMT protein expression, and simultaneous enhancement of nicotine-elevated adrenaline secretion also took place. These findings thus suggest that imidacloprid may facilitate the physiological functions of adrenal glands in mammals. Copyright © 2017 Elsevier B.V. All rights reserved.

Network analysis of transcriptomics expands regulatory landscapes in Synechococcus sp. PCC 7002

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

McClure, Ryan S.; Overall, Christopher C.; McDermott, Jason E.

Cyanobacterial regulation of gene expression must contend with a genome organization that lacks apparent functional context, as the majority of cellular processes and metabolic pathways are encoded by genes found at disparate locations across the genome. In addition, the fact that coordinated regulation of cyanobacterial cellular machinery takes place with significantly fewer transcription factors, compared to other Eubacteria, suggests the involvement of post-transcriptional mechanisms and regulatory adaptations which are not fully understood. Global transcript abundance from model cyanobacterium Synechococcus sp. PCC 7002 grown under 42 different conditions was analyzed using context-likelihood of relatedness. The resulting 903-gene network, which was organizedmore » into 11 modules, not only allowed classification of cyanobacterial responses to specific environmental variables but provided insight into the transcriptional network topology and led to the expansion of predicted regulons. When used in conjunction with genome sequence, the global transcript abundance allowed identification of putative post-transcriptional changes in expression as well as novel potential targets of both DNA binding proteins and asRNA regulators. The results offer a new perspective into the multi-level regulation that governs cellular adaptations of fast-growing physiologically robust cyanobacterium Synechococcus sp. PCC 7002 to changing environmental variables. It also extends a methodological knowledge-based framework for studying multi-scale regulatory mechanisms that operate in cyanobacteria. Finally, it provides valuable context for integrating systems-level data to enhance evidence-driven genomic annotation, especially in organisms where traditional context analyses cannot be implemented due to lack of operon-based functional organization.« less

Gene expression of antioxidant enzymes and coffee seed quality during pre- and post-physiological maturity.

PubMed

Santos, F C; Caixeta, F; Clemente, A C S; Pinho, E V; Rosa, S D V F

2014-12-19

Seeds collected at different maturation stages vary in physiological quality and patterns of protective antioxidant systems against deterioration. In this study we investigated the expression of genes that codify catalase (CAT), dismutase superoxide (SOD), and polyphenol oxidase (PPO) during the pre- and post-physiological maturation phases in whole seeds and in endosperms and embryos extracted from the seeds. Coffea arabica L. berries were collected at the green, yellowish-green, cherry, over-ripe, and dry stages, and the seeds were examined physiologically. The transcription levels of the genes were quantified by quantitative real-time polymerase chain reaction using coffee-specific primers. The highest level of SOD expression was observed in the endosperm at the cherry and over-ripe stages; in addition, these seeds presented the greatest physiological quality (assessed via germination test). The highest CAT3 transcript expression was observed at the green stage in whole seeds, and at the green and over-ripe stages in the embryos and endosperms. High expression of the PPO transcript was observed at the green and yellowish-green stages in whole seeds. In embryos and endosperms, peak expression of the PPO transcript was observed at the green stage; subsequently, peaks at the cherry and over-ripe stages were observed. We concluded that the expression patterns of the SOD and CAT3 transcripts were similar at the more advanced maturation stages, which corresponded to enhanced physiological seed quality. High expression of the PPO transcript at the over-ripe stage, also observed in the embryos and endosperms at the cherry stage, coincided with the highest physiological seed quality.

Ectopic Expression of the Wild Grape WRKY Transcription Factor VqWRKY52 in Arabidopsis thaliana Enhances Resistance to the Biotrophic Pathogen Powdery Mildew But Not to the Necrotrophic Pathogen Botrytis cinerea.

PubMed

Wang, Xianhang; Guo, Rongrong; Tu, Mingxing; Wang, Dejun; Guo, Chunlei; Wan, Ran; Li, Zhi; Wang, Xiping

2017-01-01

WRKY transcription factors are known to play important roles in plant responses to biotic stresses. We previously showed that the expression of the WRKY gene, VqWRKY52 , from Chinese wild Vitis quinquangularis was strongly induced 24 h post inoculation with powdery mildew. In this study, we analyzed the expression levels of VqWRKY52 following treatment with the defense related hormones salicylic acid (SA) and methyl jasmonate, revealing that VqWRKY52 was strongly induced by SA but not JA. We characterized the VqWRKY52 gene, which encodes a WRKY III gene family member, and found that ectopic expression in Arabidopsis thaliana enhanced resistance to powdery mildew and Pseudomonas syringae pv. tomato DC3000, but increased susceptibility to Botrytis cinerea , compared with wild type (WT) plants. The transgenic A. thaliana lines displayed strong cell death induced by the biotrophic powdery mildew pathogen, the hemibiotrophic P. syringe pathogen and the necrotrophic pathogen B. cinerea . In addition, the relative expression levels of various defense-related genes were compared between the transgenic A. thaliana lines and WT plants following the infection by different pathogens. Collectively, the results indicated that VqWRKY52 plays essential roles in the SA dependent signal transduction pathway and that it can enhance the hypersensitive response cell death triggered by microbial pathogens.

Low-level expression of human ACAT2 gene in monocytic cells is regulated by the C/EBP transcription factors

PubMed Central

Guo, Dongqing; Lu, Ming; Hu, Xihan; Xu, Jiajia; Hu, Guangjing; Zhu, Ming; Zhang, Xiaowei; Li, Qin; Chang, Catherine C. Y.; Chang, Tayuan; Song, Baoliang; Xiong, Ying; Li, Boliang

2016-01-01

Acyl-coenzyme A:cholesterol acyltransferases (ACATs) are the exclusive intracellular enzymes that catalyze the formation of cholesteryl/steryl esters (CE/SE). In our previous work, we found that the high-level expression of human ACAT2 gene with the CpG hypomethylation of its whole promoter was synergistically regulated by two transcription factors Cdx2 and HNF1α in the intestine and fetal liver. Here, we first observed that the specific CpG-hypomethylated promoter was correlated with the low expression of human ACAT2 gene in monocytic cell line THP-1. Then, two CCAAT/enhancer binding protein (C/EBP) elements within the activation domain in the specific CpG-hypomethylation promoter region were identified, and the expression of ACAT2 in THP-1 cells was evidently decreased when the C/EBP transcription factors were knock-downed using RNAi technology. Furthermore, ChIP assay confirmed that C/EBPs directly bind to their elements for low-level expression of human ACAT2 gene in THP-1 cells. Significantly, the increased expressions of ACAT2 and C/EBPs were also found in macrophages differentiated from both ATRA-treated THP-1 cells and cultured human blood monocytes. These results demonstrate that the low-level expression of human ACAT2 gene with specific CpG-hypomethylated promoter is regulated by the C/EBP transcription factors in monocytic cells, and imply that the lowly expressed ACAT2 catalyzes the synthesis of certain CE/SE that are assembled into lipoproteins for the secretion. PMID:27688151

Role of crustacean hyperglycemic hormone (CHH) in the environmental stressor-exposed intertidal copepod Tigriopus japonicus.

PubMed

Kim, Bo-Mi; Jeong, Chang-Bum; Han, Jeonghoon; Kim, Il-Chan; Rhee, Jae-Sung; Lee, Jae-Seong

2013-09-01

To identify and characterize CHH (TJ-CHH) gene in the copepod Tigriopus japonicus, we analyzed the full-length cDNA sequence, genomic structure, and promoter region. The full-length TJ-CHH cDNA was 716 bp in length, encoding 136 amino acid residues. The deduced amino acid sequences of TJ-CHH showed a high similarity of the CHH mature domain to other crustaceans. Six conserved cysteine residues and five conserved structural motifs in the CHH mature peptide domain were also observed. The genomic structure of the TJ-CHH gene contained three exons and two introns in its open reading frame (ORF), and several transcriptional elements were detected in the promoter region of the TJ-CHH gene. To investigate transcriptional change of TJ-CHH under environmental stress, T. japonicus were exposed to heat treatment, UV-B radiation, heavy metals, and water-accommodated fractions (WAFs) of Iranian crude oil. Upon heat stress, TJ-CHH transcripts were elevated at 30 °C and 35 °C for 96 h in a time-course experiment. UV-B radiation led to a decreased pattern of the TJ-CHH transcript 48 h and more after radiation (12 kJ/m(2)). After exposure of a fixed dose (12 kJ/m(2)) in a time-course experiment, TJ-CHH transcript was down-regulated in time-dependent manner with a lowest value at 12h. However, the TJ-CHH transcript level was increased in response to five heavy metal exposures for 96 h. Also, the level of the TJ-CHH transcript was significantly up-regulated at 20% of WAFs after exposure to WAFs for 48 h and then remarkably reduced in a dose-dependent manner. These findings suggest that the enhanced TJ-CHH transcript level is associated with a cellular stress response of the TJ-CHH gene as shown in decapod crustaceans. This study is also helpful for a better understanding of the detrimental effects of environmental changes on the CHH-triggered copepod metabolism. Copyright © 2013 Elsevier Inc. All rights reserved.

Bovine Foamy Virus Transactivator BTas Interacts with Cellular RelB To Enhance Viral Transcription▿

PubMed Central

Wang, Jian; Tan, Juan; Guo, Hongyan; Zhang, Qicheng; Jia, Rui; Xu, Xuan; Geng, Yunqi; Qiao, Wentao

2010-01-01

Viruses are obligate intracellular parasites that depend on cellular machinery for their efficient transcription and replication. In a previous study we reported that bovine foamy virus (BFV) is able to activate the nuclear factor κB (NF-κB) pathway through the action of its transactivator BTas to enhance viral transcription. However, the mechanism used by NF-κB to enhance BFV transcription remains elusive. To address this question, we employed a yeast two-hybrid assay to screen for BTas-interacting proteins. We found that RelB, a member of NF-κB protein family, interacts with BTas. We confirmed the putative RelB-BTas interaction in vitro and in vivo and identified the protein regions responsible for the RelB-BTas interaction. Using a luciferase reporter assay, we next showed that RelB enhances BFV transcription (BTas-induced long terminal repeat [LTR] transactivation) and that this process requires both the localization of the RelB-BTas interaction in the nucleus and the Rel homology domain of RelB. The knockdown of the cellular endogenous RelB protein using small interfering RNA (siRNA) significantly attenuated BTas-induced LTR transcription. The results of chromatin immunoprecipitation (ChIP) analysis showed that endogenous RelB binds to the viral LTR in BFV-infected cells. Together, these results suggest that BFV engages the RelB protein as a cotransactivator of BTas to enhance viral transcription. In addition, our findings indicate that BFV infection upregulates cellular RelB expression through BTas-induced NF-κB activation. Thus, this study demonstrates the existence of a positive-feedback circuit in which BFV utilizes the host's NF-κB pathway through the RelB protein for efficient viral transcription. PMID:20844054

FBI-1 enhances transcription of the nuclear factor-kappaB (NF-kappaB)-responsive E-selectin gene by nuclear localization of the p65 subunit of NF-kappaB.

PubMed

Lee, Dong-Kee; Kang, Jae-Eun; Park, Hye-Jin; Kim, Myung-Hwa; Yim, Tae-Hee; Kim, Jung-Min; Heo, Min-Kyu; Kim, Kyu-Yeun; Kwon, Ho Jeong; Hur, Man-Wook

2005-07-29

The POZ domain is a highly conserved protein-protein interaction motif found in many regulatory proteins. Nuclear factor-kappaB (NF-kappaB) plays a key role in the expression of a variety of genes in response to infection, inflammation, and stressful conditions. We found that the POZ domain of FBI-1 (factor that binds to the inducer of short transcripts of human immunodeficiency virus-1) interacted with the Rel homology domain of the p65 subunit of NF-kappaB in both in vivo and in vitro protein-protein interaction assays. FBI-1 enhanced NF-kappaB-mediated transcription of E-selectin genes in HeLa cells upon phorbol 12-myristate 13-acetate stimulation and overcame gene repression by IkappaB alpha or IkappaB beta. In contrast, the POZ domain of FBI-1, which is a dominant-negative form of FBI-1, repressed NF-kappaB-mediated transcription, and the repression was cooperative with IkappaB alpha or IkappaB beta. In contrast, the POZ domain tagged with a nuclear localization sequence polypeptide of FBI-1 enhanced NF-kappaB-responsive gene transcription, suggesting that the molecular interaction between the POZ domain and the Rel homology domain of p65 and the nuclear localization by the nuclear localization sequence are important in the transcription enhancement mediated by FBI-1. Confocal microscopy showed that FBI-1 increased NF-kappaB movement into the nucleus and increased the stability of NF-kappaB in the nucleus, which enhanced NF-kappaB-mediated transcription of the E-selectin gene. FBI-1 also interacted with IkappaB alpha and IkappaB beta.

HPV integration hijacks and multimerizes a cellular enhancer to generate a viral-cellular super-enhancer that drives high viral oncogene expression

PubMed Central

Redmond, Catherine J.; Dooley, Katharine E.; Fu, Haiqing; Gillison, Maura L.; Akagi, Keiko; Symer, David E.; Aladjem, Mirit I.

2018-01-01

Integration of human papillomavirus (HPV) genomes into cellular chromatin is common in HPV-associated cancers. Integration is random, and each site is unique depending on how and where the virus integrates. We recently showed that tandemly integrated HPV16 could result in the formation of a super-enhancer-like element that drives transcription of the viral oncogenes. Here, we characterize the chromatin landscape and genomic architecture of this integration locus to elucidate the mechanisms that promoted de novo super-enhancer formation. Using next-generation sequencing and molecular combing/fiber-FISH, we show that ~26 copies of HPV16 are integrated into an intergenic region of chromosome 2p23.2, interspersed with 25 kb of amplified, flanking cellular DNA. This interspersed, co-amplified viral-host pattern is frequent in HPV-associated cancers and here we designate it as Type III integration. An abundant viral-cellular fusion transcript encoding the viral E6/E7 oncogenes is expressed from the integration locus and the chromatin encompassing both the viral enhancer and a region in the adjacent amplified cellular sequences is strongly enriched in the super-enhancer markers H3K27ac and Brd4. Notably, the peak in the amplified cellular sequence corresponds to an epithelial-cell-type specific enhancer. Thus, HPV16 integration generated a super-enhancer-like element composed of tandem interspersed copies of the viral upstream regulatory region and a cellular enhancer, to drive high levels of oncogene expression. PMID:29364907

Transvection Goes Live-Visualizing Enhancer-Promoter Communication between Chromosomes.

PubMed

Tsai, Albert; Singer, Robert H; Crocker, Justin

2018-04-19

Lim et al. (2018) use live imaging in Drosophila embryos to show that enhancers can drive transcription from promoters on another chromosome when they are in close proximity. In addition, they show that multiple promoters can access the same enhancer without competition, potentially sharing a pool of factors in a transcriptional "hub." Copyright © 2018 Elsevier Inc. All rights reserved.

Longitudinal evaluation of leukocyte transcripts in killer whales (Orcinus Orca)

USGS Publications Warehouse

Sitt, Tatjana; Bowen, Lizabeth; Lee, Chia-Shan; Blanchard, Myra; McBain, James; Dold, Christopher; Stott, Jeffrey L.

2016-01-01

Early identification of illness and/or presence of environmental and/or social stressors in free-ranging and domestic cetaceans is a priority for marine mammal health care professionals. Incorporation of leukocyte gene transcript analysis into the diagnostic tool kit has the potential to augment classical diagnostics based upon ease of sample storage and shipment, inducible nature and well-defined roles of transcription and associated downstream actions. Development of biomarkers that could serve to identify “insults” and potentially differentiate disease etiology would be of great diagnostic value. To this end, a modest number of peripheral blood leukocyte gene transcripts were selected for application to a domestic killer whale population with a focus on broad representation of inducible immunologically relevant genes. Normalized leukocyte transcript values, longitudinally acquired from 232 blood samples derived from 26 clinically healthy whales, were not visibly influenced temporally nor by sex or the specific Park in which they resided. Stability in leukocyte transcript number during periods of health enhances their potential use in diagnostics through identification of outliers. Transcript levels of two cytokine genes, IL-4 and IL-17, were highly variable within the group as compared to the other transcripts. IL-4 transcripts were typically absent. Analysis of transcript levels on the other genes of interest, on an individual animal basis, identified more outliers than were visible when analyzed in the context of the entire population. The majority of outliers (9 samples) were low, though elevated transcripts were identified for IL-17 from 2 animals and one each for Cox-2 and IL-10. The low number of outliers was not unexpected as sample selection was intentionally directed towards animals that were clinically healthy at the time of collection. Outliers may reflect animals experiencing subclinical disease that is transient and self-limiting. The immunologic knowledge derived from longitudinal immunologic studies in killer whales, as was the target of the present study, has the potential to improve diagnostics and health related decision making for this and other domestic and free-ranging cetacean species.

Longitudinal evaluation of leukocyte transcripts in killer whales (Orcinus Orca).

PubMed

Sitt, Tatjana; Bowen, Lizabeth; Lee, Chia-Shan; Blanchard, Myra T; McBain, James; Dold, Christopher; Stott, Jeffrey L

2016-07-01

Early identification of illness and/or presence of environmental and/or social stressors in free-ranging and domestic cetaceans is a priority for marine mammal health care professionals. Incorporation of leukocyte gene transcript analysis into the diagnostic tool kit has the potential to augment classical diagnostics based upon ease of sample storage and shipment, inducible nature and well-defined roles of transcription and associated downstream actions. Development of biomarkers that could serve to identify "insults" and potentially differentiate disease etiology would be of great diagnostic value. To this end, a modest number of peripheral blood leukocyte gene transcripts were selected for application to a domestic killer whale population with a focus on broad representation of inducible immunologically relevant genes. Normalized leukocyte transcript values, longitudinally acquired from 232 blood samples derived from 26 clinically healthy whales, were not visibly influenced temporally nor by sex or the specific Park in which they resided. Stability in leukocyte transcript number during periods of health enhances their potential use in diagnostics through identification of outliers. Transcript levels of two cytokine genes, IL-4 and IL-17, were highly variable within the group as compared to the other transcripts. IL-4 transcripts were typically absent. Analysis of transcript levels on the other genes of interest, on an individual animal basis, identified more outliers than were visible when analyzed in the context of the entire population. The majority of outliers (9 samples) were low, though elevated transcripts were identified for IL-17 from 2 animals and one each for Cox-2 and IL-10. The low number of outliers was not unexpected as sample selection was intentionally directed towards animals that were clinically healthy at the time of collection. Outliers may reflect animals experiencing subclinical disease that is transient and self-limiting. The immunologic knowledge derived from longitudinal immunologic studies in killer whales, as was the target of the present study, has the potential to improve diagnostics and health related decision making for this and other domestic and free-ranging cetacean species. Copyright © 2016. Published by Elsevier B.V.

Morin, a plant derived flavonoid, modulates the expression of peroxisome proliferator-activated receptor-γ coactivator-1α mediated by AMPK pathway in hepatic stellate cells

PubMed Central

Yuan, Wei; Ahmad, Shoaib; Najar, Ajaz

2017-01-01

Morin exerts inhibitory effects on hepatic stellate cell (HSC) stimulation which is considered important step for fibrogenesis in liver. These morin-induced inhibitory effects are mediated through enhancement in the expression levels of peroxisome proliferator-activated receptor-γ (PPARγ). PPARγ plays a critical role in inhibition of HSC stimulation. Peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) acts as a co-activator for PPARγ. Hence, studies directed at examining the influence of morin on PGC-1α may help to understand the mechanisms behind the morin induced suppression of HSC stimulation and liver fibrosis via PPARγ. The current research was therefore designed to examine the effect of morin on the expression levels of PGC-1α in HSCs under in vitro conditions and to attempt to investigate the involved potential mechanisms by western blotting, RT-PCR, and transfection assays. The results revealed that morin increased the expression of PGC-1α and the effects of morin on the expression of PGC-1α were positively associated with the stimulation of adenosine monophosphate-activated protein kinase (AMPK). Additionally, morin enhanced superoxide dimutase-2 (SOD-2) transcript levels as well as the activity via AMPK/PGC-1α axis. Furthermore, PGC-1α was found to suppress α1 (I) collagen transcript levels in HSCs. Taken together, these results revealed that the effect of morin on the enhancement of the expression of PGC-1α is mediated through AMPK pathway which ultimately leads to increase in the activity of PPARγ and SOD-2. PMID:29312518

CCAAT-enhancer-binding protein β (C/EBPβ) and downstream human placental growth hormone genes are targets for dysregulation in pregnancies complicated by maternal obesity.

PubMed

Vakili, Hana; Jin, Yan; Menticoglou, Savas; Cattini, Peter A

2013-08-02

Human chorionic somatomammotropin (CS) and placental growth hormone variant (GH-V) act as metabolic adaptors in response to maternal insulin resistance, which occurs in "normal" pregnancy. Maternal obesity can exacerbate this "resistance," suggesting that CS, GH-V, or transcription factors that regulate their production might be targets. The human CS genes, hCS-A and hCS-B, flank the GH-V gene. A significant decrease in pre-term placental CS/GH-V RNA levels was observed in transgenic mice containing the CS/GH-V genes in a model of high fat diet (HFD)-induced maternal obesity. Similarly, a decrease in CS/GH-V RNA levels was detected in term placentas from obese (body mass index (BMI) ≥ 35 kg/m(2)) versus lean (BMI 20-25 kg/m(2)) women. A specific decrease in transcription factor CCAAT-enhancer-binding protein β (C/EBPβ) RNA levels was also seen with obesity; C/EBPβ is required for mouse placenta development and is expressed, like CS and GH-V, in syncytiotrophoblasts. Binding of C/EBPβ to the CS gene downstream enhancer regions, which by virtue of their position distally flank the GH-V gene, was reduced in placenta chromatin from mice on a HFD and in obese women; a corresponding decrease in RNA polymerase II associated with CS/GH-V promoters was also observed. Detection of decreased endogenous CS/GH-V RNA levels in human placental tumor cells treated with C/EBPβ siRNA is consistent with a direct effect. These data provide evidence for CS/GH-V dysregulation in acute HFD-induced obesity in mouse pregnancy and chronic obesity in human pregnancy and implicate C/EBPβ, a factor associated with CS regulation and placental development.

A dual switch controls bacterial enhancer-dependent transcription

PubMed Central

Wiesler, Simone C.; Burrows, Patricia C.; Buck, Martin

2012-01-01

Bacterial RNA polymerases (RNAPs) are targets for antibiotics. Myxopyronin binds to the RNAP switch regions to block structural rearrangements needed for formation of open promoter complexes. Bacterial RNAPs containing the major variant σ54 factor are activated by enhancer-binding proteins (bEBPs) and transcribe genes whose products are needed in pathogenicity and stress responses. We show that (i) enhancer-dependent RNAPs help Escherichia coli to survive in the presence of myxopyronin, (ii) enhancer-dependent RNAPs partially resist inhibition by myxopyronin and (iii) ATP hydrolysis catalysed by bEBPs is obligatory for functional interaction of the RNAP switch regions with the transcription start site. We demonstrate that enhancer-dependent promoters contain two barriers to full DNA opening, allowing tight regulation of transcription initiation. bEBPs engage in a dual switch to (i) allow propagation of nucleated DNA melting from an upstream DNA fork junction and (ii) complete the formation of the transcription bubble and downstream DNA fork junction at the RNA synthesis start site, resulting in switch region-dependent RNAP clamp closure and open promoter complex formation. PMID:22965125

MED12 Regulates HSC-Specific Enhancers Independently of Mediator Kinase Activity to Control Hematopoiesis.

PubMed

Aranda-Orgilles, Beatriz; Saldaña-Meyer, Ricardo; Wang, Eric; Trompouki, Eirini; Fassl, Anne; Lau, Stephanie; Mullenders, Jasper; Rocha, Pedro P; Raviram, Ramya; Guillamot, María; Sánchez-Díaz, María; Wang, Kun; Kayembe, Clarisse; Zhang, Nan; Amoasii, Leonela; Choudhuri, Avik; Skok, Jane A; Schober, Markus; Reinberg, Danny; Sicinski, Piotr; Schrewe, Heinrich; Tsirigos, Aristotelis; Zon, Leonard I; Aifantis, Iannis

2016-12-01

Hematopoietic-specific transcription factors require coactivators to communicate with the general transcription machinery and establish transcriptional programs that maintain hematopoietic stem cell (HSC) self-renewal, promote differentiation, and prevent malignant transformation. Mediator is a large coactivator complex that bridges enhancer-localized transcription factors with promoters, but little is known about Mediator function in adult stem cell self-renewal and differentiation. We show that MED12, a member of the Mediator kinase module, is an essential regulator of HSC homeostasis, as in vivo deletion of Med12 causes rapid bone marrow aplasia leading to acute lethality. Deleting other members of the Mediator kinase module does not affect HSC function, suggesting kinase-independent roles of MED12. MED12 deletion destabilizes P300 binding at lineage-specific enhancers, resulting in H3K27Ac depletion, enhancer de-activation, and consequent loss of HSC stemness signatures. As MED12 mutations have been described recently in blood malignancies, alterations in MED12-dependent enhancer regulation may control both physiological and malignant hematopoiesis. Copyright © 2016 Elsevier Inc. All rights reserved.

Deciphering the combinatorial architecture of a Drosophila homeotic gene enhancer

PubMed Central

Drewell, Robert A.; Nevarez, Michael J.; Kurata, Jessica S.; Winkler, Lauren N.; Li, Lily; Dresch, Jacqueline M.

2013-01-01

Summary In Drosophila, the 330 kb bithorax complex regulates cellular differentiation along the anterio-posterior axis during development in the thorax and abdomen and is comprised of three homeotic genes: Ultrabithorax, abdominal-A, and Abdominal-B. The expression of each of these genes is in turn controlled through interactions between transcription factors and a number of cis-regulatory modules in the neighboring intergenic regions. In this study, we examine how the sequence architecture of transcription factor binding sites mediates the functional activity of one of these cis-regulatory modules. Using computational, mathematical modeling and experimental molecular genetic approaches we investigate the IAB7b enhancer, which regulates Abdominal-B expression specifically in the presumptive seventh and ninth abdominal segments of the early embryo. A cross-species comparison of the IAB7b enhancer reveals an evolutionarily conserved signature motif containing two FUSHI-TARAZU activator transcription factor binding sites. We find that the transcriptional repressors KNIRPS, KRUPPEL and GIANT are able to restrict reporter gene expression to the posterior abdominal segments, using different molecular mechanisms including short-range repression and competitive binding. Additionally, we show the functional importance of the spacing between the two FUSHI-TARAZU binding sites and discuss the potential importance of cooperativity for transcriptional activation. Our results demonstrate that the transcriptional output of the IAB7b cis-regulatory module relies on a complex set of combinatorial inputs mediated by specific transcription factor binding and that the sequence architecture at this enhancer is critical to maintain robust regulatory function. PMID:24514265

Mode of action of the immunostimulatory effect of collagen from jellyfish.

PubMed

Nishimoto, Sogo; Goto, Yoko; Morishige, Hitoshi; Shiraishi, Ryusuke; Doi, Mikiharu; Akiyama, Koichi; Yamauchi, Satoshi; Sugahara, Takuya

2008-11-01

We have previously demonstrated that collagen from jellyfish simulated immunoglobulin and cytokine production by human-human hybridoma line HB4C5 cells and by human peripheral blood lymphocytes (hPBL). The mode of action of the collagen as an immunostimulatory factor was investigated. The expression levels of immunoglobulin mRNAs in HB4C5 cells, and those of tumor necrosis factor (TNF)-alpha, interferon (IFN)-gamma, and transforming growth factor (TGF)-beta in hPBL were up-regulated by jellyfish collagen. In addition, this collagen activated IgM production by transcription-suppressed HB4C5 cells that had been treated with actinomycin D. This collagen also enhanced IgM production by translation-suppressed HB4C5 cells that had been treated with sodium fluoride, but was ineffective in accelerating IgM production by HB4C5 cells treated with cycloheximide. Moreover, the intracellular IgM level in HB4C5 cells treated with the post-translation inhibitor, monensin, was increased by this collagen. These results suggest that collagen from jellyfish stimulated not only the transcription activity, but also the translation activity for enhanced immunoglobulin and cytokine production.

Requirement of Multiple cis-Acting Elements in the Human Cytomegalovirus Major Immediate-Early Distal Enhancer for Viral Gene Expression and Replication

PubMed Central

Meier, Jeffery L.; Keller, Michael J.; McCoy, James J.

2002-01-01

We have shown previously that the human cytomegalovirus (HCMV) major immediate-early (MIE) distal enhancer is needed for MIE promoter-dependent transcription and viral replication at low multiplicities of infection (MOI). To understand how this region works, we constructed and analyzed a series of HCMVs with various distal enhancer mutations. We show that the distal enhancer is composed of at least two parts that function independently to coordinately activate MIE promoter-dependent transcription and viral replication. One such part is contained in a 47-bp segment that has consensus binding sites for CREB/ATF, SP1, and YY1. At low MOI, these working parts likely function in cis to directly activate MIE gene expression, thus allowing viral replication to ensue. Three findings support the view that these working parts are likely cis-acting elements. (i) Deletion of either part of a bisegmented distal enhancer only slightly alters MIE gene transcription and viral replication. (ii) Reversing the distal enhancer’s orientation largely preserves MIE gene transcription and viral replication. (iii) Placement of stop codons at −300 or −345 in all reading frames does not impair MIE gene transcription and viral replication. Lastly, we show that these working parts are dispensable at high MOI, partly because of compensatory stimulation of MIE promoter activity and viral replication that is induced by a virion-associated component(s) present at a high viral particle/cell ratio. We conclude that the distal enhancer is a complex multicomponent cis-acting region that is required to augment both MIE promoter-dependent transcription and HCMV replication. PMID:11739696

Thyroid receptor β involvement in the effects of acute nicotine on hippocampus-dependent memory.

PubMed

Leach, Prescott T; Kenney, Justin W; Connor, David A; Gould, Thomas J

2015-06-01

Cigarette smoking is common despite adverse health effects. Nicotine's effects on learning may contribute to addiction by enhancing drug-context associations. Effects of nicotine on learning could be direct or could occur by altering systems that modulate cognition. Because thyroid signaling can alter cognition and nicotine/smoking may change thyroid function, nicotine could affect learning through changes in thyroid signaling. These studies investigate the functional contributions of thyroid receptor (TR) subtypes β and α1 to nicotine-enhanced learning and characterize the effects of acute nicotine and learning on thyroid hormone levels. We conducted a high throughput screen of transcription factor activity to identify novel targets that may contribute to the effects of nicotine on learning. Based on these results, which showed that combined nicotine and learning uniquely acted to increase TR activation, we identified TRs as potential targets of nicotine. Further analyses were conducted to determine the individual and combined effects of nicotine and learning on thyroid hormone levels, but no changes were seen. Next, to determine the role of TRβ and TRα1 in the effects of nicotine on learning, mice lacking the TRβ or TRα1 gene and wildtype littermates were administered acute nicotine prior to fear conditioning. Nicotine enhanced contextual fear conditioning in TRα1 knockout mice and wildtypes from both lines but TRβ knockout mice did not show nicotine-enhanced learning. This finding supports involvement of TRβ signaling in the effect of acute nicotine on hippocampus-dependent memory. Acute nicotine enhances learning and these effects may involve processes regulated by the transcription factor TRβ. Copyright © 2015 Elsevier Ltd. All rights reserved.

Ononitol monohydrate enhances PRDM16 & UCP-1 expression, mitochondrial biogenesis and insulin sensitivity via STAT6 and LTB4R in maturing adipocytes.

PubMed

Subash-Babu, P; Alshatwi, Ali A

2018-03-01

Ononitol monohydrate (OMH), a glycoside was originally isolated from Cassia tora (Linn.). Glycosides regulate lipid metabolism but scientific validation desired. Hence, we aimed to evaluate the effect of OMH on enhancing mitochondrial potential, mitochondrial biogenesis, upregulate the expression of brown adipogenesis specific genes in maturing adipocytes. In addition, we observed the inter-relation between adipocyte and T-lymphocyte; whether, OMH treated adipocyte-condition medium stimulate T-cell chemokine linked with insulin resistance. In a dose dependent manner OMH treated to preadipocyte significantly inhibited maturation and enhanced mitochondrial biogenesis, it was confirmed by Oil red 'O and Nile red stain without inducing cytotoxicity. The mRNA levels of adipocyte browning related genes such as, PR domain containing 16 (PRDM16), peroxisome proliferator activated receptor gamma coactivator 1 alpha (PPARγC1α) and uncoupling protein-1 (UCP-1) have been significantly upregulated. In addition, adipogenic transcription factors [such as proliferator activated receptor γ (PPARγ), CCAAT/enhancer binding protein (C/EBPα) and sterol regulatory element binding protein-1c (SREBP-1c)] and adipogenic genes were significantly down-regulated by treatment with OMH when compared to control cells. Protein expression levels of adiponectin have been increased; leptin, C/EBPα and leukotriene B4 receptor (LTB4R) were down regulated by OMH in mature adipocytes. In addition, adipocyte condition medium and OMH treated T-lymphocyte, significantly increased insulin signaling pathway related mRNAs, such as interlukin-4 (IL-4), signal transducer and activator of transcription 6 (STAT 6 ) and decreased leukotriene B4 (LTB 4 ). The present findings suggest that OMH increased browning factors in differentiating and maturing preadipocyte also decreased adipose tissue inflammation as well as the enhanced insulin signaling. Copyright © 2018. Published by Elsevier Masson SAS.

Metabolic evolution of Corynebacterium glutamicum for increased production of L-ornithine

PubMed Central

2013-01-01

Background L-ornithine is effective in the treatment of liver diseases and helps strengthen the heart. The commercial applications mean that efficient biotechnological production of L-ornithine has become increasingly necessary. Adaptive evolution strategies have been proven a feasible and efficient technique to achieve improved cellular properties without requiring metabolic or regulatory details of the strain. The evolved strains can be further optimised by metabolic engineering. Thus, metabolic evolution strategy was used for engineering Corynebacterium glutamicum to enhance L-ornithine production. Results A C. glutamicum strain was engineered by using a combination of gene deletions and adaptive evolution with 70 passages of growth-based selection. The metabolically evolved C. glutamicum strain, named ΔAPE6937R42, produced 24.1 g/L of L-ornithine in a 5-L bioreactor. The mechanism used by C. glutamicum ΔAPE6937R42 to produce L-ornithine was investigated by analysing transcriptional levels of select genes and NADPH contents. The upregulation of the transcription levels of genes involved in the upstream pathway of glutamate biosynthesis and the elevated NADPH concentration caused by the upregulation of the transcriptional level of the ppnK gene promoted L-ornithine production in C. glutamicum ΔAPE6937R42. Conclusions The availability of NADPH plays an important role in L-ornithine production in C. glutamicum. Our results demonstrated that the combination of growth-coupled evolution with analysis of transcript abundances provides a strategy to engineer microbial strains for improving production of target compounds. PMID:23725060

Specification of skeletal muscle differentiation by repressor element-1 silencing transcription factor (REST)-regulated Kv7.4 potassium channels

PubMed Central

Iannotti, Fabio Arturo; Barrese, Vincenzo; Formisano, Luigi; Miceli, Francesco; Taglialatela, Maurizio

2013-01-01

Changes in the expression of potassium (K+) channels is a pivotal event during skeletal muscle differentiation. In mouse C2C12 cells, similarly to human skeletal muscle cells, myotube formation increased the expression of Kv7.1, Kv7.3, and Kv7.4, the last showing the highest degree of regulation. In C2C12 cells, Kv7.4 silencing by RNA interference reduced the expression levels of differentiation markers (myogenin, myosin heavy chain, troponinT-1, and Pax3) and impaired myotube formation and multinucleation. In Kv7.4-silenced cells, the differentiation-promoting effect of the Kv7 activator N-(2-amino-4-(4-fluorobenzylamino)-phenyl)-carbamic acid ethyl ester (retigabine) was abrogated. Expression levels for the repressor element-1 silencing transcription factor (REST) declined during myotube formation. Transcript levels for Kv7.4, as well as for myogenin, troponinT-1, and Pax3, were reduced by REST overexpression and enhanced upon REST suppression by RNA interference. Four regions containing potential REST-binding sites in the 5′ untranslated region and in the first intron of the Kv7.4 gene were identified by bioinformatic analysis. Chromatin immunoprecipitation assays showed that REST binds to these regions, exhibiting a higher efficiency in myoblasts than in myotubes. These data suggest that Kv7.4 plays a permissive role in skeletal muscle differentiation and highlight REST as a crucial transcriptional regulator for this K+ channel subunit. PMID:23242999

Sleep is not just for the brain: transcriptional responses to sleep in peripheral tissues.

PubMed

Anafi, Ron C; Pellegrino, Renata; Shockley, Keith R; Romer, Micah; Tufik, Sergio; Pack, Allan I

2013-05-30

Many have assumed that the primary function of sleep is for the brain. We evaluated the molecular consequences of sleep and sleep deprivation outside the brain, in heart and lung. Using microarrays we compared gene expression in tissue from sleeping and sleep deprived mice euthanized at the same diurnal times. In each tissue, nearly two thousand genes demonstrated statistically significant differential expression as a function of sleep/wake behavioral state. To mitigate the influence of an artificial deprivation protocol, we identified a subset of these transcripts as specifically sleep-enhanced or sleep-repressed by requiring that their expression also change over the course of unperturbed sleep. 3% and 6% of the assayed transcripts showed "sleep specific" changes in the lung and heart respectively. Sleep specific transcripts in these tissues demonstrated highly significant overlap and shared temporal dynamics. Markers of cellular stress and the unfolded protein response were reduced during sleep in both tissues. These results mirror previous findings in brain. Sleep-enhanced pathways reflected the unique metabolic functions of each tissue. Transcripts related to carbohydrate and sulfur metabolic processes were enhanced by sleep in the lung, and collectively favor buffering from oxidative stress. DNA repair and protein metabolism annotations were significantly enriched among the sleep-enhanced transcripts in the heart. Our results also suggest that sleep may provide a Zeitgeber, or synchronizing cue, in the lung as a large cluster of transcripts demonstrated systematic changes in inter-animal variability as a function of both sleep duration and circadian time. Our data support the notion that the molecular consequences of sleep/wake behavioral state extend beyond the brain to include peripheral tissues. Sleep state induces a highly overlapping response in both heart and lung. We conclude that sleep enhances organ specific molecular functions and that it has a ubiquitous role in reducing cellular metabolic stress in both brain and peripheral tissues. Finally, our data suggest a novel role for sleep in synchronizing transcription in peripheral tissues.

Mediator Undergoes a Compositional Change during Transcriptional Activation.

PubMed

Petrenko, Natalia; Jin, Yi; Wong, Koon Ho; Struhl, Kevin

2016-11-03

Mediator is a transcriptional co-activator recruited to enhancers by DNA-binding activators, and it also interacts with RNA polymerase (Pol) II as part of the preinitiation complex (PIC). We demonstrate that a single Mediator complex associates with the enhancer and core promoter in vivo, indicating that it can physically bridge these transcriptional elements. However, the Mediator kinase module associates strongly with the enhancer, but not with the core promoter, and it dissociates from the enhancer upon depletion of the TFIIH kinase. Severing the kinase module from Mediator by removing the connecting subunit Med13 does not affect Mediator association at the core promoter but increases occupancy at enhancers. Thus, Mediator undergoes a compositional change in which the kinase module, recruited via Mediator to the enhancer, dissociates from Mediator to permit association with Pol II and the PIC. As such, Mediator acts as a dynamic bridge between the enhancer and core promoter. Copyright © 2016 Elsevier Inc. All rights reserved.

Silencing of ATF2 inhibits growth of pancreatic cancer cells and enhances sensitivity to chemotherapy.

PubMed

Li, Mu; Wu, Xingda; Liu, Ning; Li, Xiaoying; Meng, Fanbin; Song, Shaowei

2017-06-01

Pancreatic cancer is one of the leading causes of cancer-related death worldwide. Activating transcription factor 2 (ATF2) is a multifunctional transcription factor, and is implicated in tumor progress, yet its role in pancreatic cancer remains unclear. In the present study, the level of ATF2 in pancreatic cancer tissues and the adjacent non-tumorous tissues was detected by quantitative real-time PCR and Western blot. The roles of ATF2 in the proliferation, cell cycle, and apoptosis of pancreatic cancer cells were investigated through ATF2 silencing, and the effect of ATF2 shRNA on the sensitivity of pancreatic cancer cells to gemcitabine, an anti-tumor drug, was explored. The results of our study showed that the ATF2 level in the pancreatic cancer tissues was higher than that in the adjacent non-tumorous tissues. Silencing of ATF2 was found to inhibit proliferation, arrest cell cycle at G1 phase and induce apoptosis in pancreatic cancer cells. Moreover, ATF2 silencing enhanced gemcitabine-induced growth-inhibition and apoptosis-induction effects in pancreatic cancer cells. In summary, silencing of ATF2 inhibited the growth of pancreatic cancer cells and enhanced the anti-tumor effects of gemcitabine, suggesting that ATF2 plays a pro-survival role in pancreatic cancer. Our results also propose that a high level of ATF2 may serve as a potential biomarker of pancreatic cancer, and that ATF2 may become a potential target for anti-tumor therapy. © 2017 International Federation for Cell Biology.

MicroRNA-30b-Mediated Regulation of Catalase Expression in Human ARPE-19 Cells

PubMed Central

Haque, Rashidul; Chun, Eugene; Howell, Jennifer C.; Sengupta, Trisha; Chen, Dan; Kim, Hana

2012-01-01

Background Oxidative injury to retinal pigment epithelium (RPE) and retinal photoreceptors has been linked to a number of retinal diseases, including age-related macular degeneration (AMD). Reactive oxygen species (ROS)-mediated gene expression has been extensively studied at transcriptional levels. Also, the post-transcriptional control of gene expression at the level of translational regulation has been recently reported. However, the microRNA (miRNA/miR)-mediated post-transcriptional regulation in human RPE cells has not been thoroughly looked at. Increasing evidence points to a potential role of miRNAs in diverse physiological processes. Methodology/Principal Findings We demonstrated for the first time in a human retinal pigment epithelial cell line (ARPE-19) that the post-transcriptional control of gene expression via miRNA modulation regulates human catalase, an important and potent component of cell's antioxidant defensive network, which detoxifies hydrogen peroxide (H2O2) radicals. Exposure to several stress-inducing agents including H2O2 has been reported to alter miRNA expression profile. Here, we demonstrated that a sublethal dose of H2O2 (200 µM) up-regulated the expression of miR-30b, a member of the miR-30 family, which inhibited the expression of endogenous catalase both at the transcript and protein levels. However, antisense (antagomirs) of miR-30b was not only found to suppress the miR-30b mimics-mediated inhibitions, but also to dramatically increase the expression of catalase even under an oxidant environment. Conclusions/Significance We propose that a microRNA antisense approach could enhance cytoprotective mechanisms against oxidative stress by increasing the antioxidant defense system. PMID:22880027

Liver Transcriptome Changes in Zebrafish during Acclimation to Transport-Associated Stress

PubMed Central

Dhanasiri, Anusha K. S.; Fernandes, Jorge M. O.; Kiron, Viswanath

2013-01-01

Liver plays a key role during the stress acclimation, and liver transcriptome analysis of shipped zebrafish could reveal the molecular adjustments that occur in the organ. Transcriptional changes in liver were analyzed with a 44 K oligo array using total RNA from fish prior to transport and during a mock transport process - immediately after packing (0 h), at 48 and 72 h. Large numbers of genes related to a variety of biological processes and pathways were regulated, mainly during transport (at 48/72 h). Immediately after packing, transcripts of genes related to both gluconeogenesis and glycolysis were induced. During transport, induction of gluconeogenesis-linked genes and reduction of glycolysis-related genes may be supporting the increase in blood glucose levels. Inhibition of genes involved in fatty acid beta-oxidation may be pointing to the poor ability of fish to utilize energy from fatty acids, under transport conditions. Genes involved in some of the mechanisms that regulate body ammonia were also affected. Even though genes associated with certain transaminases were inhibited in liver, sustained glutamate deamination may have led to high ammonia accumulation in liver/body. Enhanced levels of gene transcripts in ubiquitination and MAPK signalling cascade and reduced levels of gene transcripts related to ROS generation via peroxisomal enzymes as well as xenobiotic metabolism may be signifying the importance of such cellular and tissue responses to maintain homeostasis. Furthermore, transcripts connected with stress and thyroid hormones were also regulated. Moreover, suppression of genes related to specific immune components may be denoting the deleterious impact of transport on fish health. Thus, this study has revealed the complex molecular -adjustments that occur in zebrafish when they are transported. PMID:23762281

Altered Gradients of Glutamate and Gamma-Aminobutyric Acid Transcripts in the Cortical Visuospatial Working Memory Network in Schizophrenia.

PubMed

Hoftman, Gil D; Dienel, Samuel J; Bazmi, Holly H; Zhang, Yun; Chen, Kehui; Lewis, David A

2018-04-15

Visuospatial working memory (vsWM), which is impaired in schizophrenia, requires information transfer across multiple nodes in the cerebral cortex, including visual, posterior parietal, and dorsolateral prefrontal regions. Information is conveyed across these regions via the excitatory projections of glutamatergic pyramidal neurons located in layer 3, whose activity is modulated by local inhibitory gamma-aminobutyric acidergic (GABAergic) neurons. Key properties of these neurons differ across these cortical regions. Consequently, in schizophrenia, alterations in the expression of gene products regulating these properties could disrupt vsWM function in different ways, depending on the region(s) affected. Here, we quantified the expression of markers of glutamate and GABA neurotransmission selectively in layer 3 of four cortical regions in the vsWM network from 20 matched pairs of schizophrenia and unaffected comparison subjects. In comparison subjects, levels of glutamate transcripts tended to increase, whereas GABA transcript levels tended to decrease, from caudal to rostral, across cortical regions of the vsWM network. Composite measures across all transcripts revealed a significant effect of region, with the glutamate measure lowest in the primary visual cortex and highest in the dorsolateral prefrontal cortex, whereas the GABA measure showed the opposite pattern. In schizophrenia subjects, the expression levels of many of these transcripts were altered. However, this disease effect differed across regions, such that the caudal-to-rostral increase in the glutamate measure was blunted and the caudal-to-rostral decline in the GABA measure was enhanced in the illness. Differential alterations in layer 3 glutamate and GABA neurotransmission across cortical regions may contribute to vsWM deficits in schizophrenia. Copyright © 2017 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.

Glucocorticoid suppresses the canonical Wnt signal in cultured human osteoblasts

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

Ohnaka, Keizo; Tanabe, Mizuho; Kawate, Hisaya

2005-04-01

To explore the mechanism of glucocorticoid-induced osteoporosis, we investigated the effect of glucocorticoid on canonical Wnt signaling that emerged as a novel key pathway for promoting bone formation. Wnt3a increased the T-cell factor (Tcf)/lymphoid enhancer factor (Lef)-dependent transcriptional activity in primary cultured human osteoblasts. Dexamethasone suppressed this transcriptional activity in a dose-dependent manner, while 1,25-dihydroxyvitamin D3 increased this transcriptional activity. LiCl, an inhibitor of glycogen synthase kinase-3{beta}, also enhanced the Tcf/Lef-dependent transcriptional activity, which was, however, not inhibited by dexamethasone. The addition of anti-dickkopf-1 antibody partially restored the transcriptional activity suppressed by dexamethasone. Dexamethasone decreased the cytosolic amount of {beta}-cateninmore » accumulated by Wnt3a and also inhibited the nuclear translocation of {beta}-catenin induced by Wnt3a. These data suggest that glucocorticoid suppresses the canonical Wnt signal in cultured human osteoblasts, partially through the enhancement of the dickkopf-1 production.« less

Acanthamoeba castellanii contains a ribosomal RNA enhancer binding protein which stimulates TIF-IB binding and transcription under stringent conditions.

PubMed

Yang, Q; Radebaugh, C A; Kubaska, W; Geiss, G K; Paule, M R

1995-11-11

The intergenic spacer (IGS) of Acanthamoeba castellanii rRNA genes contains repeated elements which are weak enhancers for transcription by RNA polymerase I. A protein, EBF, was identified and partially purified which binds to the enhancers and to several other sequences within the IGS, but not to other DNA fragments, including the rRNA core promoter. No consensus binding sequence could be discerned in these fragments and bound factor is in rapid equilibrium with unbound. EBF has functional characteristics similar to vertebrate upstream binding factors (UBF). Not only does it bind to the enhancer and other IGS elements, but it also stimulates binding of TIF-IB, the fundamental transcription initiation factor, to the core promoter and stimulates transcription from the promoter. Attempts to identify polypeptides with epitopes similar to rat or Xenopus laevis UBF suggest that structurally the protein from A.castellanii is not closely related to vertebrate UBF.

Acanthamoeba castellanii contains a ribosomal RNA enhancer binding protein which stimulates TIF-IB binding and transcription under stringent conditions.

PubMed Central

Yang, Q; Radebaugh, C A; Kubaska, W; Geiss, G K; Paule, M R

1995-01-01

The intergenic spacer (IGS) of Acanthamoeba castellanii rRNA genes contains repeated elements which are weak enhancers for transcription by RNA polymerase I. A protein, EBF, was identified and partially purified which binds to the enhancers and to several other sequences within the IGS, but not to other DNA fragments, including the rRNA core promoter. No consensus binding sequence could be discerned in these fragments and bound factor is in rapid equilibrium with unbound. EBF has functional characteristics similar to vertebrate upstream binding factors (UBF). Not only does it bind to the enhancer and other IGS elements, but it also stimulates binding of TIF-IB, the fundamental transcription initiation factor, to the core promoter and stimulates transcription from the promoter. Attempts to identify polypeptides with epitopes similar to rat or Xenopus laevis UBF suggest that structurally the protein from A.castellanii is not closely related to vertebrate UBF. Images PMID:7501455

Cooperative interactions enable singular olfactory receptor expression in mouse olfactory neurons

PubMed Central

Monahan, Kevin; Schieren, Ira; Cheung, Jonah; Mumbey-Wafula, Alice; Monuki, Edwin S

2017-01-01

The monogenic and monoallelic expression of only one out of >1000 mouse olfactory receptor (ORs) genes requires the formation of large heterochromatic chromatin domains that sequester the OR gene clusters. Within these domains, intergenic transcriptional enhancers evade heterochromatic silencing and converge into interchromosomal hubs that assemble over the transcriptionally active OR. The significance of this nuclear organization in OR choice remains elusive. Here, we show that transcription factors Lhx2 and Ebf specify OR enhancers by binding in a functionally cooperative fashion to stereotypically spaced motifs that defy heterochromatin. Specific displacement of Lhx2 and Ebf from OR enhancers resulted in pervasive, long-range, and trans downregulation of OR transcription, whereas pre-assembly of a multi-enhancer hub increased the frequency of OR choice in cis. Our data provide genetic support for the requirement and sufficiency of interchromosomal interactions in singular OR choice and generate general regulatory principles for stochastic, mutually exclusive gene expression programs. PMID:28933695

Expression of the serine/threonine kinase hSGK1 in chronic viral hepatitis.

PubMed

Fillon, Sophie; Klingel, Karin; Wärntges, Simone; Sauter, Martina; Gabrysch, Sabine; Pestel, Sabine; Tanneur, Valerie; Waldegger, Siegfried; Zipfel, Annette; Viebahn, Richard; Häussinger, Dieter; Bröer, Stefan; Kandolf, Reinhard; Lang, Florian

2002-01-01

The human serine/threonine kinase hSGK1 is expressed ubiquitously with highest transcript levels in pancreas and liver. This study has been performed to determine the hSGK1 distribution in normal liver and its putative role in fibrosing liver disease. HSGK1-localization was determined by in situ hybridization, regulation of hSGK1-transcription by Northern blotting, fibronectin synthesis and hSGK1 phosphorylation by Western blotting. In normal liver hSGK1 was mainly transcribed by Kupffer cells. In liver tissue from patients with chronic viral hepatitis, hSGK1 transcript levels were excessively high in numerous activated Kupffer cells and inflammatory cells localized within fibrous septum formations. HSGK1 transcripts were also detected in activated hepatic stellate cells. Accordingly, Western blotting revealed that tissue from fibrotic liver expresses excessive hSGK1 protein as compared to normal liver. TGF-beta1 (2 ng/ml) increases hSGK1 transcription in both human U937 macro-phages and HepG2 hepatoma cells. H(2)O(2) (0.3 mM) activated hSGK1 and increased fibronectin formation in HepG2 cells overexpressing hSGK1 but not in HepG2 cells expressing the inactive mutant hSGK1(K127R). In conclusion hSGK1 is upregulated by TGF-beta1 during hepatitis and may contribute to enhanced matrix formation during fibrosing liver disease. Copyright 2002 S. Karger AG, Basel

Role of the POZ Zinc Finger Transcription Factor FBI-1 in Human and Murine Adipogenesis

PubMed Central

Laudes, Matthias; Christodoulides, Constantinos; Sewter, Ciaran; Rochford, Justin J.; Considine, Robert V.; Sethi, Jaswinder K.; Vidal-Puig, Antonio; O’Rahilly, Stephen

2015-01-01

Poxvirus zinc finger (POZ) zinc finger domain transcription factors have been shown to play a role in the control of growth arrest and differentiation in several types of mesenchymal cells but not, as yet, adipocytes. We found that a POZ domain protein, factor that binds to inducer of short transcripts-1 (FBI-1), was induced during both murine and human preadipocyte differentiation with maximal expression levels seen at days 2–4. FBI-1 mRNA was expressed in human adipose tissue with the highest levels found in samples from morbidly obese subjects. Murine cell lines constitutively expressing FBI-1 showed evidence for accelerated adipogenesis with earlier induction of markers of differentiation and enhanced lipid accumulation, suggesting that FBI-1 may be an active participant in the differentiation process. Consistent with the properties of this family of proteins in other cell systems, 3T3L1 cells stably overexpressing FBI-1 showed reduced DNA synthesis and reduced expression of cyclin A, cyclin-dependent kinase 2, and p107, proteins known to be involved in the regulation of mitotic clonal expansion. In addition, FBI-1 reduced the transcriptional activity of the cyclin A promoter. Thus, FBI-1, a POZ zinc finger transcription factor, is induced during the early phases of human and murine preadipocyte differentiation where it may contribute to adipogenesis through influencing the switch from cellular proliferation to terminal differentiation. PMID:14701838

The transcriptional co-repressor TLE3 regulates myogenic differentiation by repressing the activity of the MyoD transcription factor.

PubMed

Kokabu, Shoichiro; Nakatomi, Chihiro; Matsubara, Takuma; Ono, Yusuke; Addison, William N; Lowery, Jonathan W; Urata, Mariko; Hudnall, Aaron M; Hitomi, Suzuro; Nakatomi, Mitsushiro; Sato, Tsuyoshi; Osawa, Kenji; Yoda, Tetsuya; Rosen, Vicki; Jimi, Eijiro

2017-08-04

Satellite cells are skeletal muscle stem cells that provide myonuclei for postnatal muscle growth, maintenance, and repair/regeneration in adults. Normally, satellite cells are mitotically quiescent, but they are activated in response to muscle injury, in which case they proliferate extensively and exhibit up-regulated expression of the transcription factor MyoD, a master regulator of myogenesis. MyoD forms a heterodimer with E proteins through their basic helix-loop-helix domain, binds to E boxes in the genome and thereby activates transcription at muscle-specific promoters. The central role of MyoD in muscle differentiation has increased interest in finding potential MyoD regulators. Here we identified transducin-like enhancer of split (TLE3), one of the Groucho/TLE family members, as a regulator of MyoD function during myogenesis. TLE3 was expressed in activated and proliferative satellite cells in which increased TLE3 levels suppressed myogenic differentiation, and, conversely, reduced TLE3 levels promoted myogenesis with a concomitant increase in proliferation. We found that, via its glutamine- and serine/proline-rich domains, TLE3 interferes with MyoD function by disrupting the association between the basic helix-loop-helix domain of MyoD and E proteins. Our findings indicate that TLE3 participates in skeletal muscle homeostasis by dampening satellite cell differentiation via repression of MyoD transcriptional activity. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Caffeic acid phenethyl ester down-regulates claudin-2 expression at the transcriptional and post-translational levels and enhances chemosensitivity to doxorubicin in lung adenocarcinoma A549 cells.

PubMed

Sonoki, Hiroyuki; Tanimae, Asami; Furuta, Takumi; Endo, Satoshi; Matsunaga, Toshiyuki; Ichihara, Kenji; Ikari, Akira

2018-06-01

Claudin-2 is highly expressed in human lung adenocarcinoma cells and involved in the promotion of proliferation. Here, we searched for a compound, which can decrease claudin-2 expression using lung adenocarcinoma A549 cells. In the screening using compounds included in royal jelly and propolis, the protein level of claudin-2 was dose-dependently decreased by caffeic acid phenethyl ester (CAPE), whereas the mRNA level and promoter activity were only decreased by 50 μM CAPE. These results suggest that CAPE down-regulates claudin-2 expression mediated by two different mechanisms. CAPE (50 μM) decreased the level of p-NF-κB, whereas it increased that of IκB. The CAPE-induced decrease in promoter activity of claudin-2 was blocked by the mutation in an NF-κB-binding site. The inhibition of NF-κB may be involved in the decrease in mRNA level of claudin-2. The CAPE (10 μM)-induced decrease in claudin-2 expression was inhibited by chloroquine, a lysosomal inhibitor. CAPE increased the expression and activity of protein phosphatase (PP) 1 and 2A. The CAPE-induced decrease in claudin-2 expression was blocked by cantharidin, a potent PPs inhibitor. The cell proliferation was suppressed by CAPE, which was partially rescued by ectopic expression of claudin-2. In addition, the toxicity and accumulation of doxorubicin in 3D spheroid cells were enhanced by CAPE, which was inhibited by ectopic expression of claudin-2. Taken together, CAPE down-regulates claudin-2 expression at the transcriptional and post-translational levels, and enhances sensitivity of cells to doxorubicin in 3D culture conditions. CAPE may be a useful adjunctive compound in the treatment of lung adenocarcinoma. Copyright © 2018 Elsevier Inc. All rights reserved.

Epigenetic regulation of vascular smooth muscle cell function in atherosclerosis.

PubMed

Findeisen, Hannes M; Kahles, Florian K; Bruemmer, Dennis

2013-04-01

Epigenetics involve heritable and acquired changes in gene transcription that occur independently of the DNA sequence. Epigenetic mechanisms constitute a hierarchic upper-level of transcriptional control through complex modifications of chromosomal components and nuclear structures. These modifications include, for example, DNA methylation or post-translational modifications of core histones; they are mediated by various chromatin-modifying enzymes; and ultimately they define the accessibility of a transcriptional complex to its target DNA. Integrating epigenetic mechanisms into the pathophysiologic concept of complex and multifactorial diseases such as atherosclerosis may significantly enhance our understanding of related mechanisms and provide promising therapeutic approaches. Although still in its infancy, intriguing scientific progress has begun to elucidate the role of epigenetic mechanisms in vascular biology, particularly in the control of smooth muscle cell phenotypes. In this review, we will summarize epigenetic pathways in smooth muscle cells, focusing on mechanisms involved in the regulation of vascular remodeling.

Epigenetic regulation of vascular smooth muscle cell function in atherosclerosis.

PubMed

Findeisen, Hannes M; Kahles, Florian K; Bruemmer, Dennis

2013-05-01

Epigenetics involve heritable and acquired changes in gene transcription that occur independently of the DNA sequence. Epigenetic mechanisms constitute a hierarchic upper-level of transcriptional control through complex modifications of chromosomal components and nuclear structures. These modifications include, for example, DNA methylation or post-translational modifications of core histones; they are mediated by various chromatin-modifying enzymes; and ultimately they define the accessibility of a transcriptional complex to its target DNA. Integrating epigenetic mechanisms into the pathophysiologic concept of complex and multifactorial diseases such as atherosclerosis may significantly enhance our understanding of related mechanisms and provide promising therapeutic approaches. Although still in its infancy, intriguing scientific progress has begun to elucidate the role of epigenetic mechanisms in vascular biology, particularly in the control of smooth muscle cell phenotypes. In this review, we will summarize epigenetic pathways in smooth muscle cells, focusing on mechanisms involved in the regulation of vascular remodeling.

A pause site for RNA polymerase II is associated with termination of transcription.

PubMed Central

Enriquez-Harris, P; Levitt, N; Briggs, D; Proudfoot, N J

1991-01-01

Termination of transcription by RNA polymerase II has been postulated to involve a pausing process. We have identified such a pause signal, 350 bp into the 3' flanking region of the human alpha 2 globin gene at a position where termination is thought to occur. We show that this pause signal enhances the utilization of an upstream poly(A) site which is otherwise out-competed by a stronger downstream poly(A) site. We also demonstrate that the pause site rescues a poly(A) site that is inactive due to its location within an intron. Using nuclear run-on analysis we show that elongating RNA polymerase II molecules accumulate over this pause signal. Furthermore we show that when the pause site is positioned immediately downstream of a strong poly(A) signal, significant levels of transcription termination take place. Images PMID:2050120

Epigenetic silencing of a foreign gene in nuclear transformants of Chlamydomonas.

PubMed Central

Cerutti, H; Johnson, A M; Gillham, N W; Boynton, J E

1997-01-01

The unstable expression of introduced genes poses a serious problem for the application of transgenic technology in plants. In transformants of the unicellular green alga Chlamydomonas reinhardtii, expression of a eubacterial aadA gene, conferring spectinomycin resistance, is transcriptionally suppressed by a reversible epigenetic mechanism(s). Variations in the size and frequency of colonies surviving on different concentrations of spectinomycin as well as the levels of transcriptional activity of the introduced transgene(s) suggest the existence of intermediate expression states in genetically identical cells. Gene silencing does not correlate with methylation of the integrated DNA and does not involve large alterations in its chromatin structure, as revealed by digestion with restriction endonucleases and DNase I. Transgene repression is enhanced by lower temperatures, similar to position effect variegation in Drosophila. By analogy to epigenetic phenomena in several eukaryotes, our results suggest a possible role for (hetero)chromatic chromosomal domains in transcriptional inactivation. PMID:9212467

A novel E2 box-GATA element modulates Cdc6 transcription during human cells polyploidization

PubMed Central

Vilaboa, Nuria; Bermejo, Rodrigo; Martinez, Pilar; Bornstein, Rafael; Calés, Carmela

2004-01-01

Cdc6 is a key regulator of the strict alternation of S and M phases during the mitotic cell cycle. In mammalian and plant cells that physiologically become polyploid, cdc6 is transcriptionally and post-translationally regulated. We have recently reported that Cdc6 levels are maintained in megakaryoblastic HEL cells, but severely downregulated by ectopic expression of transcriptional repressor Drosophila melanogaster escargot. Here, we show that cdc6 promoter activity is upregulated during megakaryocytic differentiation of HEL endoreplicating cells, and that Escargot interferes with such activation. Transactivation experiments showed that a 1.7 kb region located at 2800 upstream cdc6 transcription initiation site behaved as a potent enhancer in endoreplicating cells only. This activity was mainly dependent on a novel cis-regulatory element composed by an E2 box overlapping a GATA motif. Ectopic Escargot could bind this regulatory element in vitro and endogenous GATA-1 and E2A formed specific complexes in megakaryoblastic cells as well as in primary megakaryocytes. Chromatin Immunoprecipitation analysis revealed that both transcription factors were occupying the E2 box/GATA site in vivo. Altogether, these data suggest that cdc6 expression could be actively maintained during megakaryocytic differentiation through transcriptional mechanisms involving specific cis- and trans-regulatory elements. PMID:15590906

cis-antisense RNA, another level of gene regulation in bacteria.

PubMed

Georg, Jens; Hess, Wolfgang R

2011-06-01

A substantial amount of antisense transcription is a hallmark of gene expression in eukaryotes. However, antisense transcription was first demonstrated in bacteria almost 50 years ago. The transcriptomes of bacteria as different as Helicobacter pylori, Bacillus subtilis, Escherichia coli, Synechocystis sp. strain PCC6803, Mycoplasma pneumoniae, Sinorhizobium meliloti, Geobacter sulfurreducens, Vibrio cholerae, Chlamydia trachomatis, Pseudomonas syringae, and Staphylococcus aureus have now been reported to contain antisense RNA (asRNA) transcripts for a high percentage of genes. Bacterial asRNAs share functional similarities with trans-acting regulatory RNAs, but in addition, they use their own distinct mechanisms. Among their confirmed functional roles are transcription termination, codegradation, control of translation, transcriptional interference, and enhanced stability of their respective target transcripts. Here, we review recent publications indicating that asRNAs occur as frequently in simple unicellular bacteria as they do in higher organisms, and we provide a comprehensive overview of the experimentally confirmed characteristics of asRNA actions and intimately linked quantitative aspects. Emerging functional data suggest that asRNAs in bacteria mediate a plethora of effects and are involved in far more processes than were previously anticipated. Thus, the functional impact of asRNAs should be considered when developing new strategies against pathogenic bacteria and when optimizing bacterial strains for biotechnology.

cis-Antisense RNA, Another Level of Gene Regulation in Bacteria

PubMed Central

Georg, Jens; Hess, Wolfgang R.

2011-01-01

Summary: A substantial amount of antisense transcription is a hallmark of gene expression in eukaryotes. However, antisense transcription was first demonstrated in bacteria almost 50 years ago. The transcriptomes of bacteria as different as Helicobacter pylori, Bacillus subtilis, Escherichia coli, Synechocystis sp. strain PCC6803, Mycoplasma pneumoniae, Sinorhizobium meliloti, Geobacter sulfurreducens, Vibrio cholerae, Chlamydia trachomatis, Pseudomonas syringae, and Staphylococcus aureus have now been reported to contain antisense RNA (asRNA) transcripts for a high percentage of genes. Bacterial asRNAs share functional similarities with trans-acting regulatory RNAs, but in addition, they use their own distinct mechanisms. Among their confirmed functional roles are transcription termination, codegradation, control of translation, transcriptional interference, and enhanced stability of their respective target transcripts. Here, we review recent publications indicating that asRNAs occur as frequently in simple unicellular bacteria as they do in higher organisms, and we provide a comprehensive overview of the experimentally confirmed characteristics of asRNA actions and intimately linked quantitative aspects. Emerging functional data suggest that asRNAs in bacteria mediate a plethora of effects and are involved in far more processes than were previously anticipated. Thus, the functional impact of asRNAs should be considered when developing new strategies against pathogenic bacteria and when optimizing bacterial strains for biotechnology. PMID:21646430

Compensatory hyperinsulinemia in high-fat diet-induced obese mice is associated with enhanced insulin translation in islets

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

Kanno, Ayumi, E-mail: akanno@med.kobe-u.ac.jp; Asahara, Shun-ichiro, E-mail: asahara@med.kobe-u.ac.jp; Masuda, Katsuhisa, E-mail: katsuhisa.m.0707@gmail.com

A high-fat diet (HF) is associated with obesity, insulin resistance, and hyperglycemia. Animal studies have shown compensatory mechanisms in pancreatic β-cells after high fat load, such as increased pancreatic β-cell mass, enhanced insulin secretion, and exocytosis. However, the effects of high fat intake on insulin synthesis are obscure. Here, we investigated whether insulin synthesis was altered in correlation with an HF diet, for the purpose of obtaining further understanding of the compensatory mechanisms in pancreatic β-cells. Mice fed an HF diet are obese, insulin resistant, hyperinsulinemic, and glucose intolerant. In islets of mice fed an HF diet, more storage ofmore » insulin was identified. We analyzed insulin translation in mouse islets, as well as in INS-1 cells, using non-radioisotope chemicals. We found that insulin translational levels were significantly increased in islets of mice fed an HF diet to meet systemic demand, without altering its transcriptional levels. Our data showed that not only increased pancreatic β-cell mass and insulin secretion but also elevated insulin translation is the major compensatory mechanism of pancreatic β-cells. - Highlights: • More stored insulin was recognized in islets of mice fed a high-fat diet. • Insulin translation was not enhanced by fatty acids, but by insulin demand. • Insulin transcription was not altered in islets of mice fed a high-fat diet. • Insulin translation was markedly enhanced in islets of mice fed a high-fat diet. • Non-radioisotope chemicals were used to measure insulin translation in mouse islets.« less

Quantifying Temporal Autocorrelations for the Expression of Geobacter species mRNA Gene Transcripts at Variable Ammonium Levels during in situ U(VI) Bioremediation

NASA Astrophysics Data System (ADS)

Mouser, P. J.

2010-12-01

In order to develop decision-making tools for the prediction and optimization of subsurface bioremediation strategies, we must be able to link the molecular-scale activity of microorganisms involved in remediation processes with biogeochemical processes observed at the field-scale. This requires the ability to quantify changes in the in situ metabolic condition of dominant microbes and associate these changes to fluctuations in nutrient levels throughout the bioremediation process. It also necessitates a need to understand the spatiotemporal variability of the molecular-scale information to develop meaningful parameters and constraint ranges in complex bio-physio-chemical models. The expression of three Geobacter species genes (ammonium transporter (amtB), nitrogen fixation (nifD), and a housekeeping gene (recA)) were tracked at two monitoring locations that differed significantly in ammonium (NH4+) concentrations during a field-scale experiment where acetate was injected into the subsurface to simulate Geobacteraceae in a uranium-contaminated aquifer. Analysis of amtB and nifD mRNA transcript levels indicated that NH4+ was the primary form of fixed nitrogen during bioremediation. Overall expression levels of amtB were on average 8-fold higher at NH4+ concentrations of 300 μM or more than at lower NH4+ levels (average 60 μM). The degree of temporal correlation in Geobacter species mRNA expression levels was calculated at both locations using autocorrelation methods that describe the relationship between sample semi-variance and time lag. At the monitoring location with lower NH4+, a temporal correlation lag of 8 days was observed for both amtB and nifD transcript patterns. At the location where higher NH4+ levels were observed, no discernable temporal correlation lag above the sampling frequency (approximately every 2 days) was observed for amtB or nifD transcript fluctuations. Autocorrelation trends in recA expression levels at both locations indicated that while a temporal correlation in the general metabolic activity of Geobacter species may exist, considerable variability in transcript levels masked these correlations at the sampled scale. These findings suggest that when Geobacter species are dependent upon a particular nutrient such as NH4+, the time length for which their activity level relating to this nutrient condition can be predicted is significantly enhanced.

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

Balasubramanian, Sivaprakasam; Eckert, Richard L., E-mail: reckert@umaryland.edu

We have proposed that it is important to examine the impact of chemopreventive agents on the function of normal human epidermal keratinocytes since these cells comprise the barrier that protects the body from a range of environmental insults. In this context, it is widely appreciated that cancer may be retarded by consumption or topical application of naturally occurring food-derived chemopreventive agents. Our studies show that (-)-epigallocatechin-3-gallate (EGCG), a green tea-derived polyphenol, acts to enhance the differentiation of normal human keratinocytes as evidenced by its ability to increase involucrin (hINV), transglutaminase type 1 (TG1) and caspase-14 gene expression. EGCG also stimulatesmore » keratinocyte morphological differentiation. These actions of EGCG are mediated via activation of a nPKC, Ras, MEKK1, MEK3, p38{delta}-ERK1/2 signaling cascade which leads to increased activator protein 1 (AP1) and CAATT enhancer binding protein (C/EBP) transcription factor expression, increased binding of these factors to DNA, and increased gene transcription. In contrast, apigenin, a dietary flavonoid derived from plants and vegetables, and curcumin, an agent derived from turmeric, inhibit differentiation by suppressing MAPK signal transduction and reducing API transcription factor level. Curcumin also acts to enhance apoptosis, although EGCG and apigenin do not stimulate apoptosis. In addition, all of these agents inhibit keratinocyte proliferation. These findings indicate that each of these diet-derived chemopreventive agents has a profound impact on normal human keratinocyte function and that they operate via distinct and sometimes opposing mechanisms. However, all are expected to act as chemopreventive agents.« less

Enhancement of welan gum production in Sphingomonas sp. HT-1 via heterologous expression of Vitreoscilla hemoglobin gene.

PubMed

Liu, Xiaoliu; Zhu, Ping; Jiang, Ruifan; Wu, Lingtian; Feng, Xiaohai; Li, Sha; Xu, Hong

2017-01-20

Welan gum is a microbial polysaccharide produced by Sphingomonas sp. Its production is limited by the dissolved oxygen levels in the highly viscous fermentation. A strategy of heterologous expression of the Vitreoscilla hemoglobin gene in Sphingomonas sp. HT-1 was investigated to alleviate oxygen limitation and improve the yield of welan gum. Ultimately, the welan gum production increased from 25.3g/L to 34.6g/L, whereas the rheological behavior of welan gum solutions remained virtually unchanged. The transcriptional levels of the key genes in the electron transfer chain, TCA cycle and welan gum synthesis pathway, as well as ATP level revealed that the VHb expression in Sphingomonas sp. HT-1 enhanced welan gum biosynthesis by improving respiration and ATP supply. This study would pave the genetic manipulation way for enhancing welan gum yield, and it's also of great importance for the industrial applications of welan gum under harsh conditions. Copyright © 2016 Elsevier Ltd. All rights reserved.

Regulatory role of glycogen synthase kinase 3 for transcriptional activity of ADD1/SREBP1c.

PubMed

Kim, Kang Ho; Song, Min Jeong; Yoo, Eung Jae; Choe, Sung Sik; Park, Sang Dai; Kim, Jae Bum

2004-12-10

Adipocyte determination- and differentiation-dependent factor 1 (ADD1) plays important roles in lipid metabolism and insulin-dependent gene expression. Because insulin stimulates carbohydrate and lipid synthesis, it would be important to decipher how the transcriptional activity of ADD1/SREBP1c is regulated in the insulin signaling pathway. In this study, we demonstrated that glycogen synthase kinase (GSK)-3 negatively regulates the transcriptional activity of ADD1/SREBP1c. GSK3 inhibitors enhanced a transcriptional activity of ADD1/SREBP1c and expression of ADD1/SREBP1c target genes including fatty acid synthase (FAS), acetyl-CoA carboxylase 1 (ACC1), and steroyl-CoA desaturase 1 (SCD1) in adipocytes and hepatocytes. In contrast, overexpression of GSK3beta down-regulated the transcriptional activity of ADD1/SREBP1c. GSK3 inhibitor-mediated ADD1/SREBP1c target gene activation did not require de novo protein synthesis, implying that GSK3 might affect transcriptional activity of ADD1/SREBP1c at the level of post-translational modification. Additionally, we demonstrated that GSK3 efficiently phosphorylated ADD1/SREBP1c in vitro and in vivo. Therefore, these data suggest that GSK3 inactivation is crucial to confer stimulated transcriptional activity of ADD1/SREBP1c for insulin-dependent gene expression, which would coordinate lipid and glucose metabolism.

Exploration of G-quadruplex function in c-Myb gene and its transcriptional regulation by topotecan.

PubMed

Li, Fangyuan; Zhou, Jiang; Xu, Ming; Yuan, Gu

2018-02-01

Our bioinformatics research shows that there are four G-rich sequences (S1-S4) in the upstream region of the transcription start site of c-Myb gene, and we have proved that these sequences have the ability to form G-quadruplex structures. This work mainly focuses on G-quadruplex function, recognition and transcription regulation in c-Myb gene, revealing a novel regulatory element in c-Myb proximal promoter region, and its transcription regulation by G-quadruplex binder. The research has identified that the enhancer effect in c-Myb transcription was primarily affected by the G-quadruplex formed by S1 sequence, and the up-regulation effect may due to the removal of repressive progress of MZF-1 by stabilizing G-quadruplex. Attentions were being paid to the development of G-quadruplex binders for selective recognition, and topotecan was found to have high binding affinity in vitro and could effectively affect the c-Myb transcription activities in cells. The regulation of G-quadruplex with binders in transcriptional, translational levels by Q-RT-PCR and western blot was in expectation of providing a strategy for gene expression modulation. In conclusion, our study revealed a G-quadruplex structure in c-Myb proximal promoter region, which was of great importance in the regulation of c-Myb function. Copyright © 2017 Elsevier B.V. All rights reserved.

FOXM1 promotes the progression of prostate cancer by regulating PSA gene transcription.

PubMed

Liu, Youhong; Liu, Yijun; Yuan, Bowen; Yin, Linglong; Peng, Yuchong; Yu, Xiaohui; Zhou, Weibing; Gong, Zhicheng; Liu, Jianye; He, Leye; Li, Xiong

2017-03-07

Androgen/AR is the primary contributor to prostate cancer (PCa) progression by regulating Prostate Specific Antigen (PSA) gene transcription. The disease inevitably evolves to androgen-independent (AI) status. Other mechanisms by which PSA is regulated and develops to AI have not yet been fully determined. FOXM1 is a cell proliferation-specific transcription factor highly expressed in PCa cells compared to non-malignant prostate epithelial cells, suggesting that the aberrant overexpression of FOXM1 contributes to PCa development. In addition to regulating AR gene transcription and cell cycle-regulatory genes, FOXM1 selectively regulates the gene transcription of KLK2 and PSA, typical androgen responsive genes. Screening the potential FOXM1-binding sites by ChIP-PCR, we found that FOXM1 directly binds to the FHK binding motifs in the PSA promoter/enhancer regions. AI C4-2 cells have more FOXM1 binding sites than androgen dependent LNCaP cells. The depletion of FOXM1 by small molecular inhibitors significantly improves the suppression of PSA gene transcription by the anti-AR agent Cadosax. This is the first report showing that FOXM1 promotes PCa progression by regulating PSA gene transcription, particularly in AI PCa cells. The combination of anti-AR agents and FOXM1 inhibitors has the potential to greatly improve therapy for late-stage PCa patients by suppressing PSA levels.

Hepatotoxicity of high affinity gapmer antisense oligonucleotides is mediated by RNase H1 dependent promiscuous reduction of very long pre-mRNA transcripts

PubMed Central

Burel, Sebastien A.; Hart, Christopher E.; Cauntay, Patrick; Hsiao, Jill; Machemer, Todd; Katz, Melanie; Watt, Andy; Bui, Huynh-hoa; Younis, Husam; Sabripour, Mahyar; Freier, Susan M.; Hung, Gene; Dan, Amy; Prakash, T.P.; Seth, Punit P.; Swayze, Eric E.; Bennett, C. Frank; Crooke, Stanley T.; Henry, Scott P.

2016-01-01

High affinity antisense oligonucleotides (ASOs) containing bicylic modifications (BNA) such as locked nucleic acid (LNA) designed to induce target RNA cleavage have been shown to have enhanced potency along with a higher propensity to cause hepatotoxicity. In order to understand the mechanism of this hepatotoxicity, transcriptional profiles were collected from the livers of mice treated with a panel of highly efficacious hepatotoxic or non-hepatotoxic LNA ASOs. We observed highly selective transcript knockdown in mice treated with non-hepatotoxic LNA ASOs, while the levels of many unintended transcripts were reduced in mice treated with hepatotoxic LNA ASOs. This transcriptional signature was concurrent with on-target RNA reduction and preceded transaminitis. Remarkably, the mRNA transcripts commonly reduced by toxic LNA ASOs were generally not strongly associated with any particular biological process, cellular component or functional group. However, they tended to have much longer pre-mRNA transcripts. We also demonstrate that the off-target RNA knockdown and hepatotoxicity is attenuated by RNase H1 knockdown, and that this effect can be generalized to high affinity modifications beyond LNA. This suggests that for a certain set of ASOs containing high affinity modifications such as LNA, hepatotoxicity can occur as a result of unintended off-target RNase H1 dependent RNA degradation. PMID:26553810

An engineered tale-transcription factor rescues transcription of factor VII impaired by promoter mutations and enhances its endogenous expression in hepatocytes.

PubMed

Barbon, Elena; Pignani, Silvia; Branchini, Alessio; Bernardi, Francesco; Pinotti, Mirko; Bovolenta, Matteo

2016-06-24

Tailored approaches to restore defective transcription responsible for severe diseases have been poorly explored. We tested transcription activator-like effectors fused to an activation domain (TALE-TFs) in a coagulation factor VII (FVII) deficiency model. In this model, the deficiency is caused by the -94C > G or -61T > G mutation, which abrogate the binding of Sp1 or HNF-4 transcription factors. Reporter assays in hepatoma HepG2 cells naturally expressing FVII identified a single TALE-TF (TF4) that, by targeting the region between mutations, specifically trans-activated both the variant (>100-fold) and wild-type (20-40-fold) F7 promoters. Importantly, in the genomic context of transfected HepG2 and transduced primary hepatocytes, TF4 increased F7 mRNA and protein levels (2- to 3-fold) without detectable off-target effects, even for the homologous F10 gene. The ectopic F7 expression in renal HEK293 cells was modestly affected by TF4 or by TALE-TF combinations. These results provide experimental evidence for TALE-TFs as gene-specific tools useful to counteract disease-causing promoter mutations.

Potential for increased photosynthetic performance and crop productivity in response to climate change: role of CBFs and gibberellic acid

PubMed Central

Hüner, Norman P. A.; Dahal, Keshav; Kurepin, Leonid V.; Savitch, Leonid; Singh, Jas; Ivanov, Alexander G.; Kane, Khalil; Sarhan, Fathey

2014-01-01

We propose that targeting the enhanced photosynthetic performance associated with the cold acclimation of winter cultivars of rye (Secale cereale L.), wheat (Triticum aestivum L.), and Brassica napus L. may provide a novel approach to improve crop productivity under abiotic as well as biotic stress conditions. In support of this hypothesis, we provide the physiological, biochemical, and molecular evidence that the dwarf phenotype induced by cold acclimation is coupled to significant enhancement in photosynthetic performance, resistance to photoinhibition, and a decreased dependence on photoprotection through non-photochemical quenching which result in enhanced biomass production and ultimately increased seed yield. These system-wide changes at the levels of phenotype, physiology, and biochemistry appear to be governed by the family of C-repeat/dehydration-responsive family of transcription factors (CBF/DREB1). We relate this phenomenon to the semi-dwarf, gibberellic acid insensitive (GAI), cereal varieties developed during the “green revolution” of the early 1960s and 1970s. We suggest that genetic manipulation of the family of C-repeat/dehydration-responsive element binding transcription factors (CBF/DREB1) may provide a novel approach for the maintenance and perhaps even the enhancement of plant productivity under conditions of sub-optimal growth conditions predicted for our future climate. PMID:24860799

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.

Or mutation leads to photo-oxidative stress responses in cauliflower (Brassica oleracea) seedlings during de-etiolation.

PubMed

Men, Xiao; Dong, Kang

2013-11-01

The Orange (Or) gene is a gene mutation that can increase carotenoid content in plant tissues normally devoid of pigments. It affects plastid division and is involved in the differentiation of proplastids or non-colored plastids into chromoplasts. In this study, the de-etiolation process of the wild type (WT) cauliflower (Brassica oleracea L. var. botrytis) and Or mutant seedlings was investigated. We analyzed pigment content, plastid development, transcript abundance and protein levels of genes involved in the de-etiolation process. The results showed that Or can increase the carotenoid content in green tissues, although not as effectively as in non-green tissues, and this effect might be caused by the changes in biosynthetic pathway genes at both transcriptional and post-transcriptional levels. There was no significant difference in the plastid development process between the two lines. However, the increased content of antheraxanthin and anthocyanin, and higher expression levels of violaxanthin de-epoxidase gene (VDE) suggested a stress situation leading to photoinhibition and enhanced photoprotection in the Or mutant. The up-regulated expression levels of the reactive oxygen species (ROS)-induced genes, ZAT10 for salt tolerance zinc finger protein and ASCORBATE PEROXIDASE2 (APX2), suggested the existence of photo-oxidative stress in the Or mutant. In summary, abovementioned findings provide additional insight into the functions of the Or gene in different tissues and at different developmental stages.

Low-level laser irradiation at a high power intensity increased human endothelial cell exosome secretion via Wnt signaling.

PubMed

Bagheri, Hesam Saghaei; Mousavi, Monireh; Rezabakhsh, Aysa; Rezaie, Jafar; Rasta, Seyed Hossein; Nourazarian, Alireza; Avci, Çigir Biray; Tajalli, Habib; Talebi, Mehdi; Oryan, Ahmad; Khaksar, Majid; Kazemi, Masoumeh; Nassiri, Seyed Mahdi; Ghaderi, Shahrooz; Bagca, Bakiye Goker; Rahbarghazi, Reza; Sokullu, Emel

2018-03-30

The distinct role of low-level laser irradiation (LLLI) on endothelial exosome biogenesis remains unclear. We hypothesize that laser irradiation of high dose in human endothelial cells (ECs) contributes to the modulation of exosome biogenesis via Wnt signaling pathway. When human ECs were treated with LLLI at a power density of 80 J/cm 2 , the survival rate reduced. The potential of irradiated cells to release exosomes was increased significantly by expressing genes CD63, Alix, Rab27a, and b. This occurrence coincided with an enhanced acetylcholine esterase activity, pseudopodia formation, and reduced zeta potential value 24 h post-irradiation. Western blotting showed the induction of LC3 and reduced level of P62, confirming autophagy response. Flow cytometry and electron microscopy analyses revealed the health status of the mitochondrial function indicated by normal ΔΨ activity without any changes in the transcription level of PINK1 and Optineurin. When cells exposed to high power laser irradiation, p-Akt/Akt ratio and in vitro tubulogenesis capacity were blunted. PCR array and bioinformatics analyses showed the induction of transcription factors promoting Wnt signaling pathways and GTPase activity. Thus, LLLI at high power intensity increased exosome biogenesis by the induction of autophagy and Wnt signaling. LLLI at high power intensity increases exosome biogenesis by engaging the transcription factors related to Wnt signaling and autophagy stimulate.

FBXW10 is negatively regulated in transcription and expression level by protein O-GlcNAcylation.

PubMed

Feng, Zhou; Hui, Yan; Ling, Li; Xiaoyan, Liu; Yuqiu, Wang; Peng, Wang; Lianwen, Zhang

2013-08-23

Intricate cross-talks exist among multiple post-translational modifications that play critical roles in various cellular events, such as the control of gene expression and regulation of protein function. Here, the cross-talk between O-GlcNAcylation and ubiquitination was investigated in HEK293T cells. By PCR array, 84 ubiquitination-related genes were explored in transcription level in response to the elevation of total protein O-GlcNAcylation due to over-expression of OGT, inhibition of OGA or GlcN treatment. Varied genes were transcriptionally regulated by using different method. But FBXW10, an F-box protein targeting specific proteins for ubiquitination, could be negatively regulated in all ways, suggesting its regulation by protein O-GlcNAcylation. By RT-PCR and Western blot analysis, it was found that FBXW10 could be sharply down-regulated in mRNA and protein level in GlcN-treated cells in a time-dependent way, in line with the enhancement of protein O-GlcNAcylation. It was also found that endogenous FBXW10 was modified by O-GlcNAc in HEK293T cells, implying O-GlcNAcylation might regulate FBXW10 in multiple levels. These findings indicate that O-GlcNAcylation is involved in the regulation of ubiquitination-related genes, and help us understand the cross-talk between O-GlcNAcylation and ubiquitination. Copyright © 2013 Elsevier Inc. All rights reserved.

Mediator facilitates transcriptional activation and dynamic long-range contacts at the IgH locus during class switch recombination.

PubMed

Thomas-Claudepierre, Anne-Sophie; Robert, Isabelle; Rocha, Pedro P; Raviram, Ramya; Schiavo, Ebe; Heyer, Vincent; Bonneau, Richard; Luo, Vincent M; Reddy, Janardan K; Borggrefe, Tilman; Skok, Jane A; Reina-San-Martin, Bernardo

2016-03-07

Immunoglobulin (Ig) class switch recombination (CSR) is initiated by the transcription-coupled recruitment of activation-induced cytidine deaminase (AID) to Ig switch regions (S regions). During CSR, the IgH locus undergoes dynamic three-dimensional structural changes in which promoters, enhancers, and S regions are brought to close proximity. Nevertheless, little is known about the underlying mechanisms. In this study, we show that Med1 and Med12, two subunits of the mediator complex implicated in transcription initiation and long-range enhancer/promoter loop formation, are dynamically recruited to the IgH locus enhancers and the acceptor regions during CSR and that their knockdown in CH12 cells results in impaired CSR. Furthermore, we show that conditional inactivation of Med1 in B cells results in defective CSR and reduced acceptor S region transcription. Finally, we show that in B cells undergoing CSR, the dynamic long-range contacts between the IgH enhancers and the acceptor regions correlate with Med1 and Med12 binding and that they happen at a reduced frequency in Med1-deficient B cells. Our results implicate the mediator complex in the mechanism of CSR and are consistent with a model in which mediator facilitates the long-range contacts between S regions and the IgH locus enhancers during CSR and their transcriptional activation. © 2016 Thomas-Claudepierre et al.

Transcriptomics in human blood incubation reveals the importance of oxidative stress response in Saccharomyces cerevisiae clinical strains.

PubMed

Llopis, Silvia; Querol, Amparo; Heyken, Antje; Hube, Bernhard; Jespersen, Lene; Fernández-Espinar, M Teresa; Pérez-Torrado, Roberto

2012-08-23

In recent years an increasing number of yeast infections in humans have been related to certain clinical isolates of Saccharomyces cerevisiae. Some clinical strains showed in vivo and in vitro virulence traits and were able to cause death in mice whereas other clinical strains were avirulent. In this work, we studied the transcriptional profiles of two S. cerevisiae clinical strains showing virulent traits and two control non-virulent strains during a blood incubation model and detected a specific transcriptional response of clinical strains. This response involves an mRNA levels increase of amino acid biosynthesis genes and especially oxidative stress related genes. We observed that the clinical strains were more resistant to reactive oxygen species in vitro. In addition, blood survival of clinical isolates was high, reaching similar levels to pathogenic Candida albicans strain. Furthermore, a virulent strain mutant in the transcription factor Yap1p, unable to grow in oxidative stress conditions, presented decreased survival levels in human blood compared with the wild type or YAP1 reconstituted strain. Our data suggest that this enhanced oxidative stress response in virulent clinical isolates, presumably induced in response to oxidative burst from host defense cells, is important to increase survival in human blood and can help to infect and even produce death in mice models.

Smad4 suppresses the tumorigenesis and aggressiveness of neuroblastoma through repressing the expression of heparanase.

PubMed

Qu, Hongxia; Zheng, Liduan; Jiao, Wanju; Mei, Hong; Li, Dan; Song, Huajie; Fang, Erhu; Wang, Xiaojing; Li, Shiwang; Huang, Kai; Tong, Qiangsong

2016-09-06

Heparanase (HPSE) is the only endo-β-D-glucuronidase that is correlated with the progression of neuroblastoma (NB), the most common extracranial malignancy in childhood. However, the mechanisms underlying HPSE expression in NB still remain largely unknown. Herein, through analyzing cis-regulatory elements and mining public microarray datasets, we identified SMAD family member 4 (Smad4) as a crucial transcription regulator of HPSE in NB. We demonstrated that Smad4 repressed the HPSE expression at the transcriptional levels in NB cells. Mechanistically, Smad4 suppressed the HPSE expression through directly binding to its promoter and repressing the lymphoid enhancer binding factor 1 (LEF1)-facilitated transcription of HPSE via physical interaction. Gain- and loss-of-function studies demonstrated that Smad4 inhibited the growth, invasion, metastasis, and angiogenesis of NB cells in vitro and in vivo. Restoration of HPSE expression prevented the NB cells from changes in these biological features induced by Smad4. In clinical NB specimens, Smad4 was under-expressed and inversely correlated with HPSE levels, while LEF1 was highly expressed and positively correlated with HPSE expression. Patients with high Smad4 expression, low LEF1 or HPSE levels had greater survival probability. These results demonstrate that Smad4 suppresses the tumorigenesis and aggressiveness of NB through repressing the HPSE expression.

Extremely High Expression of Antisense RNA for Wilms' Tumor 1 in Active Osteoclasts: Suppression of Wilms' Tumor 1 Protein Expression during Osteoclastogenesis.

PubMed

Li, Yin-Ji; Kukita, Akiko; Kyumoto-Nakamura, Yukari; Kukita, Toshio

2016-09-01

Wilms' tumor 1 (WT1), a zinc-finger transcription regulator of the early growth response family, identified as the product of a tumor suppressor gene of Wilms' tumors, bears potential ability to induce macrophage differentiation in blood cell differentiation. Herein, we examined the involvement of WT1 in the regulation of osteoclastogenesis. We detected a high level of WT1 protein expression in osteoclast precursors; however, WT1 expression was markedly suppressed during osteoclastogenesis. We examined expression of WT1 transcripts in bone tissue by RNA in situ hybridization. We found a high level of antisense transcripts in osteoclasts actively resorbing bone in mandible of newborn rats. Expression of antisense WT1 RNA in mandible was also confirmed by Northern blot analysis and strand-specific RT-PCR. Overexpression of antisense WT1 RNA in RAW-D cells, an osteoclast precursor cell line, resulted in a marked enhancement of osteoclastogenesis, suggesting that antisense WT1 RNA functions to suppress expression of WT1 protein in osteoclastogenesis. High level expression of antisense WT1 RNA may contribute to commitment to osteoclastogenesis, and may allow osteoclasts to maintain or stabilize their differentiation state. Copyright © 2016 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Tissue-specific epigenetics in gene neighborhoods: myogenic transcription factor genes

PubMed Central

Chandra, Sruti; Terragni, Jolyon; Zhang, Guoqiang; Pradhan, Sriharsa; Haushka, Stephen; Johnston, Douglas; Baribault, Carl; Lacey, Michelle; Ehrlich, Melanie

2015-01-01

Myogenic regulatory factor (MRF) genes, MYOD1, MYOG, MYF6 and MYF5, are critical for the skeletal muscle lineage. Here, we used various epigenome profiles from human myoblasts (Mb), myotubes (Mt), muscle and diverse non-muscle samples to elucidate the involvement of multigene neighborhoods in the regulation of MRF genes. We found more far-distal enhancer chromatin associated with MRF genes in Mb and Mt than previously reported from studies in mice. For the MYF5/MYF6 gene-pair, regions of Mb-associated enhancer chromatin were located throughout the adjacent 236-kb PTPRQ gene even though Mb expressed negligible amounts of PTPRQ mRNA. Some enhancer chromatin regions inside PTPRQ in Mb were also seen in PTPRQ mRNA-expressing non-myogenic cells. This suggests dual-purpose PTPRQ enhancers that upregulate expression of PTPRQ in non-myogenic cells and MYF5/MYF6 in myogenic cells. In contrast, the myogenic enhancer chromatin regions distal to MYOD1 were intergenic and up to 19 kb long. Two of them contain small, known MYOD1 enhancers, and one displayed an unusually high level of 5-hydroxymethylcytosine in a quantitative DNA hydroxymethylation assay. Unexpectedly, three regions of MYOD1-distal enhancer chromatin in Mb and Mt overlapped enhancer chromatin in umbilical vein endothelial cells, which might upregulate a distant gene (PIK3C2A). Lastly, genes surrounding MYOG were preferentially transcribed in Mt, like MYOG itself, and exhibited nearby myogenic enhancer chromatin. These neighboring chromatin regions may be enhancers acting in concert to regulate myogenic expression of multiple adjacent genes. Our findings reveal the very different and complex organization of gene neighborhoods containing closely related transcription factor genes. PMID:26041816

Multiple transcriptional regulatory domains in the human immunodeficiency virus type 1 long terminal repeat are involved in basal and E1A/E1B-induced promoter activity.

PubMed Central

Kliewer, S; Garcia, J; Pearson, L; Soultanakis, E; Dasgupta, A; Gaynor, R

1989-01-01

The human immunodeficiency virus (HIV) type 1 long terminal repeat (LTR) is the site of activation of the HIV tat protein. However, additional transactivators, such as the adenovirus E1A and herpesvirus ICPO proteins, have also been shown to be capable of activating the HIV LTR. Analysis of adenovirus mutants indicated that complete transactivation of the HIV LTR was dependent on both the E1A and E1B proteins. To determine which regions of the HIV LTR were important for complete E1A/E1B activation, a variety of oligonucleotide-directed mutations in HIV transcriptional regulatory domains were assayed both in vivo and in vitro. S1 nuclease analysis of RNA prepared after transfection of these HIV constructs into HeLa cells infected with wild-type adenovirus indicated that the enhancer, SP1, TATA, and a portion of the transactivation-responsive element were each required for complete E1A/E1B-mediated activation of the HIV LTR. These same promoter elements were required for both basal and E1A/E1B-induced levels of transcription in in vitro transcription reactions performed with cellular extracts prepared from cells infected with dl434, an E1A/E1B deletion mutant, or wild-type adenovirus. No mutations were found that reduced only E1A/E1B-induced expression without proportionally reducing basal levels of transcription, suggesting that E1A/E1B-mediated induction of the HIV LTR requires multiple promoter elements which are also required for basal transcriptional levels. Unlike activation by the tat protein, there was not a rigid dependence on maintenance of the transactivation-responsive stem base pairing for E1A/E1B-mediated activation either in vivo or in vitro, indicating that activation occurs by a mechanism distinct from that of tat induction. Images PMID:2529378

β-Hydroxy-β-Methylbutyrate (HMB) Promotes Neurite Outgrowth in Neuro2a Cells.

PubMed

Salto, Rafael; Vílchez, Jose D; Girón, María D; Cabrera, Elena; Campos, Nefertiti; Manzano, Manuel; Rueda, Ricardo; López-Pedrosa, Jose M

2015-01-01

β-Hydroxy-β-methylbutyrate (HMB) has been shown to enhance cell survival, differentiation and protein turnover in muscle, mainly activating phosphoinositide-3-kinase/protein kinase B (PI3K/Akt) and mitogen-activated protein kinases/ extracellular-signal-regulated kinases (MAPK/ERK) signaling pathways. Since these two pathways are related to neuronal survival and differentiation, in this study, we have investigated the neurotrophic effects of HMB in mouse neuroblastoma Neuro2a cells. In Neuro2a cells, HMB promotes differentiation to neurites independent from any effects on proliferation. These effects are mediated by activation of both the PI3K/Akt and the extracellular-signal-regulated kinases (ERK1/2) signaling as demonstrated by the use of specific inhibitors of these two pathways. As myocyte-enhancer factor 2 (MEF2) family of transcription factors are involved in neuronal survival and plasticity, the transcriptional activity and protein levels of MEF2 were also evaluated. HMB promoted MEF2-dependent transcriptional activity mediated by the activation of Akt and ERK1/2 pathways. Furthermore, HMB increases the expression of brain glucose transporters 1 (GLUT1) and 3 (GLUT3), and mTOR phosphorylation, which translates in a higher protein synthesis in Neuro2a cells. Furthermore, Torin1 and rapamycin effects on MEF2 transcriptional activity and HMB-dependent neurite outgrowth support that HMB acts through mTORC2. Together, these findings provide clear evidence to support an important role of HMB in neurite outgrowth.

β-Hydroxy-β-Methylbutyrate (HMB) Promotes Neurite Outgrowth in Neuro2a Cells

PubMed Central

Girón, María D.; Cabrera, Elena; Campos, Nefertiti; Manzano, Manuel; Rueda, Ricardo; López-Pedrosa, Jose M.

2015-01-01

β-Hydroxy-β-methylbutyrate (HMB) has been shown to enhance cell survival, differentiation and protein turnover in muscle, mainly activating phosphoinositide-3-kinase/protein kinase B (PI3K/Akt) and mitogen-activated protein kinases/ extracellular-signal-regulated kinases (MAPK/ERK) signaling pathways. Since these two pathways are related to neuronal survival and differentiation, in this study, we have investigated the neurotrophic effects of HMB in mouse neuroblastoma Neuro2a cells. In Neuro2a cells, HMB promotes differentiation to neurites independent from any effects on proliferation. These effects are mediated by activation of both the PI3K/Akt and the extracellular-signal-regulated kinases (ERK1/2) signaling as demonstrated by the use of specific inhibitors of these two pathways. As myocyte-enhancer factor 2 (MEF2) family of transcription factors are involved in neuronal survival and plasticity, the transcriptional activity and protein levels of MEF2 were also evaluated. HMB promoted MEF2-dependent transcriptional activity mediated by the activation of Akt and ERK1/2 pathways. Furthermore, HMB increases the expression of brain glucose transporters 1 (GLUT1) and 3 (GLUT3), and mTOR phosphorylation, which translates in a higher protein synthesis in Neuro2a cells. Furthermore, Torin1 and rapamycin effects on MEF2 transcriptional activity and HMB-dependent neurite outgrowth support that HMB acts through mTORC2. Together, these findings provide clear evidence to support an important role of HMB in neurite outgrowth. PMID:26267903

Arabidopsis MYC Transcription Factors Are the Target of Hormonal Salicylic Acid/Jasmonic Acid Cross Talk in Response to Pieris brassicae Egg Extract1[OPEN

PubMed Central

Schmiesing, André; Gouhier-Darimont, Caroline

2016-01-01

Arabidopsis (Arabidopsis thaliana) plants recognize insect eggs and activate the salicylic acid (SA) pathway. As a consequence, expression of defense genes regulated by the jasmonic acid (JA) pathway is suppressed and larval performance is enhanced. Cross talk between defense signaling pathways is common in plant-pathogen interactions, but the molecular mechanism mediating this phenomenon is poorly understood. Here, we demonstrate that egg-induced SA/JA antagonism works independently of the APETALA2/ETHYLENE RESPONSE FACTOR (AP2/ERF) transcription factor ORA59, which controls the ERF branch of the JA pathway. In addition, treatment with egg extract did not enhance expression or stability of JASMONATE ZIM-domain transcriptional repressors, and SA/JA cross talk did not involve JASMONATE ASSOCIATED MYC2-LIKEs, which are negative regulators of the JA pathway. Investigating the stability of MYC2, MYC3, and MYC4, three basic helix-loop-helix transcription factors that additively control jasmonate-related defense responses, we found that egg extract treatment strongly diminished MYC protein levels in an SA-dependent manner. Furthermore, we identified WRKY75 as a novel and essential factor controlling SA/JA cross talk. These data indicate that insect eggs target the MYC branch of the JA pathway and uncover an unexpected modulation of SA/JA antagonism depending on the biological context in which the SA pathway is activated. PMID:26884488

7α-Hydroxycholesterol Elicits TLR6-Mediated Expression of IL-23 in Monocytic Cells.

PubMed

Seo, Hyun Chul; Kim, Sun-Mi; Eo, Seong-Kug; Rhim, Byung-Yong; Kim, Koanhoi

2015-01-01

We investigated the question of whether 7-oxygenated cholesterol derivatives could affect inflammatory and/or immune responses in atherosclerosis by examining their effects on expression of IL-23 in monocytic cells. 7α-Hydroxycholesterol (7αOHChol) induced transcription of the TLR6 gene and elevated the level of cell surface TLR6 protein in THP-1 monocytic cells. Addition of an agonist of TLR6, FSL-1, to TLR6-expressing cells by treatment with 7αOHChol resulted in enhanced production of IL-23 and transcription of genes encoding the IL-23 subunit α (p19) and the IL-12 subunit β (p40). However, treatment with 7-ketocholesterol (7K) and 7β-hydroxycholesterol (7βOHChol) did not affect TLR6 expression, and addition of FSL-1 to cells treated with either 7K or 7βOHChol did not influence transcription of the genes. Pharmacological inhibition of ERK, Akt, or PI3K resulted in attenuated transcription of TLR6 induced by 7αOHChol as well as secretion of IL-23 enhanced by 7αOHChol plus FSL-1. Inhibition of p38 MAPK or JNK resulted in attenuated secretion of IL-23. These results indicate that a certain type of 7-oxygenated cholesterol like 7αOHChol can elicit TLR6-mediated expression of IL-23 by monocytic cells via PI3K/Akt and MAPKs pathways.

7α-Hydroxycholesterol Elicits TLR6-Mediated Expression of IL-23 in Monocytic Cells

PubMed Central

Seo, Hyun Chul; Kim, Sun-Mi; Eo, Seong-Kug; Rhim, Byung-Yong; Kim, Koanhoi

2015-01-01

We investigated the question of whether 7-oxygenated cholesterol derivatives could affect inflammatory and/or immune responses in atherosclerosis by examining their effects on expression of IL-23 in monocytic cells. 7α-Hydroxycholesterol (7αOHChol) induced transcription of the TLR6 gene and elevated the level of cell surface TLR6 protein in THP-1 monocytic cells. Addition of an agonist of TLR6, FSL-1, to TLR6-expressing cells by treatment with 7αOHChol resulted in enhanced production of IL-23 and transcription of genes encoding the IL-23 subunit α (p19) and the IL-12 subunit β (p40). However, treatment with 7-ketocholesterol (7K) and 7β-hydroxycholesterol (7βOHChol) did not affect TLR6 expression, and addition of FSL-1 to cells treated with either 7K or 7βOHChol did not influence transcription of the genes. Pharmacological inhibition of ERK, Akt, or PI3K resulted in attenuated transcription of TLR6 induced by 7αOHChol as well as secretion of IL-23 enhanced by 7αOHChol plus FSL-1. Inhibition of p38 MAPK or JNK resulted in attenuated secretion of IL-23. These results indicate that a certain type of 7-oxygenated cholesterol like 7αOHChol can elicit TLR6-mediated expression of IL-23 by monocytic cells via PI3K/Akt and MAPKs pathways. PMID:25593648

Arabidopsis MYC Transcription Factors Are the Target of Hormonal Salicylic Acid/Jasmonic Acid Cross Talk in Response to Pieris brassicae Egg Extract.

PubMed

Schmiesing, André; Emonet, Aurélia; Gouhier-Darimont, Caroline; Reymond, Philippe

2016-04-01

Arabidopsis (Arabidopsis thaliana) plants recognize insect eggs and activate the salicylic acid (SA) pathway. As a consequence, expression of defense genes regulated by the jasmonic acid (JA) pathway is suppressed and larval performance is enhanced. Cross talk between defense signaling pathways is common in plant-pathogen interactions, but the molecular mechanism mediating this phenomenon is poorly understood. Here, we demonstrate that egg-induced SA/JA antagonism works independently of the APETALA2/ETHYLENE RESPONSE FACTOR (AP2/ERF) transcription factor ORA59, which controls the ERF branch of the JA pathway. In addition, treatment with egg extract did not enhance expression or stability of JASMONATE ZIM-domain transcriptional repressors, and SA/JA cross talk did not involve JASMONATE ASSOCIATED MYC2-LIKEs, which are negative regulators of the JA pathway. Investigating the stability of MYC2, MYC3, and MYC4, three basic helix-loop-helix transcription factors that additively control jasmonate-related defense responses, we found that egg extract treatment strongly diminished MYC protein levels in an SA-dependent manner. Furthermore, we identified WRKY75 as a novel and essential factor controlling SA/JA cross talk. These data indicate that insect eggs target the MYC branch of the JA pathway and uncover an unexpected modulation of SA/JA antagonism depending on the biological context in which the SA pathway is activated. © 2016 American Society of Plant Biologists. All Rights Reserved.

Multiple binding sites for transcriptional repressors can produce regular bursting and enhance noise suppression

NASA Astrophysics Data System (ADS)

Lengyel, Iván M.; Morelli, Luis G.

2017-04-01

Cells may control fluctuations in protein levels by means of negative autoregulation, where transcription factors bind DNA sites to repress their own production. Theoretical studies have assumed a single binding site for the repressor, while in most species it is found that multiple binding sites are arranged in clusters. We study a stochastic description of negative autoregulation with multiple binding sites for the repressor. We find that increasing the number of binding sites induces regular bursting of gene products. By tuning the threshold for repression, we show that multiple binding sites can also suppress fluctuations. Our results highlight possible roles for the presence of multiple binding sites of negative autoregulators.

Temporal and Spatial Variations in Transcriptional Patterns among Closely Related Marine Microbial Taxa

NASA Astrophysics Data System (ADS)

Shilova, I. N.; Robidart, J.; DeLong, E.; Zehr, J. P.

2016-02-01

Marine microbial communities are complex, and even closely related marine microbial populations are genetically and physiologically diverse. Despite such great diversity, conserved and highly synchronized rhythmic transcriptional patterns have been observed in microbial communities worldwide. The current widely used approaches analyzing high-throughput sequence data from microbiomes are not designed to differentiate transcription at strain or ecotype level. We used a novel MicroArray-inspired Gene-Centric (MAGC) bioinformatics approach to discern daily transcription by individual strains in previously analyzed metatranscriptomes from two oceanic regions, California Current System and central North Pacific. The results demonstrated that marine microbial taxa (within cyanobacteria Prochlorococcus and Synechococcus, Alphaproteobacterium Pelagibacter and picoeukaryote Ostreococcus) have unique transcription patterns and respond differentially to variability in space and time in the ocean. For example, the timing of maximum transcription for the photosynthesis and pigments genes varied among Synechococcus strains in the California Current study, likely for optimizing light utilization based on their differences in genetics and physiology. While several Prochlorococcus genotypes were present in the North Pacific study, transcription of the phosphate transporter gene, pstS, in specific genotypes was negatively correlated with phosphate concentrations. These individual transcriptional patterns underlie whole microbial community responses and may be sensitive indicators of environmental conditions, including those associated with long-term environmental change. The MAGC applied here to ocean ecosystems is a promising complementary approach that can enhance the ability to analyze metatranscriptomic data from a variety of environmental microbiomes.

Molecular stress-inducing compounds increase osteoclast formation in a heat shock factor 1 protein-dependent manner.

PubMed

Chai, Ryan C; Kouspou, Michelle M; Lang, Benjamin J; Nguyen, Chau H; van der Kraan, A Gabrielle J; Vieusseux, Jessica L; Lim, Reece C; Gillespie, Matthew T; Benjamin, Ivor J; Quinn, Julian M W; Price, John T

2014-05-09

Many anticancer therapeutic agents cause bone loss, which increases the risk of fractures that severely reduce quality of life. Thus, in drug development, it is critical to identify and understand such effects. Anticancer therapeutic and HSP90 inhibitor 17-(allylamino)-17-demethoxygeldanamycin (17-AAG) causes bone loss by increasing osteoclast formation, but the mechanism underlying this is not understood. 17-AAG activates heat shock factor 1 (Hsf1), the master transcriptional regulator of heat shock/cell stress responses, which may be involved in this negative action of 17-AAG upon bone. Using mouse bone marrow and RAW264.7 osteoclast differentiation models we found that HSP90 inhibitors that induced a heat shock response also enhanced osteoclast formation, whereas HSP90 inhibitors that did not (including coumermycin A1 and novobiocin) did not affect osteoclast formation. Pharmacological inhibition or shRNAmir knockdown of Hsf1 in RAW264.7 cells as well as the use of Hsf1 null mouse bone marrow cells demonstrated that 17-AAG-enhanced osteoclast formation was Hsf1-dependent. Moreover, ectopic overexpression of Hsf1 enhanced 17-AAG effects upon osteoclast formation. Consistent with these findings, protein levels of the essential osteoclast transcription factor microphthalmia-associated transcription factor were increased by 17-AAG in an Hsf1-dependent manner. In addition to HSP90 inhibitors, we also identified that other agents that induced cellular stress, such as ethanol, doxorubicin, and methotrexate, also directly increased osteoclast formation, potentially in an Hsf1-dependent manner. These results, therefore, indicate that cellular stress can enhance osteoclast differentiation via Hsf1-dependent mechanisms and may significantly contribute to pathological and therapeutic related bone loss.

Interleukin 6 downregulates p53 expression and activity by stimulating ribosome biogenesis: a new pathway connecting inflammation to cancer

PubMed Central

Brighenti, E; Calabrese, C; Liguori, G; Giannone, F A; Trerè, D; Montanaro, L; Derenzini, M

2014-01-01

Chronic inflammation is an established risk factor for the onset of cancer, and the inflammatory cytokine IL-6 has a role in tumorigenesis by enhancing proliferation and hindering apoptosis. As factors stimulating proliferation also downregulate p53 expression by enhancing ribosome biogenesis, we hypothesized that IL-6 may cause similar changes in inflamed tissues, thus activating a mechanism that favors neoplastic transformation. Here, we showed that IL-6 downregulated the expression and activity of p53 in transformed and untransformed human cell lines. This was the consequence of IL-6-dependent stimulation of c-MYC mRNA translation, which was responsible for the upregulation of rRNA transcription. The enhanced rRNA transcription stimulated the MDM2-mediated proteasomal degradation of p53, by reducing the availability of ribosome proteins for MDM2 binding. The p53 downregulation induced the acquisition of cellular phenotypic changes characteristic of epithelial–mesenchymal transition, such as a reduced level of E-cadherin expression, increased cell invasiveness and a decreased response to cytotoxic stresses. We found that these changes also occurred in colon epithelial cells of patients with ulcerative colitis, a very representative example of chronic inflammation at high risk for tumor development. Histochemical and immunohistochemical analysis of colon biopsy samples showed an upregulation of ribosome biogenesis, a reduced expression of p53, together with a focal reduction or absence of E-cadherin expression in chronic colitis in comparison with normal mucosa samples. These changes disappeared after treatment with anti-inflammatory drugs. Taken together, the present results highlight a new mechanism that may link chronic inflammation to cancer, based on p53 downregulation, which is activated by the enhancement of rRNA transcription upon IL-6 exposure. PMID:24531714

Human Sex Determination at the Edge of Ambiguity: INHERITED XY SEX REVERSAL DUE TO ENHANCED UBIQUITINATION AND PROTEASOMAL DEGRADATION OF A MASTER TRANSCRIPTION FACTOR.

PubMed

Racca, Joseph D; Chen, Yen-Shan; Yang, Yanwu; Phillips, Nelson B; Weiss, Michael A

2016-10-14

A general problem is posed by analysis of transcriptional thresholds governing cell fate decisions in metazoan development. A model is provided by testis determination in therian mammals. Its key step, Sertoli cell differentiation in the embryonic gonadal ridge, is initiated by SRY, a Y-encoded architectural transcription factor. Mutations in human SRY cause gonadal dysgenesis leading to XY female development (Swyer syndrome). Here, we have characterized an inherited mutation compatible with either male or female somatic phenotypes as observed in an XY father and XY daughter, respectively. The mutation (a crevice-forming substitution at a conserved back surface of the SRY high mobility group box) markedly destabilizes the domain but preserves specific DNA affinity and induced DNA bend angle. On transient transfection of diverse human and rodent cell lines, the variant SRY exhibited accelerated proteasomal degradation (relative to wild type) associated with increased ubiquitination; in vitro susceptibility to ubiquitin-independent ("default") cleavage by the 20S core proteasome was unchanged. The variant's gene regulatory activity (as assessed in a cellular model of the rat embryonic XY gonadal ridge) was reduced by 2-fold relative to wild-type SRY at similar levels of mRNA expression. Chemical proteasome inhibition restored native-like SRY expression and transcriptional activity in association with restored occupancy of a sex-specific enhancer element in principal downstream gene Sox9, demonstrating that the variant SRY exhibits essentially native activity on a per molecule basis. Our findings define a novel mechanism of impaired organogenesis, accelerated ubiquitin-directed proteasomal degradation of a master transcription factor leading to a developmental decision poised at the edge of ambiguity. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

The Transcription Factor ABI4 Is Required for the Ascorbic Acid–Dependent Regulation of Growth and Regulation of Jasmonate-Dependent Defense Signaling Pathways in Arabidopsis[C][W

PubMed Central

Kerchev, Pavel I.; Pellny, Till K.; Vivancos, Pedro Diaz; Kiddle, Guy; Hedden, Peter; Driscoll, Simon; Vanacker, Hélène; Verrier, Paul; Hancock, Robert D.; Foyer, Christine H.

2011-01-01

Cellular redox homeostasis is a hub for signal integration. Interactions between redox metabolism and the ABSCISIC ACID-INSENSITIVE-4 (ABI4) transcription factor were characterized in the Arabidopsis thaliana vitamin c defective1 (vtc1) and vtc2 mutants, which are defective in ascorbic acid synthesis and show a slow growth phenotype together with enhanced abscisic acid (ABA) levels relative to the wild type (Columbia-0). The 75% decrease in the leaf ascorbate pool in the vtc2 mutants was not sufficient to adversely affect GA metabolism. The transcriptome signatures of the abi4, vtc1, and vtc2 mutants showed significant overlap, with a large number of transcription factors or signaling components similarly repressed or induced. Moreover, lincomycin-dependent changes in LIGHT HARVESTING CHLOROPHYLL A/B BINDING PROTEIN 1.1 expression were comparable in these mutants, suggesting overlapping participation in chloroplast to nucleus signaling. The slow growth phenotype of vtc2 was absent in the abi4 vtc2 double mutant, as was the sugar-insensitive phenotype of the abi4 mutant. Octadecanoid derivative-responsive AP2/ERF-domain transcription factor 47 (ORA47) and AP3 (an ABI5 binding factor) transcripts were enhanced in vtc2 but repressed in abi4 vtc2, suggesting that ABI4 and ascorbate modulate growth and defense gene expression through jasmonate signaling. We conclude that low ascorbate triggers ABA- and jasmonate-dependent signaling pathways that together regulate growth through ABI4. Moreover, cellular redox homeostasis exerts a strong influence on sugar-dependent growth regulation. PMID:21926335

Polo-like kinase 1 expression is suppressed by CCAAT/enhancer-binding protein α to mediate colon carcinoma cell differentiation and apoptosis.

PubMed

Dasgupta, Nirmalya; Thakur, Bhupesh Kumar; Ta, Atri; Das, Sayan; Banik, George; Das, Santasabuj

2017-07-01

Human polo-like kinase 1 (PLK1), a highly conserved serine/threonine kinase is a key player in several essential cell-cycle events. PLK1 is considered an oncogene and its overexpression often correlates with poor prognosis of cancers, including colorectal cancer (CRC). However, regulation of PLK1 expression in colorectal cells was never studied earlier and it is currently unknown if PLK1 regulates differentiation and apoptosis of CRC. PLK1 expression was analyzed by real-time PCR and western blotting. Transcriptional regulation was studied by reporter assay, gene knock-down, EMSA and ChIP. PLK1 expression was down-regulated during butyrate-induced differentiation of HT-29 and other CRC cells. Also, PLK1 down-regulation mediated the role of butyrate in CRC differentiation and apoptosis. We report here a novel transcriptional regulation of PLK1 by butyrate. Transcription factors CCAAT/enhancer-binding protein α (C/EBPα) and Oct-1 share an overlapping binding site over the PLK1 promoter. Elevated levels of C/EBPα by butyrate treatment of CRC cells competed out the activator protein Oct-1 from binding to the PLK1 promoter and sequestered it. Binding of C/EBPα was associated with increased deacetylation near the transcription start site (TSS) of the PLK1 promoter, which abrogated transcription through reduced recruitment of RNA polymerase II. We also found a synergistic role between the synthetic PLK1-inhibitor SBE13 and butyrate on the apoptosis of CRC cells. This study offered a novel p53-independent regulation of PLK1 during CRC differentiation and apoptosis. Down-regulation of PLK1 is one of the mechanisms underlying the anti-cancer role of dietary fibre-derived butyrate in CRC. Copyright © 2017 Elsevier B.V. All rights reserved.

The Drosophila transcriptional network is structured by microbiota.

PubMed

Dobson, Adam J; Chaston, John M; Douglas, Angela E

2016-11-25

Resident microorganisms (microbiota) have far-reaching effects on the biology of their animal hosts, with major consequences for the host's health and fitness. A full understanding of microbiota-dependent gene regulation requires analysis of the overall architecture of the host transcriptome, by identifying suites of genes that are expressed synchronously. In this study, we investigated the impact of the microbiota on gene coexpression in Drosophila. Our transcriptomic analysis, of 17 lines representative of the global genetic diversity of Drosophila, yielded a total of 11 transcriptional modules of co-expressed genes. For seven of these modules, the strength of the transcriptional network (defined as gene-gene coexpression) differed significantly between flies bearing a defined gut microbiota (gnotobiotic flies) and flies reared under microbiologically sterile conditions (axenic flies). Furthermore, gene coexpression was uniformly stronger in these microbiota-dependent modules than in both the microbiota-independent modules in gnotobiotic flies and all modules in axenic flies, indicating that the presence of the microbiota directs gene regulation in a subset of the transcriptome. The genes constituting the microbiota-dependent transcriptional modules include regulators of growth, metabolism and neurophysiology, previously implicated in mediating phenotypic effects of microbiota on Drosophila phenotype. Together these results provide the first evidence that the microbiota enhances the coexpression of specific and functionally-related genes relative to the animal's intrinsic baseline level of coexpression. Our system-wide analysis demonstrates that the presence of microbiota enhances gene coexpression, thereby structuring the transcriptional network in the animal host. This finding has potentially major implications for understanding of the mechanisms by which microbiota affect host health and fitness, and the ways in which hosts and their resident microbiota coevolve.

Conformational Control of the Binding of the Transactivation Domain of the MLL Protein and c-Myb to the KIX Domain of CREB

PubMed Central

Korkmaz, Elif Nihal; Nussinov, Ruth; Haliloğlu, Türkan

2012-01-01

The KIX domain of CBP is a transcriptional coactivator. Concomitant binding to the activation domain of proto-oncogene protein c-Myb and the transactivation domain of the trithorax group protein mixed lineage leukemia (MLL) transcription factor lead to the biologically active ternary MLL∶KIX∶c-Myb complex which plays a role in Pol II-mediated transcription. The binding of the activation domain of MLL to KIX enhances c-Myb binding. Here we carried out molecular dynamics (MD) simulations for the MLL∶KIX∶c-Myb ternary complex, its binary components and KIX with the goal of providing a mechanistic explanation for the experimental observations. The dynamic behavior revealed that the MLL binding site is allosterically coupled to the c-Myb binding site. MLL binding redistributes the conformational ensemble of KIX, leading to higher populations of states which favor c-Myb binding. The key element in the allosteric communication pathways is the KIX loop, which acts as a control mechanism to enhance subsequent binding events. We tested this conclusion by in silico mutations of loop residues in the KIX∶MLL complex and by comparing wild type and mutant dynamics through MD simulations. The loop assumed MLL binding conformation similar to that observed in the KIX∶c-Myb state which disfavors the allosteric network. The coupling with c-Myb binding site faded, abolishing the positive cooperativity observed in the presence of MLL. Our major conclusion is that by eliciting a loop-mediated allosteric switch between the different states following the binding events, transcriptional activation can be regulated. The KIX system presents an example how nature makes use of conformational control in higher level regulation of transcriptional activity and thus cellular events. PMID:22438798

Zipper-interacting protein kinase is involved in regulation of ubiquitination of the androgen receptor, thereby contributing to dynamic transcription complex assembly.

PubMed

Felten, A; Brinckmann, D; Landsberg, G; Scheidtmann, K H

2013-10-10

We have recently identified apoptosis-antagonizing transcription factor (AATF), tumor-susceptibility gene 101 (TSG101) and zipper-interacting protein kinase (ZIPK) as novel coactivators of the androgen receptor (AR). The mechanisms of coactivation remained obscure, however. Here we investigated the interplay and interdependence between these coactivators and the AR using the endogenous prostate specific antigen (PSA) gene as model for AR-target genes. Chromatin immunoprecipitation in combination with siRNA-mediated knockdown revealed that recruitment of AATF and ZIPK to the PSA enhancer was dependent on AR, whereas recruitment of TSG101 was dependent on AATF. Association of AR and its coactivators with the PSA enhancer or promoter occurred in cycles. Dissociation of AR-transcription complexes was due to degradation because inhibition of the proteasome system by MG132 caused accumulation of AR at enhancer/promoter elements. Moreover, inhibition of degradation strongly reduced transcription, indicating that continued and efficient transcription is based on initiation, degradation and reinitiation cycles. Interestingly, knockdown of ZIPK by siRNA had a similar effect as MG132, leading to reduced transcription but enhanced accumulation of AR at androgen-response elements. In addition, knockdown of ZIPK, as well as overexpression of a dominant-negative ZIPK mutant, diminished polyubiquitination of AR. Furthermore, ZIPK cooperated with the E3 ligase Mdm2 in AR-dependent transactivation, assembled into a single complex on chromatin and phosphorylated Mdm2 in vitro. These results suggest that ZIPK has a crucial role in regulation of ubiquitination and degradation of the AR, and hence promoter clearance and efficient transcription.

Mbd3/NuRD controls lymphoid cell fate and inhibits tumorigenesis by repressing a B cell transcriptional program

PubMed Central

Hamey, Fiona K.; Errami, Youssef

2017-01-01

Differentiation of lineage-committed cells from multipotent progenitors requires the establishment of accessible chromatin at lineage-specific transcriptional enhancers and promoters, which is mediated by pioneer transcription factors that recruit activating chromatin remodeling complexes. Here we show that the Mbd3/nucleosome remodeling and deacetylation (NuRD) chromatin remodeling complex opposes this transcriptional pioneering during B cell programming of multipotent lymphoid progenitors by restricting chromatin accessibility at B cell enhancers and promoters. Mbd3/NuRD-deficient lymphoid progenitors therefore prematurely activate a B cell transcriptional program and are biased toward overproduction of pro–B cells at the expense of T cell progenitors. The striking reduction in early thymic T cell progenitors results in compensatory hyperproliferation of immature thymocytes and development of T cell lymphoma. Our results reveal that Mbd3/NuRD can regulate multilineage differentiation by constraining the activation of dormant lineage-specific enhancers and promoters. In this way, Mbd3/NuRD protects the multipotency of lymphoid progenitors, preventing B cell–programming transcription factors from prematurely enacting lineage commitment. Mbd3/NuRD therefore controls the fate of lymphoid progenitors, ensuring appropriate production of lineage-committed progeny and suppressing tumor formation. PMID:28899870

Transcriptional and post-transcriptional regulation of the ionizing radiation response by ATM and p53

PubMed Central

Venkata Narayanan, Ishwarya; Paulsen, Michelle T.; Bedi, Karan; Berg, Nathan; Ljungman, Emily A.; Francia, Sofia; Veloso, Artur; Magnuson, Brian; di Fagagna, Fabrizio d’Adda; Wilson, Thomas E.; Ljungman, Mats

2017-01-01

In response to ionizing radiation (IR), cells activate a DNA damage response (DDR) pathway to re-program gene expression. Previous studies using total cellular RNA analyses have shown that the stress kinase ATM and the transcription factor p53 are integral components required for induction of IR-induced gene expression. These studies did not distinguish between changes in RNA synthesis and RNA turnover and did not address the role of enhancer elements in DDR-mediated transcriptional regulation. To determine the contribution of synthesis and degradation of RNA and monitor the activity of enhancer elements following exposure to IR, we used the recently developed Bru-seq, BruChase-seq and BruUV-seq techniques. Our results show that ATM and p53 regulate both RNA synthesis and stability as well as enhancer element activity following exposure to IR. Importantly, many genes in the p53-signaling pathway were coordinately up-regulated by both increased synthesis and RNA stability while down-regulated genes were suppressed either by reduced synthesis or stability. Our study is the first of its kind that independently assessed the effects of ionizing radiation on transcription and post-transcriptional regulation in normal human cells. PMID:28256581

Loss of Activity-Induced Phosphorylation of MeCP2 Enhances Synaptogenesis, LTP, and Spatial Memory

PubMed Central

Li, Hongda; Zhong, Xiaofen; Chau, Kevin Fongching; Williams, Emily Cunningham; Chang, Qiang

2012-01-01

DNA methylation-dependent epigenetic mechanisms underlie the development and function of the mammalian brain. MeCP2 expresses highly in neurons, and functions as a molecular linker between DNA methylation, chromatin remodeling and transcription regulation. Previous in vitro studies showed neuronal activity-induced phosphorylation (NAIP) of MeCP2 precedes its release from the Bdnf promoter and the ensuing Bdnf transcription. However, the in vivo function of this phosphorylation event remains elusive. We generated knockin mice that lack NAIP of MeCP2, and show here the Mecp2 phospho-mutant mice perform better in hippocampus-dependent memory tests, present enhanced LTP at two synapses in the hippocampus, and show increased excitatory synaptogenesis. At the molecular level, the phospho-mutant MeCP2 protein binds more tightly to several MeCP2 target gene promoters and alters the expression of these genes. Our results supply the first genetic evidence that NAIP of MeCP2 is required in modulating dynamic functions of the adult mouse brain. PMID:21765426

Synthetic Gene Expression Circuits for Designing Precision Tools in Oncology

PubMed Central

Re, Angela

2017-01-01

Precision medicine in oncology needs to enhance its capabilities to match diagnostic and therapeutic technologies to individual patients. Synthetic biology streamlines the design and construction of functionalized devices through standardization and rational engineering of basic biological elements decoupled from their natural context. Remarkable improvements have opened the prospects for the availability of synthetic devices of enhanced mechanism clarity, robustness, sensitivity, as well as scalability and portability, which might bring new capabilities in precision cancer medicine implementations. In this review, we begin by presenting a brief overview of some of the major advances in the engineering of synthetic genetic circuits aimed to the control of gene expression and operating at the transcriptional, post-transcriptional/translational, and post-translational levels. We then focus on engineering synthetic circuits as an enabling methodology for the successful establishment of precision technologies in oncology. We describe significant advancements in our capabilities to tailor synthetic genetic circuits to specific applications in tumor diagnosis, tumor cell- and gene-based therapy, and drug delivery. PMID:28894736

Analysis of genetically modified red-fleshed apples reveals effects on growth and consumer attributes.

PubMed

Espley, Richard V; Bovy, Arnaud; Bava, Christina; Jaeger, Sara R; Tomes, Sumathi; Norling, Cara; Crawford, Jonathan; Rowan, Daryl; McGhie, Tony K; Brendolise, Cyril; Putterill, Jo; Schouten, Henk J; Hellens, Roger P; Allan, Andrew C

2013-05-01

Consumers of whole foods, such as fruits, demand consistent high quality and seek varieties with enhanced health properties, convenience or novel taste. We have raised the polyphenolic content of apple by genetic engineering of the anthocyanin pathway using the apple transcription factor MYB10. These apples have very high concentrations of foliar, flower and fruit anthocyanins, especially in the fruit peel. Independent lines were examined for impacts on tree growth, photosynthesis and fruit characteristics. Fruit were analysed for changes in metabolite and transcript levels. Fruit were also used in taste trials to study the consumer perception of such a novel apple. No negative taste attributes were associated with the elevated anthocyanins. Modification with this one gene provides near isogenic material and allows us to examine the effects on an established cultivar, with a view to enhancing consumer appeal independently of other fruit qualities. © 2012 The Authors Plant Biotechnology Journal © 2012 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd.

Effects of granulation on organic acid metabolism and its relation to mineral elements in Citrus grandis juice sacs.

PubMed

Wang, Xian-You; Wang, Ping; Qi, Yi-Ping; Zhou, Chen-Ping; Yang, Lin-Tong; Liao, Xin-Yan; Wang, Liu-Qing; Zhu, Dong-Huang; Chen, Li-Song

2014-02-15

We investigated the effects of granulation on organic acid metabolism and its relation to mineral elements in 'Guanximiyou' pummelo (Citrus grandis) juice sacs. Granulated juice sacs had decreased concentrations of citrate and isocitrate, thus lowering juice sac acidity. By contrast, malate concentration was higher in granulated juice sacs than in normal ones. The reduction in citrate concentration might be caused by increased degradation, as indicated by enhanced aconitase activity, whilst the increase in malate concentration might be caused by increased biosynthesis, as indicated by enhanced phosphoenolpyruvate carboxylase (PEPC). Real time quantitative reverse transcription PCR (qRT-PCR) analysis showed that the activities of most acid-metabolizing enzymes were regulated at the transcriptional level, whilst post-translational modifications might influence the PEPC activity. Granulation led to increased accumulation of mineral elements (especially phosphorus, magnesium, sulphur, zinc and copper) in juice sacs, which might be involved in the incidence of granulation in pummelo fruits. Copyright © 2013 Elsevier Ltd. All rights reserved.

Green tea polyphenols as potent enhancers of glucocorticoid-induced mouse mammary tumor virus gene expression.

PubMed

Abe, I; Umehara, K; Morita, R; Nemoto, K; Degawa, M; Noguchi, H

2001-02-16

The effect of natural and synthetic galloyl esters on glucocorticoid-induced gene expression was evaluated by using rat fibroblast 3Y1 cells stably transfected with a luciferase reporter gene under the transcriptional regulation of the mouse mammary tumor virus promoter. The glucocorticoid-induced gene transcription was strongly suppressed by synthetic alkyl esters; n-dodecyl gallate showed the most potent inhibition (66% inhibition at 10 microM), which was far more potent than that of crude tannic acid. n-Octyl and n-cetyl gallate also showed good inhibition, while gallic acid itself was not so active, suggesting that the presence of hydrophobic side chain is important for the suppressive effect. On the other hand, surprisingly, green tea gallocatechins, (-)-epigallocatechin-3-O-gallate and theasinensin A, potently enhanced the promoter activity (182 and 247% activity at 1 microM, respectively). The regulation of the level of the glucocorticoid-induced gene expression by the antioxidative gallates is of great interest from a therapeutic point of view.

TaNAC29, a NAC transcription factor from wheat, enhances salt and drought tolerance in transgenic Arabidopsis.

PubMed

Huang, Quanjun; Wang, Yan; Li, Bin; Chang, Junli; Chen, Mingjie; Li, Kexiu; Yang, Guangxiao; He, Guangyuan

2015-11-04

NAC (NAM, ATAF, and CUC) transcription factors play important roles in plant biological processes, including phytohormone homeostasis, plant development, and in responses to various environmental stresses. TaNAC29 was introduced into Arabidopsis using the Agrobacterium tumefaciens-mediated floral dipping method. TaNAC29-overexpression plants were subjected to salt and drought stresses for examining gene functions. To investigate tolerant mechanisms involved in the salt and drought responses, expression of related marker genes analyses were conducted, and related physiological indices were also measured. Expressions of genes were analyzed by quantitative real-time polymerase chain reaction (qRT-PCR). A novel NAC transcription factor gene, designated TaNAC29, was isolated from bread wheat (Triticum aestivum). Sequence alignment suggested that TaNAC29 might be located on chromosome 2BS. TaNAC29 was localized to the nucleus in wheat protoplasts, and proved to have transcriptional activation activities in yeast. TaNAC29 was expressed at a higher level in the leaves, and expression levels were much higher in senescent leaves, indicating that TaNAC29 might be involved in the senescence process. TaNAC29 transcripts were increased following treatments with salt, PEG6000, H2O2, and abscisic acid (ABA). To examine TaNAC29 function, transgenic Arabidopsis plants overexpressing TaNAC29 were generated. Germination and root length assays of transgenic plants demonstrated that TaNAC29 overexpression plants had enhanced tolerances to high salinity and dehydration, and exhibited an ABA-hypersensitive response. When grown in the greenhouse, TaNAC29-overexpression plants showed the same tolerance response to salt and drought stresses at both the vegetative and reproductive period, and had delayed bolting and flowering in the reproductive period. Moreover, TaNAC29 overexpression plants accumulated lesser malondialdehyde (MDA), H2O2, while had higher superoxide dismutase (SOD) and catalase (CAT) activities under high salinity and/or dehydration stress. Our results demonstrate that TaNAC29 plays important roles in the senescence process and response to salt and drought stresses. ABA signal pathway and antioxidant enzyme systems are involved in TaNAC29-mediated stress tolerance mechanisms.

Expression of geminiviral AC2 RNA silencing suppressor changes sugar and jasmonate responsive gene expression in transgenic tobacco plants

PubMed Central

2012-01-01

Background RNA-silencing is a conserved gene regulation and surveillance machinery, which in plants, is also used as major defence mechanism against viruses. Various virus-specific dsRNA structures are recognized by the silencing machinery leading to degradation of the viral RNAs or, as in case of begomoviruses, to methylation of their DNA genomes. Viruses produce specific RNA silencing suppressor (RSS) proteins to prevent these host defence mechanisms, and as these interfere with the silencing machinery they also disturb the endogenous silencing reactions. In this paper, we describe how expression of AC2 RSS, derived from African cassava mosaic geminivirus changes transcription profile in tobacco (Nicotiana tabacum) leaves and in flowers. Results Expression of AC2 RSS in transgenic tobacco plants induced clear phenotypic changes both in leaves and in flowers. Transcriptomes of these plants were strongly altered, with total of 1118 and 251 differentially expressed genes in leaves and flowers, respectively. The three most up-regulated transcript groups were related to stress, cell wall modifications and signalling, whereas the three most down-regulated groups were related to translation, photosynthesis and transcription. It appears that many of the gene expression alterations appeared to be related to enhanced biosynthesis of jasmonate and ethylene, and consequent enhancement of the genes and pathways that are regulated by these hormones, or to the retrograde signalling caused by the reduced photosynthetic activity and sugar metabolism. Comparison of these results to a previous transcriptional profiling of HC-Pro RSS-expressing plants revealed that some of same genes were induced by both RSSs, but their expression levels were typically higher in AC2 than in HC-Pro RSS expressing plants. All in all, a large number of transcript alterations were found to be specific to each of the RSS expressing transgenic plants. Conclusions AC2 RSS in transgenic tobacco plants interferes with the silencing machinery. It causes stress and defence reactions for instance via induction of the jasmonate and ethylene biosynthesis, and by consequent gene expression alteration regulated by these hormones. The changed sugar metabolism may cause significant down-regulation of genes encoding ribosomal proteins, thus reducing the general translation level. PMID:23130567

HDT701, a histone H4 deacetylase, negatively regulates plant innate immunity by modulating histone H4 acetylation of defense-related genes in rice.

PubMed

Ding, Bo; Bellizzi, Maria del Rosario; Ning, Yuese; Meyers, Blake C; Wang, Guo-Liang

2012-09-01

Histone acetylation and deacetylation play an important role in the modification of chromatin structure and regulation of gene expression in eukaryotes. Chromatin acetylation status is modulated antagonistically by histone acetyltransferases and histone deacetylases (HDACs). In this study, we characterized the function of histone deacetylase701 (HDT701), a member of the plant-specific HD2 subfamily of HDACs, in rice (Oryza sativa) innate immunity. Transcription of HDT701 is increased in the compatible reaction and decreased in the incompatible reaction after infection by the fungal pathogen Magnaporthe oryzae. Overexpression of HDT701 in transgenic rice leads to decreased levels of histone H4 acetylation and enhanced susceptibility to the rice pathogens M. oryzae and Xanthomonas oryzae pv oryzae (Xoo). By contrast, silencing of HDT701 in transgenic rice causes elevated levels of histone H4 acetylation and elevated transcription of pattern recognition receptor (PRR) and defense-related genes, increased generation of reactive oxygen species after pathogen-associated molecular pattern elicitor treatment, as well as enhanced resistance to both M. oryzae and Xoo. We also found that HDT701 can bind to defense-related genes to regulate their expression. Taken together, these results demonstrate that HDT701 negatively regulates innate immunity by modulating the levels of histone H4 acetylation of PRR and defense-related genes in rice.

Enhanced triterpene accumulation in Panax ginseng hairy roots overexpressing mevalonate-5-pyrophosphate decarboxylase and farnesyl pyrophosphate synthase.

PubMed

Kim, Yong-Kyoung; Kim, Yeon Bok; Uddin, Md Romij; Lee, Sanghyun; Kim, Soo-Un; Park, Sang Un

2014-10-17

To elucidate the function of mevalonate-5-pyrophosphate decarboxylase (MVD) and farnesyl pyrophosphate synthase (FPS) in triterpene biosynthesis, the genes governing the expression of these enzymes were transformed into Panax ginseng hairy roots. All the transgenic lines showed higher expression levels of PgMVD and PgFPS than that by the wild-type control. Among the hairy root lines transformed with PgMVD, M18 showed the highest level of transcription compared to the control (14.5-fold higher). Transcriptions of F11 and F20 transformed with PgFPS showed 11.1-fold higher level compared with control. In triterpene analysis, M25 of PgMVD produced 4.4-fold higher stigmasterol content (138.95 μg/100 mg, dry weight [DW]) than that by the control; F17 of PgFPS showed the highest total ginsenoside (36.42 mg/g DW) content, which was 2.4-fold higher compared with control. Our results indicate that metabolic engineering in P. ginseng was successfully achieved through Agrobacterium rhizogenes-mediated transformation and that the accumulation of phytosterols and ginsenosides was enhanced by introducing the PgMVD and PgFPS genes into the hairy roots of the plant. Our results suggest that PgMVD and PgFPS play an important role in the triterpene biosynthesis of P. ginseng.

Translational induction of heat shock transcription factor σ32: evidence for a built-in RNA thermosensor

PubMed Central

Morita, Miyo Terao; Tanaka, Yoshiyuki; Kodama, Takashi S.; Kyogoku, Yoshimasa; Yanagi, Hideki; Yura, Takashi

1999-01-01

Induction of heat shock proteins in Escherichia coli is primarily caused by increased cellular levels of the heat shock σ-factor σ32 encoded by the rpoH gene. Increased σ32 levels result from both enhanced synthesis and stabilization. Previous work indicated that σ32 synthesis is induced at the translational level and is mediated by the mRNA secondary structure formed within the 5′-coding sequence of rpoH, including the translation initiation region. To understand the mechanism of heat induction of σ32 synthesis further, we analyzed expression of rpoH–lacZ gene fusions with altered stability of mRNA structure before and after heat shock. A clear correlation was found between the stability and expression or the extent of heat induction. Temperature-melting profiles of mRNAs with or without mutations correlated well with the expression patterns of fusion genes carrying the corresponding mutations in vivo. Furthermore, temperature dependence of mRNA–30S ribosome–tRNAfMet complex formation with wild-type or mutant mRNAs in vitro agreed well with that of the expression of gene fusions in vivo. Our results support a novel mechanism in which partial melting of mRNA secondary structure at high temperature enhances ribosome entry and translational initiation without involvement of other cellular components, that is, intrinsic mRNA stability controls synthesis of a transcriptional regulator. PMID:10090722

An sRNA and Cold Shock Protein Homolog-Based Feedforward Loop Post-transcriptionally Controls Cell Cycle Master Regulator CtrA

PubMed Central

Robledo, Marta; Schlüter, Jan-Philip; Loehr, Lars O.; Linne, Uwe; Albaum, Stefan P.; Jiménez-Zurdo, José I.; Becker, Anke

2018-01-01

Adjustment of cell cycle progression is crucial for bacterial survival and adaptation under adverse conditions. However, the understanding of modulation of cell cycle control in response to environmental changes is rather incomplete. In α-proteobacteria, the broadly conserved cell cycle master regulator CtrA underlies multiple levels of control, including coupling of cell cycle and cell differentiation. CtrA levels are known to be tightly controlled through diverse transcriptional and post-translational mechanisms. Here, small RNA (sRNA)-mediated post-transcriptional regulation is uncovered as an additional level of CtrA fine-tuning. Computational predictions as well as transcriptome and proteome studies consistently suggested targeting of ctrA and the putative cold shock chaperone cspA5 mRNAs by the trans-encoded sRNA (trans-sRNA) GspR (formerly SmelC775) in several Sinorhizobium species. GspR strongly accumulated in the stationary growth phase, especially in minimal medium (MM) cultures. Lack of the gspR locus confers a fitness disadvantage in competition with the wild type, while its overproduction hampers cell growth, suggesting that this riboregulator interferes with cell cycle progression. An eGFP-based reporter in vivo assay, involving wild-type and mutant sRNA and mRNA pairs, experimentally confirmed GspR-dependent post-transcriptional down-regulation of ctrA and cspA5 expression, which most likely occurs through base-pairing to the respective mRNA. The energetically favored secondary structure of GspR is predicted to comprise three stem-loop domains, with stem-loop 1 and stem-loop 3 targeting ctrA and cspA5 mRNA, respectively. Moreover, this work reports evidence for post-transcriptional control of ctrA by CspA5. Thus, this regulation and GspR-mediated post-transcriptional repression of ctrA and cspA5 expression constitute a coherent feed-forward loop, which may enhance the negative effect of GspR on CtrA levels. This novel regulatory circuit involving the riboregulator GspR, CtrA, and a cold shock chaperone may contribute to fine-tuning of ctrA expression. PMID:29740411

An sRNA and Cold Shock Protein Homolog-Based Feedforward Loop Post-transcriptionally Controls Cell Cycle Master Regulator CtrA.

PubMed

Robledo, Marta; Schlüter, Jan-Philip; Loehr, Lars O; Linne, Uwe; Albaum, Stefan P; Jiménez-Zurdo, José I; Becker, Anke

2018-01-01

Adjustment of cell cycle progression is crucial for bacterial survival and adaptation under adverse conditions. However, the understanding of modulation of cell cycle control in response to environmental changes is rather incomplete. In α-proteobacteria, the broadly conserved cell cycle master regulator CtrA underlies multiple levels of control, including coupling of cell cycle and cell differentiation. CtrA levels are known to be tightly controlled through diverse transcriptional and post-translational mechanisms. Here, small RNA (sRNA)-mediated post-transcriptional regulation is uncovered as an additional level of CtrA fine-tuning. Computational predictions as well as transcriptome and proteome studies consistently suggested targeting of ctrA and the putative cold shock chaperone cspA5 mRNAs by the trans- encoded sRNA ( trans- sRNA) GspR (formerly SmelC775) in several Sinorhizobium species. GspR strongly accumulated in the stationary growth phase, especially in minimal medium (MM) cultures. Lack of the gspR locus confers a fitness disadvantage in competition with the wild type, while its overproduction hampers cell growth, suggesting that this riboregulator interferes with cell cycle progression. An eGFP-based reporter in vivo assay, involving wild-type and mutant sRNA and mRNA pairs, experimentally confirmed GspR-dependent post-transcriptional down-regulation of ctrA and cspA5 expression, which most likely occurs through base-pairing to the respective mRNA. The energetically favored secondary structure of GspR is predicted to comprise three stem-loop domains, with stem-loop 1 and stem-loop 3 targeting ctrA and cspA5 mRNA, respectively. Moreover, this work reports evidence for post-transcriptional control of ctrA by CspA5. Thus, this regulation and GspR-mediated post-transcriptional repression of ctrA and cspA5 expression constitute a coherent feed-forward loop, which may enhance the negative effect of GspR on CtrA levels. This novel regulatory circuit involving the riboregulator GspR, CtrA, and a cold shock chaperone may contribute to fine-tuning of ctrA expression.

Cyclic AMP Enhances TGFβ Responses of Breast Cancer Cells by Upregulating TGFβ Receptor I Expression

PubMed Central

Oerlecke, Ilka; Bauer, Elke; Dittmer, Angela; Leyh, Benjamin; Dittmer, Jürgen

2013-01-01

Cellular functions are regulated by complex networks of many different signaling pathways. The TGFβ and cAMP pathways are of particular importance in tumor progression. We analyzed the cross-talk between these pathways in breast cancer cells in 2D and 3D cultures. We found that cAMP potentiated TGFβ-dependent gene expression by enhancing Smad3 phosphorylation. Higher levels of total Smad3, as observed in 3D-cultured cells, blocked this effect. Two Smad3 regulating proteins, YAP (Yes-associated protein) and TβRI (TGFβ receptor 1), were responsive to cAMP. While YAP had little effect on TGFβ-dependent expression and Smad3 phosphorylation, a constitutively active form of TβRI mimicked the cAMP effect on TGFβ signaling. In 3D-cultured cells, which show much higher levels of TβRI and cAMP, TβRI was unresponsive to cAMP. Upregulation of TβRI expression by cAMP was dependent on transcription. A proximal TβRI promoter fragment was moderately, but significantly activated by cAMP suggesting that cAMP increases TβRI expression at least partially by activating TβRI transcription. Neither the cAMP-responsive element binding protein (CREB) nor the TβRI-regulating transcription factor Six1 was required for the cAMP effect. An inhibitor of histone deacetylases alone or together with cAMP increased TβRI expression by a similar extent as cAMP alone suggesting that cAMP may exert its effect by interfering with histone acetylation. Along with an additive stimulatory effect of cAMP and TGFβ on p21 expression an additive inhibitory effect of these agents on proliferation was observed. Finally, we show that mesenchymal stem cells that interact with breast cancer cells can simultaneously activate the cAMP and TGFβ pathways. In summary, these data suggest that combined effects of cAMP and TGFβ, as e.g. induced by mesenchymal stem cells, involve the upregulation of TβRI expression on the transcriptional level, likely due to changes in histone acetylation. As a consequence, cancer cell functions such as proliferation are affected. PMID:23349840

Metallothionein expression in chloroplasts enhances mercury accumulation and phytoremediation capability

PubMed Central

Ruiz, Oscar N.; Alvarez, Derry; Torres, Cesar; Roman, Laura; Daniell, Henry

2015-01-01

Summary Genetic engineering to enhance mercury phytoremediation has been accomplished by expression of the merAB genes that protects the cell by converting Hg[II] into Hg[0] which volatilizes from the cell. A drawback of this approach is that toxic Hg is released back into the environment. A better phytoremediation strategy would be to accumulate mercury inside plants for subsequent retrieval. We report here the development of a transplastomic approach to express the mouse metallothionein gene (mt1) and accumulate mercury in high concentrations within plant cells. Real-time PCR analysis showed that up to 1284 copies of the mt1 gene were found per cell when compared with 1326 copies of the 16S rrn gene, thereby attaining homoplasmy. Past studies in chloroplast transformation used qualitative Southern blots to evaluate indirectly transgene copy number, whereas we used real-time PCR for the first time to establish homoplasmy and estimate transgene copy number and transcript levels. The mt1 transcript levels were very high with 183 000 copies per ng of RNA or 41% the abundance of the 16S rrn transcripts. The transplastomic lines were resistant up to 20 μm mercury and maintained high chlorophyll content and biomass. Although the transgenic plants accumulated high concentrations of mercury in all tissues, leaves accumulated up to 106 ng, indicating active phytoremediation and translocation of mercury. Such accumulation of mercury in plant tissues facilitates proper disposal or recycling. This study reports, for the first time, the use of metallothioniens in plants for mercury phytoremediation. Chloroplast genetic engineering approach is useful to express metal-scavenging proteins for phytoremediation. PMID:21518240

Loss of Runx2 sensitises osteosarcoma to chemotherapy-induced apoptosis

PubMed Central

Roos, Alison; Satterfield, Laura; Zhao, Shuying; Fuja, Daniel; Shuck, Ryan; Hicks, M John; Donehower, Lawrence A; Yustein, Jason T

2015-01-01

Background: Osteosarcoma (OS) is the most common bone malignancy in the paediatric population, principally affecting adolescents and young adults. Minimal advancements in patient prognosis have been made over the past two decades because of the poor understanding of disease biology. Runx2, a critical transcription factor in bone development, is frequently amplified and overexpressed in OS. However, the molecular and biological consequences of Runx2 overexpression remain unclear. Methods: si/shRNA and overexpression technology to alter Runx2 levels in OS cells. In vitro assessment of doxorubicin (doxo)-induced apoptosis and in vivo chemosensitivity studies. Small-molecule inhibitor of c-Myc transcriptional activity was used to assess its role. Results: Loss of Runx2 sensitises cells to doxo-induced apoptosis both in vitro and in vivo. Furthermore, in conjunction with chemotherapy, decreasing Runx2 protein levels activates both the intrinsic and extrinsic apoptotic pathways. Transplanted tumour studies demonstrated that loss of endogenous Runx2 protein expression enhances caspase-3 cleavage and tumour necrosis in response to chemotherapy. Finally, upon doxo-treated Runx2 knockdown OS cells there was evidence of enhanced c-Myc expression and transcriptional activity. Inhibition of c-Myc under these conditions resulted in decreased activation of apoptosis, therefore insinuating a role for c-Myc in dox-induced activation of apoptotic pathways. Conclusions: Therefore, we have established a novel molecular mechanism by which Runx2 provides a chemoprotective role in OS, indicating that in conjunction to standard chemotherapy, targeting Runx2 may be a new therapeutic strategy for patients with OS. PMID:26528706

After-Ripening Induced Transcriptional Changes of Hormonal Genes in Wheat Seeds: The Cases of Brassinosteroids, Ethylene, Cytokinin and Salicylic Acid

PubMed Central

Yao, Zhen; Jordan, Mark C.; Park, Seokhoon; Ayele, Belay T.

2014-01-01

Maintenance and release of seed dormancy is regulated by plant hormones; their levels and seed sensitivity being the critical factors. This study reports transcriptional regulation of brassinosteroids (BR), ethylene (ET), cytokinin (CK) and salicylic acid (SA) related wheat genes by after-ripening, a period of dry storage that decays dormancy. Changes in the expression of hormonal genes due to seed after-ripening did not occur in the anhydrobiotic state but rather in the hydrated state. After-ripening induced dormancy decay appears to be associated with imbibition mediated increase in the synthesis and signalling of BR, via transcriptional activation of de-etiolated2, dwarf4 and brassinosteroid signaling kinase, and repression of brassinosteroid insensitive 2. Our analysis is also suggestive of the significance of increased ET production, as reflected by enhanced transcription of 1-aminocyclopropane-1-carboxylic acid oxidase in after-ripened seeds, and tight regulation of seed response to ET in regulating dormancy decay. Differential transcriptions of lonely guy, zeatin O-glucosyltransferases and cytokinin oxidases, and pseudo-response regulator between dormant and after-ripened seeds implicate CK in the regulation of seed dormancy in wheat. Our analysis also reflects the association of dormancy decay in wheat with seed SA level and NPR independent SA signaling that appear to be regulated transcriptionally by phenylalanine ammonia lyase, and whirly and suppressor of npr1 inducible1 genes, respectively. Co-expression clustering of the hormonal genes implies the significance of synergistic and antagonistic interaction between the different plant hormones in regulating wheat seed dormancy. These results contribute to further our understanding of the molecular features controlling seed dormancy in wheat. PMID:24498132

Cell division and turgor mediate enhanced plant growth in Arabidopsis plants treated with the bacterial signalling molecule lumichrome.

PubMed

Pholo, Motlalepula; Coetzee, Beatrix; Maree, Hans J; Young, Philip R; Lloyd, James R; Kossmann, Jens; Hills, Paul N

2018-05-17

Transcriptomic analysis indicates that the bacterial signalling molecule lumichrome enhances plant growth through a combination of enhanced cell division and cell enlargement, and possibly enhances photosynthesis. Lumichrome (7,8 dimethylalloxazine), a novel multitrophic signal molecule produced by Sinorhizobium meliloti bacteria, has previously been shown to elicit growth promotion in different plant species (Phillips et al. in Proc Natl Acad Sci USA 96:12275-12280, https://doi.org/10.1073/pnas.96.22.12275 , 1999). However, the molecular mechanisms that underlie this plant growth promotion remain obscure. Global transcript profiling using RNA-seq suggests that lumichrome enhances growth by inducing genes impacting on turgor driven growth and mitotic cell cycle that ensures the integration of cell division and expansion of developing leaves. The abundance of XTH9 and XPA4 transcripts was attributed to improved mediation of cell-wall loosening to allow turgor-driven cell enlargement. Mitotic CYCD3.3, CYCA1.1, SP1L3, RSW7 and PDF1 transcripts were increased in lumichrome-treated Arabidopsis thaliana plants, suggesting enhanced growth was underpinned by increased cell differentiation and expansion with a consequential increase in biomass. Synergistic ethylene-auxin cross-talk was also observed through reciprocal over-expression of ACO1 and SAUR54, in which ethylene activates the auxin signalling pathway and regulates Arabidopsis growth by both stimulating auxin biosynthesis and modulating the auxin transport machinery to the leaves. Decreased transcription of jasmonate biosynthesis and responsive-related transcripts (LOX2; LOX3; LOX6; JAL34; JR1) might contribute towards suppression of the negative effects of methyl jasmonate (MeJa) such as chlorophyll loss and decreases in RuBisCO and photosynthesis. This work contributes towards a deeper understanding of how lumichrome enhances plant growth and development.

HBS1L-MYB intergenic variants modulate fetal hemoglobin via long-range MYB enhancers

PubMed Central

Stadhouders, Ralph; Aktuna, Suleyman; Thongjuea, Supat; Aghajanirefah, Ali; Pourfarzad, Farzin; van IJcken, Wilfred; Lenhard, Boris; Rooks, Helen; Best, Steve; Menzel, Stephan; Grosveld, Frank; Thein, Swee Lay; Soler, Eric

2014-01-01

Genetic studies have identified common variants within the intergenic region (HBS1L-MYB) between GTP-binding elongation factor HBS1L and myeloblastosis oncogene MYB on chromosome 6q that are associated with elevated fetal hemoglobin (HbF) levels and alterations of other clinically important human erythroid traits. It is unclear how these noncoding sequence variants affect multiple erythrocyte characteristics. Here, we determined that several HBS1L-MYB intergenic variants affect regulatory elements that are occupied by key erythroid transcription factors within this region. These elements interact with MYB, a critical regulator of erythroid development and HbF levels. We found that several HBS1L-MYB intergenic variants reduce transcription factor binding, affecting long-range interactions with MYB and MYB expression levels. These data provide a functional explanation for the genetic association of HBS1L-MYB intergenic polymorphisms with human erythroid traits and HbF levels. Our results further designate MYB as a target for therapeutic induction of HbF to ameliorate sickle cell and β-thalassemia disease severity. PMID:24614105

Hyperhomocysteinemia associated skeletal muscle weakness involves mitochondrial dysfunction and epigenetic modifications

PubMed Central

Veeranki, Sudhakar; Winchester, Lee J; Tyagi, Suresh C

2015-01-01

HHcy has been implicated in elderly frailty, but the underlying mechanisms are poorly understood. Using C57 and CBS+/- mice and C2C12 cell line, we investigated mechanisms behind HHcy induced skeletal muscle weakness and fatigability. Possible alterations in metabolic capacity (levels of LDH, CS, MM-CK and COX-IV), in structural proteins (levels of dystrophin) and in mitochondrial function (ATP production) were examined. An exercise regimen was employed to reverse HHcy induced changes. CBS+/- mice exhibited more fatigability, and generated less contraction force. No significant changes in muscle morphology were observed. However, there is corresponding reduction in large muscle fiber number in CBS+/- mice. Excess fatigability was not due to changes in key enzymes involved in metabolism, but was due to reduced ATP levels. A marginal reduction in dystrophin levels along with a decrease in mitochondrial transcription factor A (mtTFA) were observed. There was also an increase in the mir-31, and mir-494 quantities that were implicated in dystrophin and mtTFA regulation respectively. The molecular changes elevated during HHcy, with the exception of dystrophin levels, were reversed after exercise. In addition, amount of NRF-1, one of the transcriptional regulators of mtTFA, was significantly decreased. Furthermore, there was enhancement in mir-494 levels and a concomitant decline in mtTFA protein quantity in homocysteine treated cells. These changes in C2C12 cells were also accompanied by an increase in DNMT3a and DNMT3b proteins and global DNA methylation levels. Together, these results suggest that HHcy plays a causal role in enhanced fatigability through mitochondrial dysfunction which involves epigenetic changes. PMID:25615794

Two-level inhibition of galK expression by Spot 42: Degradation of mRNA mK2 and enhanced transcription termination before the galK gene

PubMed Central

Wang, Xun; Ji, Sang Chun; Jeon, Heung Jin; Lee, Yonho; Lim, Heon M.

2015-01-01

The Escherichia coli gal operon has the structure Pgal-galE-galT-galK-galM. During early log growth, a gradient in gene expression, named type 2 polarity, is established, as follows: galE > galT > galK > galM. However, during late-log growth, type 1 polarity is established in which galK is greater than galT, as follows: galE > galK > galT > galM. We found that type 2 polarity occurs as a result of the down-regulation of galK, which is caused by two different molecular mechanisms: Spot 42-mediated degradation of the galK-specific mRNA, mK2, and Spot 42-mediated Rho-dependent transcription termination at the end of galT. Because the concentration of Spot 42 drops during the transition period of the polarity type switch, these results demonstrate that type 1 polarity is the result of alleviation of Spot 42-mediated galK down-regulation. Because the Spot 42-binding site overlaps with a putative Rho-binding site, a molecular mechanism is proposed to explain how Spot 42, possibly with Hfq, enhances Rho-mediated transcription termination at the end of galT. PMID:26045496

Evidence for transcriptional interference in a dual-luciferase reporter system.

PubMed

Wu, Guo-Qing; Wang, Xiao; Zhou, Hong-Ying; Chai, Ke-Qun; Xue, Qian; Zheng, Ai-Hong; Zhu, Xiu-Ming; Xiao, Jian-Yong; Ying, Xu-Hua; Wang, Fu-Wei; Rui, Tao; Xu, Li-Yun; Zhang, Yong-Kui; Liao, Yi-Ji; Xie, Dan; Lu, Li-Qin; Huang, Dong-Sheng

2015-12-01

The dual-luciferase reporter assay is widely used for microRNA target identification and the functional validation of predicted targets. To determine whether curcumin regulates expression of the histone methyltransferase enhancer of zeste homolog 2 (EZH2) by targeting its 3'untranslated region (3'UTR), two luciferase reporter systems containing exactly the same sequence of the EZH2 3'UTR were used to perform dual-luciferase reporter assays. Surprisingly, there were certain discrepancies between the luciferase activities derived from these two reporter constructs. We normalized luciferase activity to an internal control to determine the amount of the reporter construct successfully transfected into cells, induced a transcriptional block with flavopiridol, quantified renilla luciferase mRNA levels, and compared the absolute luciferase activity among the different groups. The results suggested that curcumin promoted the transcription of the luciferase genes located downstream of the simian vacuolating virus 40 (SV40) early enhancer/promoter, but not those located downstream of the human cytomegalovirus (CMV) immediate-early or the herpes simplex virus thymidine kinase (HSV-TK) promoters. These results explain the discrepancies between the two luciferase reporter systems. The current study underscores the importance of taking caution when interpreting the results of dual-luciferase reporter assays and provides strategies to overcome the potential pitfall accompanying dual-luciferase reporter systems.

Evidence for transcriptional interference in a dual-luciferase reporter system

PubMed Central

Wu, Guo-Qing; Wang, Xiao; Zhou, Hong-Ying; Chai, Ke-Qun; Xue, Qian; Zheng, Ai-Hong; Zhu, Xiu-Ming; Xiao, Jian-Yong; Ying, Xu-Hua; Wang, Fu-Wei; Rui, Tao; Xu, Li-Yun; Zhang, Yong-Kui; Liao, Yi-Ji; Xie, Dan; Lu, Li-Qin; Huang, Dong-Sheng

2015-01-01

The dual-luciferase reporter assay is widely used for microRNA target identification and the functional validation of predicted targets. To determine whether curcumin regulates expression of the histone methyltransferase enhancer of zeste homolog 2 (EZH2) by targeting its 3′untranslated region (3′UTR), two luciferase reporter systems containing exactly the same sequence of the EZH2 3′UTR were used to perform dual-luciferase reporter assays. Surprisingly, there were certain discrepancies between the luciferase activities derived from these two reporter constructs. We normalized luciferase activity to an internal control to determine the amount of the reporter construct successfully transfected into cells, induced a transcriptional block with flavopiridol, quantified renilla luciferase mRNA levels, and compared the absolute luciferase activity among the different groups. The results suggested that curcumin promoted the transcription of the luciferase genes located downstream of the simian vacuolating virus 40 (SV40) early enhancer/promoter, but not those located downstream of the human cytomegalovirus (CMV) immediate-early or the herpes simplex virus thymidine kinase (HSV-TK) promoters. These results explain the discrepancies between the two luciferase reporter systems. The current study underscores the importance of taking caution when interpreting the results of dual-luciferase reporter assays and provides strategies to overcome the potential pitfall accompanying dual-luciferase reporter systems. PMID:26620302

Astrocyte Elevated Gene 1 Interacts with Acetyltransferase p300 and c-Jun To Promote Tumor Aggressiveness

PubMed Central

Liu, Liping; Guan, Hongyu; Li, Yun; Ying, Zhe; Wu, Jueheng; Zhu, Xun; Song, Libing

2016-01-01

ABSTRACT Astrocyte elevated gene 1 (AEG-1) is an oncoprotein that strongly promotes the development and progression of cancers. However, the detailed underlying mechanisms through which AEG-1 enhances tumor development and progression remain to be determined. In this study, we identified c-Jun and p300 to be novel interacting partners of AEG-1 in gliomas. AEG-1 promoted c-Jun transcriptional activity by interacting with the c-Jun/p300 complex and inducing c-Jun acetylation. Furthermore, the AEG-1/c-Jun/p300 complex was found to bind the promoter of c-Jun downstream targeted genes, consequently establishing an acetylated chromatin state that favors transcriptional activation. Importantly, AEG-1/p300-mediated c-Jun acetylation resulted in the development of a more aggressive malignant phenotype in gliomas through a drastic increase in glioma cell proliferation and angiogenesis in vitro and in vivo. Consistently, the AEG-1 expression levels in clinical glioma specimens correlated with the status of c-Jun activation. Taken together, our results suggest that AEG-1 mediates a novel epigenetic mechanism that enhances c-Jun transcriptional activity to induce glioma progression and that AEG-1 might be a novel, potential target for the treatment of gliomas. PMID:27956703

TEAD and YAP regulate the enhancer network of human embryonic pancreatic progenitors.

PubMed

Cebola, Inês; Rodríguez-Seguí, Santiago A; Cho, Candy H-H; Bessa, José; Rovira, Meritxell; Luengo, Mario; Chhatriwala, Mariya; Berry, Andrew; Ponsa-Cobas, Joan; Maestro, Miguel Angel; Jennings, Rachel E; Pasquali, Lorenzo; Morán, Ignasi; Castro, Natalia; Hanley, Neil A; Gomez-Skarmeta, Jose Luis; Vallier, Ludovic; Ferrer, Jorge

2015-05-01

The genomic regulatory programmes that underlie human organogenesis are poorly understood. Pancreas development, in particular, has pivotal implications for pancreatic regeneration, cancer and diabetes. We have now characterized the regulatory landscape of embryonic multipotent progenitor cells that give rise to all pancreatic epithelial lineages. Using human embryonic pancreas and embryonic-stem-cell-derived progenitors we identify stage-specific transcripts and associated enhancers, many of which are co-occupied by transcription factors that are essential for pancreas development. We further show that TEAD1, a Hippo signalling effector, is an integral component of the transcription factor combinatorial code of pancreatic progenitor enhancers. TEAD and its coactivator YAP activate key pancreatic signalling mediators and transcription factors, and regulate the expansion of pancreatic progenitors. This work therefore uncovers a central role for TEAD and YAP as signal-responsive regulators of multipotent pancreatic progenitors, and provides a resource for the study of embryonic development of the human pancreas.

Activation of the carbohydrate response element binding protein (ChREBP) in response to anoxia in the turtle Trachemys scripta elegans.

PubMed

Krivoruchko, Anastasia; Storey, Kenneth B

2014-10-01

ChREBP (carbohydrate response element binding protein) is a glucose-responsive transcription factor that is known to be an important regulator of glycolytic and lipogenic genes in response to glucose. We hypothesized that activation of ChREBP could be relevant to anoxia survival by the anoxia-tolerant turtle, Trachemys scripta elegans. Expression of ChREBP in response to 5 and 20h of anoxia was examined using RT-PCR and Western immunoblotting. In addition, subcellular localization and DNA-binding activity of ChREBP protein were assessed and transcript levels of liver pyruvate kinase (LPK), a downstream gene under ChREBP control were quantified using RT-PCR. ChREBP was anoxia-responsive in kidney and liver, with transcript levels increasing by 1.2-1.8 fold in response to anoxia and protein levels increasing by 1.8-1.9 fold. Enhanced nuclear presence under anoxia was also observed in both tissues by 2.2-2.8 fold. A 4.2 fold increase in DNA binding activity of ChREBP was also observed in liver in response to 5h of anoxia. In addition, transcript levels of LPK increased by 2.1 fold in response to 5h of anoxia in the liver. The results suggest that activation of ChREBP in response to anoxia might be a crucial factor for anoxia survival in turtle liver by contributing to elevated glycolytic flux in the initial phases of oxygen limitation. This study provides the first demonstration of activation of ChREBP in response to anoxia in a natural model of anoxia tolerance, further improving our understanding of the molecular nature of anoxia tolerance. Copyright © 2014 Elsevier B.V. All rights reserved.

Expression Patterns of Genes Involved in Sugar Metabolism and Accumulation during Apple Fruit Development

PubMed Central

Cheng, Lailiang

2012-01-01

Both sorbitol and sucrose are imported into apple fruit from leaves. The metabolism of sorbitol and sucrose fuels fruit growth and development, and accumulation of sugars in fruit is central to the edible quality of apple. However, our understanding of the mechanisms controlling sugar metabolism and accumulation in apple remains quite limited. We identified members of various gene families encoding key enzymes or transporters involved in sugar metabolism and accumulation in apple fruit using homology searches and comparison of their expression patterns in different tissues, and analyzed the relationship of their transcripts with enzyme activities and sugar accumulation during fruit development. At the early stage of fruit development, the transcript levels of sorbitol dehydrogenase, cell wall invertase, neutral invertase, sucrose synthase, fructokinase and hexokinase are high, and the resulting high enzyme activities are responsible for the rapid utilization of the imported sorbitol and sucrose for fruit growth, with low levels of sugar accumulation. As the fruit continues to grow due to cell expansion, the transcript levels and activities of these enzymes are down-regulated, with concomitant accumulation of fructose and elevated transcript levels of tonoplast monosaccharide transporters (TMTs), MdTMT1 and MdTMT2; the excess carbon is converted into starch. At the late stage of fruit development, sucrose accumulation is enhanced, consistent with the elevated expression of sucrose-phosphate synthase (SPS), MdSPS5 and MdSPS6, and an increase in its total activity. Our data indicate that sugar metabolism and accumulation in apple fruit is developmentally regulated. This represents a comprehensive analysis of the genes involved in sugar metabolism and accumulation in apple, which will serve as a platform for further studies on the functions of these genes and subsequent manipulation of sugar metabolism and fruit quality traits related to carbohydrates. PMID:22412983

Butyric acid stimulates bone sialoprotein gene transcription.

PubMed

Yang, Li; Li, Zhengyang; Li, Xinyue; Wang, Zhitao; Wang, Shuang; Sasaki, Yoko; Takai, Hideki; Ogata, Yorimasa

2010-06-01

Butyric acid (sodium butyrate; BA) is an extracellular metabolite secreted from periodontopathic bacteria present in subgingival plaque. BA induces apoptosis of T and B cells, and acts as a potent inhibitor of histone deacetylases. Bone sialoprotein (BSP) is thought to function in the initial mineralization of bone, and may be crucial for osteoblast differentiation, bone matrix mineralization and tumor metastasis. In the present study we investigated the regulation of BSP transcription by BA in rat osteoblast-like ROS17/2.8 cells. At 12 h, BA (10(-4) M) increased the level of BSP mRNA, and enhanced the luciferase activity of the construct pLUC3, which includes the promoter sequence between nucleotides -116 and +60. Transcriptional stimulation by BA was abrogated in the pLUC3 construct which containing a 2-bp mutation in the fibroblast growth factor 2 response element (FRE). Gel shift analyses showed that BA increased the binding of nuclear protein to FRE. These data suggest that BA increases the transcription of the BSP gene mediated through FRE in the rat BSP gene promoter, and induces osteoblast activity in the early stage of bone formation.

Arabidopsis CAPRICE (MYB) and GLABRA3 (bHLH) Control Tomato (Solanum lycopersicum) Anthocyanin Biosynthesis

PubMed Central

Wada, Takuji; Kunihiro, Asuka; Tominaga-Wada, Rumi

2014-01-01

In Arabidopsis thaliana the MYB transcription factor CAPRICE (CPC) and the bHLH transcription factor GLABRA3 (GL3) are central regulators of root-hair differentiation and trichome initiation. By transforming the orthologous tomato genes SlTRY (CPC) and SlGL3 (GL3) into Arabidopsis, we demonstrated that these genes influence epidermal cell differentiation in Arabidopsis, suggesting that tomato and Arabidopsis partially use similar transcription factors for epidermal cell differentiation. CPC and GL3 are also known to be involved in anthocyanin biosynthesis. After transformation into tomato, 35S::CPC inhibited anthocyanin accumulation, whereas GL3::GL3 enhanced anthocyanin accumulation. Real-time reverse transcription PCR analyses showed that the expression of anthocyanin biosynthetic genes including Phe-ammonia lyase (PAL), the flavonoid pathway genes chalcone synthase (CHS), dihydroflavonol reductase (DFR), and anthocyanidin synthase (ANS) were repressed in 35S::CPC tomato. In contrast, the expression levels of PAL, CHS, DFR, and ANS were significantly higher in GL3::GL3 tomato compared with control plants. These results suggest that CPC and GL3 also influence anthocyanin pigment synthesis in tomato. PMID:25268379

LIN28 phosphorylation by MAPK/ERK couples signaling to the post-transcriptional control of pluripotency

PubMed Central

Tsanov, Kaloyan M.; Pearson, Daniel S.; Wu, Zhaoting; Han, Areum; Triboulet, Robinson; Seligson, Marc T.; Powers, John T.; Osborne, Jihan K.; Kane, Susan; Gygi, Steven P.; Gregory, Richard I.; Daley, George Q.

2016-01-01

Signaling and post-transcriptional gene control are both critical for the regulation of pluripotency1,2, yet how they are integrated to influence cell identity remains poorly understood. LIN28 (also known as LIN28A), a highly conserved RNA-binding protein (RBP), has emerged as a central post-transcriptional regulator of cell fate through blockade of let-7 microRNA (miRNA) biogenesis and direct modulation of mRNA translation3. Here we show that LIN28 is phosphorylated by MAPK/ERK in pluripotent stem cells (PSCs), which increases its levels via post-translational stabilization. LIN28 phosphorylation had little impact on let-7 but enhanced LIN28’s effect on its direct mRNA targets, revealing a mechanism that uncouples LIN28’s let-7-dependent and independent activities. We have linked this mechanism to the induction of pluripotency by somatic cell reprogramming and the transition from naïve to primed pluripotency. Collectively, our findings indicate that MAPK/ERK directly impacts LIN28, defining an axis that connects signaling, post-transcriptional gene control, and cell fate regulation. PMID:27992407

Ethanol induced astaxanthin accumulation and transcriptional expression of carotenogenic genes in Haematococcus pluvialis.

PubMed

Wen, Zewen; Liu, Zhiyong; Hou, Yuyong; Liu, Chenfeng; Gao, Feng; Zheng, Yubin; Chen, Fangjian

2015-10-01

Haematococcus pluvialis is one of the most promising natural sources of astaxanthin. However, inducing the accumulation process has become one of the primary obstacles in astaxanthin production. In this study, the effect of ethanol on astaxanthin accumulation was investigated. The results demonstrated that astaxanthin accumulation occurred with ethanol addition even under low-light conditions. The astaxanthin productivity could reach 11.26 mg L(-1) d(-1) at 3% (v/v) ethanol, which was 2.03 times of that of the control. The transcriptional expression patterns of eight carotenogenic genes were evaluated using real-time PCR. The results showed that ethanol greatly enhanced transcription of the isopentenyl diphosphate (IPP) isomerase genes (ipi-1 and ipi-2), which were responsible for isomerization reaction of IPP and dimethylallyl diphosphate (DMAPP). This finding suggests that ethanol induced astaxanthin biosynthesis was up-regulated mainly by ipi-1 and ipi-2 at transcriptional level, promoting isoprenoid synthesis and substrate supply to carotenoid formation. Thus ethanol has the potential to be used as an effective reagent to induce astaxanthin accumulation in H. pluvialis. Copyright © 2015 Elsevier Inc. All rights reserved.

Super elongation complex promotes early HIV transcription and its function is modulated by P-TEFb.

PubMed

Kuzmina, Alona; Krasnopolsky, Simona; Taube, Ran

2017-05-27

Early work on the control of transcription of the human immunodeficiency virus (HIV) laid the foundation for our current knowledge of how RNA Polymerase II is released from promoter-proximal pausing sites and transcription elongation is enhanced. The viral Tat activator recruits Positive Transcription Elongation Factor b (P-TEFb) and Super Elongation Complex (SEC) that jointly drive transcription elongation. While substantial progress in understanding the role of SEC in HIV gene transcription elongation has been obtained, defining of the mechanisms that govern SEC functions is still limited, and the role of SEC in controlling HIV transcription in the absence of Tat is less clear. Here we revisit the contribution of SEC in early steps of HIV gene transcription. In the absence of Tat, the AF4/FMR2 Family member 4 (AFF4) of SEC efficiently activates HIV transcription, while gene activation by its homolog AFF1 is substantially lower. Differential recruitment to the HIV promoter and association with Human Polymerase-Associated Factor complex (PAFc) play key role in this functional distinction between AFF4 and AFF1. Moreover, while depletion of cyclin T1 expression has subtle effects on HIV gene transcription in the absence of Tat, knockout (KO) of AFF1, AFF4, or both proteins slightly repress this early step of viral transcription. Upon Tat expression, HIV transcription reaches optimal levels despite KO of AFF1 or AFF4 expression. However, double AFF1/AFF4 KO completely diminishes Tat trans-activation. Significantly, our results show that P-TEFb phosphorylates AFF4 and modulates SEC assembly, AFF1/4 dimerization and recruitment to the viral promoter. We conclude that SEC promotes both early steps of HIV transcription in the absence of Tat, as well as elongation of transcription, when Tat is expressed. Significantly, SEC functions are modulated by P-TEFb.

Super elongation complex promotes early HIV transcription and its function is modulated by P-TEFb

PubMed Central

Kuzmina, Alona; Krasnopolsky, Simona; Taube, Ran

2017-01-01

ABSTRACT Early work on the control of transcription of the human immunodeficiency virus (HIV) laid the foundation for our current knowledge of how RNA Polymerase II is released from promoter-proximal pausing sites and transcription elongation is enhanced. The viral Tat activator recruits Positive Transcription Elongation Factor b (P-TEFb) and Super Elongation Complex (SEC) that jointly drive transcription elongation. While substantial progress in understanding the role of SEC in HIV gene transcription elongation has been obtained, defining of the mechanisms that govern SEC functions is still limited, and the role of SEC in controlling HIV transcription in the absence of Tat is less clear. Here we revisit the contribution of SEC in early steps of HIV gene transcription. In the absence of Tat, the AF4/FMR2 Family member 4 (AFF4) of SEC efficiently activates HIV transcription, while gene activation by its homolog AFF1 is substantially lower. Differential recruitment to the HIV promoter and association with Human Polymerase-Associated Factor complex (PAFc) play key role in this functional distinction between AFF4 and AFF1. Moreover, while depletion of cyclin T1 expression has subtle effects on HIV gene transcription in the absence of Tat, knockout (KO) of AFF1, AFF4, or both proteins slightly repress this early step of viral transcription. Upon Tat expression, HIV transcription reaches optimal levels despite KO of AFF1 or AFF4 expression. However, double AFF1/AFF4 KO completely diminishes Tat trans-activation. Significantly, our results show that P-TEFb phosphorylates AFF4 and modulates SEC assembly, AFF1/4 dimerization and recruitment to the viral promoter. We conclude that SEC promotes both early steps of HIV transcription in the absence of Tat, as well as elongation of transcription, when Tat is expressed. Significantly, SEC functions are modulated by P-TEFb. PMID:28340332

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

Increased IL-2 production in T cells by xanthohumol through enhanced NF-AT and AP-1 activity.

PubMed

Choi, Jin Myung; Kim, Hyun Jung; Lee, Kwang Youl; Choi, Hyun Jin; Lee, Ik-Soo; Kang, Bok Yun

2009-01-01

Xanthohumol (XN) is a major chalcone found in hop, which is used to add bitterness and flavor to beer. In this study, we investigated the effects of XN on the production of interlukin-2 (IL-2), a potent T cell growth factor. Treatment with XN significantly increased IL-2 production in mouse EL-4 T cells activated with phorbol 12-myristate 13-acetate (PMA) plus ionomycin (Io) in a dose-dependent manner. To further characterize its regulatory mechanism of XN on increased IL-2 production, the effects of XN on IL-2 promoter activity and the activity of several transcription factors modulating IL-2 expression were analyzed. XN enhanced activity of the IL-2 promoter, which contains distal and proximal regulatory elements in PMA/Io-activated EL-4 T cells. Furthermore, the activity of NF-AT and AP-1 was enhanced but NF-kappaB activity was not influenced by XN in PMA/Io-activated EL-4 T cells. These results suggest that XN increased IL-2 production at the transcriptional levels via the up-regulation of NF-AT and AP-1 in PMA/Io-activated EL-4 T cells.

The HSP terminator of Arabidopsis thaliana increases gene expression in plant cells.

PubMed

Nagaya, Shingo; Kawamura, Kazue; Shinmyo, Atsuhiko; Kato, Ko

2010-02-01

To express a foreign gene in plants effectively, a good expression system is required. Here we describe the identification of a transcriptional terminator that supports increased levels of expression. The terminators of several Arabidopsis genes were examined in transfected Arabidopsis T87 protoplasts. The heat shock protein 18.2 (HSP) terminator was the most effective in supporting increased levels of expression. The HSP terminator increases mRNA levels of both transiently and stably expressed transgenes approximately 2-fold more than the NOS (nopaline synthase) terminator. When combined with the HSP terminator, a translational enhancer increased gene expression levels approximately 60- to 100-fold in transgenic plants.

A Land Plant-Specific Transcription Factor Directly Enhances Transcription of a Pathogenic Noncoding RNA Template by DNA-Dependent RNA Polymerase II[OPEN

PubMed Central

Qu, Jie; Ji, Shaoyi; Wallace, Andrew J.; Wu, Jian; Li, Yi; Gopalan, Venkat; Ding, Biao

2016-01-01

Some DNA-dependent RNA polymerases (DdRPs) possess RNA-dependent RNA polymerase activity, as was first discovered in the replication of Potato spindle tuber viroid (PSTVd) RNA genome in tomato (Solanum lycopersicum). Recent studies revealed that this activity in bacteria and mammals is important for transcriptional and posttranscriptional regulatory mechanisms. Here, we used PSTVd as a model to uncover auxiliary factors essential for RNA-templated transcription by DdRP. PSTVd replication in the nucleoplasm generates (−)-PSTVd intermediates and (+)-PSTVd copies. We found that the Nicotiana benthamiana canonical 9-zinc finger (ZF) Transcription Factor IIIA (TFIIIA-9ZF) as well as its variant TFIIIA-7ZF interacted with (+)-PSTVd, but only TFIIIA-7ZF interacted with (−)-PSTVd. Suppression of TFIIIA-7ZF reduced PSTVd replication, and overexpression of TFIIIA-7ZF enhanced PSTVd replication in planta. Consistent with the locale of PSTVd replication, TFIIIA-7ZF was found in the nucleoplasm and nucleolus, in contrast to the strictly nucleolar localization of TFIIIA-9ZF. Footprinting assays revealed that only TFIIIA-7ZF bound to a region of PSTVd critical for initiating transcription. Furthermore, TFIIIA-7ZF strongly enhanced the in vitro transcription of circular (+)-PSTVd by partially purified Pol II. Together, our results identify TFIIIA-7ZF as a dedicated cellular transcription factor that acts in DdRP-catalyzed RNA-templated transcription, highlighting both the extraordinary evolutionary adaptation of viroids and the potential of DdRPs for a broader role in cellular processes. PMID:27113774

Enhancer competition between H19 and Igf2 does not mediate their imprinting

PubMed Central

Schmidt, Jennifer V.; Levorse, John M.; Tilghman, Shirley M.

1999-01-01

The linked H19 and Igf2 genes on mouse distal chromosome 7 are subject to genomic imprinting. Competition between the promoters of the genes for transcription from shared enhancers has been proposed as an explanation for the coordinate expression and reciprocal imprinting of these two genes. To test this model, we have used Cre-loxP technology to generate in mice a conditional deletion of the H19 promoter and structural gene that leaves no transcription unit in the locus. Contrary to the prediction of enhancer competition we find that transcriptional activity from the H19 promoter is not required for the imprinted silencing of the Igf2 gene. PMID:10449763

groHMM: a computational tool for identifying unannotated and cell type-specific transcription units from global run-on sequencing data.

PubMed

Chae, Minho; Danko, Charles G; Kraus, W Lee

2015-07-16

Global run-on coupled with deep sequencing (GRO-seq) provides extensive information on the location and function of coding and non-coding transcripts, including primary microRNAs (miRNAs), long non-coding RNAs (lncRNAs), and enhancer RNAs (eRNAs), as well as yet undiscovered classes of transcripts. However, few computational tools tailored toward this new type of sequencing data are available, limiting the applicability of GRO-seq data for identifying novel transcription units. Here, we present groHMM, a computational tool in R, which defines the boundaries of transcription units de novo using a two state hidden-Markov model (HMM). A systematic comparison of the performance between groHMM and two existing peak-calling methods tuned to identify broad regions (SICER and HOMER) favorably supports our approach on existing GRO-seq data from MCF-7 breast cancer cells. To demonstrate the broader utility of our approach, we have used groHMM to annotate a diverse array of transcription units (i.e., primary transcripts) from four GRO-seq data sets derived from cells representing a variety of different human tissue types, including non-transformed cells (cardiomyocytes and lung fibroblasts) and transformed cells (LNCaP and MCF-7 cancer cells), as well as non-mammalian cells (from flies and worms). As an example of the utility of groHMM and its application to questions about the transcriptome, we show how groHMM can be used to analyze cell type-specific enhancers as defined by newly annotated enhancer transcripts. Our results show that groHMM can reveal new insights into cell type-specific transcription by identifying novel transcription units, and serve as a complete and useful tool for evaluating functional genomic elements in cells.

Baicalin Ameliorates Liver Injury Induced by Chronic plus Binge Ethanol Feeding by Modulating Oxidative Stress and Inflammation via CYP2E1 and NRF2 in Mice

PubMed Central

He, Ping; Wu, Yafeng; Shun, Jianchao; Liang, Yaodong; Cheng, Mingliang

2017-01-01

Alcoholic liver injury leads to serious complication including death. The potential role of baicalin at the transcription level in mice model of alcohol injury is not known yet. In this study, we examined the effect of baicalin against chronic plus binge ethanol model in mice and understanding the mechanism of protection. Liver function, histology, steatosis, inflammation, NF-κB activity, oxidative stress sources, nuclear translocation of NRF2 transcription factor, and cell death were assessed. Treatment with baicalin ameliorated ethanol-induced oxidative stress, inflammation, and cell death. Baicalin attenuated ethanol-induced proinflammatory molecules such as TNF-α, IL-1β, MIP-2, and MCP-1 and reversed redox-sensitive transcription factor NF-κB activation. Baicalin also modulated Kupffer cell activation in vitro. Baicalin inhibited ethanol-induced expression of reactive oxygen species (ROS) generating enzymes NOX2, p67phox, xanthine oxidase, and iNOS in addition to CYP2E1 activities. Baicalin also enhanced ethanol-induced NRF2 nuclear translocation and increased downstream target gene HO-1 as antioxidant defense. Finally, baicalin reduced significant apoptotic and necrotic cell death. Our study suggests that baicalin ameliorates chronic plus binge ethanol-induced liver injury involving molecular crosstalk of multiple pathways at the transcriptional level and through upregulation of antioxidant defense mechanism. PMID:28951767

Identification of Candidate Transcriptional Regulators of Epidermal Transfer Cell Development in Vicia faba Cotyledons

PubMed Central

Arun-Chinnappa, Kiruba S.; McCurdy, David W.

2016-01-01

Transfer cells (TCs) are anatomically-specialized cells formed at apoplasmic-symplasmic bottlenecks in nutrient transport pathways in plants. TCs form invaginated wall ingrowths which provide a scaffold to amplify plasma membrane surface area and thus increase the density of nutrient transporters required to achieve enhanced nutrient flow across these bottlenecks. Despite their importance to nutrient transport in plants, little is known of the transcriptional regulation of wall ingrowth formation. Here, we used RNA-Seq to identify transcription factors putatively involved in regulating epidermal TC development in cotyledons of Vicia faba. Comparing cotyledons cultured for 0, 3, 9, and 24 h to induce trans-differentiation of epidermal TCs identified 43 transcription factors that showed either epidermal-specific or epidermal–enhanced expression, and 10 that showed epidermal-specific down regulation. Members of the WRKY and ethylene-responsive families were prominent in the cohort of transcription factors showing epidermal-specific or epidermal–enhanced expression, consistent with the initiation of TC development often representing a response to stress. Members of the MYB family were also prominent in these categories, including orthologs of MYB genes involved in localized secondary wall deposition in Arabidopsis thaliana. Among the group of transcription factors showing down regulation were various homeobox genes and members of the MADs-box and zinc-finger families of poorly defined functions. Collectively, this study identified several transcription factors showing expression characteristics and orthologous functions that indicate likely participation in transcriptional regulation of epidermal TC development in V. faba cotyledons. PMID:27252730

The relationship between the plant-encoded RNA-dependent RNA polymerase 1 and alternative oxidase in tomato basal defense against Tobacco mosaic virus.

PubMed

Liao, Yang-Wen-Ke; Liu, Ya-Ru; Liang, Jia-Yang; Wang, Wen-Ping; Zhou, Jie; Xia, Xiao-Jian; Zhou, Yan-Hong; Yu, Jing-Quan; Shi, Kai

2015-03-01

Salicylic acid (SA) plays a critical role in plant defense against pathogen attack. The SA-induced viral defense in plants is distinct from the pathways mediating bacterial and fungal defense, which is pathogenesis-related protein-independent but involves an RNA-dependent RNA polymerase 1 (RDR1)-mediated RNA silencing mechanism and/or an alternative oxidase (AOX)-associated defense pathway. However, the relationship between these two viral defense-related pathways remains unclear. In this study, Tobacco mosaic virus (TMV) inoculation onto Solanum lycopersicum (tomato) leaves induced a rapid induction of the SlAOX1a transcript level as well as the total and CN-resistant respiration at 0.5 dpi, followed by an increase in SlRDR1 gene expression at 1 dpi in the upper uninoculated leaves. Silencing SlRDR1 using virus-induced gene silencing system significantly reduced SlRDR1 expression and tomato defense against TMV but had no evident effect on SlAOX1a transcription. Conversely, silencing SlAOX1a not only effectively reduced the AOX1a transcript level, but also blocked the TMV-induced SlRDR1 expression and decreased the basal defense against TMV. Furthermore, the application of an exogenous AOX activator on empty vector-silenced control plants greatly induced the accumulation of SlRDR1 and SlAOX1a transcript and reduced TMV viral RNA accumulation, but failed to have such effects on SlRDR1-silenced plants. Moreover, RDR1-overexpressed transgenic Nicotiana benthamiana plants enhanced defense against TMV than the empty vector-transformed plants, but these effects were not affected by the exogenous AOX activator or inhibitor. These results indicate that RDR1 is involved in the AOX-mediated defense pathway against TMV infection and plays a crucial role in enhancing RNA silencing to limit virus systemic spread.

Transgenic wheat expressing Thinopyrum intermedium MYB transcription factor TiMYB2R-1 shows enhanced resistance to the take-all disease.

PubMed

Liu, Xin; Yang, Lihua; Zhou, Xianyao; Zhou, Miaoping; Lu, Yan; Ma, Lingjian; Ma, Hongxiang; Zhang, Zengyan

2013-05-01

The disease take-all, caused by the fungus Gaeumannomyces graminis, is one of the most destructive root diseases of wheat worldwide. Breeding resistant cultivars is an effective way to protect wheat from take-all. However, little progress has been made in improving the disease resistance level in commercial wheat cultivars. MYB transcription factors play important roles in plant responses to environmental stresses. In this study, an R2R3-MYB gene in Thinopyrum intermedium, TiMYB2R-1, was cloned and characterized. The gene sequence includes two exons and an intron. The expression of TiMYB2R-1 was significantly induced following G. graminis infection. An in vitro DNA binding assay proved that TiMYB2R-1 protein could bind to the MYB-binding site cis-element ACI. Subcellular localization assays revealed that TiMYB2R-1 was localized in the nucleus. TiMYB2R-1 transgenic wheat plants were generated, characterized molecularly, and evaluated for take-all resistance. PCR and Southern blot analyses confirmed that TiMYB2R-1 was integrated into the genomes of three independent transgenic wheat lines by distinct patterns and the transgene was heritable. Reverse transcription-PCR and western blot analyses revealed that TiMYB2R-1 was highly expressed in the transgenic wheat lines. Based on disease response assessments for three successive generations, the significantly enhanced resistance to take-all was observed in the three TiMYB2R-1-overexpressing transgenic wheat lines. Furthermore, the transcript levels of at least six wheat defence-related genes were significantly elevated in the TiMYB2R-1 transgenic wheat lines. These results suggest that engineering and overexpression of TiMYB2R-1 may be used for improving take-all resistance of wheat and other cereal crops.

Resveratrol upregulates Egr-1 expression and activity involving extracellular signal-regulated protein kinase and ternary complex factors

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

Rössler, Oliver G.; Glatzel, Daniel; Thiel, Gerald, E-mail: gerald.thiel@uks.eu

2015-03-01

Many intracellular functions have been attributed to resveratrol, a polyphenolic phytoalexin found in grapes and in other plants. Here, we show that resveratrol induces the expression of the transcription factor Egr-1 in human embryonic kidney cells. Using a chromosomally embedded Egr-1-responsive reporter gene, we show that the Egr-1 activity was significantly elevated in resveratrol-treated cells, indicating that the newly synthesized Egr-1 protein was biologically active. Stimulus-transcription coupling leading to the resveratrol-induced upregulation of Egr-1 expression and activity requires the protein kinases Raf and extracellular signal-regulated protein kinase ERK, while MAP kinase phosphatase-1 functions as a nuclear shut-off device that interruptsmore » the signaling cascade connecting resveratrol stimulation with enhanced Egr-1 expression. On the transcriptional level, Elk-1, a key transcriptional regulator of serum response element-driven gene transcription, connects the intracellular signaling cascade elicited by resveratrol with transcription of the Egr-1 gene. These data were corroborated by the observation that stimulation of the cells with resveratrol increased the transcriptional activation potential of Elk-1. The SRE as well as the GC-rich DNA binding site of Egr-1 function as resveratrol-responsive elements. Thus, resveratrol regulates gene transcription via activation of the stimulus-regulated protein kinases Raf and ERK and the stimulus-responsive transcription factors TCF and Egr-1. - Highlights: • The plant polyphenol resveratrol upregulates Egr-1 expression and activity. • The stimulation of Egr-1 requires the protein kinases ERK and Raf. • Resveratrol treatment upregulates the transcriptional activation potential of Elk-1. • Resveratrol-induced stimulation of Egr-1 requires ternary complex factors. • Two distinct resveratrol-responsive elements were identified.« less

Trichostatin A enhances estrogen receptor-alpha repression in MCF-7 breast cancer cells under hypoxia

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

Noh, Hyunggyun; Park, Joonwoo; Shim, Myeongguk

Estrogen receptor (ER) is a crucial determinant of resistance to endocrine therapy, which may change during the progression of breast cancer. We previously showed that hypoxia induces ESR1 gene repression and ERα protein degradation via proteasome-mediated pathway in breast cancer cells. HDAC plays important roles in the regulation of histone and non-histone protein post-translational modification. HDAC inhibitors can induce epigenetic changes and have therapeutic potential for targeting various cancers. Trichostatin A exerts potent antitumor activities against breast cancer cells in vitro and in vivo. In this report, we show that TSA augments ESR1 gene repression at the transcriptional level and downregulates ERαmore » protein expression under hypoxic conditions through a proteasome-mediated pathway. TSA-induced estrogen response element-driven reporter activity in the absence of estrogen was synergistically enhanced under hypoxia; however, TSA inhibited cell proliferation under both normoxia and hypoxia. Our data show that the hypoxia-induced repression of ESR1 and degradation of ERα are enhanced by concomitant treatment with TSA. These findings expand our understanding of hormone responsiveness in the tumor microenvironment; however, additional in-depth studies are required to elucidate the detailed mechanisms of TSA-induced ERα regulation under hypoxia. - Highlights: • TSA augments ESR1 gene repression at the transcriptional level under hypoxia. • TSA downregulates ERα protein expression under hypoxia. • TSA-induced ERα regulation under hypoxia is essential for understanding the behavior and progression of breast cancer.« less

Molecular characterization of cDNA encoding oxygen evolving enhancer protein 1 increased by salt treatment in the mangrove Bruguiera gymnorrhiza.

PubMed

Sugihara, K; Hanagata, N; Dubinsky, Z; Baba, S; Karube, I

2000-11-01

Young plants of the common Okinawa mangrove species Bruguiera gymnorrhiza were transferred from freshwater to a medium with seawater salt level (500 mM NaCl). Two-dimensional gel electrophoresis revealed in the leaf extract of the plant a 33 kDa protein with pI 5.2, whose quantity increased as a result of NaCl treatment. The N-terminal amino acids sequence of this protein had a significant homology with mature region of oxygen evolving enhancer protein 1 (OEE1) precursor. The cloning of OEE1 precursor cDNA fragment was carried out by means of reverse transcription-PCR (RT-PCR) using degenerated primers. Both 3'- and 5'-regions were isolated by rapid amplification of cDNA ends (RACE) method. The deduced amino acid sequence consisted of 322 amino acids and was 87% identical to that of Nicotiana tabacum. In B. gymnorrhiza, the predicted amino acid sequence of the mature protein starts at the residue number 85 of the open reading frame. The first 84-amino acid residues correspond to a typical transit sequence for the signal directing OEE1 to its appropriate compartment of chloroplast. The expression of OEE1 was analyzed together with other OEE subunits and D1 protein of photosystem II. The transcript levels of all the three OEEs were enhanced by NaCl treatment, but the significant increase of D1 protein was not observed.

Additional sex combs interacts with enhancer of zeste and trithorax and modulates levels of trimethylation on histone H3K4 and H3K27 during transcription of hsp70.

PubMed

Li, Taosui; Hodgson, Jacob W; Petruk, Svetlana; Mazo, Alexander; Brock, Hugh W

2017-09-19

Maintenance of cell fate determination requires the Polycomb group for repression; the trithorax group for gene activation; and the enhancer of trithorax and Polycomb (ETP) group for both repression and activation. Additional sex combs (Asx) is a genetically identified ETP for the Hox loci, but the molecular basis of its dual function is unclear. We show that in vitro, Asx binds directly to the SET domains of the histone methyltransferases (HMT) enhancer of zeste [E(z)] (H3K27me3) and Trx (H3K4me3) through a bipartite interaction site separated by 846 amino acid residues. In Drosophila S2 cell nuclei, Asx interacts with E(z) and Trx in vivo. Drosophila Asx is required for repression of heat-shock gene hsp70 and is recruited downstream of the hsp70 promoter. Changes in the levels of H3K4me3 and H3K27me3 downstream of the hsp70 promoter in Asx mutants relative to wild type show that Asx regulates H3K4 and H3K27 trimethylation. We propose that during transcription Asx modulates the ratio of H3K4me3 to H3K27me3 by selectively recruiting the antagonistic HMTs, E(z) and Trx or other nucleosome-modifying enzymes to hsp70.

TGF-beta1 stimulates expression of the aromatase (CYP19) gene in human osteoblast-like cells and THP-1 cells.

PubMed

Shozu, M; Zhao, Y; Simpson, E R

2000-02-25

Recent evidence has shown that bone is not only a target of estrogen action but also a source of local estrogen production. Bone cells such as osteoblasts express aromatase (P450arom) and the expression of P450arom in osteoblasts is positively regulated in a tissue specific fashion, as in the case of other tissues which express P450arom. To clarify the physiological factors regulating expression of P450arom in bone, we tested TGF-beta1 using osteoblast-like cells obtained from human fetuses as well as THP-1 cells. TGF-beta1 increased IL-1beta+DEX- induced aromatase activity in osteoblast-like cells, while it inhibited activity in skin fibroblasts. Similar enhancement of aromatase activity by TGF-beta1 was found in DEX-stimulated THP-1 cells and this cell line was used for further experiments. In THP-1 cells, TGF-beta1 enhanced DEX-induced aromatase activity almost linearly by 12 h and thereafter. Increased levels of P450arom transcripts were also demonstrated by RT-PCR at 3 h of TGF-beta1 treatment and thereafter. Cyclohexamide abolished enhancement of activity but did not inhibit the accumulation of P450arom transcripts induced by TGF-beta1. Increase in P450arom expression by TGF-beta1 was attributable to expression driven by promoter I.4. TGF-beta1 did not change the half life of P450arom transcripts. To identify the cis-acting elements responsible for TGF-beta1 action on aromatase expression, transient transfection assays were performed using a series of deletion constructs for promoter I.4 (P450-I.4/Luc). Two constructs (-410/+14 and-340/+14) that contain a functional glucocorticoid response element (GRE) and downstream sequence showed significant increase of luciferase activity in response to TGF-beta1. Deletion and mutation of the GRE in P450-I.4/Luc (-340/+14) abolished the TGF-beta1. The luciferase activity of a (GRE)(1)-SV40/Luc construct was also stimulated by TGF-beta1. These results indicate that TGF-beta1 increases the expression of P450arom at the level of transcription through promoter I.4, at least in part via an enhancement of transactivation activity of the GR in THP-1 cells. TGF-beta1 is suggested to be one of the physiological up-regulatory factors of bone aromatase.

Super-Enhancer-Driven Long Non-Coding RNA LINC01503, Regulated by TP63, Is Over-Expressed and Oncogenic in Squamous Cell Carcinoma.

PubMed

Xie, Jian-Jun; Jiang, Yan-Yi; Jiang, Yuan; Li, Chun-Quan; Lim, Mei-Chee; An, Omer; Mayakonda, Anand; Ding, Ling-Wen; Long, Lin; Sun, Chun; Lin, Le-Hang; Chen, Li; Wu, Jian-Yi; Wu, Zhi-Yong; Cao, Qi; Fang, Wang-Kai; Yang, Wei; Soukiasian, Harmik; Meltzer, Stephen J; Yang, Henry; Fullwood, Melissa; Xu, Li-Yan; Li, En-Min; Lin, De-Chen; Koeffler, H Phillip

2018-06-01

Long non-coding RNAs (lncRNAs) are expressed in tissue-specific pattern, but it is not clear how these are regulated. We aimed to identify squamous cell carcinoma (SCC)-specific lncRNAs and investigate mechanisms that control their expression and function. We studied expression patterns and functions of 4 SCC-specific lncRNAs. We obtained 113 esophageal SCC (ESCC) and matched non-tumor esophageal tissues from a hospital in Shantou City, China, and performed quantitative reverse transcription polymerase chain reaction assays to measure expression levels of LINC01503. We collected clinical data from patients and compared expression levels with survival times. LINC01503 was knocked down using small interfering RNAs and oligonucleotides in TE7, TE5, and KYSE510 cell lines and overexpressed in KYSE30 cells. Cells were analyzed by chromatin immunoprecipitation sequencing, luciferase reporter assays, colony formation, migration and invasion, and mass spectrometry analyses. Cells were injected into nude mice and growth of xenograft tumors was measured. LINC01503 interaction with proteins was studied using fluorescence in situ hybridization, RNA pulldown, and RNA immunoprecipitation analyses. We identified a lncRNA, LINC01503, which is regulated by a super enhancer and is expressed at significantly higher levels in esophageal and head and neck SCCs than in non-tumor tissues. High levels in SCCs correlated with shorter survival times of patients. The transcription factor TP63 bound to the super enhancer at the LINC01503 locus and activated its transcription. Expression of LINC01503 in ESCC cell lines increased their proliferation, colony formation, migration, and invasion. Knockdown of LINC01503 in SCC cells reduced their proliferation, colony formation, migration, and invasion, and the growth of xenograft tumors in nude mice. Expression of LINC01503 in ESCC cell lines reduced ERK2 dephosphorylation by DUSP6, leading to activation of ERK signaling via MAPK. LINC01503 disrupted the interaction between EBP1 and the p85 subunit of PI3K, increasing AKT signaling. We identified an lncRNA, LINC01503, which is increased in SCC cells compared with non-tumor cells. Increased expression of LINC01503 promotes ESCC cell proliferation, migration, invasion, and growth of xenograft tumors. It might be developed as a biomarker of aggressive SCCs in patients. Copyright © 2018 AGA Institute. Published by Elsevier Inc. All rights reserved.

Inhibition of DNA-PKcs enhances radiosensitivity and increases the levels of ATM and ATR in NSCLC cells exposed to carbon ion irradiation.

PubMed

Yang, Lina; Liu, Yuanyuan; Sun, Chao; Yang, Xinrui; Yang, Zhen; Ran, Juntao; Zhang, Qiuning; Zhang, Hong; Wang, Xinyu; Wang, Xiaohu

2015-11-01

Non-small cell lung cancer (NSCLC) exhibits radioresistance to conventional rays, due to its DNA damage repair systems. NSCLC may potentially be sensitized to radiation treatment by reducing those factors that continuously enhance the repair of damaged DNA. In the present study, normal lung fibroblast MRC-5 and lung cancer A549 cells were treated with NU7026 and CGK733, which are inhibitors of the DNA-dependent protein kinase catalytic subunit (PKcs) and ataxia telangiectasia mutated (ATM) and ataxia telangiectasia and Rad3-related (ATR), respectively, followed by exposure to X-rays and carbon ion irradiation. The cytotoxic activity, cell survival rate, DNA damage repair ability, cell cycle arrest and apoptosis rate of the treated cells were analyzed with MTT assay, colony formation assay, immunofluorescence and flow cytometry, respectively. The transcription and translation levels of the ATM, ATR and DNA-PKcs genes were detected by reverse transcription-quantitative polymerase chain reaction and western blotting, respectively. The results indicated that the radiosensitivity and DNA repair ability of A549 cells were reduced, and the percentages of apoptotic cells and those arrested at the G 2 /M phase of the cell cycle were significantly increased, following ionizing radiation with inhibitor-pretreatment. The expression levels of ATM, ATR, DNA-PKcs and phosphorylated histone H2AX, a biomarker for DNA double-strand breaks, were all upregulated at the transcriptional or translational level in A549 cells treated with carbon ion irradiation, compared with the control and X-rays-treated cells. In addition, the treatment with 5-50 µM NU7026 or CGK733 did not produce any obvious cytotoxicity in MRC-5 cells, and the effect of the DNA-PKcs-inhibitor on enhancing the radiosensitivity of A549 cells was stronger than that observed for the ATM and ATR-inhibitor. These findings demonstrated a minor role for ATM and ATR in radiation-induced cell death, since the upregulation of ATM and ATR did not rescue the A549 cells subjected to ionizing irradiation. Therefore, future studies on DNA-PKcs, ATM and ATR may lead to novel specific therapies that supplement general radiotherapy for the treatment of lung cancer.

CacyBP/SIP as a regulator of transcriptional responses in brain cells

PubMed Central

Kilanczyk, Ewa; Filipek, Anna; Hetman, Michal

2014-01-01

Summary The Calcyclin-Binding Protein/Siah-1-Interacting Protein (CacyBP/SIP) is highly expressed in the brain and was shown to regulate the β-catenin-driven transcription in thymocytes. Therefore, it was investigated whether in brain cells CacyBP/SIP might play a role as a transcriptional regulator. In BDNF- or forskolin-stimulated rat primary cortical neurons, overexpression of CacyBP/SIP enhanced transcriptional activity of the cAMP-response element (CRE). In addition, overexpressed CacyBP/SIP enhanced BDNF-mediated activation of the Nuclear Factor of Activated T-cells (NFAT) but not the Serum Response Element (SRE). These stimulatory effects required an intact C-terminal domain of CacyBP/SIP. Moreover, in C6 rat glioma cells, the overexpressed CacyBP/SIP enhanced activation of CRE- or NFAT- following forskolin- or serum stimulation, respectively. Conversely, knockdown of endogenous CacyBP/SIP reduced activation of CRE- and NFAT but not SRE. Taken together, these results indicate that CacyBP/SIP is a novel regulator of CRE- and NFAT-driven transcription. PMID:25163685

Synergism between a half-site and an imperfect estrogen-responsive element, and cooperation with COUP-TFI are required for estrogen receptor (ER) to achieve a maximal estrogen-stimulation of rainbow trout ER gene.

PubMed

Petit, F G; Métivier, R; Valotaire, Y; Pakdel, F

1999-01-01

In all oviparous, liver represents one of the main E2-target tissues where estrogen receptor (ER) constitutes the key mediator of estrogen action. The rainbow trout estrogen receptor (rtER) gene expression is markedly up-regulated by estrogens and the sequences responsible for this autoregulation have been located in a 0.2 kb upstream transcription start site within - 40/- 248 enhancer region. Absence of interference with steroid hormone receptors and tissue-specific factors and a conserved basal transcriptional machinery between yeast and higher eukaryotes, make yeast a simple assay system that will enable determination of important cis-acting regulatory sequences within rtER gene promoter and identification of transcription factors implicated in the regulation of this gene. Deletion analysis allowed to show a synergistic effect between an imperfect estrogen-responsive element (ERE) and a consensus half-ERE to achieve a high hormone-dependent transcriptional activation of the rtER gene promoter in the presence of stably expressed rtER. As in mammalian cells, here we observed a positive regulation of the rtER gene promoter by the chicken ovalbumin upstream promoter-transcription factor I (COUP-TFI) through enhancing autoregulation. Using a point mutation COUP-TFI mutant unable to bind DNA demonstrates that enhancement of rtER gene autoregulation requires the interaction of COUP-TFI to the DNA. Moreover, this enhancement of transcriptional activation by COUP-TFI requires specifically the AF-1 transactivation function of ER and can be observed in the presence of E2 or 4-hydroxytamoxifen but not ICI 164384. Thus, this paper describes the reconstitution of a hormone-responsive transcription unit in yeast in which the regulation of rtER gene promoter could be enhanced by the participation of cis-elements and/or trans-acting factors, such as ER itself or COUP-TF.

A20 Regulates Atherogenic Interferon (IFN)-γ Signaling in Vascular Cells by Modulating Basal IFNβ Levels*

PubMed Central

Moll, Herwig P.; Lee, Andy; Minussi, Darlan C.; da Silva, Cleide G.; Csizmadia, Eva; Bhasin, Manoj; Ferran, Christiane

2014-01-01

IFNγ signaling in endothelial (EC) and smooth muscle cells (SMC) is a key culprit of pathologic vascular remodeling. The impact of NF-κB inhibitory protein A20 on IFNγ signaling in vascular cells remains unknown. In gain- and loss-of-function studies, A20 inversely regulated expression of IFNγ-induced atherogenic genes in human EC and SMC by modulating STAT1 transcription. In vivo, inadequate A20 expression in A20 heterozygote mice aggravated intimal hyperplasia following partial carotid artery ligation. This outcome uniquely associated with increased levels of Stat1 and super-induction of Ifnγ-dependent genes. Transcriptome analysis of the aortic media from A20 heterozygote versus wild-type mice revealed increased basal Ifnβ signaling as the likely cause for higher Stat1 transcription. We confirmed higher basal IFNβ levels in A20-silenced human SMC and showed that neutralization or knockdown of IFNβ abrogates heightened STAT1 levels in these cells. Upstream of IFNβ, A20-silenced EC and SMC demonstrated higher levels of phosphorylated/activated TANK-binding kinase-1 (TBK1), a regulator of IFNβ transcription. This suggested that A20 knockdown increased STAT1 transcription by enhancing TBK1 activation and subsequently basal IFNβ levels. Altogether, these results uncover A20 as a key physiologic regulator of atherogenic IFNγ/STAT1 signaling. This novel function of A20 added to its ability to inhibit nuclear factor-κB (NF-κB) activation solidifies its promise as an ideal therapeutic candidate for treatment and prevention of vascular diseases. In light of recently discovered A20/TNFAIP3 (TNFα-induced protein 3) single nucleotide polymorphisms that impart lower A20 expression or function, these results also qualify A20 as a reliable clinical biomarker for vascular risk assessment. PMID:25217635

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

Lee, Sungsoo, E-mail: sungsoo.lee@utsouthwestern.edu; Wang, Ping-Yuan; Jeong, Yangsik

Oxysterol binding protein related protein 1S (ORP1S) is a member of a family of sterol transport proteins. Here we present evidence that ORP1S translocates from the cytoplasm to the nucleus in response to sterol binding. The sterols that best promote nuclear import of ORP1S also activate the liver X receptor (LXR) transcription factors and we show that ORP1S binds to LXRs, promotes binding of LXRs to LXR response elements (LXREs) and specifically enhances LXR-dependent transcription via the ME.1 and ME.2 enhancer elements of the apoE gene. We propose that ORP1S is a cytoplasmic sterol sensor, which transports sterols to themore » nucleus and promotes LXR-dependent gene transcription through select enhancer elements. -- Highlights: Black-Right-Pointing-Pointer ORP1S translocates to the nucleus in response to sterol binding. Black-Right-Pointing-Pointer The sterols that best promote nuclear import of ORP1S are LXR agonists. Black-Right-Pointing-Pointer ORP1S binds to LXRs, enhances binding of LXRs to LXREs and promotes LXR-dependent transcription of apoE.« less

Def1 interacts with TFIIH and modulates RNA polymerase II transcription.

PubMed

Damodaren, Nivedita; Van Eeuwen, Trevor; Zamel, Joanna; Lin-Shiao, Enrique; Kalisman, Nir; Murakami, Kenji

2017-12-12

The DNA damage response is an essential process for the survival of living cells. In a subset of stress-responsive genes in humans, Elongin controls transcription in response to multiple stimuli, such as DNA damage, oxidative stress, and heat shock. Yeast Elongin (Ela1-Elc1), along with Def1, is known to facilitate ubiquitylation and degradation of RNA polymerase II (pol II) in response to multiple stimuli, yet transcription activity has not been examined. We have found that Def1 copurifies from yeast whole-cell extract with TFIIH, the largest general transcription factor required for transcription initiation and nucleotide excision repair. The addition of recombinant Def1 and Ela1-Elc1 enhanced transcription initiation in an in vitro reconstituted system including pol II, the general transcription factors, and TFIIS. Def1 also enhanced transcription restart from TFIIS-induced cleavage in a pol II transcribing complex. In the Δdef1 strain, heat shock genes were misregulated, indicating that Def1 is required for induction of some stress-responsive genes in yeast. Taken together, our results extend the understanding of the molecular mechanism of transcription regulation on cellular stress and reveal functional similarities to the mammalian system.

Integrative analysis with ChIP-seq advances the limits of transcript quantification from RNA-seq.

PubMed

Liu, Peng; Sanalkumar, Rajendran; Bresnick, Emery H; Keleş, Sündüz; Dewey, Colin N

2016-08-01

RNA-seq is currently the technology of choice for global measurement of transcript abundances in cells. Despite its successes, isoform-level quantification remains difficult because short RNA-seq reads are often compatible with multiple alternatively spliced isoforms. Existing methods rely heavily on uniquely mapping reads, which are not available for numerous isoforms that lack regions of unique sequence. To improve quantification accuracy in such difficult cases, we developed a novel computational method, prior-enhanced RSEM (pRSEM), which uses a complementary data type in addition to RNA-seq data. We found that ChIP-seq data of RNA polymerase II and histone modifications were particularly informative in this approach. In qRT-PCR validations, pRSEM was shown to be superior than competing methods in estimating relative isoform abundances within or across conditions. Data-driven simulations suggested that pRSEM has a greatly decreased false-positive rate at the expense of a small increase in false-negative rate. In aggregate, our study demonstrates that pRSEM transforms existing capacity to precisely estimate transcript abundances, especially at the isoform level. © 2016 Liu et al.; Published by Cold Spring Harbor Laboratory Press.

Blue light alters miR167 expression and microRNA-targeted auxin response factor genes in Arabidopsis thaliana plants.

PubMed

Pashkovskiy, Pavel P; Kartashov, Alexander V; Zlobin, Ilya E; Pogosyan, Sergei I; Kuznetsov, Vladimir V

2016-07-01

The effect of blue LED (450 nm) on the photomorphogenesis of Arabidopsis thaliana Col-0 plants and the transcript levels of several genes, including miRNAs, photoreceptors and auxin response factors (ARF) was investigated. It was observed that blue light accelerated the generative development, reduced the rosette leaf number, significantly reduced the leaf area, dry biomass and led to the disruption of conductive tissue formation. The blue LED differentially influenced the transcript levels of several phytochromes (PHY a, b, c, d, and e), cryptochromes (CRY 1 and 2) and phototropins (PHOT 1 and 2). At the same time, the blue LED significantly increased miR167 expression compared to a fluorescent lamp or white LEDs. This increase likely resulted in the enhanced transcription of the auxin response factor genes ARF4 and ARF8, which are regulated by this miRNA. These findings support the hypothesis that the effects of blue light on A. thaliana are mediated by auxin signalling pathway involving miRNA-dependent regulation of ARF gene expression. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Genome-wide Expression Analysis and Metabolite Profiling Elucidate Transcriptional Regulation of Flavonoid Biosynthesis and Modulation under Abiotic Stresses in Banana

PubMed Central

Pandey, Ashutosh; Alok, Anshu; Lakhwani, Deepika; Singh, Jagdeep; Asif, Mehar H.; Trivedi, Prabodh K.

2016-01-01

Flavonoid biosynthesis is largely regulated at the transcriptional level due to the modulated expression of genes related to the phenylpropanoid pathway in plants. Although accumulation of different flavonoids has been reported in banana, a staple fruit crop, no detailed information is available on regulation of the biosynthesis in this important plant. We carried out genome-wide analysis of banana (Musa acuminata, AAA genome) and identified 28 genes belonging to 9 gene families associated with flavonoid biosynthesis. Expression analysis suggested spatial and temporal regulation of the identified genes in different tissues of banana. Analysis revealed enhanced expression of genes related to flavonol and proanthocyanidin (PA) biosynthesis in peel and pulp at the early developmental stages of fruit. Genes involved in anthocyanin biosynthesis were highly expressed during banana fruit ripening. In general, higher accumulation of metabolites was observed in the peel as compared to pulp tissue. A correlation between expression of genes and metabolite content was observed at the early stage of fruit development. Furthermore, this study also suggests regulation of flavonoid biosynthesis, at transcriptional level, under light and dark exposures as well as methyl jasmonate (MJ) treatment in banana. PMID:27539368

Genome-wide Expression Analysis and Metabolite Profiling Elucidate Transcriptional Regulation of Flavonoid Biosynthesis and Modulation under Abiotic Stresses in Banana.

PubMed

Pandey, Ashutosh; Alok, Anshu; Lakhwani, Deepika; Singh, Jagdeep; Asif, Mehar H; Trivedi, Prabodh K

2016-08-19

Flavonoid biosynthesis is largely regulated at the transcriptional level due to the modulated expression of genes related to the phenylpropanoid pathway in plants. Although accumulation of different flavonoids has been reported in banana, a staple fruit crop, no detailed information is available on regulation of the biosynthesis in this important plant. We carried out genome-wide analysis of banana (Musa acuminata, AAA genome) and identified 28 genes belonging to 9 gene families associated with flavonoid biosynthesis. Expression analysis suggested spatial and temporal regulation of the identified genes in different tissues of banana. Analysis revealed enhanced expression of genes related to flavonol and proanthocyanidin (PA) biosynthesis in peel and pulp at the early developmental stages of fruit. Genes involved in anthocyanin biosynthesis were highly expressed during banana fruit ripening. In general, higher accumulation of metabolites was observed in the peel as compared to pulp tissue. A correlation between expression of genes and metabolite content was observed at the early stage of fruit development. Furthermore, this study also suggests regulation of flavonoid biosynthesis, at transcriptional level, under light and dark exposures as well as methyl jasmonate (MJ) treatment in banana.

Tissue contaminants and associated transcriptional response in trout liver from high elevation lakes of Washington

USGS Publications Warehouse

Moran, P.W.; Aluru, N.; Black, R.W.; Vijayan, M.M.

2007-01-01

The consistent cold temperatures and large amount of precipitation in the Olympic and Cascade ranges of Washington State are thought to enhance atmospheric deposition of contaminants. However, little is known about contaminant levels in organisms residing in these remote high elevation lakes. We measured total mercury and 28 organochlorine compounds in trout collected from 14 remote lakes in the Olympic, Mt. Rainer, and North Cascades National Parks. Mercury was detected in trout from all lakes sampled (15 to 262 ??g/kg ww), while two organochlorines, total polychlorinated biphenyls (tPCB) and dichlorodiphenyldichloroethylene (DDE), were also detected in these fish tissues (<25 ??g/kg ww). In sediments, organochlorine levels were below detection, while median total and methyl mercury were 30.4 and 0.34 ??g/ kg dry weight (ww), respectively. Using fish from two lakes, representing different contaminant loading levels (Wilcox lake: high; Skymo lake: low), we examined transcriptional response in the liver using a custom-made low-density targeted rainbow trout cDNA microarray. We detected significant differences in liver transcriptional response, including significant changes in metabolic, endocrine, and immune-related genes, in fish collected from Wilcox Lake compared to Skymo Lake. Overall, our results suggest that local urban areas contribute to the observed contaminant patterns in these high elevation lakes, while the transcriptional changes point to a biological response associated with exposure to these contaminants in fish. Specifically, the gene expression pattern leads us to hypothesize a role for mercury in disrupting the metabolic and reproductive pathways in fish from high elevation lakes in western Washington. ?? 2007 American Chemical Society.

Induction of antiviral genes, Mx and vig-1, by dsRNA and Chum salmon reovirus in rainbow trout monocyte/macrophage and fibroblast cell lines.

PubMed

DeWitte-Orr, Stephanie J; Leong, Jo-Ann C; Bols, Niels C

2007-09-01

The expression of potential antiviral genes, Mx1, Mx2, Mx3 and vig-1, was studied in two rainbow trout cell lines: monocyte/macrophage RTS11 and fibroblast-like RTG-2. Transcripts were monitored by RT-PCR; Mx protein by Western blotting. In unstimulated cultures Mx1 and vig-1 transcripts were seen occasionally in RTS11 but rarely in RTG-2. A low level of Mx protein was seen in unstimulated RTS11 but not in RTG-2. In both cell lines, Mx and vig-1 transcripts were induced by a dsRNA, poly inosinic: poly cytidylic acid (poly IC), and by Chum salmon reovirus (CSV). Medium conditioned by cells previously exposed to poly IC or CSV and assumed to contain interferon (IFN) induced the antiviral genes in RTS11. However, RTG-2 responded only to medium conditioned by RTG-2 exposed previously to CSV. In both cell lines, poly IC and CSV induced Mx transcripts in the presence of cycloheximide, suggesting a direct induction mechanism, independent of IFN, was also possible. For CSV, ribavirin blocked induction in RTS11 but not in RTG-2, suggesting viral RNA synthesis was required for induction only in RTS11. In both RTS11 and RTG-2 cultures, Mx protein showed enhanced accumulation by 24h after exposure to poly IC and CSV, but subsequently Mx protein levels declined back to control levels in RTS11 but not in RTG-2. These results suggest that Mx can be regulated differently in macrophages and fibroblasts.

Caveolin-1–mediated Suppression of Cyclooxygenase-2 via a β-catenin-Tcf/Lef–dependent Transcriptional Mechanism Reduced Prostaglandin E2 Production and Survivin Expression

PubMed Central

Rodriguez, Diego A.; Tapia, Julio C.; Fernandez, Jaime G.; Torres, Vicente A.; Muñoz, Nicolas; Galleguillos, Daniela; Leyton, Lisette

2009-01-01

Augmented expression of cyclooxygenase-2 (COX-2) and enhanced production of prostaglandin E2 (PGE2) are associated with increased tumor cell survival and malignancy. Caveolin-1 is a scaffold protein that has been proposed to function as a tumor suppressor in human cancer cells, although mechanisms underlying this ability remain controversial. Intriguingly, the possibility that caveolin-1 regulates the expression of COX-2 has not been explored. Here we show that augmented caveolin-1 expression in cells with low basal levels of this protein, such as human colon cancer (HT29, DLD-1), breast cancer (ZR75), and embryonic kidney (HEK293T) cells reduced COX-2 mRNA and protein levels and β-catenin-Tcf/Lef and COX-2 gene reporter activity, as well as the production of PGE2 and cell proliferation. Moreover, COX-2 overexpression or PGE2 supplementation increased levels of the inhibitor of apoptosis protein survivin by a transcriptional mechanism, as determined by PCR analysis, survivin gene reporter assays and Western blotting. Furthermore, addition of PGE2 to the medium prevented effects attributed to caveolin-1–mediated inhibition of β-catenin-Tcf/Lef–dependent transcription. Finally, PGE2 reduced the coimmunoprecipitation of caveolin-1 with β-catenin and their colocalization at the plasma membrane. Thus, by reducing COX-2 expression, caveolin-1 interrupts a feedback amplification loop involving PGE2-induced signaling events linked to β-catenin/Tcf/Lef–dependent transcription of tumor survival genes including cox-2 itself and survivin. PMID:19244345

Sleep is not just for the brain: transcriptional responses to sleep in peripheral tissues

PubMed Central

2013-01-01

Background Many have assumed that the primary function of sleep is for the brain. We evaluated the molecular consequences of sleep and sleep deprivation outside the brain, in heart and lung. Using microarrays we compared gene expression in tissue from sleeping and sleep deprived mice euthanized at the same diurnal times. Results In each tissue, nearly two thousand genes demonstrated statistically significant differential expression as a function of sleep/wake behavioral state. To mitigate the influence of an artificial deprivation protocol, we identified a subset of these transcripts as specifically sleep-enhanced or sleep-repressed by requiring that their expression also change over the course of unperturbed sleep. 3% and 6% of the assayed transcripts showed “sleep specific” changes in the lung and heart respectively. Sleep specific transcripts in these tissues demonstrated highly significant overlap and shared temporal dynamics. Markers of cellular stress and the unfolded protein response were reduced during sleep in both tissues. These results mirror previous findings in brain. Sleep-enhanced pathways reflected the unique metabolic functions of each tissue. Transcripts related to carbohydrate and sulfur metabolic processes were enhanced by sleep in the lung, and collectively favor buffering from oxidative stress. DNA repair and protein metabolism annotations were significantly enriched among the sleep-enhanced transcripts in the heart. Our results also suggest that sleep may provide a Zeitgeber, or synchronizing cue, in the lung as a large cluster of transcripts demonstrated systematic changes in inter-animal variability as a function of both sleep duration and circadian time. Conclusion Our data support the notion that the molecular consequences of sleep/wake behavioral state extend beyond the brain to include peripheral tissues. Sleep state induces a highly overlapping response in both heart and lung. We conclude that sleep enhances organ specific molecular functions and that it has a ubiquitous role in reducing cellular metabolic stress in both brain and peripheral tissues. Finally, our data suggest a novel role for sleep in synchronizing transcription in peripheral tissues. PMID:23721503

Quantifying Intrinsic and Extrinsic Variability in Stochastic Gene Expression Models

PubMed Central

Singh, Abhyudai; Soltani, Mohammad

2013-01-01

Genetically identical cell populations exhibit considerable intercellular variation in the level of a given protein or mRNA. Both intrinsic and extrinsic sources of noise drive this variability in gene expression. More specifically, extrinsic noise is the expression variability that arises from cell-to-cell differences in cell-specific factors such as enzyme levels, cell size and cell cycle stage. In contrast, intrinsic noise is the expression variability that is not accounted for by extrinsic noise, and typically arises from the inherent stochastic nature of biochemical processes. Two-color reporter experiments are employed to decompose expression variability into its intrinsic and extrinsic noise components. Analytical formulas for intrinsic and extrinsic noise are derived for a class of stochastic gene expression models, where variations in cell-specific factors cause fluctuations in model parameters, in particular, transcription and/or translation rate fluctuations. Assuming mRNA production occurs in random bursts, transcription rate is represented by either the burst frequency (how often the bursts occur) or the burst size (number of mRNAs produced in each burst). Our analysis shows that fluctuations in the transcription burst frequency enhance extrinsic noise but do not affect the intrinsic noise. On the contrary, fluctuations in the transcription burst size or mRNA translation rate dramatically increase both intrinsic and extrinsic noise components. Interestingly, simultaneous fluctuations in transcription and translation rates arising from randomness in ATP abundance can decrease intrinsic noise measured in a two-color reporter assay. Finally, we discuss how these formulas can be combined with single-cell gene expression data from two-color reporter experiments for estimating model parameters. PMID:24391934

Quantifying intrinsic and extrinsic variability in stochastic gene expression models.

PubMed

Singh, Abhyudai; Soltani, Mohammad

2013-01-01

Genetically identical cell populations exhibit considerable intercellular variation in the level of a given protein or mRNA. Both intrinsic and extrinsic sources of noise drive this variability in gene expression. More specifically, extrinsic noise is the expression variability that arises from cell-to-cell differences in cell-specific factors such as enzyme levels, cell size and cell cycle stage. In contrast, intrinsic noise is the expression variability that is not accounted for by extrinsic noise, and typically arises from the inherent stochastic nature of biochemical processes. Two-color reporter experiments are employed to decompose expression variability into its intrinsic and extrinsic noise components. Analytical formulas for intrinsic and extrinsic noise are derived for a class of stochastic gene expression models, where variations in cell-specific factors cause fluctuations in model parameters, in particular, transcription and/or translation rate fluctuations. Assuming mRNA production occurs in random bursts, transcription rate is represented by either the burst frequency (how often the bursts occur) or the burst size (number of mRNAs produced in each burst). Our analysis shows that fluctuations in the transcription burst frequency enhance extrinsic noise but do not affect the intrinsic noise. On the contrary, fluctuations in the transcription burst size or mRNA translation rate dramatically increase both intrinsic and extrinsic noise components. Interestingly, simultaneous fluctuations in transcription and translation rates arising from randomness in ATP abundance can decrease intrinsic noise measured in a two-color reporter assay. Finally, we discuss how these formulas can be combined with single-cell gene expression data from two-color reporter experiments for estimating model parameters.

PmRunt regulated by Pm-miR-183 participates in nacre formation possibly through promoting the expression of collagen VI-like and Nacrein in pearl oyster Pinctada martensii.

PubMed

Zheng, Zhe; Du, Xiaodong; Xiong, Xinwei; Jiao, Yu; Deng, Yuewen; Wang, Qingheng; Huang, Ronglian

2017-01-01

Heterodimeric PEBP2/CBFs are key regulators in diverse biological processes, such as haematopoietic stem-cell generation, bone formation and cancers. In this work, we cloned runt-like transcriptional factor (designated as PmRunt) and CBF β (designated as PmCBF) gene, which comprise the heterodimeric transcriptional factor in Pinctada martensii. PmRunt was identified with an open reading frame that encodes 545 amino acids and has typical Runt domain. Phylogenetic analysis results speculated that runt-like transcriptional factors (RDs) in vertebrates and invertebrates are separated into two branches. In molluscs, PmRunt and other RDs are clustered in one of these branches. Direct interaction between PmRunt and PmCBF was evidenced by yeast two-hybrid assay results. Gene repression by RNA interference decreased the expression level of PmRunt, and subsequent observation of the inner surface of the nacre by scanning electron microscopy demonstrated disordered growth. The luciferase activities of reporters that contain promoter regions of Collagen VI-like (PmColVI) and PmNacrein were enhanced by PmRunt. Meanwhile, Pm-miR-183 apparently inhibited the relative luciferase activity of reporters containing the 3'-UTR of PmRunt. The expression level of PmRunt was repressed after Pm-miR-183 was overexpressed in the mantle tissue. Therefore, we proposed that PmRunt could be targeted by Pm-miR-183 and regulate the transcription of PmColVI and PmNacrein by increasing their transcriptional activity, thereby governing nacre formation.

Heat Shock Protein-90 Inhibitors Enhance Antigen Expression on Melanomas and Increase T Cell Recognition of Tumor Cells

PubMed Central

Haggerty, Timothy J.; Dunn, Ian S.; Rose, Lenora B.; Newton, Estelle E.; Pandolfi, Franco; Kurnick, James T.

2014-01-01

In an effort to enhance antigen-specific T cell recognition of cancer cells, we have examined numerous modulators of antigen-expression. In this report we demonstrate that twelve different Hsp90 inhibitors (iHsp90) share the ability to increase the expression of differentiation antigens and MHC Class I antigens. These iHsp90 are active in several molecular and cellular assays on a series of tumor cell lines, including eleven human melanomas, a murine B16 melanoma, and two human glioma-derived cell lines. Intra-cytoplasmic antibody staining showed that all of the tested iHsp90 increased expression of the melanocyte differentiation antigens Melan-A/MART-1, gp100, and TRP-2, as well as MHC Class I. The gliomas showed enhanced gp100 and MHC staining. Quantitative analysis of mRNA levels showed a parallel increase in message transcription, and a reporter assay shows induction of promoter activity for Melan-A/MART-1 gene. In addition, iHsp90 increased recognition of tumor cells by T cells specific for Melan-A/MART-1. In contrast to direct Hsp90 client proteins, the increased levels of full-length differentiation antigens that result from iHsp90 treatment are most likely the result of transcriptional activation of their encoding genes. In combination, these results suggest that iHsp90 improve recognition of tumor cells by T cells specific for a melanoma-associated antigen as a result of increasing the expressed intracellular antigen pool available for processing and presentation by MHC Class I, along with increased levels of MHC Class I itself. As these Hsp90 inhibitors do not interfere with T cell function, they could have potential for use in immunotherapy of cancer. PMID:25503774

The ctenidium of the giant clam, Tridacna squamosa, expresses an ammonium transporter 1 that displays light-suppressed gene and protein expression and may be involved in ammonia excretion.

PubMed

Boo, Mel V; Hiong, Kum C; Goh, Enan J K; Choo, Celine Y L; Wong, Wai P; Chew, Shit F; Ip, Yuen K

2018-04-24

Ammonium transporters (AMTs) can participate in ammonia uptake or excretion across the plasma membrane of prokaryotic, plant and invertebrate cells. The giant clam, Tridacna squamosa, harbors nitrogen-deficient symbiotic zooxanthellae, and normally conducts light-enhanced ammonia absorption to benefit the symbionts. Nonetheless, it can excrete ammonia when there is a supply of exogenous nitrogen or exposed to continuous darkness. This study aimed to elucidate the role of AMT1 in the ctenidium of T. squamosa by cloning and characterizing the AMT1/AMT1, determining its subcellular localization, and examining changes in its transcript and protein expression levels in response to light exposure. The cDNA coding sequence of AMT1 from T. squamosa consisted of 1527 bp and encoded 508 amino acids of 54.6 kDa. AMT1-immunofluorescence was detected mainly at the apical epithelium of ctenidial filaments, and it decreased significantly after 12 h of exposure to light. By contrast, the epithelial cells surrounding the tertiary water channels in the ctentidium, which are known to exhibit light-enhanced glutamine synthetase expression and take part in the assimilation of exogenous ammonia in light, did not display any AMT1-immunolabelling. Furthermore, the transcript level and protein abundance of ctenidial AMT1/AMT1 decreased significantly at the 6th and 12th h of light exposure. Taken together, these results indicate that AMT1 might participate in ammonia excretion instead of ammonia absorption and assimilation in T. squamosa. It is probable that the expression levels of AMT1/AMT1 need to be down-regulated during light exposure to achieve light-enhanced ammonia uptake.

Insertion of transposon in the vicinity of SSK2 confers enhanced tolerance to furfural in Saccharomyces cerevisiae.

PubMed

Kim, Hyun-Soo; Kim, Na-Rae; Kim, Wankee; Choi, Wonja

2012-07-01

Furfural is one of the major inhibitors generated during sugar production from cellulosic materials and, as an aldehyde, inhibits various cellular activities of microorganisms used, leading to prolonged lag time during ethanologenic fermentation. Since Saccharomyces cerevisiae strains tolerant to furfural are of great economic benefit in producing bioethanol, much effort to obtain more efficient strains continues to be made. In this study, we examined the furfural tolerance of transposon mutant strains (Tn 1-5) with enhanced ethanol tolerance and found that one of them (Tn 2), in which SSK2 is downregulated at the transcriptional level, displayed improved furfural tolerance. Such phenotype was abolished by complementation of the entire open reading frame of SSK2, which encodes a mitogen-activated protein (MAP) kinase kinase kinase of the high osmolarity glycerol (HOG) signaling pathway, suggesting an inhibitory effect of SSK2 in coping with furfural stress. Tn 2 showed a significant decrease in the intracellular level of reactive oxygen species (ROS) and early and high activation of Hog1p, a MAP kinase integral to the HOG pathway in response to furfural. The transcriptional levels of CTT1 and GLR1, two of known Hog1p downstream target genes whose protein products are involved in reducing ROS, were increased by 43 % and 56 % respectively compared with a control strain, probably resulting in the ROS decrease. Tn 2 also showed a shortened lag time during fermentation in the presence of furfural, resulting from efficient conversion of furfural to non-toxic (or less toxic) furfuryl alcohol. Taken together, the enhanced furfural tolerance of Tn 2 is suggested to be conferred by the combined effect of an early event of less ROS accumulation and a late event of efficient detoxification of furfural.

The role of DNA methylation in catechol-enhanced erythroid differentiation of K562 cells

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

Li, Xiao-Fei; Wu, Xiao-Rong; Xue, Ming

2012-11-15

Catechol is one of phenolic metabolites of benzene in vivo. Catechol is also widely used in pharmaceutical and chemical industries. In addition, fruits, vegetables and cigarette smoke also contain catechol. Our precious study showed that several benzene metabolites (phenol, hydroquinone, and 1,2,4-benzenetriol) inhibited erythroid differentiation of K562 cells. In present study, the effect of catechol on erythroid differentiation of K562 cells was investigated. Moreover, to address the role of DNA methylation in catechol-induced effect on erythroid differentiation in K562 cells, methylation levels of erythroid-specific genes were analyzed by Quantitative MassARRAY methylation analysis platform. Benzidine staining showed that exposure to catecholmore » enhanced hemin-induced hemoglobin accumulation in K562 cells in concentration- and time-dependent manners. The mRNA expression of erythroid specific genes, including α-globin, β-globin, γ-globin, erythroid 5-aminolevulinate synthase, erythroid porphobilinogen deaminase, and transcription factor GATA-1 genes, showed a significant concentration-dependent increase in catechol-treated K562 cells. The exposure to catechol caused a decrease in DNA methylation levels at a few CpG sites in some erythroid specific genes including α-globin, β-globin and erythroid porphobilinogen deaminase genes. These results indicated that catechol improved erythroid differentiation potency of K562 cells at least partly via up-regulating transcription of some erythroid related genes, and suggested that inhibition of DNA methylation might be involved in up-regulated expression of some erythroid related genes. -- Highlights: ► Catechol enhanced hemin-induced hemoglobin accumulation. ► Exposure to catechol resulted in up-regulated expression of erythroid genes. ► Catechol reduced methylation levels at some CpG sites in erythroid genes.« less

Oxidized low density lipoprotein increases RANKL level in human vascular cells. Involvement of oxidative stress

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

Mazière, Cécile, E-mail: maziere.cecile@chu-amiens.fr; Salle, Valéry; INSERM U1088

Highlights: •Oxidized LDL enhances RANKL level in human smooth muscle cells. •The effect of OxLDL is mediated by the transcription factor NFAT. •UVA, H{sub 2}O{sub 2} and buthionine sulfoximine also increase RANKL level. •All these effects are observed in human fibroblasts and endothelial cells. -- Abstract: Receptor Activator of NFκB Ligand (RANKL) and its decoy receptor osteoprotegerin (OPG) have been shown to play a role not only in bone remodeling but also in inflammation, arterial calcification and atherosclerotic plaque rupture. In human smooth muscle cells, Cu{sup 2+}-oxidized LDL (CuLDL) 10–50 μg/ml increased reactive oxygen species (ROS) and RANKL level inmore » a dose-dependent manner, whereas OPG level was not affected. The lipid extract of CuLDL reproduced the effects of the whole particle. Vivit, an inhibitor of the transcription factor NFAT, reduced the CuLDL-induced increase in RANKL, whereas PKA and NFκB inhibitors were ineffective. LDL oxidized by myeloperoxidase (MPO-LDL), or other pro-oxidant conditions such as ultraviolet A (UVA) irradiation, incubation with H{sub 2}O{sub 2} or with buthionine sulfoximine (BSO), an inhibitor of glutathione synthesis{sub ,} also induced an oxidative stress and enhanced RANKL level. The increase in RANKL in pro-oxidant conditions was also observed in fibroblasts and endothelial cells. Since RANKL is involved in myocardial inflammation, vascular calcification and plaque rupture, this study highlights a new mechanism whereby OxLDL might, by generation of an oxidative stress, exert a deleterious effect on different cell types of the arterial wall.« less

Commensal Microbiota Enhance Both Osteoclast and Osteoblast Activities.

PubMed

Uchida, Yoko; Irie, Koichiro; Fukuhara, Daiki; Kataoka, Kota; Hattori, Takako; Ono, Mitsuaki; Ekuni, Daisuke; Kubota, Satoshi; Morita, Manabu

2018-06-23

Recent studies suggest that the commensal microbiota affects not only host energy metabolism and development of immunity but also bone remodeling by positive regulation of osteoclast activity. However, the mechanism of regulation of bone cells by the commensal microbiota has not been elucidated. In this study, 8-week-old specific pathogen-free (SPF) and germ-free (GF) mice were compared in terms of alveolar bones and primary osteoblasts isolated from calvarias. Micro-CT analysis showed that SPF mice had larger body size associated with lower bone mineral density and bone volume fraction in alveolar bones compared with GF mice. Greater numbers of osteoclasts in alveolar bone and higher serum levels of tartrate-resistant acid phosphatase 5b were observed in SPF mice. Tissue extracts from SPF alveolar bone showed higher levels of cathepsin K, indicating higher osteoclast activity. SPF alveolar extracts also showed elevated levels of γ-carboxylated glutamic acid⁻osteocalcin as a marker of mature osteoblasts compared with GF mice. Polymerase chain reaction (PCR) array analysis of RNA directly isolated from alveolar bone showed that in SPF mice, expression of mRNA of osteocalcin , which also acts as an inhibitor of bone mineralization, was strongly enhanced compared with GF mice. Cultured calvarial osteoblasts from SPF mice showed reduced mineralization but significantly enhanced expression of mRNAs of osteocalcin, alkaline phosphatase, insulin-like growth factor-I/II , and decreased ratio of osteoprotegerin/receptor activator of nuclear factor-kappa B ligand compared with GF mice. Furthermore, PCR array analyses of transcription factors in cultured calvarial osteoblasts showed strongly upregulated expression of Forkhead box g1 . In contrast, Gata-binding protein 3 was strongly downregulated in SPF osteoblasts. These results suggest that the commensal microbiota prevents excessive mineralization possibly by stimulating osteocalcin expression in osteoblasts, and enhances both osteoblast and osteoclast activity by regulating specific transcription factors.

Downregulated long non-coding RNA MEG3 in breast cancer regulates proliferation, migration and invasion by depending on p53’s transcriptional activity

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

Sun, Lin; Li, Yu; Yang, Bangxiang, E-mail: b19933009@qq.coom

Long non-coding RNAs (lncRNAs) was found to play critical roles in tumorigenesis, hence, screen of tumor-related lncRNAs, identification of their biological roles is important for understanding the processes of tumorigenesis. In this study, we identified the expressing difference of several tumor-related lncRNAs in breast cancer samples and found that, MEG3, which is downregulated in non-small cell lung cancer (NSCLC) tumor tissues, is also downregulated in breast cancer samples compared with adjacent tissues. For figuring out the effect of MEG3 in breast cancer cells MCF7 and MB231, we overexpressed MEG3 in these cells, and found that it resulted the inhibition ofmore » proliferation, colony formation, migration and invasion capacities by enhancing p53’s transcriptional activity on its target genes, including p21, Maspin and KAI1. MEG3 presented similar effects in MB157, which is a p53-null breast cancer cell line, when functional p53 but not p53R273H mutant, which lacks transcriptional activity, was introduced. Surprisingly, overexpression of MEG3 activates p53’s transcriptional activity by decreasing MDM2’s transcription level, and thus stabilizes and accumulates P53. Taken together, our findings indicate that MEG3 is downregulated in breast cancer tissues and affects breast cancer cells’ malignant behaviors, which indicate MEG3 a potential therapeutic target for breast cancer. - Highlights: • MEG3 RNA is widely downregulated in breast tumor tissue. • MEG3 regulates P53 indirectly through transcriptional regulation of MDM2. • Under unstressed condition, MEG3-related P53 accumulation transcriptionally activates p53’s target genes. • MEG3 expression level tightly regulates proliferation, colony formation, migration and invasion in breast tumor cells.« less

Matrix Metalloproteinase (MMP) 9 Transcription in Mouse Brain Induced by Fear Learning*

PubMed Central

Ganguly, Krishnendu; Rejmak, Emilia; Mikosz, Marta; Nikolaev, Evgeni; Knapska, Ewelina; Kaczmarek, Leszek

2013-01-01

Memory formation requires learning-based molecular and structural changes in neurons, whereas matrix metalloproteinase (MMP) 9 is involved in the synaptic plasticity by cleaving extracellular matrix proteins and, thus, is associated with learning processes in the mammalian brain. Because the mechanisms of MMP-9 transcription in the brain are poorly understood, this study aimed to elucidate regulation of MMP-9 gene expression in the mouse brain after fear learning. We show here that contextual fear conditioning markedly increases MMP-9 transcription, followed by enhanced enzymatic levels in the three major brain structures implicated in fear learning, i.e. the amygdala, hippocampus, and prefrontal cortex. To reveal the role of AP-1 transcription factor in MMP-9 gene expression, we have used reporter gene constructs with specifically mutated AP-1 gene promoter sites. The constructs were introduced into the medial prefrontal cortex of neonatal mouse pups by electroporation, and the regulation of MMP-9 transcription was studied after contextual fear conditioning in the adult animals. Specifically, −42/-50- and −478/-486-bp AP-1 binding motifs of the mouse MMP-9 promoter sequence have been found to play a major role in MMP-9 gene activation. Furthermore, increases in MMP-9 gene promoter binding by the AP-1 transcription factor proteins c-Fos and c-Jun have been demonstrated in all three brain structures under investigation. Hence, our results suggest that AP-1 acts as a positive regulator of MMP-9 transcription in the brain following fear learning. PMID:23720741

Matrix metalloproteinase (MMP) 9 transcription in mouse brain induced by fear learning.

PubMed

Ganguly, Krishnendu; Rejmak, Emilia; Mikosz, Marta; Nikolaev, Evgeni; Knapska, Ewelina; Kaczmarek, Leszek

2013-07-19

Memory formation requires learning-based molecular and structural changes in neurons, whereas matrix metalloproteinase (MMP) 9 is involved in the synaptic plasticity by cleaving extracellular matrix proteins and, thus, is associated with learning processes in the mammalian brain. Because the mechanisms of MMP-9 transcription in the brain are poorly understood, this study aimed to elucidate regulation of MMP-9 gene expression in the mouse brain after fear learning. We show here that contextual fear conditioning markedly increases MMP-9 transcription, followed by enhanced enzymatic levels in the three major brain structures implicated in fear learning, i.e. the amygdala, hippocampus, and prefrontal cortex. To reveal the role of AP-1 transcription factor in MMP-9 gene expression, we have used reporter gene constructs with specifically mutated AP-1 gene promoter sites. The constructs were introduced into the medial prefrontal cortex of neonatal mouse pups by electroporation, and the regulation of MMP-9 transcription was studied after contextual fear conditioning in the adult animals. Specifically, -42/-50- and -478/-486-bp AP-1 binding motifs of the mouse MMP-9 promoter sequence have been found to play a major role in MMP-9 gene activation. Furthermore, increases in MMP-9 gene promoter binding by the AP-1 transcription factor proteins c-Fos and c-Jun have been demonstrated in all three brain structures under investigation. Hence, our results suggest that AP-1 acts as a positive regulator of MMP-9 transcription in the brain following fear learning.

The oncoprotein HBXIP upregulates PDGFB via activating transcription factor Sp1 to promote the proliferation of breast cancer cells

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

Zhang, Yingyi; Zhao, Yu; Li, Leilei

2013-05-03

Highlights: •HBXIP is able to upregulate the expression of PDGFB in breast cancer cells. •HBXIP serves as a coactivator of activating transcription factor Sp1. •HBXIP stimulates the PDGFB promoter via activating transcription factor Sp1. •HBXIP promotes the proliferation of breast cancer cell via upregulating PDGFB. -- Abstract: We have reported that the oncoprotein hepatitis B virus X-interacting protein (HBXIP) acts as a novel transcriptional coactivator to promote proliferation and migration of breast cancer cells. Previously, we showed that HBXIP was able to activate nuclear factor-κB (NF-κB) in breast cancer cells. As an oncogene, the platelet-derived growth factor beta polypeptide (PDGFB)more » plays crucial roles in carcinogenesis. In the present study, we found that both HBXIP and PDGFB were highly expressed in breast cancer cell lines. Interestingly, HBXIP was able to increase transcriptional activity of NF-κB through PDGFB, suggesting that HBXIP is associated with PDGFB in the cells. Moreover, HBXIP was able to upregulate PDGFB at the levels of mRNA, protein and promoter in the cells. Then, we identified that HBXIP stimulated the promoter of PDGFB through activating transcription factor Sp1. In function, HBXIP enhanced the proliferation of breast cancer cells through PDGFB in vitro. Thus, we conclude that HBXIP upregulates PDGFB via activating transcription factor Sp1 to promote proliferation of breast cancer cells.« less

Leptin potentiates Prevotella intermedia lipopolysaccharide-induced production of TNF-alpha in monocyte-derived macrophages.

PubMed

Kim, Sung-Jo

2010-06-01

In addition to regulating body weight, leptin is also recognized for its role in the regulation of immune function and inflammation. The purpose of this study was to investigate the effect of leptin on Prevotella (P.) intermedia lipopolysaccharide (LPS)-induced tumor necrosis factor (TNF)-alpha production in differentiated THP-1 cells, a human monocytic cell line. LPS from P. intermedia ATCC 25611 was prepared by the standard hot phenol-water method. THP-1 cells were incubated in the medium supplemented with phorbol myristate acetate to induce differentiation into macrophage-like cells. The amount of TNF-alpha and interleukin-8 secreted into the culture medium was determined by enzyme-linked immunosorbent assay (ELISA). TNF-alpha and Ob-R mRNA expression levels were determined by semi-quantitative reverse transcription-polymerase chain reaction analysis. Leptin enhanced P. intermedia LPS-induced TNF-alpha production in a dose-dependent manner. Leptin modulated P. intermedia LPS-induced TNF-alpha expression predominantly at the transcriptional level. Effect of leptin on P. intermedia LPS-induced TNF-alpha production was not mediated by the leptin receptor. The ability of leptin to enhance P. intermedia LPS-induced TNF-alpha production may be important in the establishment of chronic lesion accompanied by osseous tissue destruction observed in inflammatory periodontal disease.

Insights into RNA polymerase catalysis and adaptive evolution gained from mutational analysis of a locus conferring rifampicin resistance

PubMed Central

Yurieva, Olga; Nikiforov, Vadim; O’Donnell, Michael

2017-01-01

Abstract S531 of Escherichia coli RNA polymerase (RNAP) β subunit is a part of RNA binding domain in transcription complex. While highly conserved, S531 is not involved in interactions within the transcription complex as suggested by X-ray analysis. To understand the basis for S531 conservation we performed systematic mutagenesis of this residue. We find that the most of the mutations significantly decreased initiation-to-elongation transition by RNAP. Surprisingly, some changes enhanced the production of full-size transcripts by suppressing abortive loss of short RNAs. S531-R increased transcript retention by establishing a salt bridge with RNA, thereby explaining the R substitution at the equivalent position in extremophilic organisms, in which short RNAs retention is likely to be an issue. Generally, the substitutions had the same effect on bacterial doubling time when measured at 20°. Raising growth temperature to 37° ablated the positive influence of some mutations on the growth rate in contrast to their in vitro action, reflecting secondary effects of cellular environment on transcription and complex involvement of 531 locus in the cell biology. The properties of generated RNAP variants revealed an RNA/protein interaction network that is crucial for transcription, thereby explaining the details of initiation-to-elongation transition on atomic level. PMID:29036608

Temporal expression of the human alcohol dehydrogenase gene family during liver development correlates with differential promoter activation by hepatocyte nuclear factor 1, CCAAT/enhancer-binding protein alpha, liver activator protein, and D-element-binding protein.

PubMed Central

van Ooij, C; Snyder, R C; Paeper, B W; Duester, G

1992-01-01

The human class I alcohol dehydrogenase (ADH) gene family consists of ADH1, ADH2, and ADH3, which are sequentially activated in early fetal, late fetal, and postnatal liver, respectively. Analysis of ADH promoters revealed differential activation by several factors previously shown to control liver transcription. In cotransfection assays, the ADH1 promoter, but not the ADH2 or ADH3 promoter, was shown to respond to hepatocyte nuclear factor 1 (HNF-1), which has previously been shown to regulate transcription in early liver development. The ADH2 promoter, but not the ADH1 or ADH3 promoter, was shown to respond to CCAAT/enhancer-binding protein alpha (C/EBP alpha), a transcription factor particularly active during late fetal liver and early postnatal liver development. The ADH1, ADH2, and ADH3 promoters all responded to the liver transcription factors liver activator protein (LAP) and D-element-binding protein (DBP), which are most active in postnatal liver. For all three promoters, the activation by LAP or DBP was higher than that seen by HNF-1 or C/EBP alpha, and a significant synergism between C/EBP alpha and LAP was noticed for the ADH2 and ADH3 promoters when both factors were simultaneously cotransfected. A hierarchy of ADH promoter responsiveness to C/EBP alpha and LAP homo- and heterodimers is suggested. In all three ADH genes, LAP bound to the same four sites previously reported for C/EBP alpha (i.e., -160, -120, -40, and -20 bp), but DBP bound strongly only to the site located at -40 bp relative to the transcriptional start. Mutational analysis of ADH2 indicated that the -40 bp element accounts for most of the promoter regulation by the bZIP factors analyzed. These studies suggest that HNF-1 and C/EBP alpha help establish ADH gene family transcription in fetal liver and that LAP and DBP help maintain high-level ADH gene family transcription in postnatal liver. Images PMID:1620113

6-Mercaptopurine, an activator of Nur77, enhances transcriptional activity of HIF-1alpha resulting in new vessel formation.

PubMed

Yoo, Y-G; Na, T-Y; Yang, W-K; Kim, H-J; Lee, I-K; Kong, G; Chung, J-H; Lee, M-O

2007-05-31

Hypoxia-inducible factor-1alpha (HIF-1alpha) plays a central role in oxygen homeostasis. Previously, we reported that the orphan nuclear receptor Nur77 functions in stabilizing HIF-1alpha. Here, we demonstrate that 6-mercaptopurine (6-MP), an activator of the NR4A family members, enhances transcriptional activity of HIF-1. 6-MP enhanced the protein-level of HIF-1alpha as well as vascular endothelial growth factor (VEGF) in a dose- and time-dependent manner. The induction of HIF-1alpha was abolished by the transfection of either a dominant-negative Nur77 mutant or si-Nur77, indicating a critical role of Nur77 in the 6-MP action. The HIF-1alpha protein level remained up to 60 min in the presence of 6-MP when de novo protein synthesis was blocked by cycloheximide, suggesting that 6-MP induces stabilization of the HIF-1alpha protein. The fact that 6-MP decreased the association of HIF-1alpha with von Hippel-Lindau protein and the acetylation of HIF-1alpha, may explain how 6-MP induced stability of HIF-1alpha. Further, 6-MP induced the transactivation function of HIF-1alpha by recruiting co-activator cyclic-AMP-response-element-binding protein. Finally, 6-MP enhanced the expression of HIF-1alpha and VEGF, and the formation of capillary tubes in human umbilical vascular endothelial cells. Together, our results provide a new insight for 6-MP action in the stabilization of HIF-1alpha and imply a potential application of 6-MP in hypoxia-associated human vascular diseases.

Glucocorticoids enhance activation of the human type II 3beta-hydroxysteroid dehydrogenase/Delta5-Delta4 isomerase gene.

PubMed

Feltus, F Alex; Cote, Stephanie; Simard, Jacques; Gingras, Sebastien; Kovacs, William J; Nicholson, Wendell E; Clark, Barbara J; Melner, Michael H

2002-09-01

Glucocorticoids indirectly alter adrenocortical steroid output through the inhibition of ACTH secretion by the anterior pituitary. However, previous studies suggest that glucocorticoids can directly affect adrenocortical steroid production. Therefore, we have investigated the ability of glucocorticoids to affect transcription of adrenocortical steroid biosynthetic enzymes. One potential target of glucocorticoid action in the adrenal is an enzyme critical for adrenocortical steroid production: 3beta-hydroxysteroid dehydrogenase/Delta5-Delta4 isomerase (3beta-HSD). Treatment of the adrenocortical cell line (H295R) with the glucocorticoid agonist dexamethasone (DEX) increased cortisol production and 3beta-HSD mRNA levels alone or in conjunction with phorbol ester. This increase in 3beta-HSD mRNA was paralleled by increases in Steroidogenic Acute Regulatory Protein (StAR) mRNA levels. The human type II 3beta-HSD promoter lacks a consensus palindromic glucocorticoid response element (GRE) but does contain a Stat5 response element (Stat5RE) suggesting that glucocorticoids could affect type II 3beta-HSD transcription via interaction with Stat5. Transfection experiments show enhancement of human type II 3beta-HSD promoter activity by coexpression of the glucocorticoid receptor (GR) and Stat5A and treatment with 100nM dexamethasone. Furthermore, removal of the Stat5RE either by truncation of the 5' flanking sequence in the promoter or introduction of point mutations to the Stat5RE abolished the ability of DEX to enhance 3beta-HSD promoter activity. These studies demonstrate the ability of glucocorticoids to directly enhance the expression of an adrenal steroidogenic enzyme gene albeit independent of a consensus palindromic glucocorticoid response element.

Heregulin/ErbB3 Signaling Enhances CXCR4-Driven Rac1 Activation and Breast Cancer Cell Motility via Hypoxia-Inducible Factor 1α

PubMed Central

Lopez-Haber, Cynthia; Barrio-Real, Laura; Casado-Medrano, Victoria

2016-01-01

The growth factor heregulin (HRG), a ligand of ErbB3 and ErbB4 receptors, contributes to breast cancer development and the promotion of metastatic disease, and its expression in breast tumors has been associated with poor clinical outcome and resistance to therapy. In this study, we found that breast cancer cells exposed to sustained HRG treatment show markedly enhanced Rac1 activation and migratory activity in response to the CXCR4 ligand SDF-1/CXCL12, effects mediated by P-Rex1, a Rac-guanine nucleotide exchange factor (GEF) aberrantly expressed in breast cancer. Notably, HRG treatment upregulates surface expression levels of CXCR4, a G protein-coupled receptor (GPCR) implicated in breast cancer metastasis and an indicator of poor prognosis in breast cancer patients. A detailed mechanistic analysis revealed that CXCR4 upregulation and sensitization of the Rac response/motility by HRG are mediated by the transcription factor hypoxia-inducible factor 1α (HIF-1α) via ErbB3 and independently of ErbB4. HRG caused prominent induction in the nuclear expression of HIF-1α, which transcriptionally activates the CXCR4 gene via binding to a responsive element located in positions −1376 to −1372 in the CXCR4 promoter, as revealed by mutagenesis analysis and chromatin immunoprecipitation (ChIP). Our results uncovered a novel function for ErbB3 in enhancing breast cancer cell motility and sensitization of the P-Rex1/Rac1 pathway through HIF-1α-mediated transcriptional induction of CXCR4. PMID:27185877

Transcription activator-like (TAL) effectors targeting OsSWEET genes enhance virulence on diverse rice (Oryza sativa) varieties when expressed individually in a TAL effector-deficient strain of Xanthomonas oryzae.

PubMed

Verdier, Valérie; Triplett, Lindsay R; Hummel, Aaron W; Corral, Rene; Cernadas, R Andres; Schmidt, Clarice L; Bogdanove, Adam J; Leach, Jan E

2012-12-01

Genomes of the rice (Oryza sativa) xylem and mesophyll pathogens Xanthomonas oryzae pv. oryzae (Xoo) and pv. oryzicola (Xoc) encode numerous secreted transcription factors called transcription activator-like (TAL) effectors. In a few studied rice varieties, some of these contribute to virulence by activating corresponding host susceptibility genes. Some activate disease resistance genes. The roles of X. oryzae TAL effectors in diverse rice backgrounds, however, are poorly understood. Xoo TAL effectors that promote infection by activating SWEET sucrose transporter genes were expressed in TAL effector-deficient X. oryzae strain X11-5A, and assessed in 21 rice varieties. Some were also tested in Xoc on variety Nipponbare. Several Xoc TAL effectors were tested in X11-5A on four rice varieties. Xoo TAL effectors enhanced X11-5A virulence on most varieties, but to varying extents depending on the effector and variety. SWEET genes were activated in all tested varieties, but increased virulence did not correlate with activation level. SWEET activators also enhanced Xoc virulence on Nipponbare. Xoc TAL effectors did not alter X11-5A virulence. SWEET-targeting TAL effectors contribute broadly and non-tissue-specifically to virulence in rice, and their function is affected by host differences besides target sequences. Further, the utility of X11-5A for characterizing individual TAL effectors in rice was established. © 2012 The Authors. New Phytologist © 2012 New Phytologist Trust.

Role of Architecture in the Function and Specificity of Two Notch-Regulated Transcriptional Enhancer Modules

PubMed Central

Liu, Feng; Posakony, James W.

2012-01-01

In Drosophila melanogaster, cis-regulatory modules that are activated by the Notch cell–cell signaling pathway all contain two types of transcription factor binding sites: those for the pathway's transducing factor Suppressor of Hairless [Su(H)] and those for one or more tissue- or cell type–specific factors called “local activators.” The use of different “Su(H) plus local activator” motif combinations, or codes, is critical to ensure that only the correct subset of the broadly utilized Notch pathway's target genes are activated in each developmental context. However, much less is known about the role of enhancer “architecture”—the number, order, spacing, and orientation of its component transcription factor binding motifs—in determining the module's specificity. Here we investigate the relationship between architecture and function for two Notch-regulated enhancers with spatially distinct activities, each of which includes five high-affinity Su(H) sites. We find that the first, which is active specifically in the socket cells of external sensory organs, is largely resistant to perturbations of its architecture. By contrast, the second enhancer, active in the “non-SOP” cells of the proneural clusters from which neural precursors arise, is sensitive to even simple rearrangements of its transcription factor binding sites, responding with both loss of normal specificity and striking ectopic activity. Thus, diverse cryptic specificities can be inherent in an enhancer's particular combination of transcription factor binding motifs. We propose that for certain types of enhancer, architecture plays an essential role in determining specificity, not only by permitting factor–factor synergies necessary to generate the desired activity, but also by preventing other activator synergies that would otherwise lead to unwanted specificities. PMID:22792075

Identification of an Enhancer That Increases miR-200b~200a~429 Gene Expression in Breast Cancer Cells

PubMed Central

Attema, Joanne L.; Bert, Andrew G.; Lim, Yat-Yuen; Kolesnikoff, Natasha; Lawrence, David M.; Pillman, Katherine A.; Smith, Eric; Drew, Paul A.; Khew-Goodall, Yeesim; Shannon, Frances; Goodall, Gregory J.

2013-01-01

The miR-200b~200a~429 gene cluster is a key regulator of EMT and cancer metastasis, however the transcription-based mechanisms controlling its expression during this process are not well understood. We have analyzed the miR-200b~200a~429 locus for epigenetic modifications in breast epithelial and mesenchymal cell lines using chromatin immunoprecipitation assays and DNA methylation analysis. We discovered a novel enhancer located approximately 5.1kb upstream of the miR-200b~200a~429 transcriptional start site. This region was associated with the active enhancer chromatin signature comprising H3K4me1, H3K27ac, RNA polymerase II and CpG dinucleotide hypomethylation. Luciferase reporter assays revealed the upstream enhancer stimulated the transcription of the miR-200b~200a~429 minimal promoter region approximately 27-fold in breast epithelial cells. Furthermore, we found that a region of the enhancer was transcribed, producing a short, GC-rich, mainly nuclear, non-polyadenylated RNA transcript designated miR-200b eRNA. Over-expression of miR-200b eRNA had little effect on miR-200b~200a~429 promoter activity and its production did not correlate with miR-200b~200a~429 gene expression. While additional investigations of miR-200b eRNA function will be necessary, it is possible that miR-200b eRNA may be involved in the regulation of miR-200b~200a~429 gene expression and silencing. Taken together, these findings reveal the presence of a novel enhancer, which contributes to miR-200b~200a~429 transcriptional regulation in epithelial cells. PMID:24086551

Functional importance of cardiac enhancer-associated noncoding RNAs in heart development and disease

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

Ounzain, Samir; Pezzuto, Iole; Micheletti, Rudi

We report here that the key information processing units within gene regulatory networks are enhancers. Enhancer activity is associated with the production of tissue-specific noncoding RNAs, yet the existence of such transcripts during cardiac development has not been established. Using an integrated genomic approach, we demonstrate that fetal cardiac enhancers generate long noncoding RNAs (lncRNAs) during cardiac differentiation and morphogenesis. Enhancer expression correlates with the emergence of active enhancer chromatin states, the initiation of RNA polymerase II at enhancer loci and expression of target genes. Orthologous human sequences are also transcribed in fetal human hearts and cardiac progenitor cells. Throughmore » a systematic bioinformatic analysis, we identified and characterized, for the first time, a catalog of lncRNAs that are expressed during embryonic stem cell differentiation into cardiomyocytes and associated with active cardiac enhancer sequences. RNA-sequencing demonstrates that many of these transcripts are polyadenylated, multi-exonic long noncoding RNAs. Moreover, knockdown of two enhancer-associated lncRNAs resulted in the specific downregulation of their predicted target genes. Interestingly, the reactivation of the fetal gene program, a hallmark of the stress response in the adult heart, is accompanied by increased expression of fetal cardiac enhancer transcripts. Altogether, these findings demonstrate that the activity of cardiac enhancers and expression of their target genes are associated with the production of enhancer-derived lncRNAs.« less

Functional importance of cardiac enhancer-associated noncoding RNAs in heart development and disease

DOE PAGES

Ounzain, Samir; Pezzuto, Iole; Micheletti, Rudi; ...

2014-08-19

We report here that the key information processing units within gene regulatory networks are enhancers. Enhancer activity is associated with the production of tissue-specific noncoding RNAs, yet the existence of such transcripts during cardiac development has not been established. Using an integrated genomic approach, we demonstrate that fetal cardiac enhancers generate long noncoding RNAs (lncRNAs) during cardiac differentiation and morphogenesis. Enhancer expression correlates with the emergence of active enhancer chromatin states, the initiation of RNA polymerase II at enhancer loci and expression of target genes. Orthologous human sequences are also transcribed in fetal human hearts and cardiac progenitor cells. Throughmore » a systematic bioinformatic analysis, we identified and characterized, for the first time, a catalog of lncRNAs that are expressed during embryonic stem cell differentiation into cardiomyocytes and associated with active cardiac enhancer sequences. RNA-sequencing demonstrates that many of these transcripts are polyadenylated, multi-exonic long noncoding RNAs. Moreover, knockdown of two enhancer-associated lncRNAs resulted in the specific downregulation of their predicted target genes. Interestingly, the reactivation of the fetal gene program, a hallmark of the stress response in the adult heart, is accompanied by increased expression of fetal cardiac enhancer transcripts. Altogether, these findings demonstrate that the activity of cardiac enhancers and expression of their target genes are associated with the production of enhancer-derived lncRNAs.« less

Adjacent DNA sequences modulate Sox9 transcriptional activation at paired Sox sites in three chondrocyte-specific enhancer elements

PubMed Central

Bridgewater, Laura C.; Walker, Marlan D.; Miller, Gwen C.; Ellison, Trevor A.; Holsinger, L. Daniel; Potter, Jennifer L.; Jackson, Todd L.; Chen, Reuben K.; Winkel, Vicki L.; Zhang, Zhaoping; McKinney, Sandra; de Crombrugghe, Benoit

2003-01-01

Expression of the type XI collagen gene Col11a2 is directed to cartilage by at least three chondrocyte-specific enhancer elements, two in the 5′ region and one in the first intron of the gene. The three enhancers each contain two heptameric sites with homology to the Sox protein-binding consensus sequence. The two sites are separated by 3 or 4 bp and arranged in opposite orientation to each other. Targeted mutational analyses of these three enhancers showed that in the intronic enhancer, as in the other two enhancers, both Sox sites in a pair are essential for enhancer activity. The transcription factor Sox9 binds as a dimer at the paired sites, and the introduction of insertion mutations between the sites demonstrated that physical interactions between the adjacently bound proteins are essential for enhancer activity. Additional mutational analyses demonstrated that although Sox9 binding at the paired Sox sites is necessary for enhancer activity, it alone is not sufficient. Adjacent DNA sequences in each enhancer are also required, and mutation of those sequences can eliminate enhancer activity without preventing Sox9 binding. The data suggest a new model in which adjacently bound proteins affect the DNA bend angle produced by Sox9, which in turn determines whether an active transcriptional enhancer complex is assembled. PMID:12595563

Co-operation of the transcription factor hepatocyte nuclear factor-4 with Sp1 or Sp3 leads to transcriptional activation of the human haem oxygenase-1 gene promoter in a hepatoma cell line.

PubMed Central

Takahashi, Shigeru; Matsuura, Naomi; Kurokawa, Takako; Takahashi, Yuji; Miura, Takashi

2002-01-01

We reported previously that the 5'-flanking region (nucleotides -1976 to -1655) of the human haem oxygenase-1 ( hHO-1 ) gene enhances hHO-1 promoter activity in human hepatoma HepG2 cells, but not in HeLa cells [Takahashi, Takahashi, Ito, Nagano, Shibahara and Miura (1999) Biochim. Biophys. Acta 1447, 231-235]. To define more precisely the regulatory elements involved, in the present study we have functionally dissected this region and localized the enhancer to a 50 bp fragment (-1793 to -1744). Site-direct mutagenesis analysis revealed that two regions were responsible for this enhancer activity, i.e. a hepatocyte nuclear factor-4 (HNF-4) homologous region and a GC box motif homologous region. Mutation in either region alone moderately decreased enhancer activity. However, mutations in both regions reduced promoter activity to the basal level. Electrophoretic mobility-shift assays demonstrated that the P5-2 fragment (-1793 to -1744) interacted with at least two nuclear factors, i.e. HNF-4 and Sp1/Sp3. Co-transfection experiments using Drosophila SL2 cells revealed that HNF-4 and Sp1/Sp3 synergistically stimulated the enhancer activity of the P5-2 fragment. These results indicate that co-operation of HNF-4 with Sp1 or Sp3 leads to the activation of hHO-1 gene expression in hepatoma cells. PMID:12133007

Co-operation of the transcription factor hepatocyte nuclear factor-4 with Sp1 or Sp3 leads to transcriptional activation of the human haem oxygenase-1 gene promoter in a hepatoma cell line.

PubMed

Takahashi, Shigeru; Matsuura, Naomi; Kurokawa, Takako; Takahashi, Yuji; Miura, Takashi

2002-11-01

We reported previously that the 5'-flanking region (nucleotides -1976 to -1655) of the human haem oxygenase-1 ( hHO-1 ) gene enhances hHO-1 promoter activity in human hepatoma HepG2 cells, but not in HeLa cells [Takahashi, Takahashi, Ito, Nagano, Shibahara and Miura (1999) Biochim. Biophys. Acta 1447, 231-235]. To define more precisely the regulatory elements involved, in the present study we have functionally dissected this region and localized the enhancer to a 50 bp fragment (-1793 to -1744). Site-direct mutagenesis analysis revealed that two regions were responsible for this enhancer activity, i.e. a hepatocyte nuclear factor-4 (HNF-4) homologous region and a GC box motif homologous region. Mutation in either region alone moderately decreased enhancer activity. However, mutations in both regions reduced promoter activity to the basal level. Electrophoretic mobility-shift assays demonstrated that the P5-2 fragment (-1793 to -1744) interacted with at least two nuclear factors, i.e. HNF-4 and Sp1/Sp3. Co-transfection experiments using Drosophila SL2 cells revealed that HNF-4 and Sp1/Sp3 synergistically stimulated the enhancer activity of the P5-2 fragment. These results indicate that co-operation of HNF-4 with Sp1 or Sp3 leads to the activation of hHO-1 gene expression in hepatoma cells.