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Sample records for oct4 gene regulatory

  1. Oct4 Targets Regulatory Nodes to Modulate Stem Cell Function

    PubMed Central

    Campbell, Pearl A.; Perez-Iratxeta, Carolina; Andrade-Navarro, Miguel A.; Rudnicki, Michael A.

    2007-01-01

    Stem cells are characterized by two defining features, the ability to self-renew and to differentiate into highly specialized cell types. The POU homeodomain transcription factor Oct4 (Pou5f1) is an essential mediator of the embryonic stem cell state and has been implicated in lineage specific differentiation, adult stem cell identity, and cancer. Recent description of the regulatory networks which maintain ‘ES’ have highlighted a dual role for Oct4 in the transcriptional activation of genes required to maintain self-renewal and pluripotency while concomitantly repressing genes which facilitate lineage specific differentiation. However, the molecular mechanism by which Oct4 mediates differential activation or repression at these loci to either maintain stem cell identity or facilitate the emergence of alternate transcriptional programs required for the realization of lineage remains to be elucidated. To further investigate Oct4 function, we employed gene expression profiling together with a robust statistical analysis to identify genes highly correlated to Oct4. Gene Ontology analysis to categorize overrepresented genes has led to the identification of themes which may prove essential to stem cell identity, including chromatin structure, nuclear architecture, cell cycle control, DNA repair, and apoptosis. Our experiments have identified previously unappreciated roles for Oct4 for firstly, regulating chromatin structure in a state consistent with self-renewal and pluripotency, and secondly, facilitating the expression of genes that keeps the cell poised to respond to cues that lead to differentiation. Together, these data define the mechanism by which Oct4 orchestrates cellular regulatory pathways to enforce the stem cell state and provides important insight into stem cell function and cancer. PMID:17579724

  2. Stem cell regulatory function mediated by expression of a novel mouse Oct4 pseudogene

    SciTech Connect

    Lin, Huey; Shabbir, Arsalan; Molnar, Merced; Lee, Techung . E-mail: chunglee@buffalo.edu

    2007-03-30

    Multiple pseudogenes have been proposed for embryonic stem (ES) cell-specific genes, and their abundance suggests that some of these potential pseudogenes may be functional. ES cell-specific expression of Oct4 regulates stem cell pluripotency and self-renewing state. Although Oct4 expression has been reported in adult tissues during gene reprogramming, the detected Oct4 signal might be contributed by Oct4 pseudogenes. Among the multiple Oct4 transcripts characterized here is a {approx}1 kb clone derived from P19 embryonal carcinoma stem cells, which shares a {approx}87% sequence homology with the parent Oct4 gene, and has the potential of encoding an 80-amino acid product (designated as Oct4P1). Adenoviral expression of Oct4P1 in mesenchymal stem cells promotes their proliferation and inhibits their osteochondral differentiation. These dual effects of Oct4P1 are reminiscent of the stem cell regulatory function of the parent Oct4, and suggest that Oct4P1 may be a functional pseudogene or a novel Oct4-related gene with a unique function in stem cells.

  3. [Expression of regulatory genes Oct-4, Pax-6, Prox-1, Ptx-2 at the initial stages of differentiation of embryonic stem cells in vitro].

    PubMed

    Gordeeva, O F; Manuilova, E S; Grivennikov, I A; Smirnova, Iu A; Krasnikova, N Iu; Zinov'eva, R D; Khrushchov, N G

    2003-01-01

    The expression of regulatory genes of the POU, Pax, Prox, and Ptx gene families was studied at the initial stages of differentiation of murine embryonic stem cells of R1 line. mRNAs were isolated from undifferentiated embryonic stem cells and embryoid bodies formed at the early stages of in vitro differentiation and cDNA sequences were synthesized for comparative PCR analysis of the expression of studied genes. The levels of expression of the gene Oct-4 involved in maintenance of the pluripotent status of embryonic stem cells proved to be practically indistinguishable in undifferentiated cells and embryoid bodies, while the expression of Pax-6 markedly increased in the latter. The levels and patterns of expression of the homeobox transcription factors Prox-1 and Ptx-2 were compared on this cell model for the first time. A probable role of these genes in differentiation of the murine embryonic stem cells is discussed.

  4. The pioneer factor OCT4 requires the chromatin remodeller BRG1 to support gene regulatory element function in mouse embryonic stem cells

    PubMed Central

    King, Hamish W; Klose, Robert J

    2017-01-01

    Pioneer transcription factors recognise and bind their target sequences in inaccessible chromatin to establish new transcriptional networks throughout development and cellular reprogramming. During this process, pioneer factors establish an accessible chromatin state to facilitate additional transcription factor binding, yet it remains unclear how different pioneer factors achieve this. Here, we discover that the pluripotency-associated pioneer factor OCT4 binds chromatin to shape accessibility, transcription factor co-binding, and regulatory element function in mouse embryonic stem cells. Chromatin accessibility at OCT4-bound sites requires the chromatin remodeller BRG1, which is recruited to these sites by OCT4 to support additional transcription factor binding and expression of the pluripotency-associated transcriptome. Furthermore, the requirement for BRG1 in shaping OCT4 binding reflects how these target sites are used during cellular reprogramming and early mouse development. Together this reveals a distinct requirement for a chromatin remodeller in promoting the activity of the pioneer factor OCT4 and regulating the pluripotency network. DOI: http://dx.doi.org/10.7554/eLife.22631.001 PMID:28287392

  5. Functional similarities among genes regulated by OCT4 in human mesenchymal and embryonic stem cells.

    PubMed

    Greco, Steven J; Liu, Katherine; Rameshwar, Pranela

    2007-12-01

    OCT4 is a master transcriptional regulator, which mediates pluripotency in ESCs through inhibition of tissue-specific and promotion of stem cell-specific genes. Suppression of OCT4, along with other regulators of pluripotency, such as SOX2 and NANOG, has been correlated with cell-fate specification and lineage-specific differentiation. Recent reports have shown the expression of OCT4 in adult MSCs but have not ascribed functional homology with ESCs. MSCs are mesoderm-derived cells, primarily resident in adult bone marrow, that undergo lineage-specific differentiation to generate specialized cells such as stroma, fat, bone, and cartilage. We have previously demonstrated the plasticity of MSCs through their ability to generate neuronal cells. Here, we show that OCT4 provides similar regulatory circuitries in human MSCs and ESCs, using chromatin immunoprecipitation-DNA selection and ligation technology and loss-of-function studies. MSCs were found to express the embryonic transcription factors OCT4, NANOG, and SOX2. In addition, OCT4 was found to (a) target similar genes in MSCs and ESCs, (b) promote the expression of MSC-specific genes, and (c) regulate MSC cell cycle progression. The results suggest similar regulatory mechanisms for OCT4 in MSCs and ESCs and have implications regarding MSC plasticity. Disclosure of potential conflicts of interest is found at the end of this article.

  6. Nuclear actin activates human transcription factor genes including the OCT4 gene.

    PubMed

    Yamazaki, Shota; Yamamoto, Koji; Tokunaga, Makio; Sakata-Sogawa, Kumiko; Harata, Masahiko

    2015-01-01

    RNA microarray analyses revealed that nuclear actin activated many human transcription factor genes including OCT4, which is required for gene reprogramming. Oct4 is known to be activated by nuclear actin in Xenopus oocytes. Our findings imply that this process of OCT4 activation is conserved in vertebrates and among cell types and could be used for gene reprogramming of human cells.

  7. In silico identification of a core regulatory network of OCT4 in human embryonic stem cells using an integrated approach

    PubMed Central

    Chavez, Lukas; Bais, Abha S; Vingron, Martin; Lehrach, Hans; Adjaye, James; Herwig, Ralf

    2009-01-01

    Background The transcription factor OCT4 is highly expressed in pluripotent embryonic stem cells which are derived from the inner cell mass of mammalian blastocysts. Pluripotency and self renewal are controlled by a transcription regulatory network governed by the transcription factors OCT4, SOX2 and NANOG. Recent studies on reprogramming somatic cells to induced pluripotent stem cells highlight OCT4 as a key regulator of pluripotency. Results We have carried out an integrated analysis of high-throughput data (ChIP-on-chip and RNAi experiments along with promoter sequence analysis of putative target genes) and identified a core OCT4 regulatory network in human embryonic stem cells consisting of 33 target genes. Enrichment analysis with these target genes revealed that this integrative analysis increases the functional information content by factors of 1.3 – 4.7 compared to the individual studies. In order to identify potential regulatory co-factors of OCT4, we performed a de novo motif analysis. In addition to known validated OCT4 motifs we obtained binding sites similar to motifs recognized by further regulators of pluripotency and development; e.g. the heterodimer of the transcription factors C-MYC and MAX, a prerequisite for C-MYC transcriptional activity that leads to cell growth and proliferation. Conclusion Our analysis shows how heterogeneous functional information can be integrated in order to reconstruct gene regulatory networks. As a test case we identified a core OCT4-regulated network that is important for the analysis of stem cell characteristics and cellular differentiation. Functional information is largely enriched using different experimental results. The de novo motif discovery identified well-known regulators closely connected to the OCT4 network as well as potential new regulators of pluripotency and differentiation. These results provide the basis for further targeted functional studies. PMID:19604364

  8. The EWS–Oct-4 fusion gene encodes a transforming gene

    PubMed Central

    Lee, Jungwoon; Kim, Ja Young; Kang, In Young; Kim, Hye Kyoung; Han, Yong-Mahn; Kim, Jungho

    2007-01-01

    The t(6;22)(p21;q12) translocation associated with human bone and soft-tissue tumours results in a chimaeric molecule fusing the NTD (N-terminal domain) of the EWS (Ewing's sarcoma) gene to the CTD (C-terminal domain) of the Oct-4 (octamer-4) embryonic gene. Since the N-terminal domains of EWS and Oct-4 are structurally different, in the present study we have assessed the functional consequences of the EWS–Oct-4 fusion. We find that this chimaeric gene encodes a nuclear protein which binds DNA with the same sequence specificity as the parental Oct-4 protein. Comparison of the transactivation properties of EWS–Oct-4 and Oct-4 indicates that the former has higher transactivation activity for a known target reporter gene containing Oct-4 binding. Deletion analysis of the functional domains of EWS–Oct-4 indicates that the EWS (NTD), the POU domain and the CTD of EWS–Oct-4 are necessary for full transactivation potential. EWS–Oct-4 induced the expression of fgf-4 (fibroblast growth factor 4) and nanog, which are potent mitogens as well as Oct-4 downstream target genes whose promoters contain potential Oct-4-binding sites. Finally, ectopic expression of EWS–Oct-4 in Oct-4-null ZHBTc4 ES (embryonic stem) cells resulted in increased tumorigenic growth potential in nude mice. These results suggest that the oncogenic effect of the t(6;22) translocation is due to the EWS–Oct-4 chimaeric protein and that fusion of the EWS NTD to the Oct-4 DNA-binding domain produces a transforming chimaeric product. PMID:17564582

  9. The BET family member BRD4 interacts with OCT4 and regulates pluripotency gene expression.

    PubMed

    Wu, Tao; Pinto, Hugo Borges; Kamikawa, Yasunao F; Donohoe, Mary E

    2015-03-10

    Embryonic stem cell (ESC) pluripotency is controlled by defined transcription factors. During cellular differentiation, ESCs undergo a global epigenetic reprogramming. Female ESCs exemplify this process as one of the two X-chromosomes is globally silenced during X chromosome inactivation (XCI) to balance the X-linked gene disparity with XY males. The pluripotent factor OCT4 regulates XCI by triggering X chromosome pairing and counting. OCT4 directly binds Xite and Tsix, which encode two long noncoding RNAs (lncRNAs) that suppress the silencer lncRNA, Xist. To control its activity as a master regulator in pluripotency and XCI, OCT4 must have chromatin protein partners. Here we show that BRD4, a member of the BET protein subfamily, interacts with OCT4. BRD4 occupies the regulatory regions of pluripotent genes and the lncRNAs of XCI. BET inhibition or depletion of BRD4 reduces the expression of many pluripotent genes and shifts cellular fate showing that BRD4 is pivotal for transcription in ESCs. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  10. Analysis of nuclear reprogramming in cloned miniature pig embryos by expression of Oct-4 and Oct-4 related genes

    SciTech Connect

    Lee, Eugine; Lee, So Hyun; Kim, Sue

    2006-10-06

    Xenotransplantation is a rapidly expanding field of research and cloned miniature pigs have been considered as a model animal for it. However, the efficiency of somatic cell nuclear transfer (SCNT) is extremely low, with most clones resulting in early lethality and several kinds of aberrant development. A possible explanation for the developmental failure of SCNT embryos is insufficient reprogramming of the somatic cell nucleus by the oocyte. In order to test this, we analyzed the reprogramming capacity of differentiated fibroblast cell nuclei and embryonic germ cell nuclei with Oct-4 and Oct-4 related genes (Ndp5211, Dppa2, Dppa3, and Dppa5), which are important for embryonic development, Hand1 and GATA-4, which are important for placental development, as molecular markers using RT-PCR. The Oct-4 expression level was significantly lower (P < 0.05) in cloned hatched blastocysts derived from fibroblasts and many of fibroblast-derived clones failed to reactivate at least one of the tested genes, while most of the germ cell clones and control embryos correctly expressed these genes. In conclusion, our results suggest that the reprogramming of fibroblast-derived cloned embryos is highly aberrant and this improper reprogramming could be one reason of the early lethality and post-implantation anomalies of somatic cell-derived clones.

  11. EWS-Oct-4B, an alternative EWS-Oct-4 fusion gene, is a potent oncogene linked to human epithelial tumours

    PubMed Central

    Kim, S; Lim, B; Kim, J

    2010-01-01

    Background: Characterisation of EWS-Oct-4 translocation fusion product in bone and soft-tissue tumours revealed a chimeric gene resulting from an in-frame fusion between EWS (Ewing's sarcoma gene) exons 1–6 and Oct-4 exons 1–4. Recently, an alternative form of the fusion protein between the EWS and Oct-4 genes, named EWS-Oct-4B, was reported in two types of epithelial tumours, a hidradenoma of the skin and a mucoepidermoid carcinoma of the salivary glands. As the N-terminal and POU domains of the EWS-Oct-4 and EWS-Oct-4B proteins are not structurally identical, we decided to investigate the functional consequences of the EWS-Oct-4B fusion. Methods: In this report, we have characterised the EWS-Oct-4B fusion protein. To investigate how the EWS-Oct-4B protein contributes to tumourigenesis in human cancers, we analysed its DNA-binding activity, subcellular localisation, transcriptional activation behaviour, and oncogenic properties. Results: We found that this new chimeric gene encodes a nuclear protein that binds DNA with the same sequence specificity as the parental Oct-4 protein or the fusion EWS-Oct-4 protein. We show that the nuclear localisation signal of EWS-Oct-4B is dependent on the POU DNA-binding domain, and we identified a cluster of basic amino acids, 269RKRKR273, in the POU domain that specifically mediates the nuclear localisation of EWS-Oct-4B. Comparison of the properties of EWS-Oct-4B and EWS-Oct-4 indicated that EWS-Oct-4B is a less-potent transcriptional activator of a reporter construct carrying the Oct-4-binding sites. Deletion analysis of the functional domains of EWS-Oct-4B revealed that the EWS N-terminal domain (NTD)B, POU, and C-terminal domain (CTD) are necessary for its full transactivation potential. Despite its reduced activity as a transcriptional activator, EWS-Oct-4B regulated the expression of fgf-4 (fibroblast growth factor-4) and nanog, which are potent mitogens, as well as of Oct-4 downstream target genes, the promoters of

  12. NonO binds to the CpG island of oct4 promoter and functions as a transcriptional activator of oct4 gene expression.

    PubMed

    Park, Yoojin; Lee, Ja-Myong; Hwang, Min-Young; Son, Gi-hoon; Geum, Dongho

    2013-01-01

    We investigated the relationship between oct4 gene expression patterns and CpG sites methylation profiles during ES cell differentiation into neurons, and identified relevant binding factor. The oct4 gene expression level gradually declined as ES cell differentiation progressed, and the CpG sites in the oct4 proximal enhancer (PE) and promoter regions were methylated in concert with ES cell differentiation. An electro-mobility shift assay (EMSA) showed that putative proteins bind to CpG sites in the oct4 PE/promoter. We purified CpG binding proteins with DNAbinding purification method, and NonO was identified by liquid chromatography-mass spectrometry. EMSA with specific competitors revealed that NonO specifically binds to the conserved CCGGTGAC sequence in the oct4 promoter. Methylation at a specific cytosine residue (CC* GGTGAC) reduced the binding affinity of NonO for the recognition sequence. Chromatin immunoprecipitation analysis confirmed that NonO binds to the unmethylated oct4 promoter. There were no changes in the NonO mRNA and protein levels between ES cells and differentiated cells. The transcriptional role of NonO in oct4 gene expression was evaluated by luciferase assays and knockdown experiments. The luciferase activity significantly increased threefold when the NonO expression vector was cotransfected with the NonO recognition sequence, indicating that NonO has a transcription activator effect on oct4 gene expression. In accordance with this effect, when NonO expression was inhibited by siRNA treatment, oct4 expression was also significantly reduced. In summary, we purified NonO, a novel protein that binds to the CpG island of oct4 promoter, and positively regulates oct4 gene expression in ES cells.

  13. [Investigation of transcriptional regulation of Oct4 (POU5F1) gene with distal enhancer].

    PubMed

    Nazarov, I B; Krasnoborova, V A; Mittenberg, A G; Chikhirzhina, E V; Davydov-Sinitsyn, A P; Liskovykh, M A; Tomilin, A N

    2013-01-01

    Investigations of transcriptional regulation of Oct4 gene in mouse embryonic stem cells have revealed an important cis-element--the distal enhancer (DE). DE consists of two functionally significant elements--DEa and DEb. Both elements are necessary to complete the DE-mediated expression of Oct4 gene in pluripotent cells. The most likely candidates for the binding site DEb are Oct4 itself in complex with Sox2 protein. It remains unclear which transcriptional proteins bind to the DEa site and what is the mechanism of the co-operation between the DEa and the DEb. Through the use of using the EMSA and chromatographic fractionation of proteins from extracts of mouse embryonic stem cells and mouse tissues, were isolated proteins specifically interacting with the sequence DEa Oct4 gene.

  14. Tethering of Lsh at the Oct4 locus promotes gene repression associated with epigenetic changes.

    PubMed

    Ren, Jianke; Hathaway, Nathaniel A; Crabtree, Gerald R; Muegge, Kathrin

    2017-06-16

    Lsh is a chromatin remodeling factor that regulates DNA methylation and chromatin function in mammals. The dynamics of these chromatin changes and whether they are directly controlled by Lsh remain unclear. To understand the molecular mechanisms of Lsh chromatin controlled regulation of gene expression, we established a tethering system that recruits a Gal4-Lsh fusion protein to an engineered Oct4 locus through Gal4 binding sites in murine embryonic stem (ES) cells. We examined the molecular epigenetic events induced by Lsh binding including: histone modification, DNA methylation and chromatin accessibility to determine nucleosome occupancy before and after embryonic stem cell differentiation. Our results indicate that Lsh assists gene repression upon binding to the Oct4 promoter region. Furthermore, we detected less chromatin accessibility and reduced active histone modifications at the tethered site in undifferentiated ES, while GFP reporter gene expression and DNA methylation patterns remained unchanged at this stage. Upon differentiation, association of Lsh promotes transcriptional repression of the reporter gene accompanied by the increase of repressive histone marks and a gain of DNA methylation at distal and proximal Oct4 enhancer sites. Taken together, this approach allowed us to examine Lsh mediated epigenetic regulation as a dynamic process and revealed chromatin accessibility changes as the primary consequence of Lsh function.

  15. CIP2A is an Oct4 target gene involved in head and neck squamous cell cancer oncogenicity and radioresistance

    PubMed Central

    Ventelä, Sami; Sittig, Eleonora; Mannermaa, Leni; Mäkelä, Juho-Antti; Kulmala, Jarmo; Löyttyniemi, Eliisa; Strauss, Leena; Cárpen, Olli; Toppari, Jorma; Grénman, Reidar; Westermarck, Jukka

    2015-01-01

    Radiotherapy is a mainstay for treatment of many human cancer types, including head and neck squamous cell carcinoma (HNSCC). Thereby, it is clinically very relevant to understand the mechanisms determining radioresistance. Here, we identify CIP2A as an Oct4 target gene and provide evidence that they co-operate in radioresistance. Oct4 positively regulates CIP2A expression both in testicular cancer cell lines as well as in embryonic stem cells. To expand the relevance of these findings we show that Oct4 and CIP2A are co-expressed in CD24 positive side-population of patient-derived HNSCC cell lines. Most importantly, all Oct4 positive HNSCC patient samples were CIP2A positive and this double positivity was linked to poor differentiation level, and predicted for decreased patient survival among radiotherapy treated HNSCC patients. Oct4 and CIP2A expression was also linked with increased aggressiveness and radioresistancy in HNSCC cell lines. Together we demonstrate that CIP2A is a novel Oct4 target gene in stem cells and in human cancer cell lines. Clinically these results suggest that diagnostic evaluation of HNSCC tumors for Oct4 or Oct4/CIP2A positivity might help to predict HNSCC tumor radioresistancy. These results also identify both Oct4 and CIP2A as potential targets for radiosensitation. PMID:25474139

  16. Upregulation of Nanog and Sox-2 genes following ectopic expression of Oct-4 in amniotic fluid mesenchymal stem cells.

    PubMed

    Wang, Kai-Hung; Kao, An-Pei; Chang, Chia-Cheng; Lin, Ta-Chin; Kuo, Tsung-Cheng

    2015-01-01

    Octamer-binding transcription factor 4 (Oct-4), an important gene regulating stem cell pluripotency, is well-known for its ability to reprogram somatic cells in vitro, either alone or in concert with other factors. The aim of this study was to assess the effect of ectopic expression of Oct human amniotic fluid stem cells. We developed a novel method for isolation of putative human amniotic fluid-derived multipotent stem cells. These cells showing mesenchymal stem cell phenotypes (human amniotic fluid-derived mesenchymal stem cells, hAFMSCs) were transfected with a plasmid carrying genes for Oct-4 and the green fluorescent protein (GFP). The stably transfected cells, hAFMSCs-Oct4/GFP, were selected by using G418 and found to express the GFP reporter gene under the control of Oct-4 promoter. We found that hAFMSCs developed by our method possess very high self-renewal ability (about 78 cumulative population doublings) and multilineage differentiation potency. Significantly, the hAFMSCs-Oct4/GFP cells showed enhanced expression of the three major pluripotency genes Oct-4, Nanog, and Sox-2, and increased colony-forming ability and growth rate compared with the parental hAFMSCs. We demonstrated that the ectopic expression of Oct-4 gene in hAFMSCs with high self-renewal ability could upregulate Nanog and Sox-2 gene expression and enhance cell growth rate and colony-forming efficiency. Therefore, the ectopic expression of Oct-4 could be a strategy to develop pluripotency in hAFMSCs for clinical applications. © 2014 International Union of Biochemistry and Molecular Biology, Inc.

  17. Global Oct4 target gene analysis reveals novel downstream PTEN and TNC genes required for drug-resistance and metastasis in lung cancer

    PubMed Central

    Tang, Yen-An; Chen, Chi-Hsin; Sun, H. Sunny; Cheng, Chun-Pei; Tseng, Vincent S.; Hsu, Han-Shui; Su, Wu-Chou; Lai, Wu-Wei; Wang, Yi-Ching

    2015-01-01

    Overexpression of Oct4, a stemness gene encoding a transcription factor, has been reported in several cancers. However, the mechanism by which Oct4 directs transcriptional program that leads to somatic cancer progression remains unclear. In this study, we provide mechanistic insight into Oct4-driven transcriptional network promoting drug-resistance and metastasis in lung cancer cell, animal and clinical studies. Through an integrative approach combining our Oct4 chromatin-immunoprecipitation sequencing and ENCODE datasets, we identified the genome-wide binding regions of Oct4 in lung cancer at promoter and enhancer of numerous genes involved in critical pathways which promote tumorigenesis. Notably, PTEN and TNC were previously undefined targets of Oct4. In addition, novel Oct4-binding motifs were found to overlap with DNA elements for Sp1 transcription factor. We provided evidence that Oct4 suppressed PTEN in an Sp1-dependent manner by recruitment of HDAC1/2, leading to activation of AKT signaling and drug-resistance. In contrast, Oct4 transactivated TNC independent of Sp1 and resulted in cancer metastasis. Clinically, lung cancer patients with Oct4 high, PTEN low and TNC high expression profile significantly correlated with poor disease-free survival. Our study reveals a critical Oct4-driven transcriptional program that promotes lung cancer progression, illustrating the therapeutic potential of targeting Oc4 transcriptionally regulated genes. PMID:25609695

  18. Forced expression of OCT4 influences the expression of pluripotent genes in human mesenchymal stem cells and fibroblasts.

    PubMed

    Palma, C S; Tannous, M A; Malta, T M; Russo, E M S; Covas, D T; Picanço-Castro, V

    2013-04-02

    Genetic reprogramming of adult cells to generate induced pluripotent stem (iPS) cells is a new and important step in sidestepping some of the ethical issues and risks involved in the use of embryonic stem cells. iPS cells can be generated by introduction of transcription factors, such as OCT4, SOX2, KLF4, and CMYC. iPS cells resemble embryonic stem cells in their properties and differentiation potential. The mechanisms that lead to induced pluripotency and the effect of each transcription factor are not completely understood. We performed a critical evaluation of the effect of overexpressing OCT4 in mesenchymal stem cells and fibroblasts and found that OCT4 can activate the expression of other stemness genes, such as SOX2, NANOG, CMYC, FOXD3, KLF4, and βCATENIN, which are not normally or are very weakly expressed in mesenchymal stem cells. Transient expression of OCT4 was also performed to evaluate whether these genes are affected by its overexpression in the first 48 h. Transfected fibroblast cells expressed around 275-fold more OCT4 than non-transfected cells. In transient expression, in which cells were analyzed after 48 h, we detected only the up-regulation of FOXD3, SOX2, and KLF4 genes, suggesting that these genes are the earlier targets of OCT4 in this cellular type. We conclude that forced expression of OCT4 can alter cell status and activate the pluripotent network. Knowledge gained through study of these systems may help us to understand the kinetics and mechanism of cell reprogramming.

  19. Direct activation of human and mouse Oct4 genes using engineered TALE and Cas9 transcription factors

    PubMed Central

    Hu, Jiabiao; Lei, Yong; Wong, Wing-Ki; Liu, Senquan; Lee, Kai-Chuen; He, Xiangjun; You, Wenxing; Zhou, Rui; Guo, Jun-Tao; Chen, Xiongfong; Peng, Xianlu; Sun, Hao; Huang, He; Zhao, Hui; Feng, Bo

    2014-01-01

    The newly developed transcription activator-like effector protein (TALE) and clustered regularly interspaced short palindromic repeats/Cas9 transcription factors (TF) offered a powerful and precise approach for modulating gene expression. In this article, we systematically investigated the potential of these new tools in activating the stringently silenced pluripotency gene Oct4 (Pou5f1) in mouse and human somatic cells. First, with a number of TALEs and sgRNAs targeting various regions in the mouse and human Oct4 promoters, we found that the most efficient TALE-VP64s bound around −120 to −80 bp, while highly effective sgRNAs targeted from −147 to −89-bp upstream of the transcription start sites to induce high activity of luciferase reporters. In addition, we observed significant transcriptional synergy when multiple TFs were applied simultaneously. Although individual TFs exhibited marginal activity to up-regulate endogenous gene expression, optimized combinations of TALE-VP64s could enhance endogenous Oct4 transcription up to 30-fold in mouse NIH3T3 cells and 20-fold in human HEK293T cells. More importantly, the enhancement of OCT4 transcription ultimately generated OCT4 proteins. Furthermore, examination of different epigenetic modifiers showed that histone acetyltransferase p300 could enhance both TALE-VP64 and sgRNA/dCas9-VP64 induced transcription of endogenous OCT4. Taken together, our study suggested that engineered TALE-TF and dCas9-TF are useful tools for modulating gene expression in mammalian cells. PMID:24500196

  20. Direct activation of human and mouse Oct4 genes using engineered TALE and Cas9 transcription factors.

    PubMed

    Hu, Jiabiao; Lei, Yong; Wong, Wing-Ki; Liu, Senquan; Lee, Kai-Chuen; He, Xiangjun; You, Wenxing; Zhou, Rui; Guo, Jun-Tao; Chen, Xiongfong; Peng, Xianlu; Sun, Hao; Huang, He; Zhao, Hui; Feng, Bo

    2014-04-01

    The newly developed transcription activator-like effector protein (TALE) and clustered regularly interspaced short palindromic repeats/Cas9 transcription factors (TF) offered a powerful and precise approach for modulating gene expression. In this article, we systematically investigated the potential of these new tools in activating the stringently silenced pluripotency gene Oct4 (Pou5f1) in mouse and human somatic cells. First, with a number of TALEs and sgRNAs targeting various regions in the mouse and human Oct4 promoters, we found that the most efficient TALE-VP64s bound around -120 to -80 bp, while highly effective sgRNAs targeted from -147 to -89-bp upstream of the transcription start sites to induce high activity of luciferase reporters. In addition, we observed significant transcriptional synergy when multiple TFs were applied simultaneously. Although individual TFs exhibited marginal activity to up-regulate endogenous gene expression, optimized combinations of TALE-VP64s could enhance endogenous Oct4 transcription up to 30-fold in mouse NIH3T3 cells and 20-fold in human HEK293T cells. More importantly, the enhancement of OCT4 transcription ultimately generated OCT4 proteins. Furthermore, examination of different epigenetic modifiers showed that histone acetyltransferase p300 could enhance both TALE-VP64 and sgRNA/dCas9-VP64 induced transcription of endogenous OCT4. Taken together, our study suggested that engineered TALE-TF and dCas9-TF are useful tools for modulating gene expression in mammalian cells.

  1. Oct4 was a novel target of Wnt signaling pathway.

    PubMed

    Li, Jun; Li, Jingyi; Chen, Bingbo

    2012-03-01

    The specific expression of Oct4 during early mouse development is required for the correct maintenance of pluripotent cells, and the regulatory control of the Oct4 expression is important. Wnt signaling could have multiple and/or complex effects on embryonic stem (ES) cells characteristics. Elucidation of the molecular mechanisms affecting Wnt signaling in ES cells could provide a better understanding of how these effects occur. The purpose of this study was to determine whether Oct4 was regulated by Wnt signaling in undifferentiated ES cells. Here, we report Oct4 as a novel target of β-catenin-mediated transcription. First, we observe that Wnt signaling pathway is activated in undifferentiated mouse ES cells. In 239T cells, Oct4 promoter was regulated by β-catenin. Through promoter mapping and chromatin immuno-precipitation assays, we found that Oct4 is a direct target of β-catenin/TCF-mediated transcription and the binding site at -875/-881 of Oct4 promoter is critical for b-catenin/TCF-dependent expression regulation. We further detect the expression of Oct4 in treatment with glycogen syntheses kinase (GSK)-3-specific inhibitor in mouse ES cells and HepG2 cells. We found that GSK-3-specific inhibitor can maintain the expression of Oct4 in ES cells and can enhance the expression of Oct4 in HepG2 cells. Our results suggest that Oct4 might be a novel target of β-catenin/TCF-mediated downstream gene in Wnt-activated cells.

  2. Manganese Superoxide Dismutase Gene Expression Is Induced by Nanog and Oct4, Essential Pluripotent Stem Cells' Transcription Factors.

    PubMed

    Solari, Claudia; Vázquez Echegaray, Camila; Cosentino, María Soledad; Petrone, María Victoria; Waisman, Ariel; Luzzani, Carlos; Francia, Marcos; Villodre, Emilly; Lenz, Guido; Miriuka, Santiago; Barañao, Lino; Guberman, Alejandra

    2015-01-01

    Pluripotent stem cells possess complex systems that protect them from oxidative stress and ensure genomic stability, vital for their role in development. Even though it has been reported that antioxidant activity diminishes along stem cell differentiation, little is known about the transcriptional regulation of the involved genes. The reported modulation of some of these genes led us to hypothesize that some of them could be regulated by the transcription factors critical for self-renewal and pluripotency in embryonic stem cells (ESCs) and in induced pluripotent stem cells (iPSCs). In this work, we studied the expression profile of multiple genes involved in antioxidant defense systems in both ESCs and iPSCs. We found that Manganese superoxide dismutase gene (Mn-Sod/Sod2) was repressed during diverse differentiation protocols showing an expression pattern similar to Nanog gene. Moreover, Sod2 promoter activity was induced by Oct4 and Nanog when we performed a transactivation assay using two different reporter constructions. Finally, we studied Sod2 gene regulation by modulating the expression of Oct4 and Nanog in ESCs by shRNAs and found that downregulation of any of them reduced Sod2 expression. Our results indicate that pluripotency transcription factors positively modulate Sod2 gene transcription.

  3. Manganese Superoxide Dismutase Gene Expression Is Induced by Nanog and Oct4, Essential Pluripotent Stem Cells’ Transcription Factors

    PubMed Central

    Solari, Claudia; Vázquez Echegaray, Camila; Cosentino, María Soledad; Petrone, María Victoria; Waisman, Ariel; Luzzani, Carlos; Francia, Marcos; Villodre, Emilly; Lenz, Guido; Miriuka, Santiago; Barañao, Lino; Guberman, Alejandra

    2015-01-01

    Pluripotent stem cells possess complex systems that protect them from oxidative stress and ensure genomic stability, vital for their role in development. Even though it has been reported that antioxidant activity diminishes along stem cell differentiation, little is known about the transcriptional regulation of the involved genes. The reported modulation of some of these genes led us to hypothesize that some of them could be regulated by the transcription factors critical for self-renewal and pluripotency in embryonic stem cells (ESCs) and in induced pluripotent stem cells (iPSCs). In this work, we studied the expression profile of multiple genes involved in antioxidant defense systems in both ESCs and iPSCs. We found that Manganese superoxide dismutase gene (Mn-Sod/Sod2) was repressed during diverse differentiation protocols showing an expression pattern similar to Nanog gene. Moreover, Sod2 promoter activity was induced by Oct4 and Nanog when we performed a transactivation assay using two different reporter constructions. Finally, we studied Sod2 gene regulation by modulating the expression of Oct4 and Nanog in ESCs by shRNAs and found that downregulation of any of them reduced Sod2 expression. Our results indicate that pluripotency transcription factors positively modulate Sod2 gene transcription. PMID:26642061

  4. From adult stem cells to cancer stem cells: Oct-4 Gene, cell-cell communication, and hormones during tumor promotion.

    PubMed

    Trosko, James E

    2006-11-01

    Carcinogenesis is characterized by "initiation," "promotion," and "progression" phases. The "stem cell theory" and "de-differentiation" theories are used to explain the origin of cancer. Growth control for stem cells, which lack functional gap junctional intercellular communication (GJIC), involves negative soluble or niche factors, while for progenitor cells, it involves GJIC. Tumor promoters, hormones, and growth factors inhibit GJIC reversibly. Oncogenes stably inhibit GJIC. Cancer cells, which lack growth control and the ability to terminally differentiate and to apoptose, lack GJIC. The Oct3/4 gene, a POU (Pit-Oct-Unc) family of transcription factors was thought to be expressed only in embryonic stem cells and in tumor cells. With the availability of normal adult human stem cells, tests for the expression of Oct3/4 gene and the stem cell theory in human carcinogenesis became possible. Human breast, liver, pancreas, kidney, mesenchyme, and gastric stem cells, HeLa and MCF-7 cells, and canine tumors were tested with antibodies and polymerase chain reaction (PCR) primers for Oct3/4. Adult human breast stem cells, immortalized nontumorigenic and tumor cell lines, but not the normal differentiated cells, expressed Oct3/4. Adult human differentiated cells lose their Oct-4 expression. Oct3/4 is expressed in a few cells found in the basal layer of human skin epidermis. The data demonstrate that normal adult stem cells and cancer stem cells maintain expression of Oct3/4, consistent with the stem cell hypothesis of carcinogenesis. These Oct-4 positive cells might represent the "cancer stem cells." A strategy to target "cancer stem cells" is to suppress the Oct-4 gene in order to cause the cells to differentiate.

  5. Investigating the functionality of an OCT4-short response element in human induced pluripotent stem cells

    PubMed Central

    Vega-Crespo, Agustin; Truong, Brian; Hermann, Kip J; Awe, Jason P; Chang, Katherine M; Lee, Patrick C; Schoenberg, Benjamen E; Wu, Lily; Byrne, James A; Lipshutz, Gerald S

    2016-01-01

    Pluripotent stem cells offer great therapeutic promise for personalized treatment platforms for numerous injuries, disorders, and diseases. Octamer-binding transcription factor 4 (OCT4) is a key regulatory gene maintaining pluripotency and self-renewal of mammalian cells. With site-specific integration for gene correction in cellular therapeutics, use of the OCT4 promoter may have advantages when expressing a suicide gene if pluripotency remains. However, the human OCT4 promoter region is 4 kb in size, limiting the capacity of therapeutic genes and other regulatory components for viral vectors, and decreasing the efficiency of homologous recombination. The purpose of this investigation was to characterize the functionality of a novel 967bp OCT4-short response element during pluripotency and to examine the OCT4 titer-dependent response during differentiation to human derivatives not expressing OCT4. Our findings demonstrate that the OCT4-short response element is active in pluripotency and this activity is in high correlation with transgene expression in vitro, and the OCT4-short response element is inactivated when pluripotent cells differentiate. These studies demonstrate that this shortened OCT4 regulatory element is functional and may be useful as part of an optimized safety component in a site-specific gene transferring system that could be used as an efficient and clinically applicable safety platform for gene transfer in cellular therapeutics. PMID:27500178

  6. Development and gene expression of porcine cloned embryos derived from bone marrow stem cells with overexpressing Oct4 and Sox2.

    PubMed

    Lee, Jeong-Hyeon; Lee, Won-Jae; Jeon, Ryoung-Hoon; Lee, Yeon-Mi; Jang, Si-Jung; Lee, Sung-Lim; Jeon, Byung-Geun; Ock, Sun-A; King, W Allen; Rho, Gyu-Jin

    2014-12-01

    The present study compared the potential of porcine bone marrow mesenchymal stem cells (pBMSCs) at different passages as nuclear transfer (NT) donors and the developmental efficiency of NT embryos from donor cells transfected with/without Oct4 and Sox2. Early-passage pBMSCs showed higher proliferation and expression of Oct4 and Sox2 and differentiation potential into mesenchymal lineages than middle- and late-passage pBMSCs. Cleavage rate did not differ among pBMSCs at different passages, but NT embryos with early-passage pBMSCs and middle-passage pBMSCs transfected with Oct4 (Oct4-pBMSCs) had significantly (p<0.05) higher blastocyst development than those with middle-passage pBMSCs. The incidence of apoptotic bodies in NT blastocysts from late-passage pBMSCs and Sox2-transfected middle-passage pBMSCs (Sox2-pBMSCs) was significantly (p<0.05) higher than others. The transcriptional levels of Oct4, Sox2, Nanog, Cdx2, Dnmt3a, and Igf2r genes were significantly (p<0.05) higher in Oct4- and Sox2-pBMSCs NT embryos. Middle-passage pBMSCs NT embryos revealed lower transcriptional levels of Bcl2 than others, except Sox2-pBMSCs NT embryos. The transcriptional level of Bax increased gradually in NT embryos derived from pBMSCs following extended passages and was significantly (p<0.05) higher in Sox2-pBMSCs NT embryos. Our results demonstrated that early-passage pBMSCs are more potent in expressing transcription factors and displayed higher differentiation ability, and middle-passage pBMSCs transfected with Oct4 improved the developmental efficiency of NT embryos, suggesting that high Oct4 expression cells are more efficient as NT donors.

  7. Development and Gene Expression of Porcine Cloned Embryos Derived from Bone Marrow Stem Cells with Overexpressing Oct4 and Sox2

    PubMed Central

    Lee, Jeong-Hyeon; Lee, Won-Jae; Jeon, Ryoung-Hoon; Lee, Yeon-Mi; Jang, Si-Jung; Lee, Sung-Lim; Jeon, Byung-Geun; Ock, Sun-A; King, W. Allen

    2014-01-01

    Abstract The present study compared the potential of porcine bone marrow mesenchymal stem cells (pBMSCs) at different passages as nuclear transfer (NT) donors and the developmental efficiency of NT embryos from donor cells transfected with/without Oct4 and Sox2. Early-passage pBMSCs showed higher proliferation and expression of Oct4 and Sox2 and differentiation potential into mesenchymal lineages than middle- and late-passage pBMSCs. Cleavage rate did not differ among pBMSCs at different passages, but NT embryos with early-passage pBMSCs and middle-passage pBMSCs transfected with Oct4 (Oct4-pBMSCs) had significantly (p<0.05) higher blastocyst development than those with middle-passage pBMSCs. The incidence of apoptotic bodies in NT blastocysts from late-passage pBMSCs and Sox2-transfected middle-passage pBMSCs (Sox2-pBMSCs) was significantly (p<0.05) higher than others. The transcriptional levels of Oct4, Sox2, Nanog, Cdx2, Dnmt3a, and Igf2r genes were significantly (p<0.05) higher in Oct4- and Sox2-pBMSCs NT embryos. Middle-passage pBMSCs NT embryos revealed lower transcriptional levels of Bcl2 than others, except Sox2-pBMSCs NT embryos. The transcriptional level of Bax increased gradually in NT embryos derived from pBMSCs following extended passages and was significantly (p<0.05) higher in Sox2-pBMSCs NT embryos. Our results demonstrated that early-passage pBMSCs are more potent in expressing transcription factors and displayed higher differentiation ability, and middle-passage pBMSCs transfected with Oct4 improved the developmental efficiency of NT embryos, suggesting that high Oct4 expression cells are more efficient as NT donors. PMID:25437870

  8. [Effect of down-regulation of Oct4 gene on biological characteristics of MDA-MB-231 breast cancer stem cells].

    PubMed

    2015-04-01

    To investigate the effect and significance of down-regulation of Oct4 gene on biological characteristics of MDA-MB-231 breast cancer stem cells. Breast cancer cell line MDA-MB-231 cells were used in this study. Breast cancer stem cells were isolated and enriched by serum-free culture. The obtained stem cells were identified through calculating the percentages of CD44 and CD24 stem cells by FACS and evaluating the paclitaxel resistance in vitro and tumorigenicity in mice. RT-PCR, real-time PCR (qPCR) and Western blot were used to detect Oct4 expression. RNA interference was applied to induce Oct4 down-regulation. The interference experiment set up a control group (no siRNA transfection), negative control group (negative siRNA group, transfection of siRNA sequences without any interfering effect on the cells) and Oct4 siRNA group (transfection of siRNA with interfering effect on the Oct4 gene). Methyl thiazolyl tetrazolium (MTT) and Transwell chamber tests were conducted to detect the proliferation and invasion ability of MDA-MB-231 breast cancer stem cells after Oct4 knock-down, and paclitaxel inhibition test was applied to evaluate drug resistance of MDA-MB-231 breast cancer stem cells after Oct4 knock-down. MDA-MB-231 breast cancer stem cells grew as spheres cultured in serum-free suspension. MDA-MB-231 breast cancer stem cells showed a higher percentage of CD44+/CD24-/low cells (97.2%) than that in MDA-MB-231 breast cancer cells (76.6%) (P < 0.05). The tumor size in mice inoculated with MDA-MB-231 breast cancer stem cells was (124.60 ± 13.65) mm3, significantly larger than that of mice inoculated with breast cancer cells (68.20 ± 9.99 mm3) (P = 0.0007). MDA-MB-231 breast cancer stem cells were less sensitive to paclitaxel inhibition than MDA-MB-231 breast cancer cells showing by 50% inhibitory concentration (IC50) [(4.40 ± 0.48) µg/ml vs. (8.20 ± 0.34) µg/m, P < 0.05]. However, the expression of transcriptional factors Oct4 was higher in MDA-MB-231 breast

  9. Nanog, Oct4 and Tet1 interplay in establishing pluripotency

    PubMed Central

    Olariu, Victor; Lövkvist, Cecilia; Sneppen, Kim

    2016-01-01

    A few central transcription factors inside mouse embryonic stem (ES) cells and induced pluripotent stem (iPS) cells are believed to control the cells’ pluripotency. Characterizations of pluripotent state were put forward on both transcription factor and epigenetic levels. Whereas core players have been identified, it is desirable to map out gene regulatory networks which govern the reprogramming of somatic cells as well as the early developmental decisions. Here we propose a multiple level model where the regulatory network of Oct4, Nanog and Tet1 includes positive feedback loops involving DNA-demethylation around the promoters of Oct4 and Tet1. We put forward a mechanistic understanding of the regulatory dynamics which account for i) Oct4 overexpression is sufficient to induce pluripotency in somatic cell types expressing the other Yamanaka reprogramming factors endogenously; ii) Tet1 can replace Oct4 in reprogramming cocktail; iii) Nanog is not necessary for reprogramming however its over-expression leads to enhanced self-renewal; iv) DNA methylation is the key to the regulation of pluripotency genes; v) Lif withdrawal leads to loss of pluripotency. Overall, our paper proposes a novel framework combining transcription regulation with DNA methylation modifications which, takes into account the multi-layer nature of regulatory mechanisms governing pluripotency acquisition through reprogramming. PMID:27146218

  10. Nanog, Oct4 and Tet1 interplay in establishing pluripotency.

    PubMed

    Olariu, Victor; Lövkvist, Cecilia; Sneppen, Kim

    2016-05-05

    A few central transcription factors inside mouse embryonic stem (ES) cells and induced pluripotent stem (iPS) cells are believed to control the cells' pluripotency. Characterizations of pluripotent state were put forward on both transcription factor and epigenetic levels. Whereas core players have been identified, it is desirable to map out gene regulatory networks which govern the reprogramming of somatic cells as well as the early developmental decisions. Here we propose a multiple level model where the regulatory network of Oct4, Nanog and Tet1 includes positive feedback loops involving DNA-demethylation around the promoters of Oct4 and Tet1. We put forward a mechanistic understanding of the regulatory dynamics which account for i) Oct4 overexpression is sufficient to induce pluripotency in somatic cell types expressing the other Yamanaka reprogramming factors endogenously; ii) Tet1 can replace Oct4 in reprogramming cocktail; iii) Nanog is not necessary for reprogramming however its over-expression leads to enhanced self-renewal; iv) DNA methylation is the key to the regulation of pluripotency genes; v) Lif withdrawal leads to loss of pluripotency. Overall, our paper proposes a novel framework combining transcription regulation with DNA methylation modifications which, takes into account the multi-layer nature of regulatory mechanisms governing pluripotency acquisition through reprogramming.

  11. Identification of a novel 12-bp insertion/deletion (indel) of iPS-related Oct4 gene and its association with reproductive traits in male piglets.

    PubMed

    Ren, Fa; Yu, Shuai; Chen, Rui; Lv, Xiaoyan; Pan, Chuanying

    2017-03-01

    As a key factor of cellular reprogramming, Oct4 is one of vital transcription factors for induced pluripotent stem cells (iPSCs). Loss of its function or deletion causes apoptosis in primordial germ cells (PGCs), which affect reproductive traits in mammals. In this study, a novel 12-bp insertion/deletion (indel) polymorphism (NC_010449:g.2759-2760insGGTTTTTGTCTA) within the Oct4 gene was identified in 442 pigs of Large White (LW) and Landrace (LD) breeds, showing three genotypes designated as II, ID, and DD. The frequencies of allele "I" in LW and LD pigs were 0.587 and 0.648, respectively. The male piglets with homozygous II or DD genotypes of Oct4 gene exhibited better reproductive traits than those with heterozygous ID genotype. Moreover, there were two significant associations between this 12-bp indel polymorphism and testis long circumference (TLC) (P=0.005) and testis short girth (TSG) (P=0.003) as well as 15-day testis weight (TW) (P=0.013) in the LW male piglets. These findings suggest that the 12-bp indel polymorphism of the Oct4 gene might be a potential DNA marker for selecting preferred individuals in relation to reproductive traits in pig marker-assisted selection (MAS) breeding, which could contribute to the breeding and genetics in male piglets.

  12. Reduced Oct4 Expression Directs a Robust Pluripotent State with Distinct Signaling Activity and Increased Enhancer Occupancy by Oct4 and Nanog

    PubMed Central

    Karwacki-Neisius, Violetta; Göke, Jonathan; Osorno, Rodrigo; Halbritter, Florian; Ng, Jia Hui; Weiße, Andrea Y.; Wong, Frederick C.K.; Gagliardi, Alessia; Mullin, Nicholas P.; Festuccia, Nicola; Colby, Douglas; Tomlinson, Simon R.; Ng, Huck-Hui; Chambers, Ian

    2013-01-01

    Summary Embryonic stem cell (ESC) pluripotency is governed by a gene regulatory network centered on the transcription factors Oct4 and Nanog. To date, robust self-renewing ESC states have only been obtained through the chemical inhibition of signaling pathways or enforced transgene expression. Here, we show that ESCs with reduced Oct4 expression resulting from heterozygosity also exhibit a stabilized pluripotent state. Despite having reduced Oct4 expression, Oct4+/− ESCs show increased genome-wide binding of Oct4, particularly at pluripotency-associated enhancers, homogeneous expression of pluripotency transcription factors, enhanced self-renewal efficiency, and delayed differentiation kinetics. Cells also exhibit increased Wnt expression, enhanced leukemia inhibitory factor (LIF) sensitivity, and reduced responsiveness to fibroblast growth factor. Although they are able to maintain pluripotency in the absence of bone morphogenetic protein, removal of LIF destabilizes pluripotency. Our findings suggest that cells with a reduced Oct4 concentration range are maintained in a robust pluripotent state and that the wild-type Oct4 concentration range enables effective differentiation. PMID:23642364

  13. Functional interplay between MYCN, NCYM, and OCT4 promotes aggressiveness of human neuroblastomas

    PubMed Central

    Kaneko, Yoshiki; Suenaga, Yusuke; Islam, S M Rafiqul; Matsumoto, Daisuke; Nakamura, Yohko; Ohira, Miki; Yokoi, Sana; Nakagawara, Akira

    2015-01-01

    Neuroblastoma is a pediatric solid tumor that originates from embryonic neural crest cells. The MYCN gene locus is frequently amplified in unfavorable neuroblastomas, and the gene product promotes the progression of neuroblastomas. However, the molecular mechanisms by which MYCN amplification contributes to stem cell-like states of neuroblastoma remain elusive. In this study, we show that MYCN and its cis-antisense gene, NCYM, form a positive feedback loop with OCT4, a core regulatory gene maintaining a multipotent state of neural stem cells. We previously reported that NCYM is co-amplified with the MYCN gene in primary human neuroblastomas and that the gene product promotes aggressiveness of neuroblastoma by stabilization of MYCN. In 36 MYCN-amplified primary human neuroblastomas, OCT4 mRNA expression was associated with unfavorable prognosis and was correlated with that of NCYM. The OCT4 protein induced both NCYM and MYCN in human neuroblastoma cells, whereas NCYM stabilized MYCN to induce OCT4 and stem cell-related genes, including NANOG, SOX2, and LIN28. In sharp contrast to MYCN, enforced expression of c-MYC did not enhance OCT4 expression in human neuroblastoma cells. All-trans retinoic acid treatment reduced MYCN, NCYM, and OCT4 expression, accompanied by the decreased amount of OCT4 recruited onto the intron 1 region of MYCN. Knockdown of NCYM or OCT4 inhibited formation of spheres of neuroblastoma cells and promoted asymmetric cell division in MYCN-amplified human neuroblastoma cells. These results suggest that the functional interplay between MYCN, NCYM, and OCT4 contributes to aggressiveness of MYCN-amplified human neuroblastomas. PMID:25880909

  14. The developmental dismantling of pluripotency is reversed by ectopic Oct4 expression

    PubMed Central

    Osorno, Rodrigo; Tsakiridis, Anestis; Wong, Frederick; Cambray, Noemí; Economou, Constantinos; Wilkie, Ronald; Blin, Guillaume; Scotting, Paul J.; Chambers, Ian; Wilson, Valerie

    2012-01-01

    The transcription factors Nanog and Oct4 regulate pluripotency in the pre-implantation epiblast and in derivative embryonic stem cells. During post-implantation development, the precise timing and mechanism of the loss of pluripotency is unknown. Here, we show that in the mouse, pluripotency is extinguished at the onset of somitogenesis, coincident with reduced expression and chromatin accessibility of Oct4 and Nanog regulatory regions. Prior to somitogenesis expression of both Nanog and Oct4 is regionalized. We show that pluripotency tracks the in vivo level of Oct4 and not Nanog by assessing the ability to reactivate or maintain Nanog expression in cell culture. Enforced Oct4 expression in somitogenesis-stage tissue provokes rapid reopening of Oct4 and Nanog chromatin, Nanog re-expression and resuscitates moribund pluripotency. Our data suggest that decreasing Oct4 expression is converted to a sudden drop in competence to maintain pluripotency gene regulatory network activity that is subsequently stabilized by epigenetic locks. PMID:22669820

  15. Differential Expression of OCT4 Pseudogenes in Pluripotent and Tumor Cell Lines

    PubMed Central

    Poursani, Ensieh M.; Mohammad Soltani, Bahram; Mowla, Seyed Javad

    2016-01-01

    Objective The human OCT4 gene, the most important pluripotency marker, can generate at least three different transcripts (OCT4A, OCT4B, and OCT4B1) by alternative splicing. OCT4A is the main isoform responsible for the stemness property of embryonic stem (ES) cells. There also exist eight processed OCT4 pseudogenes in the human genome with high homology to the OCT4A, some of which are transcribed in various cancers. Recent conflicting reports on OCT4 expression in tumor cells and tissues emphasize the need to discriminate the expression of OCT4A from other variants as well as OCT4 pseudogenes. Materials and Methods In this experimental study, DNA sequencing confirmed the authenticity of transcripts of OCT4 pseudogenes and their expression patterns were investigated in a panel of different human cell lines by reverse transcription-polymerase chain reaction (RT-PCR). Results Differential expression of OCT4 pseudogenes in various human cancer and pluripotent cell lines was observed. Moreover, the expression pattern of OCT4-pseudogene 3 (OCT4-pg3) followed that of OCT4A during neural differentiation of the pluripotent cell line of NTERA-2 (NT2). Although OCT4-pg3 was highly expressed in undifferentiated NT2 cells, its expression was rapidly down-regulated upon induction of neural differentiation. Analysis of protein expression of OCT4A, OCT4-pg1, OCT4-pg3, and OCT4-pg4 by Western blotting indicated that OCT4 pseudogenes cannot produce stable proteins. Consistent with a newly proposed competitive role of pseudogene microRNA docking sites, we detected miR-145 binding sites on all transcripts of OCT4 and OCT4 pseudogenes. Conclusion Our study suggests a potential coding-independent function for OCT4 pseudogenes during differentiation or tumorigenesis. PMID:27054116

  16. Acquired cancer stem cell phenotypes through Oct4-mediated dedifferentiation

    PubMed Central

    Kumar, Suresh M.; Liu, Shujing; Lu, Hezhe; Zhang, Hongtao; Zhang, Paul J.; Gimotty, Phyllis A.; Guerra, Matthew; Guo, Wei; Xu, Xiaowei

    2012-01-01

    There is enormous interest to target cancer stem cells (CSCs) for clinical treatment because these cells are highly tumorigenic and resistant to chemotherapy. Oct4 is expressed by CSC-like cells in different types of cancer. However, function of Oct4 in tumor cells is unclear. In this study, we showed that expression of Oct4 gene or transmembrane delivery of Oct4 protein promoted dedifferentiation of melanoma cells to CSC-like cells. The dedifferentiated melanoma cells showed significantly decreased expression of melanocytic markers and acquired the ability to form tumor spheroids. They showed markedly increased resistance to chemotherapeutic agents and hypoxic injury. In the subcutaneous xenograft and tail vein injection assays, these cells had significantly increased tumorigenic capacity. The dedifferentiated melanoma cells acquired features associated with CSCs such as multipotent differentiation capacity and expression of melanoma CSC markers such as ABCB5 and CD271. Mechanistically, Oct4 induced dedifferentiation was associated with increased expression of endogenous Oct4, Nanog and Klf4, and global gene expression changes that enriched for transcription factors. RNAi mediated knockdown of Oct4 in dedifferentiated cells led to diminished CSC phenotypes. Oct4 expression in melanoma was regulated by hypoxia and its expression was detected in a subpopulation of melanoma cells in clinical samples. Our data indicate that Oct4 is a positive regulator of tumor dedifferentiation. The results suggest that CSC phenotype is dynamic and may be acquired through dedifferentiation. Oct4 mediated tumor cell dedifferentiation may play an important role during tumor progression. PMID:22286766

  17. Concurrent Expression of Oct4 and Nanog Maintains Mesenchymal Stem-Like Property of Human Dental Pulp Cells

    PubMed Central

    Huang, Chuan-En; Hu, Fang-Wei; Yu, Chuan-Hang; Tsai, Lo-Lin; Lee, Tzu-Hsin; Chou, Ming-Yung; Yu, Cheng-Chia

    2014-01-01

    Human dental pulp stem cells (DPSCs), unique mesenchymal stem cells (MSCs) type, exhibit the characteristics of self-renewal and multi-lineage differentiation capacity. Oct4 and Nanog are pluripotent genes. The aim of this study was to determine the physiological functions of Oct4 and Nanog expression in DPSCs. Herein, we determined the critical role of an Oct4/Nanog axis modulating MSCs properties of DPSCs by lentiviral-mediated co-overexpression or co-knockdown of Oct4/Nanog in DPSCs. MSCs properties including osteogenic/chondrogenic/adipogenic induction differentiation was assayed for expression of osteogenic/chondrogenic/adipogenic markers by quantitative real-time RT-PCR analysis. Initially, we observed that the expression profile of Oct4 and Nanog in dental pulp cells, which exerted properties of MSCs, was significantly up-regulated compared to that of STRO-1−CD146− dental pulp cells. Down-regulation of Oct4 and Nanog co-expression significantly reduced the cell proliferation, osteogenic differentiation capability, STRO-1, CD146, and Alkaline phosphatase (ALP) activity of DPSCs. In contrast, co-overexpression of Oct4 and Nanog enhanced the expression level of STRO-1 and CD146, proliferation rate and osteogenic/chondrogenic/adipogenic induction differentiation capability, and expression of osteogenic/chondrogenic/adipogenic induction differentiation markers. Our results suggest that Oct4-Nanog signaling is a regulatory switch to maintain properties in DPSCs. PMID:25322154

  18. Oct4 is required for primordial germ cell survival

    PubMed Central

    Kehler, James; Tolkunova, Elena; Koschorz, Birgit; Pesce, Maurizio; Gentile, Luca; Boiani, Michele; Lomelí, Hilda; Nagy, Andras; McLaughlin, K John; Schöler, Hans R; Tomilin, Alexey

    2004-01-01

    Previous studies have shown that Oct4 has an essential role in maintaining pluripotency of cells of the inner cell mass (ICM) and embryonic stem cells. However, Oct4 null homozygous embryos die around the time of implantation, thus precluding further analysis of gene function during development. We have used the conditional Cre/loxP gene targeting strategy to assess Oct4 function in primordial germ cells (PGCs). Loss of Oct4 function leads to apoptosis of PGCs rather than to differentiation into a trophectodermal lineage, as has been described for Oct4-deficient ICM cells. These new results suggest a previously unknown function of Oct4 in maintaining viability of mammalian germline. PMID:15486564

  19. Epigenetic silencing of Oct4 by a complex containing SUV39H1 and Oct4 pseudogene lncRNA

    PubMed Central

    Scarola, Michele; Comisso, Elisa; Pascolo, Rhena; Chiaradia, Riccardo; Maria Marion, Rosa; Schneider, Claudio; Blasco, Maria A.; Schoeftner, Stefan; Benetti, Roberta

    2015-01-01

    Pseudogene-derived, long non-coding RNAs (lncRNAs) act as epigenetic regulators of gene expression. Here we present a panel of new mouse Oct4 pseudogenes and demonstrate that the X-linked Oct4 pseudogene Oct4P4 critically impacts mouse embryonic stem cells (mESCs) self-renewal. Sense Oct4P4 transcription produces a spliced, nuclear-restricted lncRNA that is efficiently upregulated during mESC differentiation. Oct4P4 lncRNA forms a complex with the SUV39H1 HMTase to direct the imposition of H3K9me3 and HP1α to the promoter of the ancestral Oct4 gene, located on chromosome 17, leading to gene silencing and reduced mESC self-renewal. Targeting Oct4P4 expression in primary mouse embryonic fibroblasts causes the re-acquisition of self-renewing features of mESC. We demonstrate that Oct4P4 lncRNA plays an important role in inducing and maintaining silencing of the ancestral Oct4 gene in differentiating mESCs. Our data introduces a sense pseudogene–lncRNA-based mechanism of epigenetic gene regulation that controls the cross-talk between pseudogenes and their ancestral genes. PMID:26158551

  20. Phosphorylation regulates human OCT4.

    PubMed

    Brumbaugh, Justin; Hou, Zhonggang; Russell, Jason D; Howden, Sara E; Yu, Pengzhi; Ledvina, Aaron R; Coon, Joshua J; Thomson, James A

    2012-05-08

    The transcription factor OCT4 is fundamental to maintaining pluripotency and self-renewal. To better understand protein-level regulation of OCT4, we applied liquid chromatography-MS to identify 14 localized sites of phosphorylation, 11 of which were previously unknown. Functional analysis of two sites, T234 and S235, suggested that phosphorylation within the homeobox region of OCT4 negatively regulates its activity by interrupting sequence-specific DNA binding. Mutating T234 and S235 to mimic constitutive phosphorylation at these sites reduces transcriptional activation from an OCT4-responsive reporter and decreases reprogramming efficiency. We also cataloged 144 unique phosphopeptides on known OCT4 interacting partners, including SOX2 and SALL4, that copurified during immunoprecipitation. These proteins were enriched for phosphorylation at motifs associated with ERK signaling. Likewise, OCT4 harbored several putative ERK phosphorylation sites. Kinase assays confirmed that ERK2 phosphorylated these sites in vitro, providing a direct link between ERK signaling and the transcriptional machinery that governs pluripotency.

  1. Phosphorylation regulates human OCT4

    PubMed Central

    Brumbaugh, Justin; Russell, Jason D.; Howden, Sara E.; Yu, Pengzhi; Ledvina, Aaron R.; Coon, Joshua J.; Thomson, James A.

    2012-01-01

    The transcription factor OCT4 is fundamental to maintaining pluripotency and self-renewal. To better understand protein-level regulation of OCT4, we applied liquid chromatography–MS to identify 14 localized sites of phosphorylation, 11 of which were previously unknown. Functional analysis of two sites, T234 and S235, suggested that phosphorylation within the homeobox region of OCT4 negatively regulates its activity by interrupting sequence-specific DNA binding. Mutating T234 and S235 to mimic constitutive phosphorylation at these sites reduces transcriptional activation from an OCT4-responsive reporter and decreases reprogramming efficiency. We also cataloged 144 unique phosphopeptides on known OCT4 interacting partners, including SOX2 and SALL4, that copurified during immunoprecipitation. These proteins were enriched for phosphorylation at motifs associated with ERK signaling. Likewise, OCT4 harbored several putative ERK phosphorylation sites. Kinase assays confirmed that ERK2 phosphorylated these sites in vitro, providing a direct link between ERK signaling and the transcriptional machinery that governs pluripotency. PMID:22474382

  2. Pseudogene OCT4-pg4 functions as a natural micro RNA sponge to regulate OCT4 expression by competing for miR-145 in hepatocellular carcinoma.

    PubMed

    Wang, Lei; Guo, Zhang-Yan; Zhang, Rui; Xin, Bo; Chen, Rui; Zhao, Jing; Wang, Tao; Wen, Wei-Hong; Jia, Lin-Tao; Yao, Li-Bo; Yang, An-Gang

    2013-08-01

    The POU transcription factor OCT4 is a pleiotropic regulator of gene expression in embryonic stem cells. Recent studies demonstrated that OCT4 is aberrantly expressed in multiple types of human cancer; however, the underlying molecular mechanism remains largely unknown. In this study, we report that OCT4-pg4, a pseudogene of OCT4, is abnormally activated in hepatocellular carcinoma (HCC). The expression level of OCT4-pg4 is positively correlated with that of OCT4, and both gene transcripts can be directly targeted by a tumor-suppressive micro RNA miR-145. We find that the non-coding RNA OCT4-pg4 is biologically active, as it can upregulate OCT4 protein level in HCC. Mechanistic analysis revealed that OCT4-pg4 functions as a natural micro RNA sponge to protect OCT4 transcript from being inhibited by miR-145. In addition, our study also showed that OCT4-pg4 can promote growth and tumorigenicity of HCC cells, thus exerting an oncogenic role in hepatocarcinogenesis. Furthermore, survival analysis suggests that high OCT4-pg4 level is significantly correlated with poor prognosis of HCC patients. Taken together, our finding adds a new layer of post-transcriptional regulation of OCT4 and sheds new light on the treatment of human HCC.

  3. Simultaneous overexpression of Oct4 and Nanog abrogates terminal myogenesis.

    PubMed

    Lang, Kuan Chih; Lin, I Hsuan; Teng, Han Fang; Huang, Yi Cheng; Li, Chung Leung; Tang, Kam Tsun; Chen, Shen Liang

    2009-07-01

    Oct4 and Nanog are two embryonic stem (ES) cell-specific transcription factors that play critical roles in the maintenance of ES cell pluripotency. In this study, we investigated the effects of Oct4 and Nanog expression on the differentiation state of myogenic cells, which is sustained by a strong positive feedback loop. Oct4 and Nanog, either independently or simultaneously, were overexpressed in C2C12 myoblasts, and the expression of myogenic lineage-specific genes and terminal differentiation was observed by RT-PCR. Overexpression of Oct4 in C2C12 cultures repressed, while exogenous Nanog did not significantly alter C2C12 terminal differentiation. The expression of Pax7 was reduced in all Oct4-overexpressing myoblasts, and we identified a major Oct4-binding site in the Pax7 promoter. Simultaneous expression of Oct4 and Nanog in myoblasts inhibited the formation of myotubes, concomitant with a reduction in the endogenous levels of hallmark myogenic markers. Furthermore, overexpression of Oct4 and Nanog induced the expression of their endogenous counterparts along with the expression of Sox2. Using mammalian two-hybrid assays, we confirmed that Oct4 functions as a transcriptional repressor whereas Nanog functions as a transcriptional activator during muscle terminal differentiation. Importantly, in nonobese diabetic (NOD) severe combined immunodeficiency (SCID) mice, the pluripotency of C2C12 cells was conferred by overexpression of Oct4 and Nanog. These results suggest that Oct4 in cooperation with Nanog strongly suppresses the myogenic differentiation program and promotes pluripotency in myoblasts.

  4. Acquired cancer stem cell phenotypes through Oct4-mediated dedifferentiation.

    PubMed

    Kumar, S M; Liu, S; Lu, H; Zhang, H; Zhang, P J; Gimotty, P A; Guerra, M; Guo, W; Xu, X

    2012-11-22

    There is enormous interest to target cancer stem cells (CSCs) for clinical treatment because these cells are highly tumorigenic and resistant to chemotherapy. Oct4 is expressed by CSC-like cells in different types of cancer. However, function of Oct4 in tumor cells is unclear. In this study, we showed that expression of Oct4 gene or transmembrane delivery of Oct4 protein promoted dedifferentiation of melanoma cells to CSC-like cells. The dedifferentiated melanoma cells showed significantly decreased expression of melanocytic markers and acquired the ability to form tumor spheroids. They showed markedly increased resistance to chemotherapeutic agents and hypoxic injury. In the subcutaneous xenograft and tail vein injection assays, these cells had significantly increased tumorigenic capacity. The dedifferentiated melanoma cells acquired features associated with CSCs such as multipotent differentiation capacity and expression of melanoma CSC markers such as ABCB5 and CD271. Mechanistically, Oct4-induced dedifferentiation was associated with increased expression of endogenous Oct4, Nanog and Klf4, and global gene expression changes that enriched for transcription factors. RNAi-mediated knockdown of Oct4 in dedifferentiated cells led to diminished CSC phenotypes. Oct4 expression in melanoma was regulated by hypoxia and its expression was detected in a sub-population of melanoma cells in clinical samples. Our data indicate that Oct4 is a positive regulator of tumor dedifferentiation. The results suggest that CSC phenotype is dynamic and may be acquired through dedifferentiation. Oct4-mediated tumor cell dedifferentiation may have an important role during tumor progression.

  5. Oct4 switches partnering from Sox2 to Sox17 to reinterpret the enhancer code and specify endoderm.

    PubMed

    Aksoy, Irene; Jauch, Ralf; Chen, Jiaxuan; Dyla, Mateusz; Divakar, Ushashree; Bogu, Gireesh K; Teo, Roy; Leng Ng, Calista Keow; Herath, Wishva; Lili, Sun; Hutchins, Andrew P; Robson, Paul; Kolatkar, Prasanna R; Stanton, Lawrence W

    2013-04-03

    How regulatory information is encoded in the genome is poorly understood and poses a challenge when studying biological processes. We demonstrate here that genomic redistribution of Oct4 by alternative partnering with Sox2 and Sox17 is a fundamental regulatory event of endodermal specification. We show that Sox17 partners with Oct4 and binds to a unique 'compressed' Sox/Oct motif that earmarks endodermal genes. This is in contrast to the pluripotent state where Oct4 selectively partners with Sox2 at 'canonical' binding sites. The distinct selection of binding sites by alternative Sox/Oct partnering is underscored by our demonstration that rationally point-mutated Sox17 partners with Oct4 on pluripotency genes earmarked by the canonical Sox/Oct motif. In an endodermal differentiation assay, we demonstrate that the compressed motif is required for proper expression of endodermal genes. Evidently, Oct4 drives alternative developmental programs by switching Sox partners that affects enhancer selection, leading to either an endodermal or pluripotent cell fate. This work provides insights in understanding cell fate transcriptional regulation by highlighting the direct link between the DNA sequence of an enhancer and a developmental outcome.

  6. Oct4 cell-autonomously promotes primitive endoderm development in the mouse blastocyst

    PubMed Central

    Frum, Tristan; Halbisen, Michael A.; Wang, Chaoyang; Amiri, Hossein; Robson, Paul; Ralston, Amy

    2014-01-01

    Summary In embryonic stem (ES) cells and in early mouse embryos, the transcription factor Oct4 is an essential regulator of pluripotency. Oct4 transcriptional targets have been described in ES cell lines; however, the molecular mechanisms by which Oct4 regulates establishment of pluripotency in the epiblast (EPI) have not been fully elucidated. Here we show that neither maternal nor zygotic Oct4 are required for formation of EPI cells in the blastocyst. Rather, Oct4 is first required for development of the primitive endoderm (PE), an extraembryonic lineage. EPI cells promote PE fate in neighboring cells by secreting Fgf4, and Oct4 is required for expression of Fgf4, but we show that Oct4 promotes PE development cell-autonomously, downstream of Fgf4 and Mapk. Finally, we show that Oct4 is required for expression of multiple EPI and PE genes, as well as multiple metabolic pathways essential for the continued growth of the preimplantation embryo. PMID:23747191

  7. An Expanded Oct4 Interaction Network: Implications for Stem Cell Biology, Development, and Disease

    PubMed Central

    Pardo, Mercedes; Lang, Benjamin; Yu, Lu; Prosser, Haydn; Bradley, Allan; Babu, M. Madan; Choudhary, Jyoti

    2010-01-01

    Summary The transcription factor Oct4 is key in embryonic stem cell identity and reprogramming. Insight into its partners should illuminate how the pluripotent state is established and regulated. Here, we identify a considerably expanded set of Oct4-binding proteins in mouse embryonic stem cells. We find that Oct4 associates with a varied set of proteins including regulators of gene expression and modulators of Oct4 function. Half of its partners are transcriptionally regulated by Oct4 itself or other stem cell transcription factors, whereas one-third display a significant change in expression upon cell differentiation. The majority of Oct4-associated proteins studied to date show an early lethal phenotype when mutated. A fraction of the human orthologs is associated with inherited developmental disorders or causative of cancer. The Oct4 interactome provides a resource for dissecting mechanisms of Oct4 function, enlightening the basis of pluripotency and development, and identifying potential additional reprogramming factors. PMID:20362542

  8. Yin Yang 1 is associated with cancer stem cell transcription factors (SOX2, OCT4, BMI1) and clinical implication.

    PubMed

    Kaufhold, Samantha; Garbán, Hermes; Bonavida, Benjamin

    2016-05-25

    The transcription factor Yin Yang 1 (YY1) is frequently overexpressed in cancerous tissues compared to normal tissues and has regulatory roles in cell proliferation, cell viability, epithelial-mesenchymal transition, metastasis and drug/immune resistance. YY1 shares many properties with cancer stem cells (CSCs) that drive tumorigenesis, metastasis and drug resistance and are regulated by overexpression of certain transcription factors, including SOX2, OCT4 (POU5F1), BMI1 and NANOG. Based on these similarities, it was expected that YY1 expression would be associated with SOX2, OCT4, BMI1, and NANOG's expressions and activities. Data mining from the proteomic tissue-based datasets from the Human Protein Atlas were used for protein expression patterns of YY1 and the four CSC markers in 17 types of cancer, including both solid and hematological malignancies. A close association was revealed between the frequency of expressions of YY1 and SOX2 as well as SOX2 and OCT4 in all cancers analyzed. Two types of dynamics were identified based on the nature of their association, namely, inverse or direct, between YY1 and SOX2. These two dynamics define distinctive patterns of BMI1 and OCT4 expressions. The relationship between YY1 and SOX2 expressions as well as the expressions of BMI1 and OCT4 resulted in the classification of four groups of cancers with distinct molecular signatures: (1) Prostate, lung, cervical, endometrial, ovarian and glioma cancers (YY1(lo)SOX2(hi)BMI1(hi)OCT4(hi)) (2) Skin, testis and breast cancers (YY1(hi)SOX2(lo)BMI1(hi)OCT4(hi)) (3) Liver, stomach, renal, pancreatic and urothelial cancers (YY1(lo)SOX2(lo)BMI1(hi)OCT4(hi)) and (4) Colorectal cancer, lymphoma and melanoma (YY1(hi)SOX2(hi)BMI1(lo)OCT4(hi)). A regulatory loop is proposed consisting of the cross-talk between the NF-kB/PI3K/AKT pathways and the downstream inter-regulation of target gene products YY1, OCT4, SOX2 and BMI1.

  9. Oct-4: the almighty POUripotent regulator?

    PubMed

    Buitrago, William; Roop, Dennis R

    2007-02-01

    Oct-4 plays an essential role as a central regulator of the undifferentiated state. Grinnell et al. demonstrate for the first time that Oct-4 by itself has the ability to reprogram committed somatic cells, inducing their dedifferentiation by reverting them to a more developmentally potent state. This study provides evidence that Oct-4 might be the master regulator of the pluripotent state in mammalian cells.

  10. Genome-wide analysis of OCT4 binding sites in glioblastoma cancer cells.

    PubMed

    Fang, Xue-feng; Zhang, Wei-yi; Zhao, Na; Yu, Wei; Ding, Dong; Hong, Xu; Li, Li-sha; Zhang, Hua-rong; Zheng, Shu; Lin, Biao-yang

    2011-10-01

    OCT4, a member of the POU family of gene products, is an octamer motif-binding transcription factor. As it is known to play a crucial role in cancer processes including proliferation, invasion, and chemoradioresistance, it is important to identify the direct targets of OCT4 in living cancer cells. Here, chromatin immunoprecipitation-sequencing (ChIP-seq) was used to identify OCT4 binding sites in glioblastoma cancer cells. The results showed that 5438 OCT4 binding sites were localized in the glioblastoma cancer genome and that these sites contained a consensus sequence TTTkswTw (k=T or G, s=C or G, w=A or T), which occurred 3931 times in 2312 OCT4 binding regions. Furthermore, binding motifs of some other transcription factors were identified in OCT4 binding regions. Our results provide a valuable dataset for understanding gene regulation mechanisms underlying the function of OCT4 in glioblastoma cancer.

  11. Spatiotemporal dynamics of OCT4 protein localization during preimplantation development in mice

    PubMed Central

    Fukuda, Atsushi; Mitani, Atsushi; Miyashita, Toshiyuki; Kobayashi, Hisato; Umezawa, Akihiro

    2016-01-01

    Spatiotemporal expression of transcription factors is crucial for genomic reprogramming. Pou5f1 (Oct4) is an essential transcription factor for reprogramming. A recent study reported that OCT4A, which is crucial for establishment and maintenance of pluripotent cells, is expressed in oocytes, but maternal OCT4A is dispensable for totipotency induction. Whereas another study reported that OCT4B, which is not related to pluripotency, is predominantly expressed instead of OCT4A during early preimplantation phases in mice. To determine the expression states of OCT4 in murine preimplantation embryos, we conducted in-depth expression and functional analyses. We found that pluripotency-related OCT4 mainly localizes to the cytoplasm in early preimplantation phases, with no major nuclear localization until the 8–16-cell stage despite high expression in both oocytes and early embryos. RNA-sequencing analysis using oocytes and early preimplantation embryos could not identify the splice variants creating alternative forms of OCT4 protein. Forced expression of OCT4 in zygotes by the injection of polyadenylated mRNA clearly showed nuclear localization of OCT4 protein around 3–5-fold greater than physiological levels and impaired developmental competency in a dose-dependent manner. Embryos with modest overexpression of OCT4 could develop to the 16-cell stage; however, more than 50% of the embryos were arrested at this stage, similar to the results for OCT4 depletion. In contrast, extensive overexpression of OCT4 resulted in complete arrest at the 2-cell stage accompanied by downregulation of zygotically activated genes and repetitive elements related to the totipotent state. These results demonstrated that OCT4 protein localization was spatiotemporally altered during preimplantation development, and strict control of Oct4 protein levels was essential for proper totipotential reprogramming. PMID:27495230

  12. OCT4 promotes tumorigenesis and inhibits apoptosis of cervical cancer cells by miR-125b/BAK1 pathway

    PubMed Central

    Wang, Y-D; Cai, N; Wu, X-L; Cao, H-Z; Xie, L-L; Zheng, P-S

    2013-01-01

    Octamer-binding transcription factor 4 (OCT4) is a key regulatory gene that maintains the pluripotency and self-renewal properties of embryonic stem cells. Although there is emerging evidence that it can function as oncogene in several cancers, the role in mediating cervical cancer remains unexplored. Here we found that OCT4 protein expression showed a pattern of gradual increase from normal cervix to cervical carcinoma in situ and then to invasive cervical cancer. Overexpression of OCT4 in two types of cervical cancer cells promotes the carcinogenesis, and inhibits cancer cell apoptosis. OCT4 induces upregulation of miR-125b through directly binding to the promoter of miR-125b-1 confirmed by chromatin immunoprecipitation analysis. MiRNA-125b overexpression suppressed apoptosis and expression of BAK1 protein. In contrast, miR-125b sponge impaired the anti-apoptotic effect of OCT4, along with the upregulated expression of BAK1. Significantly, Luciferase assay showed that the activity of the wild-type BAK1 3′-untranslated region reporter was suppressed and this suppression was diminished when the miR-125b response element was mutated or deleted. In addition, we observed negative correlation between levels of BAK1 and OCT4, and positive between OCT4 and miR-125b in primary cervical cancers. These findings suggest an undescribed regulatory pathway in cervical cancer, by which OCT4 directly induces expression of miR-125b, which inhibits its direct target BAK1, leading to suppression of cervical cancer cell apoptosis. PMID:23928699

  13. The emerging roles of Oct4 in tumor-initiating cells

    PubMed Central

    Herlyn, Meenhard

    2015-01-01

    Octamer-binding transcription factor 4 (Oct4), a homeodomain transcription factor, is well established as a master factor controlling the self-renewal and pluripotency of pluripotent stem cells. Also, a large body of research has documented the detection of Oct4 in tumor cells and tissues and has indicated its enrichment in a subpopulation of undifferentiated tumor-initiating cells (TICs) that critically account for tumor initiation, metastasis, and resistance to anticancer therapies. There is circumstantial evidence for low-level expression of Oct4 in cancer cells and TICs, and the participation of Oct4 in various TIC functions such as its self-renewal and survival, epithelial-mesenchymal transition (EMT) and metastasis, and drug resistance development is implicated from considerable Oct4 knockdown and overexpression-based studies. In a few studies, efforts have been made to identify Oct4 target genes in TICs of different sources. Based on such information, Oct4 in TICs appears to act via mechanisms quite distinct from those in pluripotent stem cells, and a main challenge for future studies is to unravel the molecular mechanisms of action of Oct4, particularly to address the question on how such low levels of Oct4 may exert its functions in TICs. Acquiring cells from their native microenvironment that are of high enough quantity and purity is the key to reliably analyze Oct4 functions and its target genes in TICs, and the information gained may greatly facilitate targeting and eradicating those cells. PMID:26447206

  14. Phenotypical and ultrastructural features of Oct4-positive cells in the adult mouse lung

    PubMed Central

    Galiger, Celimene; Kostin, Sawa; Golec, Anita; Ahlbrecht, Katrin; Becker, Sven; Gherghiceanu, Mihaela; Popescu, Laurentiu M; Morty, Rory E; Seeger, Werner; Voswinckel, Robert

    2014-01-01

    Octamer binding trascription factor 4 (Oct4) is a transcription factor of POU family specifically expressed in embryonic stem cells (ESCs). A role for maintaining pluripotency and self-renewal of ESCs is assigned to Oct4 as a pluripotency marker. Oct4 can also be detected in adult stem cells such as bone marrow-derived mesenchymal stem cells. Several studies suggest a role for Oct4 in sustaining self-renewal capacity of adult stem cells. However, Oct4 gene ablation in adult stem cells revealed no abnormalities in tissue turnover or regenerative capacity. In the present study we have conspicuously found pulmonary Oct4-positive cells closely resembling the morphology of telocytes (TCs). These cells were found in the perivascular and peribronchial areas and their presence and location were confirmed by electron microscopy. Moreover, we have used Oct4-GFP transgenic mice which revealed a similar localization of the Oct4-GFP signal. We also found that Oct4 co-localized with several described TC markers such as vimentin, Sca-1, platelet-derived growth factor receptor-beta C-kit and VEGF. By flow cytometry analyses carried out with Oct4-GFP reporter mice, we described a population of EpCAMneg/CD45neg/Oct4-GFPpos that in culture displayed TC features. These results were supported by qRT-PCR with mRNA isolated from lungs by using laser capture microdissection. In addition, Oct4-positive cells were found to express Nanog and Klf4 mRNA. It is concluded for the first time that TCs in adult lung mouse tissue comprise Oct4-positive cells, which express pluripotency-related genes and represent therefore a population of adult stem cells which might contribute to lung regeneration. PMID:24889158

  15. A defined Oct4 level governs cell state transitions of pluripotency entry and differentiation into all embryonic lineages

    PubMed Central

    Radzisheuskaya, Aliaksandra; Chia, Gloryn Le Bin; dos Santos, Rodrigo L.; Theunissen, Thorold W.; Castro, L. Filipe C.; Nichols, Jennifer; Silva, José C. R.

    2013-01-01

    Oct4 is considered a master transcription factor for pluripotent cell self-renewal, but its biology remains poorly understood. Here, we investigated the role of Oct4 using the process of induced pluripotency. We found that a defined embryonic stem cell (ESC) level of Oct4 is required for pluripotency entry. However, once pluripotency is established, the Oct4 level can be decreased up to sevenfold without loss of self-renewal. Unexpectedly, cells constitutively expressing Oct4 at an ESC level robustly differentiated into all embryonic lineages and germline. In contrast, cells with low Oct4 levels were deficient in differentiation, exhibiting expression of naive pluripotency genes in the absence of pluripotency culture requisites. The restoration of Oct4 expression to an ESC level rescued the ability of these to restrict naive pluripotent gene expression and to differentiate. In conclusion, a defined Oct4 level controls the establishment of naive pluripotency as well as commitment to all embryonic lineages. PMID:23629142

  16. A defined Oct4 level governs cell state transitions of pluripotency entry and differentiation into all embryonic lineages.

    PubMed

    Radzisheuskaya, Aliaksandra; Chia, Gloryn Le Bin; dos Santos, Rodrigo L; Theunissen, Thorold W; Castro, L Filipe C; Nichols, Jennifer; Silva, José C R

    2013-06-01

    Oct4 is considered a master transcription factor for pluripotent cell self-renewal, but its biology remains poorly understood. Here, we investigated the role of Oct4 using the process of induced pluripotency. We found that a defined embryonic stem cell (ESC) level of Oct4 is required for pluripotency entry. However, once pluripotency is established, the Oct4 level can be decreased up to sevenfold without loss of self-renewal. Unexpectedly, cells constitutively expressing Oct4 at an ESC level robustly differentiated into all embryonic lineages and germline. In contrast, cells with low Oct4 levels were deficient in differentiation, exhibiting expression of naive pluripotency genes in the absence of pluripotency culture requisites. The restoration of Oct4 expression to an ESC level rescued the ability of these to restrict naive pluripotent gene expression and to differentiate. In conclusion, a defined Oct4 level controls the establishment of naive pluripotency as well as commitment to all embryonic lineages.

  17. Regulation of adipose tissue stromal cells behaviors by endogenic Oct4 expression control.

    PubMed

    Kim, Jung Hwan; Jee, Min Ki; Lee, So Young; Han, Tae Hee; Kim, Bong Sun; Kang, Kyung Sun; Kang, Soo Kyung

    2009-09-24

    To clarify the role of the POU domain transcription factor Oct4 in Adipose Tissue Stromal Cells (ATSCs), we investigated the regulation of Oct4 expression and other embryonic genes in fully differentiated cells, in addition to identifying expression at the gene and protein levels. The ATSCs and several immature cells were routinely expressing Oct4 protein before and after differentiating into specific lineages. Here, we demonstrated the role of Oct4 in ATSCs on cell proliferation and differentiation. Exogenous Oct4 improves adult ATSCs cell proliferation and differentiation potencies through epigenetic reprogramming of stemness genes such as Oct4, Nanog, Sox2, and Rex1. Oct4 directly or indirectly induces ATSCs reprogramming along with the activation of JAK/STAT3 and ERK1/2. Exogenic Oct4 introduced a transdifferentiation priority into the neural lineage than mesodermal lineage. Global gene expression analysis results showed that Oct4 regulated target genes which could be characterized as differentially regulated genes such as pluripotency markers NANOG, SOX2, and KLF4 and markers of undifferentiated stem cells FOXD1, CDC2, and EPHB1. The negatively regulated genes included FAS, TNFR, COL6A1, JAM2, FOXQ1, FOXO1, NESTIN, SMAD3, SLIT3, DKK1, WNT5A, BMP1, and GLIS3 which are implicated in differentiation processes as well as a number of novel genes. Finally we have demonstrated the therapeutic utility of Oct4/ATSCs were introduced into the mouse traumatic brain, engrafted cells was more effectively induces regeneration activity with high therapeutic modality than that of control ATSCs. Engrafted Oct4/ATSCs efficiently migrated and transdifferentiated into action potential carrying, functionally neurons in the hippocampus and promoting the amelioration of lesion cavities.

  18. Regulation of Adipose Tissue Stromal Cells Behaviors by Endogenic Oct4 Expression Control

    PubMed Central

    Lee, So Young; Han, Tae Hee; Kim, Bong Sun; Kang, Kyung Sun; Kang, Soo Kyung

    2009-01-01

    Background To clarify the role of the POU domain transcription factor Oct4 in Adipose Tissue Stromal Cells (ATSCs), we investigated the regulation of Oct4 expression and other embryonic genes in fully differentiated cells, in addition to identifying expression at the gene and protein levels. The ATSCs and several immature cells were routinely expressing Oct4 protein before and after differentiating into specific lineages. Methodology/Principal Findings and Conclusions Here, we demonstrated the role of Oct4 in ATSCs on cell proliferation and differentiation. Exogenous Oct4 improves adult ATSCs cell proliferation and differentiation potencies through epigenetic reprogramming of stemness genes such as Oct4, Nanog, Sox2, and Rex1. Oct4 directly or indirectly induces ATSCs reprogramming along with the activation of JAK/STAT3 and ERK1/2. Exogenic Oct4 introduced a transdifferentiation priority into the neural lineage than mesodermal lineage. Global gene expression analysis results showed that Oct4 regulated target genes which could be characterized as differentially regulated genes such as pluripotency markers NANOG, SOX2, and KLF4 and markers of undifferentiated stem cells FOXD1, CDC2, and EPHB1. The negatively regulated genes included FAS, TNFR, COL6A1, JAM2, FOXQ1, FOXO1, NESTIN, SMAD3, SLIT3, DKK1, WNT5A, BMP1, and GLIS3 which are implicated in differentiation processes as well as a number of novel genes. Finally we have demonstrated the therapeutic utility of Oct4/ATSCs were introduced into the mouse traumatic brain, engrafted cells was more effectively induces regeneration activity with high therapeutic modality than that of control ATSCs. Engrafted Oct4/ATSCs efficiently migrated and transdifferentiated into action potential carrying, functionally neurons in the hippocampus and promoting the amelioration of lesion cavities. PMID:19777066

  19. Induced overexpression of OCT4A in human embryonic stem cells increases cloning efficiency.

    PubMed

    Tsai, Steven C; Chang, David F; Hong, Chang-Mu; Xia, Ping; Senadheera, Dinithi; Trump, Lisa; Mishra, Suparna; Lutzko, Carolyn

    2014-06-15

    Our knowledge of the molecular mechanisms underlying human embryonic stem cell (hESC) self-renewal and differentiation is incomplete. The level of octamer-binding transcription factor 4 (Oct4), a critical regulator of pluripotency, is precisely controlled in mouse embryonic stem cells. However, studies of human OCT4 are often confounded by the presence of three isoforms and six expressed pseudogenes, which has complicated the interpretation of results. Using an inducible lentiviral overexpression and knockdown system to manipulate OCT4A above or below physiological levels, we specifically examine the functional role of the OCT4A isoform in hESC. (We also designed and generated a comparable series of vectors, which were not functional, for the overexpression and knockdown of OCT4B.) We show that specific knockdown of OCT4A results in hESC differentiation, as indicated by morphology changes, cell surface antigen expression, and upregulation of ectodermal genes. In contrast, inducible overexpression of OCT4A in hESC leads to a transient instability of the hESC phenotype, as indicated by changes in morphology, cell surface antigen expression, and transcriptional profile, that returns to baseline within 5 days. Interestingly, sustained expression of OCT4A past 5 days enhances hESC cloning efficiency, suggesting that higher levels of OCT4A can support self-renewal. Overall, our results indicate that high levels of OCT4A increase hESC cloning efficiency and do not induce differentiation (whereas OCT4B expression cannot be induced in hESC), highlighting the importance of isoform-specific studies in a stable and inducible expression system for human OCT4. Additionally, we demonstrate the utility of an efficient method for conditional gene expression in hESC.

  20. A Conserved Oct4/POUV-Dependent Network Links Adhesion and Migration to Progenitor Maintenance

    PubMed Central

    Livigni, Alessandra; Peradziryi, Hanna; Sharov, Alexei A.; Chia, Gloryn; Hammachi, Fella; Migueles, Rosa Portero; Sukparangsi, Woranop; Pernagallo, Salvatore; Bradley, Mark; Nichols, Jennifer; Ko, Minoru S.H.; Brickman, Joshua M.

    2013-01-01

    Summary Background The class V POU domain transcription factor Oct4 (Pou5f1) is a pivotal regulator of embryonic stem cell (ESC) self-renewal and reprogramming of somatic cells to induced pluripotent stem (iPS) cells. Oct4 is also an important evolutionarily conserved regulator of progenitor cell differentiation during embryonic development. Results Here we examine the function of Oct4 homologs in Xenopus embryos and compare this to the role of Oct4 in maintaining mammalian embryo-derived stem cells. Based on a combination of expression profiling of Oct4/POUV-depleted Xenopus embryos and in silico analysis of existing mammalian Oct4 target data sets, we defined a set of evolutionary-conserved Oct4/POUV targets. Most of these targets were regulators of cell adhesion. This is consistent with Oct4/POUV phenotypes observed in the adherens junctions in Xenopus ectoderm, mouse embryonic, and epiblast stem cells. A number of these targets could rescue both Oct4/POUV phenotypes in cellular adhesion and multipotent progenitor cell maintenance, whereas expression of cadherins on their own could only transiently support adhesion and block differentiation in both ESC and Xenopus embryos. Conclusions Currently, the list of Oct4 transcriptional targets contains thousands of genes. Using evolutionary conservation, we identified a core set of functionally relevant factors that linked the maintenance of adhesion to Oct4/POUV. We found that the regulation of adhesion by the Oct4/POUV network occurred at both transcriptional and posttranslational levels and was required for pluripotency. PMID:24210613

  1. Pluripotency Transcription Factor Oct4 Mediates Stepwise Nucleosome Demethylation and Depletion

    PubMed Central

    Shakya, Arvind; Callister, Catherine; Goren, Alon; Yosef, Nir; Garg, Neha; Khoddami, Vahid; Nix, David; Regev, Aviv

    2015-01-01

    The mechanisms whereby the crucial pluripotency transcription factor Oct4 regulates target gene expression are incompletely understood. Using an assay system based on partially differentiated embryonic stem cells, we show that Oct4 opposes the accumulation of local H3K9me2 and subsequent Dnmt3a-mediated DNA methylation. Upon binding DNA, Oct4 recruits the histone lysine demethylase Jmjd1c. Chromatin immunoprecipitation (ChIP) time course experiments identify a stepwise Oct4 mechanism involving Jmjd1c recruitment and H3K9me2 demethylation, transient FACT (facilitates chromatin transactions) complex recruitment, and nucleosome depletion. Genome-wide and targeted ChIP confirms binding of newly synthesized Oct4, together with Jmjd1c and FACT, to the Pou5f1 enhancer and a small number of other Oct4 targets, including the Nanog promoter. Histone demethylation is required for both FACT recruitment and H3 depletion. Jmjd1c is required to induce endogenous Oct4 expression and fully reprogram fibroblasts to pluripotency, indicating that the assay system identifies functional Oct4 cofactors. These findings indicate that Oct4 sequentially recruits activities that catalyze histone demethylation and depletion. PMID:25582194

  2. Structural Mechanism behind Distinct Efficiency of Oct4/Sox2 Proteins in Differentially Spaced DNA Complexes

    PubMed Central

    Yesudhas, Dhanusha; Anwar, Muhammad Ayaz; Panneerselvam, Suresh; Durai, Prasannavenkatesh; Shah, Masaud; Choi, Sangdun

    2016-01-01

    The octamer-binding transcription factor 4 (Oct4) and sex-determining region Y (SRY)-box 2 (Sox2) proteins induce various transcriptional regulators to maintain cellular pluripotency. Most Oct4/Sox2 complexes have either 0 base pairs (Oct4/Sox20bp) or 3 base pairs (Oct4/Sox23bp) separation between their DNA-binding sites. Results from previous biochemical studies have shown that the complexes separated by 0 base pairs are associated with a higher pluripotency rate than those separated by 3 base pairs. Here, we performed molecular dynamics (MD) simulations and calculations to determine the binding free energy and per-residue free energy for the Oct4/Sox20bp and Oct4/Sox23bp complexes to identify structural differences that contribute to differences in induction rate. Our MD simulation results showed substantial differences in Oct4/Sox2 domain movements, as well as secondary-structure changes in the Oct4 linker region, suggesting a potential reason underlying the distinct efficiencies of these complexes during reprogramming. Moreover, we identified key residues and hydrogen bonds that potentially facilitate protein-protein and protein-DNA interactions, in agreement with previous experimental findings. Consequently, our results confess that differential spacing of the Oct4/Sox2 DNA binding sites can determine the magnitude of transcription of the targeted genes during reprogramming. PMID:26790000

  3. Otx2 and Oct4 Drive Early Enhancer Activation during Embryonic Stem Cell Transition from Naive Pluripotency

    PubMed Central

    Yang, Shen-Hsi; Kalkan, Tüzer; Morissroe, Claire; Marks, Hendrik; Stunnenberg, Hendrik; Smith, Austin; Sharrocks, Andrew D.

    2014-01-01

    Summary Embryonic stem cells (ESCs) are unique in that they have the capacity to differentiate into all of the cell types in the body. We know a lot about the complex transcriptional control circuits that maintain the naive pluripotent state under self-renewing conditions but comparatively less about how cells exit from this state in response to differentiation stimuli. Here, we examined the role of Otx2 in this process in mouse ESCs and demonstrate that it plays a leading role in remodeling the gene regulatory networks as cells exit from ground state pluripotency. Otx2 drives enhancer activation through affecting chromatin marks and the activity of associated genes. Mechanistically, Oct4 is required for Otx2 expression, and reciprocally, Otx2 is required for efficient Oct4 recruitment to many enhancer regions. Therefore, the Oct4-Otx2 regulatory axis actively establishes a new regulatory chromatin landscape during the early events that accompany exit from ground state pluripotency. PMID:24931607

  4. Transcriptional Activation by Oct4 Is Sufficient for the Maintenance and Induction of Pluripotency

    PubMed Central

    Hammachi, Fella; Morrison, Gillian M.; Sharov, Alexei A.; Livigni, Alessandra; Narayan, Santosh; Papapetrou, Eirini P.; O'Malley, James; Kaji, Keisuke; Ko, Minoru S.H.; Ptashne, Mark; Brickman, Joshua M.

    2012-01-01

    Summary Oct4 is an essential regulator of pluripotency in vivo and in vitro in embryonic stem cells, as well as a key mediator of the reprogramming of somatic cells into induced pluripotent stem cells. It is not known whether activation and/or repression of specific genes by Oct4 is relevant to these functions. Here, we show that fusion proteins containing the coding sequence of Oct4 or Xlpou91 (the Xenopus homolog of Oct4) fused to activating regions, but not those fused to repressing regions, behave as Oct4, suppressing differentiation and promoting maintenance of undifferentiated phenotypes in vivo and in vitro. An Oct4 activation domain fusion supported embryonic stem cell self-renewal in vitro at lower concentrations than that required for Oct4 while alleviating the ordinary requirement for the cytokine LIF. At still lower levels of the fusion, LIF dependence was restored. We conclude that the necessary and sufficient function of Oct4 in promoting pluripotency is to activate specific target genes. PMID:22832160

  5. Networks of Transcription Factors for Oct4 Expression in Mice.

    PubMed

    Li, Yu-Qiang

    2017-09-01

    The present review aimed to assess the networks of transcription factors regulating the Oct4 expression in mice. Through a comprehensive analysis of the binding sites and the interrelationships of the transcription factors of Oct4, it is found that transcription factors of Oct4 form three regulating complexes centered by Oct4-Sox2, Nanog, and Lrh1. They bind on CR4, CR2, and CR1 regions of Oct4 promoter/enhancer, respectively, to activate Oct4 transcription synergistically. This article also discusses the mechanisms of fine-tuning the Oct4 expression. These findings have important implications in the field of stem cell and developmental biology.

  6. BAY11 enhances OCT4 synthetic mRNA expression in adult human skin cells.

    PubMed

    Awe, Jason P; Crespo, Agustin Vega; Li, You; Kiledjian, Megerditch; Byrne, James A

    2013-02-06

    The OCT4 transcription factor is involved in many cellular processes, including development, reprogramming, maintaining pluripotency and differentiation. Synthetic OCT4 mRNA was recently used (in conjunction with other reprogramming factors) to generate human induced pluripotent stem cells. Here, we discovered that BAY 11-7082 (BAY11), at least partially through an NF-κB-inhibition based mechanism, could significantly increase the expression of OCT4 following transfection of synthetic mRNA (synRNA) into adult human skin cells. We tested various chemical and molecular small molecules on their ability to suppress the innate immune response seen upon synthetic mRNA transfection. Three molecules - B18R, BX795, and BAY11 - were used in immunocytochemical and proliferation-based assays. We also utilized global transcriptional meta-analysis coupled with quantitative PCR to identify relative gene expression downstream of OCT4. We found that human skin cells cultured in the presence of BAY11 resulted in reproducible increased expression of OCT4 that did not inhibit normal cell proliferation. The increased levels of OCT4 resulted in significantly increased expression of genes downstream of OCT4, including the previously identified SPP1, DUSP4 and GADD45G, suggesting the expressed OCT4 was functional. We also discovered a novel OCT4 putative downstream target gene SLC16A9 which demonstrated significantly increased expression following elevation of OCT4 levels. For the first time we have shown that small molecule-based stabilization of synthetic mRNA expression can be achieved with use of BAY11. This small molecule-based inhibition of innate immune responses and subsequent robust expression of transfected synthetic mRNAs may have multiple applications for future cell-based research and therapeutics.

  7. BAY11 enhances OCT4 synthetic mRNA expression in adult human skin cells

    PubMed Central

    2013-01-01

    Introduction The OCT4 transcription factor is involved in many cellular processes, including development, reprogramming, maintaining pluripotency and differentiation. Synthetic OCT4 mRNA was recently used (in conjunction with other reprogramming factors) to generate human induced pluripotent stem cells. Here, we discovered that BAY 11-7082 (BAY11), at least partially through an NF-κB-inhibition based mechanism, could significantly increase the expression of OCT4 following transfection of synthetic mRNA (synRNA) into adult human skin cells. Methods We tested various chemical and molecular small molecules on their ability to suppress the innate immune response seen upon synthetic mRNA transfection. Three molecules - B18R, BX795, and BAY11 - were used in immunocytochemical and proliferation-based assays. We also utilized global transcriptional meta-analysis coupled with quantitative PCR to identify relative gene expression downstream of OCT4. Results We found that human skin cells cultured in the presence of BAY11 resulted in reproducible increased expression of OCT4 that did not inhibit normal cell proliferation. The increased levels of OCT4 resulted in significantly increased expression of genes downstream of OCT4, including the previously identified SPP1, DUSP4 and GADD45G, suggesting the expressed OCT4 was functional. We also discovered a novel OCT4 putative downstream target gene SLC16A9 which demonstrated significantly increased expression following elevation of OCT4 levels. Conclusions For the first time we have shown that small molecule-based stabilization of synthetic mRNA expression can be achieved with use of BAY11. This small molecule-based inhibition of innate immune responses and subsequent robust expression of transfected synthetic mRNAs may have multiple applications for future cell-based research and therapeutics. PMID:23388106

  8. Oct-4 knockdown induces similar patterns of endoderm and trophoblast differentiation markers in human and mouse embryonic stem cells.

    PubMed

    Hay, David C; Sutherland, Linda; Clark, John; Burdon, Tom

    2004-01-01

    The transcription factor Oct-4 is a marker of pluripotency in mouse and human embryonic stem (ES) cells. Previous studies using a tetracycline-regulated Oct-4 transgene in the ZHBTc4 cell line demonstrated that downregulation of Oct-4 expression induced dedifferentiation into trophoblast, a lineage mouse ES cells do not normally generate. We found that transfection of Oct-4-specific short interfering RNA significantly reduced expression and functional activity of Oct-4 in mouse and human ES cells, enabling its role to be compared in both cell types. In mouse ES cells, Oct-4 knockdown produced a pattern of morphological differentiation and increase in expression of the trophoblast-associated transcription factor Cdx2, similar to that triggered by suppressing the Oct-4 transgene in the ZHBTc4 cell line. In addition, downregulation of Oct-4 was accompanied by increased expression of the endoderm-associated genes Gata6 and alpha-fetoprotein, and a gene trap associated with primitive liver/yolk sac differentiation. In human ES cells, Oct-4 knockdown also induced morphological differentiation coincident with the upregulation of Gata6. The induction of Cdx2 and other trophoblast-associated genes, however, was dependent on the culture conditions. These results establish the general requirement for Oct-4 in maintaining pluripotency in ES cells. Moreover, the upregulation of endoderm-associated markers in both mouse and human ES cells points to overlap between development of trophoblast and endoderm differentiation.

  9. Transient Pairing of Homologous Oct4 Alleles Accompanies the Onset of Embryonic Stem Cell Differentiation

    PubMed Central

    Hogan, Megan S.; Parfitt, David-Emlyn; Zepeda-Mendoza, Cinthya J.; Shen, Michael M.; Spector, David L.

    2015-01-01

    SUMMARY The relationship between chromatin organization and transcriptional regulation is an area of intense investigation. We have characterized the spatial relationships between alleles of the Oct4, Sox2, and Nanog genes in single cells during the earliest stages of mouse embryonic stem cell (ESC) differentiation and during embryonic development. We describe homologous pairing of the Oct4 alleles during ESC differentiation and embryogenesis, and present evidence that pairing is correlated with the kinetics of ESC differentiation. Importantly, we identify critical DNA elements within the Oct4 promoter/enhancer region that mediate pairing of Oct4 alleles. Finally, we show that mutation of OCT4/SOX2 binding sites within this region abolishes inter-chromosomal interactions and affects accumulation of the repressive H3K9me2 modification at the Oct4 enhancer. Our findings demonstrate that chromatin organization and transcriptional programs are intimately connected in ESCs, and that the dynamic positioning of the Oct4 alleles is associated with the transition from pluripotency to lineage specification. PMID:25748933

  10. Development: sketch for a theory of Oct4.

    PubMed

    Wagner, Ryan T; Zwaka, Thomas P

    2013-11-18

    How is it that Oct4, a transcription factor that controls pluripotency in stem cells, also controls lineage specification? A recent study investigating common Oct4 targets in vertebrate species indicates an evolutionarily conserved role in mediating cell adhesion. This finding may help decipher Oct4's versatility in governing stem cell behaviors.

  11. Functional interplay between the RK motif and linker segment dictates Oct4–DNA recognition

    PubMed Central

    Kong, Xiangqian; Liu, Jian; Li, Lianchun; Yue, Liyan; Zhang, Lihong; Jiang, Hualiang; Xie, Xin; Luo, Cheng

    2015-01-01

    The POU family transcription factor Oct4 plays pivotal roles in regulating pluripotency and somatic cell reprogramming. Previous studies have indicated an important role for major groove contacts in Oct4–DNA recognition; however, the contributions of the RK motif in the POUh domain and the linker segment joining the two DNA-binding domains remain poorly understood. Here, by combining molecular modelling and functional assays, we find that the RK motif is essential for Oct4–DNA association by recognizing the narrowed DNA minor groove. Intriguingly, computational simulations reveal that the function of the RK motif may be finely tuned by H-bond interactions with the partially disordered linker segment and that breaking these interactions significantly enhances the DNA binding and reprogramming activities of Oct4. These findings uncover a self-regulatory mechanism for specific Oct4–DNA recognition and provide insights into the functional crosstalk at the molecular level that may illuminate mechanistic studies of the Oct protein family and possibly transcription factors in the POU family. Our gain-of-function Oct4 mutants might also be useful tools for use in reprogramming and regenerative medicine. PMID:25870414

  12. Overexpression of OCT4A ortholog elevates endogenous XIST in porcine parthenogenic blastocysts

    PubMed Central

    HWANG, Jae Yeon; CHOI, Kwang-Hwan; LEE, Dong-Kyung; KIM, Seung-Hun; KIM, Eun Bae; HYUN, Sang-Hwan; LEE, Chang-Kyu

    2015-01-01

    X-chromosome inactivation (XCI) is an epigenetic process that equalizes expression of X-borne genes between male and female eutherians. This process is observed in early eutherian embryo development in a species-specific manner. Until recently, various pluripotent factors have been suggested to regulate the process of XCI by repressing XIST expression, which is the master inducer for XCI. Recent insights into the process and its regulation have been restricted in mouse species despite the evolutionary diversity of the process and molecular mechanism among the species. OCT4A is one of the represented pluripotent factors, the gate-keeper for maintaining pluripotency, and an XIST repressor. Therefore, in here, we examined the relation between OCT4A and X-linked genes in porcine preimplantation embryos. Three X-linked genes, XIST, LOC102165544, and RLIM, were selected in present study because their orthologues have been known to regulate XCI in mice. Expression levels of OCT4A were positively correlated with XIST and LOC102165544 in female blastocysts. Furthermore, overexpression of exogenous human OCT4A in cleaved parthenotes generated blastocysts with increased XIST expression levels. However, increased XIST expression was not observed when exogenous OCT4A was obtained from early blastocysts. These results suggest the possibility that OCT4A would be directly or indirectly involved in XIST expression in earlier stage porcine embryos rather than blastocysts. PMID:26255835

  13. Novel variant of OCT4B4 is differentially expressed in human embryonic stem and embryonic carcinoma cells.

    PubMed

    Poursani, Ensieh M; Mehravar, Majid; Soltani, Bahram Mohammad; Mowla, Seyed Javad

    2017-09-05

    POU domain proteins are an important family of transcription factors that regulates cell type-specific gene expression. One of the most crucial members of this family that maintains pluripotency and self-renewal of embryonic stem cells is POU5F1/OCT4. The OCT4 gene can generate several variants under different situations/cell types includes OCT4A that is the major factor sustains pluripotency in embryonic stem and embryonic carcinoma cells, and also OCT4B and OCT4B1, which are transcribed from a different potential promoter located in intron1 and are expressed in various tissues and cell types. In present study, during expression check of OCT4B1 in embryonic carcinoma cells (NT2), we discovered a novel OCT4 transcript for the first time and designated it as OCT4B4. This variant is expressed in various human pluripotent cells and its expression is down-regulated upon induction of differentiation. Moreover, knocking down of OCT4B4 by shRNA resulted in increased accumulation of transfected cells in G0/G1 phase compared to the mock-transfected control cells. Copyright © 2017. Published by Elsevier B.V.

  14. Unravelling the pluripotency paradox in fetal and placental mesenchymal stem cells: Oct-4 expression and the case of The Emperor's New Clothes.

    PubMed

    Ryan, Jennifer M; Pettit, Allison R; Guillot, Pascale V; Chan, Jerry K Y; Fisk, Nicholas M

    2013-08-01

    Mesenchymal stem cells (MSC) from fetal-placental tissues have translational advantages over their adult counterparts, and have variably been reported to express pluripotency markers. OCT-4 expression in fetal-placental MSC has been documented in some studies, paradoxically without tumourogenicity in vivo. It is possible that OCT-4 expression is insufficient to induce true "stemness", but this issue is important for the translational safety of fetal-derived MSC. To clarify this, we undertook a systematic literature review on OCT-4 in fetal or adnexal MSC to show that most studies report OCT-4 message or protein expression, but no study provides definitive evidence of true OCT-4A expression. Discrepant findings were attributable not to different culture conditions, tissue sources, or gestational ages but instead to techniques used. In assessing OCT-4 as a pluripotency marker, we highlight the challenges in detecting the correct OCT-4 isoform (OCT-4A) associated with pluripotency. Although specific detection of OCT-4A mRNA is achievable, it appears unlikely that any antibody can reliably distinguish between OCT-4A and the pseudogene OCT-4B. Finally, using five robust techniques we demonstrate that fetal derived-MSC do not express OCT-4A (or by default OCT-4B). Reports suggesting OCT-4 expression in fetal-derived MSC warrant reassessment, paying attention to gene and protein isoforms, pseudogenes, and antibody choice as well as primer design. Critical examination of the OCT-4 literature leads us to suggest that OCT-4 expression in fetal MSC may be a case of "The Emperor's New Clothes" with early reports of (false) positive expression amplified in subsequent studies without critical attention to emerging refinements in knowledge and methodology.

  15. Dynamic methylation and expression of Oct4 in early neural stem cells

    PubMed Central

    Lee, Shih-Han; Jeyapalan, Jennie N; Appleby, Vanessa; Mohamed Noor, Dzul Azri; Sottile, Virginie; Scotting, Paul J

    2010-01-01

    Neural stem cells are a multipotent population of tissue-specific stem cells with a broad but limited differentiation potential. However, recent studies have shown that over-expression of the pluripotency gene, Oct4, alone is sufficient to initiate a process by which these can form ‘induced pluripotent stem cells’ (iPS cells) with the same broad potential as embryonic stem cells. This led us to examine the expression of Oct4 in endogenous neural stem cells, as data regarding its expression in neural stem cells in vivo are contradictory and incomplete. In this study we have therefore analysed the expression of Oct4 and other genes associated with pluripotency throughout development of the mouse CNS and in neural stem cells grown in vitro. We find that Oct4 is still expressed in the CNS by E8.5, but that this expression declines rapidly until it is undetectable by E15.5. This decline is coincident with the gradual methylation of the Oct4 promoter and proximal enhancer. Immunostaining suggests that the Oct4 protein is predominantly cytoplasmic in location. We also found that neural stem cells from all ages expressed the pluripotency associated genes, Sox2, c-Myc, Klf4 and Nanog. These data provide an explanation for the varying behaviour of cells from the early neuroepithelium at different stages of development. The expression of these genes also provides an indication of why Oct4 alone is sufficient to induce iPS formation in neural stem cells at later stages. PMID:20646110

  16. TGF-βI Regulates Cell Migration through Pluripotent Transcription Factor OCT4 in Endometriosis

    PubMed Central

    Au, Heng-Kien; Chang, Jui-Hung; Wu, Yu-Chih; Kuo, Yung-Che; Chen, Yu-Hsi; Lee, Wei-Chin; Chang, Te-Sheng; Lan, Pei-Chi; Kuo, Hung-Chih; Lee, Kha-Liang; Lee, Mei-Tsu; Tzeng, Chii-Ruey; Huang, Yen-Hua

    2015-01-01

    Transforming growth factor (TGF-β)/TGF-β receptor signal is known to promote cell migration. Up-regulation of TGF-β in serum/peritoneal fluid and increased levels of pluripotent transcription factor OCT4 in endometriotic tissues are frequently observed in patients with endometriosis. However, the mechanisms underlying how TGF-β/TGF-β receptor and OCT4 affect endometriotic cell migration still remain largely unknown. Therefore, endometriotic tissue with high cell migratory capacity were collected from patients with adenomyotic myometrium (n = 23) and chocolate cyst (n = 24); and endometrial tissue with low cell migratory capacity in normal endometrium or hyperplastic endometrium (n = 8) were collected as the controls. We found the mRNA levels of TGF-β receptor I (TGF-β RI) and OCT4 were significantly higher in the high-migratory ectopic endometriotic tissues than those of the low-migratory normal or hyperplastic endometrium. Positive correlations between TGF-β RI and OCT4, and either TGF-β RI or OCT4 with migration-related genes (SNAIL, SLUG and TWIST) regarding the mRNA levels were observed in human endometriotic tissues. TGF-βI dose-dependently increased the gene and protein levels of OCT4, SNAIL and N-Cadherin (N-CAD) and silencing of endogenous OCT4 significantly suppressed the TGF-βI-induced expressions of N-CAD and SNAIL in primary human endometriotic stromal cells and human endometrial carcinoma cell lines RL95-2 and HEC1A. Furthermore, TGF-βI significantly increased the migration ability of endometriotic cells and silencing of OCT4 dramatically suppressed the TGF-βI-induced cell migration activity evidenced by wound-closure assay, transwell assay, and confocal image of F-actin cellular distribution. In conclusion, the present findings demonstrate that the niche TGF-β plays a critical role in initiating expressions of pluripotent transcription factor OCT4 which may contribute to the ectopic endometrial growth by stimulating endometrial cell

  17. TGF-βI Regulates Cell Migration through Pluripotent Transcription Factor OCT4 in Endometriosis.

    PubMed

    Au, Heng-Kien; Chang, Jui-Hung; Wu, Yu-Chih; Kuo, Yung-Che; Chen, Yu-Hsi; Lee, Wei-Chin; Chang, Te-Sheng; Lan, Pei-Chi; Kuo, Hung-Chih; Lee, Kha-Liang; Lee, Mei-Tsu; Tzeng, Chii-Ruey; Huang, Yen-Hua

    2015-01-01

    Transforming growth factor (TGF-β)/TGF-β receptor signal is known to promote cell migration. Up-regulation of TGF-β in serum/peritoneal fluid and increased levels of pluripotent transcription factor OCT4 in endometriotic tissues are frequently observed in patients with endometriosis. However, the mechanisms underlying how TGF-β/TGF-β receptor and OCT4 affect endometriotic cell migration still remain largely unknown. Therefore, endometriotic tissue with high cell migratory capacity were collected from patients with adenomyotic myometrium (n = 23) and chocolate cyst (n = 24); and endometrial tissue with low cell migratory capacity in normal endometrium or hyperplastic endometrium (n = 8) were collected as the controls. We found the mRNA levels of TGF-β receptor I (TGF-β RI) and OCT4 were significantly higher in the high-migratory ectopic endometriotic tissues than those of the low-migratory normal or hyperplastic endometrium. Positive correlations between TGF-β RI and OCT4, and either TGF-β RI or OCT4 with migration-related genes (SNAIL, SLUG and TWIST) regarding the mRNA levels were observed in human endometriotic tissues. TGF-βI dose-dependently increased the gene and protein levels of OCT4, SNAIL and N-Cadherin (N-CAD) and silencing of endogenous OCT4 significantly suppressed the TGF-βI-induced expressions of N-CAD and SNAIL in primary human endometriotic stromal cells and human endometrial carcinoma cell lines RL95-2 and HEC1A. Furthermore, TGF-βI significantly increased the migration ability of endometriotic cells and silencing of OCT4 dramatically suppressed the TGF-βI-induced cell migration activity evidenced by wound-closure assay, transwell assay, and confocal image of F-actin cellular distribution. In conclusion, the present findings demonstrate that the niche TGF-β plays a critical role in initiating expressions of pluripotent transcription factor OCT4 which may contribute to the ectopic endometrial growth by stimulating endometrial cell

  18. Oct4 resetting by Aurkb–PP1 cell cycle axis determines the identity of mouse embryonic stem cells

    PubMed Central

    Shin, Jihoon; Youn, Hong-Duk

    2016-01-01

    In embryonic stem cells (ESCs), cell cycle regulation is deeply connected to pluripotency. Especially, core transcription factors (CTFs) which are essential to maintaining the pluripotency transcription programs should be reset during M/G1 transition. However, it remains unknown about how CTFs are governed during cell cycle progression. Here, we describe that the regulation of Oct4 by Aurora kinase b (Aurkb)/protein phosphatase 1 (PP1) axis during the cell cycle is important for resetting Oct4 to pluripotency and cell cycle related target genes in determining the identity of ESCs. Aurkb starts to phosphorylate Oct4(S229) at the onset of G2/M phase, inducing the dissociation of Oct4 from chromatin, whereas PP1 binds Oct4 and dephosphorylates Oct4(S229) during M/G1 transition, which resets Oct4-driven transcription for pluripotency and the cell cycle. Furthermore, Aurkb phosphormimetic and PP1 binding-deficient mutations in Oct4 disrupt the pluripotent cell cycle, lead to the loss of pluripotency in ESCs, and decrease the efficiency of somatic cell reprogramming. Based on our findings, we suggest that the cell cycle is directly linked to pluripotency programs in ESCs. [BMB Reports 2016; 49(10): 527-528] PMID:27697110

  19. Oct4 resetting by Aurkb-PP1 cell cycle axis determines the identity of mouse embryonic stem cells.

    PubMed

    Shin, Jihoon; Youn, Hong-Duk

    2016-10-01

    In embryonic stem cells (ESCs), cell cycle regulation is deeply connected to pluripotency. Especially, core transcription factors (CTFs) which are essential to maintaining the pluripotency transcription programs should be reset during M/G1 transition. However, it remains unknown about how CTFs are governed during cell cycle progression. Here, we describe that the regulation of Oct4 by Aurora kinase b (Aurkb)/protein phosphatase 1 (PP1) axis during the cell cycle is important for resetting Oct4 to pluripotency and cell cycle related target genes in determining the identity of ESCs. Aurkb starts to phosphorylate Oct4(S229) at the onset of G2/M phase, inducing the dissociation of Oct4 from chromatin, whereas PP1 binds Oct4 and dephosphorylates Oct4(S229) during M/G1 transition, which resets Oct4-driven transcription for pluripotency and the cell cycle. Furthermore, Aurkb phosphormimetic and PP1 binding-deficient mutations in Oct4 disrupt the pluripotent cell cycle, lead to the loss of pluripotency in ESCs, and decrease the efficiency of somatic cell reprogramming. Based on our findings, we suggest that the cell cycle is directly linked to pluripotency programs in ESCs. [BMB Reports 2016; 49(10): 527-528].

  20. Aurkb/PP1-mediated resetting of Oct4 during the cell cycle determines the identity of embryonic stem cells

    PubMed Central

    Shin, Jihoon; Kim, Tae Wan; Kim, Hyunsoo; Kim, Hye Ji; Suh, Min Young; Lee, Sangho; Lee, Han-Teo; Kwak, Sojung; Lee, Sang-Eun; Lee, Jong-Hyuk; Jang, Hyonchol; Cho, Eun-Jung; Youn, Hong-Duk

    2016-01-01

    Pluripotency transcription programs by core transcription factors (CTFs) might be reset during M/G1 transition to maintain the pluripotency of embryonic stem cells (ESCs). However, little is known about how CTFs are governed during cell cycle progression. Here, we demonstrate that the regulation of Oct4 by Aurora kinase b (Aurkb)/protein phosphatase 1 (PP1) during the cell cycle is important for resetting Oct4 to pluripotency and cell cycle genes in determining the identity of ESCs. Aurkb phosphorylates Oct4(S229) during G2/M phase, leading to the dissociation of Oct4 from chromatin, whereas PP1 binds Oct4 and dephosphorylates Oct4(S229) during M/G1 transition, which resets Oct4-driven transcription for pluripotency and the cell cycle. Aurkb phosphor-mimetic and PP1 binding-deficient mutations in Oct4 alter the cell cycle, effect the loss of pluripotency in ESCs, and decrease the efficiency of somatic cell reprogramming. Our findings provide evidence that the cell cycle is linked directly to pluripotency programs in ESCs. DOI: http://dx.doi.org/10.7554/eLife.10877.001 PMID:26880562

  1. Aurkb/PP1-mediated resetting of Oct4 during the cell cycle determines the identity of embryonic stem cells.

    PubMed

    Shin, Jihoon; Kim, Tae Wan; Kim, Hyunsoo; Kim, Hye Ji; Suh, Min Young; Lee, Sangho; Lee, Han-Teo; Kwak, Sojung; Lee, Sang-Eun; Lee, Jong-Hyuk; Jang, Hyonchol; Cho, Eun-Jung; Youn, Hong-Duk

    2016-02-15

    Pluripotency transcription programs by core transcription factors (CTFs) might be reset during M/G1 transition to maintain the pluripotency of embryonic stem cells (ESCs). However, little is known about how CTFs are governed during cell cycle progression. Here, we demonstrate that the regulation of Oct4 by Aurora kinase b (Aurkb)/protein phosphatase 1 (PP1) during the cell cycle is important for resetting Oct4 to pluripotency and cell cycle genes in determining the identity of ESCs. Aurkb phosphorylates Oct4(S229) during G2/M phase, leading to the dissociation of Oct4 from chromatin, whereas PP1 binds Oct4 and dephosphorylates Oct4(S229) during M/G1 transition, which resets Oct4-driven transcription for pluripotency and the cell cycle. Aurkb phosphor-mimetic and PP1 binding-deficient mutations in Oct4 alter the cell cycle, effect the loss of pluripotency in ESCs, and decrease the efficiency of somatic cell reprogramming. Our findings provide evidence that the cell cycle is linked directly to pluripotency programs in ESCs.

  2. Of Mice and Snakes: A Tail of Oct4.

    PubMed

    Shylo, Natalia A; Weatherbee, Scott D

    2016-08-08

    The vertebrate axial skeleton comprises regions of specialized vertebrae, which vary in length between lineages. Aires et al. (2016) uncover a key role for Oct4 in determining trunk length in mice. Additionally, a heterochronic shift in Oct4 expression may underlie the extreme elongation of the trunk in snakes. Copyright © 2016 Elsevier Inc. All rights reserved.

  3. Site-specific Disruption of the Oct4/Sox2 Protein Interaction Reveals Coordinated Mesendodermal Differentiation and the Epithelial-Mesenchymal Transition*

    PubMed Central

    Pan, Xiao; Cang, Xiaohui; Dan, Songsong; Li, Jingchao; Cheng, Jie; Kang, Bo; Duan, Xiaotao; Shen, Binghui; Wang, Ying-Jie

    2016-01-01

    Although the Oct4/Sox2 complex is crucial for maintaining the pluripotency of stem cells, the molecular basis underlying its regulation during lineage-specific differentiation remains unknown. Here, we revealed that the highly conserved Oct4/Lys-156 is important for maintaining the stability of the Oct4 protein and the intermolecular salt bridge between Oct4/Lys-151 and Sox2/Asp-107 that contributes to the Oct4/Sox2 interaction. Post-translational modifications at Lys-156 and K156N, a somatic mutation detected in bladder cancer patients, both impaired the Lys-151–Asp-107 salt bridge and the Oct4/Sox2 interaction. When produced as a recombinant protein or overexpressed in pluripotent stem cells, Oct4/K156N, with reduced binding to Sox2, significantly down-regulated the stemness genes that are cooperatively controlled by the Oct4/Sox2 complex and specifically up-regulated the mesendodermal genes and the SNAIL family genes that promote the epithelial-mesenchymal transition. Thus, we conclude that Oct4/Lys-156-modulated Oct4/Sox2 interaction coordinately controls the epithelial-mesenchymal transition and mesendoderm specification induced by specific differentiation signals. PMID:27369080

  4. ZIC2-dependent OCT4 activation drives self-renewal of human liver cancer stem cells

    PubMed Central

    Zhu, Pingping; Wang, Yanying; He, Lei; Huang, Guanling; Du, Ying; Zhang, Geng; Yan, Xinlong; Xia, Pengyan; Ye, Buqing; Wang, Shuo; Hao, Lu; Wu, Jiayi; Fan, Zusen

    2015-01-01

    Liver cancer stem cells (CSCs) have been identified and shown to have self-renewal and differentiation properties; however, the biology of these hepatic CSCs remains largely unknown. Here, we analyzed transcriptome gene expression profiles of liver CSCs and non-CSCs from hepatocellular carcinoma (HCC) cells lines and found that the transcription factor (TF) ZIC2 is highly expressed in liver CSCs. ZIC2 was required for the self-renewal maintenance of liver CSCs, as ZIC2 depletion reduced sphere formation and xenograft tumor growth in mice. We determined that ZIC2 acts upstream of the TF OCT4 and that ZIC2 recruits the nuclear remodeling factor (NURF) complex to the OCT4 promoter, thereby initiating OCT4 activation. In HCC patients, expression levels of the NURF complex were consistent with clinical severity and prognosis. Moreover, ZIC2 and OCT4 levels positively correlated to the clinicopathological stages of HCC patients. Altogether, our results indicate that levels of ZIC2, OCT4, and the NURF complex can be detected and used for diagnosis and prognosis prediction of HCC patients. Moreover, these factors may be potential therapeutic targets for eradicating liver CSCs. PMID:26426078

  5. Survivin Improves Reprogramming Efficiency of Human Neural Progenitors by Single Molecule OCT4

    PubMed Central

    Liu, Yinan; Feng, Ruopeng; Wang, Caiyun; Jiang, Sibo; Zhang, Xiaoyan

    2016-01-01

    Induced pluripotent stem (iPS) cells have been generated from human somatic cells by ectopic expression of four Yamanaka factors. Here, we report that Survivin, an apoptosis inhibitor, can enhance iPS cells generation from human neural progenitor cells (NPCs) together with one factor OCT4 (1F-OCT4-Survivin). Compared with 1F-OCT4, Survivin accelerates the process of reprogramming from human NPCs. The neurocyte-originated induced pluripotent stem (NiPS) cells generated from 1F-OCT4-Survivin resemble human embryonic stem (hES) cells in morphology, surface markers, global gene expression profiling, and epigenetic status. Survivin keeps high expression in both iPS and ES cells. During the process of NiPS cell to neural cell differentiation, the expression of Survivin is rapidly decreased in protein level. The mechanism of Survivin promotion of reprogramming efficiency from NPCs may be associated with stabilization of β-catenin in WNT signaling pathway. This hypothesis is supported by experiments of RT-PCR, chromatin immune-precipitation, and Western blot in human ES cells. Our results showed overexpression of Survivin could improve the efficiency of reprogramming from NPCs to iPS cells by one factor OCT4 through stabilization of the key molecule, β-catenin. PMID:27974895

  6. Oct4-enhanced green fluorescent protein transgenic pigs: a new large animal model for reprogramming studies.

    PubMed

    Nowak-Imialek, Monika; Kues, Wilfried A; Petersen, Bjoern; Lucas-Hahn, Andrea; Herrmann, Doris; Haridoss, Srividyameena; Oropeza, Marianne; Lemme, Erika; Schöler, Hans R; Carnwath, Joseph W; Niemann, Heiner

    2011-09-01

    The domesticated pig has emerged as an important tool for development of surgical techniques, advancement of xenotransplantation, creation of important disease models, and preclinical testing of novel cell therapies. However, germ line-competent pluripotent porcine stem cells have not yet been derived. This has been a major obstacle to genetic modification of pigs. The transcription factor Oct4 is essential for the maintenance of pluripotency and for reprogramming somatic cells to a pluripotent state. Here, we report the production of transgenic pigs carrying an 18 kb genomic sequence of the murine Oct4 gene fused to the enhanced green fluorescent protein (EGFP) cDNA (OG2 construct) to allow identification of pluripotent cells by monitoring Oct4 expression by EGFP fluorescence. Eleven viable transgenic piglets were produced by somatic cell nuclear transfer. Expression of the EGFP reporter construct was confined to germ line cells, the inner cell mass and trophectoderm of blastocysts, and testicular germ cells. Reprogramming of fibroblasts from these animals by fusion with pluripotent murine embryonic stem cells or viral transduction with human OCT4, SOX2, KLF4, and c-MYC cDNAs resulted in Oct4-EGFP reactivation. The OG2 pigs have thus proved useful for monitoring reprogramming and the induction and maintenance of pluripotency in porcine cells. In conclusion, the OG2 transgenic pigs are a new large animal model for studying the derivation and maintenance of pluripotent cells, and will be valuable for the development of cell therapy.

  7. Transcription factor Oct4 promotes osteosarcoma by regulating lncRNA AK055347

    PubMed Central

    Fan, Hongwu; Liu, Guangyao; Zhao, Changfu; Li, Xuefeng; Yang, Xiaoyu

    2017-01-01

    Osteosarcoma is the most common primary bone tumor in children and adolescents, typically presenting with a poor prognosis. Octamer-binding transcription factor 4 (Oct4) protein, encoded by the POU class 5 homeobox 1 gene, is important in maintaining self-renewal of pluripotent stem cells, and is closely associated with cancer. However, its role in osteosarcoma remains to be elucidated. The present study observed Oct4 was markedly increased in osteosarcoma cell lines and in human osteosarcoma tissue samples. Following Oct4 downregulation by small interfering RNA (siRNA) in osteosarcoma F5M2 cells, the cells exhibited significant decreases in proliferation and invasion ability, and an increase in cell apoptosis. Notably, downregulation of Oct4 decreased the expression of AK055347, a newly identified long noncoding RNA (lncRNA) in human tissues. The downregulation of AK055347 by siRNA resulted in a significant suppressive effect on proliferative and invasive ability, and promotion of cell apoptosis in osteosarcoma cells. Thus, the current study suggests Oct4 exerts a promoting effect in osteosarcoma, and identifies a novel lncRNA in osteosarcoma progression. PMID:28123573

  8. Comparison of Cellular Transforming Activity of OCT4, NANOG, and SOX2 in Immortalized Astrocytes.

    PubMed

    Seo, Sunyoung; Jeon, Hee-Young; Kim, Hyunggee

    2017-09-21

    Embryonic stem cell factors-OCT4, NANOG, and SOX2-contribute to the maintenance of stem cell properties and malignant progression in various cancers, including glioblastoma. Although functional roles of each of these genes are well documented in stem cell and cancer biology, no study has directly compared their cellular transforming activity under same experimental conditions. In this study, we compared the cellular transforming activity of OCT4, NANOG, and SOX2 using human immortalized astrocytes cultured under serum-free stem cell culture conditions. We found that SOX2 exhibited the strongest transforming activities, such as cell proliferation, neurosphere formation, resistance to cytotoxic drug, and cell migration/invasion, which may be associated with the activation of the nuclear factor kappa B (NFκB) signaling pathway. Thus, OCT4, NANOG, and SOX2, known to be frequently activated in various cancers and cancer stem cells, may play a distinct role in the regulation of cellular transformation.

  9. Knockdown of stem cell regulator Oct4A in ovarian cancer reveals cellular reprogramming associated with key regulators of cytoskeleton-extracellular matrix remodelling

    PubMed Central

    Samardzija, Chantel; Greening, David W.; Escalona, Ruth; Chen, Maoshan; Bilandzic, Maree; Luwor, Rodney; Kannourakis, George; Findlay, Jock K.; Ahmed, Nuzhat

    2017-01-01

    Oct4A is a master regulator of self-renewal and pluripotency in embryonic stem cells. It is a well-established marker for cancer stem cell (CSC) in malignancies. Recently, using a loss of function studies, we have demonstrated key roles for Oct4A in tumor cell survival, metastasis and chemoresistance in in vitro and in vivo models of ovarian cancer. In an effort to understand the regulatory role of Oct4A in tumor biology, we employed the use of an ovarian cancer shRNA Oct4A knockdown cell line (HEY Oct4A KD) and a global mass spectrometry (MS)-based proteomic analysis to investigate novel biological targets of Oct4A in HEY samples (cell lysates, secretomes and mouse tumor xenografts). Based on significant differential expression, pathway and protein network analyses, and comprehensive literature search we identified key proteins involved with biologically relevant functions of Oct4A in tumor biology. Across all preparations of HEY Oct4A KD samples significant alterations in protein networks associated with cytoskeleton, extracellular matrix (ECM), proliferation, adhesion, metabolism, epithelial-mesenchymal transition (EMT), cancer stem cells (CSCs) and drug resistance was observed. This comprehensive proteomics study for the first time presents the Oct4A associated proteome and expands our understanding on the biological role of this stem cell regulator in carcinomas. PMID:28406185

  10. A cohesin–OCT4 complex mediates Sox enhancers to prime an early embryonic lineage

    PubMed Central

    Abboud, Nesrine; Moore-Morris, Thomas; Hiriart, Emilye; Yang, Henry; Bezerra, Hudson; Gualazzi, Maria-Giovanna; Stefanovic, Sonia; Guénantin, Anne-Claire; Evans, Sylvia M.; Pucéat, Michel

    2017-01-01

    Short- and long-scales intra-and inter-chromosomal interactions are linked to gene transcription, but the molecular events underlying these structures and how they affect cell fate decision during embryonic development are poorly understood. One of the first embryonic cell fate decisions (that is, mesendoderm determination) is driven by the POU factor OCT4, acting in concert with the high-mobility group genes Sox-2 and Sox-17. Here we report a chromatin-remodelling mechanism and enhancer function that mediate cell fate switching. OCT4 alters the higher-order chromatin structure at both Sox-2 and Sox-17 loci. OCT4 titrates out cohesin and switches the Sox-17 enhancer from a locked (within an interchromosomal Sox-2 enhancer/CCCTC-binding factor CTCF/cohesin loop) to an active (within an intra-chromosomal Sox-17 promoter/enhancer/cohesin loop) state. SALL4 concomitantly mobilizes the polycomb complexes at the Soxs loci. Thus, OCT4/SALL4-driven cohesin- and polycombs-mediated changes in higher-order chromatin structure mediate instruction of early cell fate in embryonic cells. PMID:25851587

  11. The Oct4 homologue PouV and Nanog regulate pluripotency in chicken embryonic stem cells.

    PubMed

    Lavial, Fabrice; Acloque, Hervé; Bertocchini, Federica; Macleod, David J; Boast, Sharon; Bachelard, Elodie; Montillet, Guillaume; Thenot, Sandrine; Sang, Helen M; Stern, Claudio D; Samarut, Jacques; Pain, Bertrand

    2007-10-01

    Embryonic stem cells (ESC) have been isolated from pregastrulation mammalian embryos. The maintenance of their pluripotency and ability to self-renew has been shown to be governed by the transcription factors Oct4 (Pou5f1) and Nanog. Oct4 appears to control cell-fate decisions of ESC in vitro and the choice between embryonic and trophectoderm cell fates in vivo. In non-mammalian vertebrates, the existence and functions of these factors are still under debate, although the identification of the zebrafish pou2 (spg; pou5f1) and Xenopus Pou91 (XlPou91) genes, which have important roles in maintaining uncommitted putative stem cell populations during early development, has suggested that these factors have common functions in all vertebrates. Using chicken ESC (cESC), which display similar properties of pluripotency and long-term self-renewal to mammalian ESC, we demonstrated the existence of an avian homologue of Oct4 that we call chicken PouV (cPouV). We established that cPouV and the chicken Nanog gene are required for the maintenance of pluripotency and self-renewal of cESC. These findings show that the mechanisms by which Oct4 and Nanog regulate pluripotency and self-renewal are not exclusive to mammals.

  12. Intercellular Transport of Oct4 in Mammalian Cells: A Basic Principle to Expand a Stem Cell Niche?

    PubMed Central

    Rolf, Hans J.; Niebert, Sabine; Niebert, Marcus; Gaus, Lena; Schliephake, Henning; Wiese, K. Günter

    2012-01-01

    Background The octamer-binding transcription factor 4 (Oct4) was originally described as a marker of embryonic stem cells. Recently, the role of Oct4 as a key regulator in pluripotency was shown by its ability to reprogram somatic cells in vitro, either alone or in concert with other factors. While artificial induction of pluripotency using transcription factors is possible in mammalian cell culture, it remains unknown whether a potential natural transfer mechanism might be of functional relevance in vivo. The stem cell based regeneration of deer antlers is a unique model for rapid and complete tissue regeneration in mammals and therefore most suitable to study such mechanisms. Here, the transfer of pluripotency factors from resident stem cell niche cells to differentiated cells could recruit more stem cells and start rapid tissue regeneration. Methodology/Principal Findings We report on the ability of STRO-1+ deer antlerogenic mesenchymal stem cells (DaMSCs) to transport Oct4 via direct cell-to-cell connections. Upon cultivation in stem cell expansion medium, we observed nuclear Oct4 expression in nearly all cells. A number of these cells exhibit Oct4 expression not only in the nucleus, but also with perinuclear localisation and within far-ranging intercellular connections. Furthermore, many cells showed intercellular connections containing both F-actin and α-tubulin and through which transport could be observed. To proof that intercellular Oct4-transfer has functional consequences in recipient cells we used a co-culture approach with STRO-1+ DaMSCs and a murine embryonic fibroblast indicator cell line (Oct4-GFP MEF). In this cell line a reporter gene (GFP) under the control of an Oct4 responsive element is only expressed in the presence of Oct4. GFP expression in Oct4-GFP cells started after 24 hours of co-culture providing evidence of Oct4 transfer from STRO-1+ DaMSCs to Oct4-GFP MEF target cells. Conclusions Our findings indicate a possible mechanism for the

  13. Glutamine Metabolism Regulates the Pluripotency Transcription Factor OCT4

    PubMed Central

    Marsboom, Glenn; Zhang, Guo-Fang; Pohl-Avila, Nicole; Zhang, Yanmin; Yuan, Yang; Kang, Hojin; Hao, Bo; Brunengraber, Henri; Malik, Asrar B.; Rehman, Jalees

    2016-01-01

    SUMMARY The molecular mechanisms underlying the regulation of pluripotency by cellular metabolism in human embryonic stem cells (hESCs) are not fully understood. We found that high levels of glutamine metabolism are essential to prevent degradation of OCT4, a key transcription factor regulating hESC pluripotency. Glutamine withdrawal depletes the endogenous anti-oxidant glutathione, which results in the oxidation of OCT4 cysteine residues required for its DNA binding and enhanced OCT4 degradation. The emergence of the OCT4lo cell population following glutamine withdrawal did not result in greater propensity for cell death. Instead, glutamine withdrawal during vascular differentiation of hESCs generated cells with greater angiogenic capacity, thus indicating that modulating glutamine metabolism enhances the differentiation and functional maturation of cells. These findings demonstrate that the pluripotency transcription factor OCT4 can serve as a metabolic-redox sensor in hESCs and that metabolic cues can act in concert with growth factor signaling to orchestrate stem cell differentiation. PMID:27346346

  14. Expression of OCT-4 and SOX-2 in Bone Marrow-Derived Human Mesenchymal Stem Cells during Osteogenic Differentiation

    PubMed Central

    Matic, Igor; Antunovic, Maja; Brkic, Sime; Josipovic, Pavle; Mihalic, Katarina Caput; Karlak, Ivan; Ivkovic, Alan; Marijanovic, Inga

    2016-01-01

    AIM: Determine the levels of expression of pluripotency genes OCT-4 and SOX-2 before and after osteogenic differentiation of human mesenchymal stem cells (hMSCs). METHODS: Human MSCs were derived from the bone marrow and differentiated into osteoblasts. The analyses were performed on days 0 and 14 of the cell culture. In vitro differentiation was evaluated due to bone markers – alkaline phosphatase (AP) activity and the messenger RNA (mRNA) expression of AP and bone sialoprotein (BSP). The OCT-4 and SOX-2 expression was evaluated at mRNA level by real-time qPCR and at protein level by immunocytochemistry. RESULTS: In vitro cultures on day 14 showed an increase in AP activity and upregulation of AP and BSP gene expression. OCT-4 and SOX-2 in undifferentiated hMSCs on day 0 is detectable and very low compared to tumor cell lines as a positive control. Immunocytochemistry detected OCT-4 in the cell nuclei prior (day 0) and post differentiation (day 14). On the same time points, cultures were negative for SOX-2 protein. CONCLUSION: Messenger RNA for pluripotency markers OCT-4 and SOX-2 isolated from hMSCs was less present, while OCT-4 protein was detected in cell nuclei prior and post differentiation into osteoblast lineage. PMID:27275321

  15. Evolution of the mammalian embryonic pluripotency gene regulatory network

    PubMed Central

    Fernandez-Tresguerres, Beatriz; Cañon, Susana; Rayon, Teresa; Pernaute, Barbara; Crespo, Miguel; Torroja, Carlos; Manzanares, Miguel

    2010-01-01

    Embryonic pluripotency in the mouse is established and maintained by a gene-regulatory network under the control of a core set of transcription factors that include octamer-binding protein 4 (Oct4; official name POU domain, class 5, transcription factor 1, Pou5f1), sex-determining region Y (SRY)-box containing gene 2 (Sox2), and homeobox protein Nanog. Although this network is largely conserved in eutherian mammals, very little information is available regarding its evolutionary conservation in other vertebrates. We have compared the embryonic pluripotency networks in mouse and chick by means of expression analysis in the pregastrulation chicken embryo, genomic comparisons, and functional assays of pluripotency-related regulatory elements in ES cells and blastocysts. We find that multiple components of the network are either novel to mammals or have acquired novel expression domains in early developmental stages of the mouse. We also find that the downstream action of the mouse core pluripotency factors is mediated largely by genomic sequence elements nonconserved with chick. In the case of Sox2 and Fgf4, we find that elements driving expression in embryonic pluripotent cells have evolved by a small number of nucleotide changes that create novel binding sites for core factors. Our results show that the network in charge of embryonic pluripotency is an evolutionary novelty of mammals that is related to the comparatively extended period during which mammalian embryonic cells need to be maintained in an undetermined state before engaging in early differentiation events. PMID:21048080

  16. Evolution of the mammalian embryonic pluripotency gene regulatory network.

    PubMed

    Fernandez-Tresguerres, Beatriz; Cañon, Susana; Rayon, Teresa; Pernaute, Barbara; Crespo, Miguel; Torroja, Carlos; Manzanares, Miguel

    2010-11-16

    Embryonic pluripotency in the mouse is established and maintained by a gene-regulatory network under the control of a core set of transcription factors that include octamer-binding protein 4 (Oct4; official name POU domain, class 5, transcription factor 1, Pou5f1), sex-determining region Y (SRY)-box containing gene 2 (Sox2), and homeobox protein Nanog. Although this network is largely conserved in eutherian mammals, very little information is available regarding its evolutionary conservation in other vertebrates. We have compared the embryonic pluripotency networks in mouse and chick by means of expression analysis in the pregastrulation chicken embryo, genomic comparisons, and functional assays of pluripotency-related regulatory elements in ES cells and blastocysts. We find that multiple components of the network are either novel to mammals or have acquired novel expression domains in early developmental stages of the mouse. We also find that the downstream action of the mouse core pluripotency factors is mediated largely by genomic sequence elements nonconserved with chick. In the case of Sox2 and Fgf4, we find that elements driving expression in embryonic pluripotent cells have evolved by a small number of nucleotide changes that create novel binding sites for core factors. Our results show that the network in charge of embryonic pluripotency is an evolutionary novelty of mammals that is related to the comparatively extended period during which mammalian embryonic cells need to be maintained in an undetermined state before engaging in early differentiation events.

  17. Oct-4 expression in adult human differentiated cells challenges its role as a pure stem cell marker.

    PubMed

    Zangrossi, Stefano; Marabese, Mirko; Broggini, Massimo; Giordano, Rosaria; D'Erasmo, Marco; Montelatici, Elisa; Intini, Daniela; Neri, Antonino; Pesce, Maurizio; Rebulla, Paolo; Lazzari, Lorenza

    2007-07-01

    The Oct-4 transcription factor, a member of the POU family that is also known as Oct-3 and Oct3/4, is expressed in totipotent embryonic stem cells (ES) and germ cells, and it has a unique role in development and in the determination of pluripotency. ES may have their postnatal counterpart in the adult stem cells, recently described in various mammalian tissues, and Oct-4 expression in putative stem cells purified from adult tissues has been considered a real marker of stemness. In this context, normal mature adult cells would not be expected to show Oct-4 expression. On the contrary, we demonstrated, using reverse transcription-polymerase chain reaction (PCR) (total RNA, Poly A+), real-time PCR, immunoprecipitation, Western blotting, band shift, and immunofluorescence, that human peripheral blood mononuclear cells, genetically stable and mainly terminally differentiated cells with well defined functions and a limited lifespan, express Oct-4. These observations raise the question as to whether the role of Oct-4 as a marker of pluripotency should be challenged. Our findings suggest that the presence of Oct-4 is not sufficient to define a cell as pluripotent, and that additional measures should be used to avoid misleading results in the case of an embryonic-specific gene with a large number of pseudogenes that may contribute to false identification of Oct-4 in adult stem cells. These unexpected findings may provide new insights into the role of Oct-4 in fully differentiated cells. Disclosure of potential conflicts of interest is found at the end of this article.

  18. Genome editing reveals a role for OCT4 in human embryogenesis.

    PubMed

    Fogarty, Norah M E; McCarthy, Afshan; Snijders, Kirsten E; Powell, Benjamin E; Kubikova, Nada; Blakeley, Paul; Lea, Rebecca; Elder, Kay; Wamaitha, Sissy E; Kim, Daesik; Maciulyte, Valdone; Kleinjung, Jens; Kim, Jin-Soo; Wells, Dagan; Vallier, Ludovic; Bertero, Alessandro; Turner, James M A; Niakan, Kathy K

    2017-10-05

    Despite their fundamental biological and clinical importance, the molecular mechanisms that regulate the first cell fate decisions in the human embryo are not well understood. Here we use CRISPR-Cas9-mediated genome editing to investigate the function of the pluripotency transcription factor OCT4 during human embryogenesis. We identified an efficient OCT4-targeting guide RNA using an inducible human embryonic stem cell-based system and microinjection of mouse zygotes. Using these refined methods, we efficiently and specifically targeted the gene encoding OCT4 (POU5F1) in diploid human zygotes and found that blastocyst development was compromised. Transcriptomics analysis revealed that, in POU5F1-null cells, gene expression was downregulated not only for extra-embryonic trophectoderm genes, such as CDX2, but also for regulators of the pluripotent epiblast, including NANOG. By contrast, Pou5f1-null mouse embryos maintained the expression of orthologous genes, and blastocyst development was established, but maintenance was compromised. We conclude that CRISPR-Cas9-mediated genome editing is a powerful method for investigating gene function in the context of human development.

  19. Polymorphism in regulatory gene sequences

    PubMed Central

    Mitchison, N A

    2001-01-01

    The extensive polymorphism revealed in non-coding gene-regulatory sequences, particularly in the immune system, suggests that this type of genetic variation is functionally and evolutionarily far more important than has been suspected, and provides a lead to new therapeutic strategies. PMID:11178274

  20. Positive Feedback Loop of OCT4 and c-JUN Expedites Cancer Stemness in Liver Cancer.

    PubMed

    Kuo, Kung-Kai; Lee, King-Teh; Chen, Ker-Kong; Yang, Ya-Han; Lin, Ying-Chu; Tsai, Ming-Ho; Wuputra, Kenly; Lee, Yen-Liang; Ku, Chia-Chen; Miyoshi, Hiroyuki; Nakamura, Yukio; Saito, Shigeo; Wu, Chun-Chieh; Chai, Chee-Yin; Eckner, Richard; Steve Lin, Chen-Lung; Wang, Sophie S-W; Wu, Deng-Chyang; Lin, Chang-Shen; Yokoyama, Kazunari K

    2016-06-24

    The network of stemness genes and oncogenes in human patient-specific reprogrammed cancer stem cells (CSCs) remains elusive, especially in liver cancer. HepG2-derived induced pluripotent stem cell-like cells (HepG2-iPS-like cells) were generated by introducing Yamanaka factors and the knockdown vector shTP53. They exhibited features of stemness and a higher tumorigenesis after xenograft transplantation compared with HepG2 cells. The cancerous mass of severe combined immunodeficiency (SCID) mice derived from one colony was dissected and cultured to establish reprogrammed HepG2-derived CSC-like cells (designated rG2-DC-1C). A single colony exhibited 42% occurrence of tumors with higher proliferation capacities. rG2-DC-1C showed continuous expression of the OCT4 stemness gene and of representative tumor markers, potentiated chemoresistance characteristics, and invasion activities. The sphere-colony formation ability and the invasion activity of rG2-DC-1C were also higher than those of HepG2 cells. Moreover, the expression of the OCT4 gene and the c-JUN oncogene, but not of c-MYC, was significantly elevated in rG2-DC-1C, whereas no c-JUN expression was observed in HepG2 cells. The positive-feedback regulation via OCT4-mediated transactivation of the c-JUN promoter and the c-JUN-mediated transactivation of the OCT4 promoter were crucial for promoting cancer development and maintaining cancer stemness in rG2-DC-1C. Increased expression of OCT4 and c-JUN was detected in the early stage of human liver cancer. Therefore, the positive feedback regulation of OCT4 and c-JUN, resulting in the continuous expression of oncogenes such as c-JUN, seems to play a critical role in the determination of the cell fate decision from iPS cells to CSCs in liver cancer. Stem Cells 2016.

  1. Luteolin and apigenin activate the Oct-4/Sox2 signal via NFATc1 in human periodontal ligament cells.

    PubMed

    Liu, Lu; Peng, Zhengjun; Huang, Haoquan; Xu, Zhezhen; Wei, Xi

    2016-10-01

    Identifying small molecules to activate the Oct-4/Sox2-derived pluripotency network represents a hopeful and safe method to pluripotency without genetic manipulation. Luteolin and apigenin, two major bioactive flavonoids, enhance reprogramming efficiency and increase expression of Oct-4/Sox2/c-Myc, albeit the detailed mechanism regulating pluripotency in dental-derived cells remains unknown. In the present study, to elucidate the effect of luteolin/apigenin on pluripotency of periodontal ligament cells (PDLCs) through interaction with downstream signals, we examined cell cycle, proliferation, apoptosis, expression of Oct-4/Sox2/c-Myc, and multilineage differentiation of PDLCs with luteolin/apigenin treatment. Moreover, we profiled the differentially expressed pluripotency genes by PCR arrays. Our results demonstrated that luteolin/apigenin restrained cell proliferation, increased apoptosis, and arrested PDLCs in G2/M and S phase. Luteolin and apigenin activated expression of Oct-4, Sox2, and c-Myc in a time- and dose-dependent pattern, and repressed lineage-specific differentiation. PCR arrays profiled multiple signals in PDLCs with luteolin/apigenin treatment, among which NFATc1 was the major upregulated gene. Notably, blocking of the NFATc1 signal with INCA-6 significantly decreased mRNA and protein expression of Oct-4, Sox2, and c-Myc in PDLCs with luteolin/apigenin treatment, indicating that NFATc1 may act as an upstream modulator of Oct-4/Sox2 signal. Taken together, this study showed that luteolin and apigenin effectively maintain pluripotency of PDLCs through activation of Oct-4/Sox2 signal via NFATc1.

  2. Plant Evolution: Evolving Antagonistic Gene Regulatory Networks.

    PubMed

    Cooper, Endymion D

    2016-06-20

    Developing a structurally complex phenotype requires a complex regulatory network. A new study shows how gene duplication provides a potential source of antagonistic interactions, an important component of gene regulatory networks.

  3. Gene regulatory networks mediating canonical Wnt signal-directed control of pluripotency and differentiation in embryo stem cells.

    PubMed

    Zhang, Xiaoxiao; Peterson, Kevin A; Liu, X Shirley; McMahon, Andrew P; Ohba, Shinsuke

    2013-12-01

    Canonical Wnt signaling supports the pluripotency of embryonic stem cells (ESCs) but also promotes differentiation of early mammalian cell lineages. To explain these paradoxical observations, we explored the gene regulatory networks at play. Canonical Wnt signaling is intertwined with the pluripotency network comprising Nanog, Oct4, and Sox2 in mouse ESCs. In defined media supporting the derivation and propagation of ESCs, Tcf3 and β-catenin interact with Oct4; Tcf3 binds to Sox motif within Oct-Sox composite motifs that are also bound by Oct4-Sox2 complexes. Furthermore, canonical Wnt signaling upregulates the activity of the Pou5f1 distal enhancer via the Sox motif in ESCs. When viewed in the context of published studies on Tcf3 and β-catenin mutants, our findings suggest Tcf3 counters pluripotency by competition with Sox2 at these sites, and Tcf3 inhibition is blocked by β-catenin entry into this complex. Wnt pathway stimulation also triggers β-catenin association at regulatory elements with classic Lef/Tcf motifs associated with differentiation programs. The failure to activate these targets in the presence of a mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) inhibitor essential for ESC culture suggests MEK/ERK signaling and canonical Wnt signaling combine to promote ESC differentiation.

  4. OCT4A contributes to the stemness and multi-potency of human umbilical cord blood-derived multipotent stem cells (hUCB-MSCs)

    SciTech Connect

    Seo, Kwang-Won; Lee, Sae-Rom; Bhandari, Dilli Ram; Roh, Kyoung-Hwan; Park, Sang-Bum; So, Ah-Young; Jung, Ji-Won; Seo, Min-Soo; Kang, Soo-Kyung; Lee, Yong-Soon; Kang, Kyung-Sun

    2009-06-19

    The OCT4A gene, a POU homeodomain transcription factor, has been shown to be expressed in embryonic stem cells (ESC) as well as hUCB-MSCs. In this study, the roles played by OCT4A in hUCB-MSCs were determined by stably inhibiting OCT4A with lenti-viral vector-based small hairpin RNA (shRNA). A decreased rate of cell proliferation was observed in OCT4-inhibited hUCB-MSCs. Down-regulation of CCNA2 expression in OCT4-inhibited hUCB-MSCs was confirmed by RT-PCR and real-time RT-PCR analysis in three genetically independent hUCB-MSC clones. Adipogenic differentiation was also suppressed in OCT4-inhibited hUCB-MSCs. The up-regulation of DTX1 and down-regulation of HDAC1, 2, and 4 expressions may be related to this differentiation deformity. The expression of other transcription factors, including SOX2, REX1 and c-MYC, was also affected by OCT4 inhibition in hUCB-MSCs. In conclusion, these finding suggest that OCT4A performs functionally conserved roles in hUCB-MSCs, making its expression biologically important for ex vivo culture of hUCB-MSCs.

  5. Pontin functions as an essential coactivator for Oct4-dependent lincRNA expression in mouse embryonic stem cells

    PubMed Central

    Boo, Kyungjin; Bhin, Jinhyuk; Jeon, Yoon; Kim, Joomyung; Shin, Hi-Jai R.; Park, Jong-Eun; Kim, Kyeongkyu; Kim, Chang Rok; Jang, Hyonchol; Kim, In-Hoo; Kim, V. Narry; Hwang, Daehee; Lee, Ho; Baek, Sung Hee

    2015-01-01

    The actions of transcription factors, chromatin modifiers and noncoding RNAs are crucial for the programming of cell states. Although the importance of various epigenetic machineries for controlling pluripotency of embryonic stem (ES) cells has been previously studied, how chromatin modifiers cooperate with specific transcription factors still remains largely elusive. Here, we find that Pontin chromatin remodelling factor plays an essential role as a coactivator for Oct4 for maintenance of pluripotency in mouse ES cells. Genome-wide analyses reveal that Pontin and Oct4 share a substantial set of target genes involved in ES cell maintenance. Intriguingly, we find that the Oct4-dependent coactivator function of Pontin extends to the transcription of large intergenic noncoding RNAs (lincRNAs) and in particular linc1253, a lineage programme repressing lincRNA, is a Pontin-dependent Oct4 target lincRNA. Together, our findings demonstrate that the Oct4-Pontin module plays critical roles in the regulation of genes involved in ES cell fate determination. PMID:25857206

  6. The expressions of stem cell markers: Oct4, Nanog, Sox2, nucleostemin, Bmi, Zfx, Tcl1, Tbx3, Dppa4, and Esrrb in bladder, colon, and prostate cancer, and certain cancer cell lines.

    PubMed

    Amini, Sabrieh; Fathi, Fardin; Mobalegi, Jafar; Sofimajidpour, Heshmatollah; Ghadimi, Tayyeb

    2014-03-01

    Uncontrolled self-renewal plays a direct function in the progression of different types of carcinomas. The same molecular pathway that manages self-renewal in normal stem cells also seems to manage cancer stem cells. Here, we examine the expressions of self-renewal regulatory factors Oct4, Nanog, Sox2, nucleostemin, Zfx, Esrrb, Tcl1, Tbx3, and Dppa4 in tissue samples of colon, prostate, and bladder carcinomas as well as cancer cell lines HT-29, Caco-2, HT-1376, LNCaP, and HepG2. We used reverse transcriptase polymerase chain reaction to examine expressions of the above mentioned regulatory factors in cancer cell lines HT-29, Caco-2, HT-1376, LNCaP, and HepG2 and in 20 tumor tissue samples. Total RNA was isolated by the ISOGEN method. RNA integrity was checked by agarose gel electrophoresis and spectrophotometry. Expressions of Oct4 and nucleostemin at the protein level were determined by immunocytochemistry. A significant relationship was found between tumor grade and self-renewal gene expression. Expressions of stem cell specific marker genes were detected in all examined cancer cell lines, in 40% to 100% of bladder cancer samples, and in 60% to 100% of colon and prostate cancer samples. Oct4 expressed in 100% of tumor tissue samples. Our data show that stem cell markers Oct4, Nanog, Sox2, nucleostemin, Bmi, Zfx, Esrrb, Tcl1, Tbx3, and Dppa4 significantly express in cancer cell lines and cancer tissues. Hence, these markers might be useful as potential tumor markers in the diagnosis and/or prognosis of tumors.

  7. Identification of novel OCT4 genetic variant associated with the risk of chronic hepatitis B in a Korean population.

    PubMed

    Shin, Joong-Gon; Cheong, Hyun Sub; Lee, Kwanghyun; Ju, Bong-Gun; Lee, Jeong-Hoon; Yu, Su Jong; Yoon, Jung-Hwan; Cheong, Jae Youn; Cho, Sung Won; Park, Neung Hwa; Namgoong, Suhg; Kim, Lyoung Hyo; Kim, Yoon Jun; Shin, Hyoung Doo

    2017-03-01

    Hepatitis B viral infection is a serious risk factor for chronic hepatitis B (CHB), cirrhosis and hepatocellular carcinoma. Recently, several genome-wide association studies (GWASs) have been conducted to identify important genetic variant associated with the risk of CHB. In our previous GWAS, TCF19 was identified as one of the susceptibility genes for CHB risk (P=4.2×10(-9) at rs1419881). In order to discover possible additional causal variants around TCF19, we performed an association study by genotyping single nucleotide polymorphisms (SNPs) in OCT4, a nearby gene to TCF19. Nineteen OCT4 genetic variants were selected and genotyped in 3902 subjects (1046 CHB patients and 2856 population controls). Logistic regression analysis revealed that OCT4 rs1265163 showed the most significant association signal for the risk of CHB (OR=1.46, P=4.78×10(-12) ). Linkage disequilibrium and conditional analysis confirmed rs1265163 in OCT4 as a novel genetic marker for CHB susceptibility. The genetic risk scores (GRSs) were calculated to visualize the combined genetic effects of all known CHB-associated loci, including OCT4 rs1265163, which had been identified in this study. Individuals with higher cumulative GRSs showed significantly increased ORs. The luciferase activity of rs885952, a tagging SNP of rs1265163, showed that OCT4 promoter activity was significantly different between the wild-type and SNP mutant form (P<.05). This follow-up study to our previous GWAS identified a possible causal genetic variant associated with the risk of CHB, and findings from this study may prove useful in the understanding of genetic susceptibility to CHB. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  8. Differential Recruitment of Methyl CpG-Binding Domain Factors and DNA Methyltransferases by the Orphan Receptor Germ Cell Nuclear Factor Initiates the Repression and Silencing of Oct4

    PubMed Central

    Gu, Peili; Xu, Xueping; Le Menuet, Damien; Chung, Arthur C-K; Cooney, Austin J

    2011-01-01

    The pluripotency gene Oct4 encodes a key transcription factor that maintains self-renewal of embryonic stem cell (ESC) and is downregulated upon differentiation of ESCs and silenced in somatic cells. A combination of cis elements, transcription factors, and epigenetic modifications, such as DNA methylation, mediates Oct4 gene expression. Here, we show that the orphan nuclear receptor germ cell nuclear factor (GCNF) initiates Oct4 repression and DNA methylation by the differential recruitment of methyl-CpG binding domain (MBD) and DNA methyltransferases (Dnmts) to the Oct4 promoter. When compared with wild-type ESCs and gastrulating embryos, Oct4 repression is lost and its proximal promoter is significantly hypomethylated in retinoic acid (RA)-differentiated GCNF−/− ESCs and GCNF−/− embryos. Efforts to characterize mediators of GCNF's repressive function and DNA methylation of the Oct4 promoter identified MBD3, MBD2, and de novo Dnmts as GCNF interacting factors. Upon differentiation, endogenous GCNF binds to the Oct4 proximal promoter and differentially recruits MBD3 and MBD2 as well as Dnmt3A. In differentiated GCNF−/− ESCs, recruitment of MBD3 and MBD2 as well as Dnmt3A to Oct4 promoter is lost and subsequently Oct4 repression and DNA methylation failed to occur. Hypomethylation of the Oct4 promoter is also observed in RA-differentiated MBD3−/− and Dnmt3A−/− ESCs, but not in MBD2−/− and Dnmt3B−/− ESCs. Thus, recruitment of MBD3, MBD2, and Dnmt3A by GCNF links two events: gene-specific repression and DNA methylation, which occur differentially at the Oct4 promoter. GCNF initiates the repression and epigenetic modification of Oct4 gene during ESC differentiation. Stem Cells 2011;29:1041–1051 PMID:21608077

  9. On attractors in gene regulatory systems

    NASA Astrophysics Data System (ADS)

    Brokan, E.; Sadyrbaev, F. Zh.

    2017-02-01

    We describe attracting sets for differential systems appearing in mathematical models of gene regulatory systems. The relation of elements in such systems can be described by regulatory matrices containing elements -1, 0 or 1 corresponding to inhibition, no relation or activation respectively. Four types of regulatory matrices are considered. The respective examples are discussed and the attractive sets are described.

  10. Bcl3 Bridges LIF-STAT3 to Oct4 Signaling in the Maintenance of Naïve Pluripotency.

    PubMed

    Chen, Chen-Yun; Lee, Desy S; Yan, Yu-Ting; Shen, Chia-Ning; Hwang, Shiaw-Min; Lee, Sho Tone; Hsieh, Patrick C H

    2015-12-01

    Leukemia inhibitory factor (LIF) regulates mouse embryonic stem cell (mESC) pluripotency through STAT3 activation, but the downstream signaling remains largely unelucidated. Using cDNA microarrays, we verified B cell leukemia/lymphoma 3 (Bcl3) as the most significantly downregulated factor following LIF withdrawal in mESCs. Bcl3 knockdown altered mESC morphology, reduced expression of pluripotency genes including Oct4, Sox2, and Nanog, and downregulated DNA binding of acetylated histone 3 and RNA polymerase II on the Oct4 promoter. Conversely, Bcl3 overexpression partially prevented cell differentiation and promoted Oct4 and Nanog promoter activities. Furthermore, coimmunoprecipitation and chromatin immunoprecipitation experiments demonstrated that Bcl3 regulation of mESC pluripotency may be through its association with Oct4 and β-catenin and its promoter binding capability. These results establish that Bcl3 positively regulates pluripotency genes and thus shed light on the mechanism of Bcl3 as a downstream molecule of LIF/STAT3 signaling in pluripotency maintenance. © 2015 AlphaMed Press.

  11. Oct4 expression in adult human stem cells: evidence in support of the stem cell theory of carcinogenesis.

    PubMed

    Tai, Mei-Hui; Chang, Chia-Cheng; Kiupel, Matti; Webster, Joshua D; Olson, L Karl; Trosko, James E

    2005-02-01

    The Oct3/4 gene, a POU family transcription factor, has been noted as being specifically expressed in embryonic stem cells and in tumor cells but not in cells of differentiated tissues. With the ability to isolate adult human stem cells it became possible to test for the expression of Oct3/4 gene in adult stem cells and to test the stem cell theory of carcinogenesis. Using antibodies and PCR primers we tested human breast, liver, pancreas, kidney, mesenchyme and gastric stem cells, the cancer cell lines HeLa and MCF-7 and human, dog and rat tumors for Oct4 expression. The results indicate that adult human stem cells, immortalized non-tumorigenic cells and tumor cells and cell lines, but not differentiated cells, express Oct4. Oct4 is expressed in a few cells found in the basal layer of human skin epidermis. The data demonstrate that adult stem cells maintain expression of Oct4, consistent with the stem cell hypothesis of carcinogenesis.

  12. OCT4 spliced variants are highly expressed in brain cancer tissues and inhibition of OCT4B1 causes G2/M arrest in brain cancer cells.

    PubMed

    Asadi, Malek Hossein; Khalifeh, Khosrow; Mowla, Seyed Javad

    2016-12-01

    The new claim about the origin of cancer known as Cancer Stem Cell theory states that a somatic differentiated cell can dedifferentiated or reprogrammed for regaining the cancer cell features. It has been recently shown that expression of stemness factors such as Oct4, Sox2, Nanog and Klf4, in a variety of somatic cancers can leads to development of tumorogenesis. Here, the expression of Oct4 variants were evaluated in brain tumor tissues by quantitative RT-PCR and immunohistochemical (IHC) analysis. In next phase of our study, the expression of Oct4B1 was knock-down in brain cancer cell lines and its effect on cell cycle was assessed. Finally, in order to get insights into sequence-structure-function relationships of Oct4 isofroms, their sequences were analysed using bioinformatic tools. Our data revealed that all three variants of Oct4 are expressed in different types of brain cancer. The expression level of Oct4B1, in contast to Oct4B, was much higher in high-grade brain tumors compared with low-grade ones. In line with qPCR, the expression of Oct4A and B isofroms was confirmed with IHC in different types of brain tumors. Moreover, as a result of the suppression of Oct4B1 expression, the brain cancer cells were arrested in G2/M phase of cell cycle. Bioinfromatics data indicated that the predicted Oct4B1 protein have DNA binding properties. All together, our findings suggest that Oct4B1 has a potential role in tumorigenesis of brain cancer and can be considered as a new tumor marker with potential value in diagnosis and treatment of brain cancer.

  13. Transdifferentiation of Human Hair Follicle Mesenchymal Stem Cells into Red Blood Cells by OCT4.

    PubMed

    Liu, Zhijing; Lu, Shi-Jiang; Lu, Yan; Tan, Xiaohua; Zhang, Xiaowei; Yang, Minlan; Zhang, Fuming; Li, Yulin; Quan, Chengshi

    2015-01-01

    Shortage of red blood cells (RBCs, erythrocytes) can have potentially life-threatening consequences for rare or unusual blood type patients with massive blood loss resulting from various conditions. Erythrocytes have been derived from human pluripotent stem cells (PSCs), but the risk of potential tumorigenicity cannot be ignored, and a majority of these cells produced from PSCs express embryonic ε- and fetal γ-globins with little or no adult β-globin and remain nucleated. Here we report a method to generate erythrocytes from human hair follicle mesenchymal stem cells (hHFMSCs) by enforcing OCT4 gene expression and cytokine stimulation. Cells generated from hHFMSCs expressed mainly the adult β-globin chain with minimum level of the fetal γ-globin chain. Furthermore, these cells also underwent multiple maturation events and formed enucleated erythrocytes with a biconcave disc shape. Gene expression analyses showed that OCT4 regulated the expression of genes associated with both pluripotency and erythroid development during hHFMSC transdifferentiation toward erythroid cells. These findings show that mature erythrocytes can be generated from adult somatic cells, which may serve as an alternative source of RBCs for potential autologous transfusion.

  14. Effect of neuronal induction on NSE, Tau, and Oct4 promoter methylation in bone marrow mesenchymal stem cells.

    PubMed

    Duan, Ping; Zhang, Ying; Han, Xuefei; Liu, Junling; Yan, Wenhai; Xing, Ying

    2012-04-01

    Cell differentiation involves widespread epigenetic reprogramming, including modulation of DNA methylation patterns. The differentiation potential differences in DNA methylation patterns might function in pluripotency restriction, while tissue-specific differences might work in lineage restriction. To investigate the effects of neuronal induction on promoter methylation pattern in rat bone marrow mesenchymal stem cells (MSCs), we used bisulfite sequencing to analyze the methylation status of the promoter regions in neuron-specific enolase (NSE), microtubule-associated protein Tau, and Oct4 genes in MSCs pre- and post-chemical induction. Neurocytes from the newborn rat brains were used as control. Data showed that NSE and Tau were abundantly expressed in the brain cells and MSC-derived neurocyte-like cells as well but not in the MSCs. However, both NSE promoter (-214~+57 bp) and Tau promoter (-239~+131 bp) were hypomethylated (<4 % CpG methylation). Oct4 was expressed in MSCs, and the Oct4 promoter (-293~-85 bp) was hypermethylated (>79 % CpG methylation). Interestingly, it was found that the methylation of the locus -113 bp upstream of Oct4 transcription start site was specifically enhanced in the process of MSCs' neuronal differentiation. Further experiments in hepatocytes derived from MSCs and hepar tissue proved that the -113 bp locus methylation increased also in non-neurogenic lineages. Tfsitescan prediction showed that AP-2-alpha/gamma and Sp1 might regulate Oct4 transcription upon MSC differentiation by binding the -113 bp locus. So, we conclude that promoter methylation modifies pluripotency-specific gene, rather than regulates the expression of neural-specific genes when MSCs differentiate into neurocyte-like cells.

  15. Regulatory genes in the ancestral chordate genomes.

    PubMed

    Satou, Yutaka; Wada, Shuichi; Sasakura, Yasunori; Satoh, Nori

    2008-12-01

    Changes or innovations in gene regulatory networks for the developmental program in the ancestral chordate genome appear to be a major component in the evolutionary process in which tadpole-type larvae, a unique characteristic of chordates, arose. These alterations may include new genetic interactions as well as the acquisition of new regulatory genes. Previous analyses of the Ciona genome revealed that many genes may have emerged after the divergence of the tunicate and vertebrate lineages. In this paper, we examined this possibility by examining a second non-vertebrate chordate genome. We conclude from this analysis that the ancient chordate included almost the same repertory of regulatory genes, but less redundancy than extant vertebrates, and that approximately 10% of vertebrate regulatory genes were innovated after the emergence of vertebrates. Thus, refined regulatory networks arose during vertebrate evolution mainly as preexisting regulatory genes multiplied rather than by generating new regulatory genes. The inferred regulatory gene sets of the ancestral chordate would be an important foundation for understanding how tadpole-type larvae, a unique characteristic of chordates, evolved.

  16. Chemotherapeutics-induced Oct4 expression contributes to drug resistance and tumor recurrence in bladder cancer

    PubMed Central

    Su, Bing-Hua; Su, Yu-Chu; Chen, Yi-Cheng; Su, Wu-Chou; Wu, Pensee; Yang, Wen-Horng; Shiau, Ai-Li; Wu, Chao-Liang

    2017-01-01

    Cancer cells initially characterized as sensitive to chemotherapy may acquire resistance to chemotherapy and lead to tumor recurrence through the expansion of drug-resistant population. Acquisition of drug resistance to conventional chemotherapy is a major obstacle in the treatment of recurrent cancer. Here we investigated whether anticancer drugs induced Oct4 expression, thereby contributing to acquired drug resistance and tumor recurrence in bladder cancer. We identified a positive correlation of Oct4 expression with tumor recurrence in 122 clinical specimens of superficial high-grade (stages T1-2) bladder transitional cell carcinoma (TCC). Increased Oct4 levels in bladder tumors were associated with short recurrence-free intervals in the patients. Chemotherapy induced Oct4 expression in bladder cancer cells. Notably, treatment with cisplatin increased CD44-positive bladder cancer cells expressing Oct4, representing cancer stem-like cell subpopulation. Forced expression of Oct4 reduced, whereas knockdown of Oct4 enhanced, drug sensitivity in bladder cancer cells. Furthermore, tumor cells overexpressing Oct4 responded poorly to cisplatin in vivo. In regard to clinical relevance, inhibition of Oct4 by all-trans retinoic acid (ATRA) synergistically increased sensitivity to cisplatin in bladder cancer cells. Furthermore, the combination of cisplatin and ATRA was superior to cisplatin alone in suppressing tumor growth. Therefore, our results provide evidence that Oct4 increases drug resistance and implicate that inhibition of Oct4 may be a therapeutic strategy to circumvent drug resistance. PMID:27244887

  17. Oct4 and klf4 reprogram dermal papilla cells into induced pluripotent stem cells.

    PubMed

    Tsai, Su-Yi; Clavel, Carlos; Kim, Soo; Ang, Yen-Sin; Grisanti, Laura; Lee, Dung-Fang; Kelley, Kevin; Rendl, Michael

    2010-02-01

    Direct reprogramming of somatic cells into induced pluripotent stem (iPS) cells by only four transcription factors (Oct4, Sox2, Klf4, and c-Myc) has great potential for tissue-specific regenerative therapies, eliminating the ethical issues surrounding the use of embryonic stem cells and the rejection problems of using non-autologous cells. The reprogramming efficiency generally is very low, however, and the problems surrounding the introduction of viral genetic material are only partially investigated. Recent efforts to reduce the number of virally expressed transcription factors succeeded at reprogramming neural stem cells into iPS cells by overexpressing Oct4 alone. However, the relative inaccessibility and difficulty of obtaining neural cells in humans remains to be resolved. Here we report that dermal papilla (DP) cells, which are specialized skin fibroblasts thought to instruct hair follicle stem cells, endogenously express high levels of Sox2 and c-Myc, and that these cells can be reprogrammed into iPS cells with only Oct4 and Klf4. Moreover, we show that DP cells are reprogrammed more efficiently than skin and embryonic fibroblasts. iPS cells derived from DP cells expressed pluripotency genes and differentiated into cells from all germ layers in vitro and widely contributed to chimeric mice in vivo, including the germline. Our work establishes DP cells as an easily accessible source to generate iPS cells with efficiency and with less genetic material. This opens up the possibility of streamlined generation of skin-derived, patient-specific pluripotent stem cells and of ultimately replacing the remaining two factors with small molecules for safe generation of transplantable cells.

  18. The intracellular distribution of the ES cell totipotent markers OCT4 and Sox2 in adult stem cells differs dramatically according to commercial antibody used.

    PubMed

    Zuk, Patricia A

    2009-04-01

    To characterize ES cells, researchers have at their disposal a list of pluripotent markers, such as OCT4. In their quest to determine if adult stem cell populations, such as MSCs and ASCs, are pluripotent, several groups have begun to report the expression of these markers in these cells. Consistent with this, human ASCs (hASCs) are shown in this study to express a plethora of ES pluripotent markers at the gene and protein level, including OCT4, Sox2, and Nanog. When intracellular distribution is examined in hASCs, both OCT4 and Sox2 are expressed within the nuclei of hASCs, consistent with their expression patterns in ES cells. However, a significant amount of expression can be noted within the hASC cytoplasm and a complete absence of nuclear expression is observed for Nanog. Recent descriptions of OCT4 transcript variants may explain the cytoplasmic expression of OCT4 in hASCs and consistent with this, hASCs do express both the OCT4A and 4B transcript variants at the gene level. However, discrepancies arise when these three pluripotent markers are studied at the protein level. Specifically, distinct differences in intracellular expression patterns were noted for OCT4, Sox2, and Nanog from commercial antibody to commercial antibody. These antibody discrepancies persisted when hMSCs and rat ASCs and MSCs were examined. Therefore, confirming the expression of OCT4, Sox2, and Nanog in adult stem cells with today's commercial antibodies must be carefully considered before the designation of pluripotent can be granted.

  19. Evolving Robust Gene Regulatory Networks

    PubMed Central

    Noman, Nasimul; Monjo, Taku; Moscato, Pablo; Iba, Hitoshi

    2015-01-01

    Design and implementation of robust network modules is essential for construction of complex biological systems through hierarchical assembly of ‘parts’ and ‘devices’. The robustness of gene regulatory networks (GRNs) is ascribed chiefly to the underlying topology. The automatic designing capability of GRN topology that can exhibit robust behavior can dramatically change the current practice in synthetic biology. A recent study shows that Darwinian evolution can gradually develop higher topological robustness. Subsequently, this work presents an evolutionary algorithm that simulates natural evolution in silico, for identifying network topologies that are robust to perturbations. We present a Monte Carlo based method for quantifying topological robustness and designed a fitness approximation approach for efficient calculation of topological robustness which is computationally very intensive. The proposed framework was verified using two classic GRN behaviors: oscillation and bistability, although the framework is generalized for evolving other types of responses. The algorithm identified robust GRN architectures which were verified using different analysis and comparison. Analysis of the results also shed light on the relationship among robustness, cooperativity and complexity. This study also shows that nature has already evolved very robust architectures for its crucial systems; hence simulation of this natural process can be very valuable for designing robust biological systems. PMID:25616055

  20. Modeling of hysteresis in gene regulatory networks.

    PubMed

    Hu, J; Qin, K R; Xiang, C; Lee, T H

    2012-08-01

    Hysteresis, observed in many gene regulatory networks, has a pivotal impact on biological systems, which enhances the robustness of cell functions. In this paper, a general model is proposed to describe the hysteretic gene regulatory network by combining the hysteresis component and the transient dynamics. The Bouc-Wen hysteresis model is modified to describe the hysteresis component in the mammalian gene regulatory networks. Rigorous mathematical analysis on the dynamical properties of the model is presented to ensure the bounded-input-bounded-output (BIBO) stability and demonstrates that the original Bouc-Wen model can only generate a clockwise hysteresis loop while the modified model can describe both clockwise and counter clockwise hysteresis loops. Simulation studies have shown that the hysteresis loops from our model are consistent with the experimental observations in three mammalian gene regulatory networks and two E.coli gene regulatory networks, which demonstrate the ability and accuracy of the mathematical model to emulate natural gene expression behavior with hysteresis. A comparison study has also been conducted to show that this model fits the experiment data significantly better than previous ones in the literature. The successful modeling of the hysteresis in all the five hysteretic gene regulatory networks suggests that the new model has the potential to be a unified framework for modeling hysteresis in gene regulatory networks and provide better understanding of the general mechanism that drives the hysteretic function.

  1. Comparative SRY incorporation on the regulatory regions of pluripotency/differentiation genes in human embryonic carcinoma cells after retinoic acid induction.

    PubMed

    Kakhki, Sara Ashrafi; Shahhoseini, Maryam; Salekdeh, Ghasem Hosseini

    2013-04-01

    Members of the SOX (SRY box) family proteins play critical roles in multiple aspects of development. SRY, as a founder member of SOX family, has been long believed to be involved in the development of sexual gonads by triggering signaling cascades which lead to the formation of testis or ovary from bipotential gonads. However, less is known about other potential regulatory roles of SRY in the development and differentiation. In order to gain further insight into the possible roles of SRY during development, we looked into possible SRY-regulated genes and their levels of expression in a human embryonic carcinoma cell line, named NTera2, before and after induction of differentiation. For this respect, SRY incorporation on the regulatory regions of two groups of genes including OCT4, NANOG, and SOX2 as pluripotency marker genes, and NESTIN and PAX6 as differentiation marker genes were evaluated quantitatively. Chromatin immunoprecipitation using SRY antibody was performed on chromatin extract of a human embryonic carcinoma cell line, NT2/NTERA-2, before and after onset of differentiation. The results showed that incorporation of SRY in both groups of genes was increased after induction of differentiation. Besides, lower expression of OCT4, SOX2, and NANOG and higher expression of PAX6 and NESTIN genes in differentiated cells suggest that SRY may act as a transcription repressor for pluripotency-associated genes and as a transcription activator for differentiation-related genes.

  2. Ethanol Alters the Balance of Sox2, Oct4, and Nanog Expression in Distinct Subpopulations During Differentiation of Embryonic Stem Cells

    PubMed Central

    Ogony, Joshua W.; Malahias, Evangelia

    2013-01-01

    The transcription factors Sox2, Oct4, and Nanog regulate within a narrow dose-range embryonic stem (ES) cell pluripotency and cell lineage commitment. Excess of Oct4 relative to Sox2 guides cells to mesoendoderm (ME), while abundance of Sox2 promotes neuroectoderm (NE) formation. Literature does not address whether ethanol interferes with these regulatory interactions during neural development. We hypothesized that ethanol exposure of ES cells in early differentiation causes an imbalance of Oct4 and Sox2 that diverts cells away from NE to ME lineage, consistent with the teratogenesis effects caused by prenatal alcohol exposure. Mouse ES cells were exposed to ethanol (0, 25, 50, and 100 mM) during retinoic acid (10 nM)-directed differentiation to NE for 0–6 days, and the expression of Sox2, Oct4, and Nanog was measured in single live cells by multiparametric flow cytometry, and the cellular phenotype was characterized by immunocytochemistry. Our data showed an ethanol dose- and time-dependent asymmetric modulation of Oct4 and Sox2 expression, as early as after 2 days of exposure. Single-cell analysis of the correlated expression of Sox2, Oct4, and Nanog revealed that ethanol promoted distinct subpopulations with a high Oct4/Sox2 ratio. Ethanol-exposed cells differentiated to fewer β-III tubulin-immunoreactive cells with an immature neuronal phenotype by 4 days. We interpret these data as suggesting that ethanol diverted cells in early differentiation from the NE fate toward the ME lineage. Our results provide a novel insight into the mode of ethanol action and opportunities for discovery of prenatal biomarkers at early stages. PMID:23470161

  3. Single-cell duplex RT-LATE-PCR reveals Oct4 and Xist RNA gradients in 8-cell embryos

    PubMed Central

    Hartshorn, Cristina; Eckert, Judith J; Hartung, Odelya; Wangh, Lawrence J

    2007-01-01

    Background The formation of two distinctive cell lineages in preimplantation mouse embryos is characterized by differential gene expression. The cells of the inner cell mass are pluripotent and express high levels of Oct4 mRNA, which is down-regulated in the surrounding trophectoderm. In contrast, the trophectoderm of female embryos contains Xist mRNA, which is absent from cells of the inner mass. Prior to blastocyst formation, all blastomeres of female embryos still express both of these RNAs. We, thus, postulated that simultaneous quantification of Oct4 and Xist transcripts in individual blastomeres at the 8-cell stage could be informative as to their subsequent fate. Testing this hypothesis, however, presented numerous technical challenges. We overcame these difficulties by combining PurAmp, a single-tube method for RNA preparation and quantification, with LATE-PCR, an advanced form of asymmetric PCR. Results We constructed a duplex RT-LATE-PCR assay for real-time measurement of Oct4 and Xist templates and confirmed its specificity and quantitative accuracy with different methods. We then undertook analysis of sets of blastomeres isolated from embryos at the 8-cell stage. At this stage, all cells in the embryo are still pluripotent and morphologically equivalent. Our results demonstrate, however, that both Oct4 and Xist RNA levels vary in individual blastomeres comprising the same embryo, with some cells having particularly elevated levels of either transcript. Analysis of multiple embryos also shows that Xist and Oct4 expression levels are not correlated at the 8-cell stage, although transcription of both genes is up-regulated at this time in development. In addition, comparison of data from males and females allowed us to determine that the efficiency of the Oct4/Xist assay is unaffected by sex-related differences in gene expression. Conclusion This paper describes the first example of multiplex RT-LATE-PCR and its utility, when combined with PurAmp sample

  4. ASSEMBLING NEURAL CREST REGULATORY CIRCUITS INTO A GENE REGULATORY NETWORK

    PubMed Central

    Betancur, Paola; Bronner-Fraser, Marianne; Sauka-Spengler, Tatjana

    2014-01-01

    The neural crest is a multipotent stem cell--like population that gives rise to a wide range of derivatives in vertebrate embryo including elements of the craniofacial skeleton and peripheral nervous system as well as melanocytes. The neural crest forms in a series of regulatory steps that include induction and specification of the prospective neural crest territory--neural plate border, specification of bona fide neural crest progenitors, and differentiation into diverse derivatives. These individual processes during neural crest ontogeny are controlled by regulatory circuits that can be assembled into a hierarchical gene regulatory network (GRN). Here we present an overview of the GRN that orchestrates the formation of cranial neural crest cells. Formulation of this network relies on information largely inferred from gene perturbation studies performed in several vertebrate model organisms. Our representation of the cranial neural crest GRN also includes information about direct regulatory interactions obtained from the cis-regulatory analyses performed to date, which increases the resolution of the architectural circuitry within the network. PMID:19575671

  5. Cobalt and Nickel Stabilize Stem Cell Transcription Factor OCT4 through Modulating Its Sumoylation and Ubiquitination

    PubMed Central

    Yao, Yixin; Lu, Yinghua; Chen, Wen-chi; Jiang, Yongping; Cheng, Tao; Ma, Yupo; Lu, Lou; Dai, Wei

    2014-01-01

    Stem cell research can lead to the development of treatments for a wide range of ailments including diabetes, heart disease, aging, neurodegenerative diseases, spinal cord injury, and cancer. OCT4 is a master regulator of self-renewal of undifferentiated embryonic stem cells. OCT4 also plays a crucial role in reprogramming of somatic cells into induced pluripotent stem (iPS) cells. Given known vivo reproductive toxicity of cobalt and nickel metals, we examined the effect of these metals on expression of several stem cell factors in embryonic Tera-1 cells, as well as stem cells. Cobalt and nickel induced a concentration-dependent increase of OCT4 and HIF-1α, but not NANOG or KLF4. OCT4 induced by cobalt and nickel was due primarily to protein stabilization because MG132 stabilized OCT4 in cells treated with either metals and because neither nickel nor cobalt significantly modulated its steady-state mRNA level. OCT4 stabilization by cobalt and nickel was mediated largely through reactive oxygen species (ROS) as co-treatment with ascorbic acid abolished OCT4 increase. Moreover, nickel and cobalt treatment increased sumoylation and mono-ubiquitination of OCT4 and K123 was crucial for mediating these modifications. Combined, our observations suggest that nickel and cobalt may exert their reproductive toxicity through perturbing OCT4 activity in the stem cell compartment. PMID:24497960

  6. Oct4 Is Required ∼E7.5 for Proliferation in the Primitive Streak

    PubMed Central

    DeVeale, Brian; Brokhman, Irina; Mohseni, Paria; Babak, Tomas; Yoon, Charles; Lin, Anthony; Onishi, Kento; Tomilin, Alexey; Pevny, Larysa; Zandstra, Peter W.; Nagy, Andras; van der Kooy, Derek

    2013-01-01

    Oct4 is a widely recognized pluripotency factor as it maintains Embryonic Stem (ES) cells in a pluripotent state, and, in vivo, prevents the inner cell mass (ICM) in murine embryos from differentiating into trophectoderm. However, its function in somatic tissue after this developmental stage is not well characterized. Using a tamoxifen-inducible Cre recombinase and floxed alleles of Oct4, we investigated the effect of depleting Oct4 in mouse embryos between the pre-streak and headfold stages, ∼E6.0–E8.0, when Oct4 is found in dynamic patterns throughout the embryonic compartment of the mouse egg cylinder. We found that depletion of Oct4 ∼E7.5 resulted in a severe phenotype, comprised of craniorachischisis, random heart tube orientation, failed turning, defective somitogenesis and posterior truncation. Unlike in ES cells, depletion of the pluripotency factors Sox2 and Oct4 after E7.0 does not phenocopy, suggesting that ∼E7.5 Oct4 is required within a network that is altered relative to the pluripotency network. Oct4 is not required in extraembryonic tissue for these processes, but is required to maintain cell viability in the embryo and normal proliferation within the primitive streak. Impaired expansion of the primitive streak occurs coincident with Oct4 depletion ∼E7.5 and precedes deficient convergent extension which contributes to several aspects of the phenotype. PMID:24244203

  7. Structural basis for the SOX-dependent genomic redistribution of OCT4 in stem cell differentiation.

    PubMed

    Merino, Felipe; Ng, Calista Keow Leng; Veerapandian, Veeramohan; Schöler, Hans Robert; Jauch, Ralf; Cojocaru, Vlad

    2014-09-02

    In pluripotent cells, OCT4 associates with SOX2 to maintain pluripotency or with SOX17 to induce primitive endoderm commitment. The OCT4-SOX2 and OCT4-SOX17 combinations bind mutually exclusive to two distinct composite DNA elements, known as the "canonical" and "compressed" motifs, respectively. The structural basis for the OCT4-SOX17 cooperativity is unknown. Whereas SOX17 has been engineered to replace SOX2 in the pluripotency circuitry, all generated SOX2 mutants have failed to act like SOX17. From molecular simulations, we revealed the OCT4-SOX17 interaction interface and elucidated the SOX-dependent motif preference of OCT4. Moreover, we designed a SOX2 mutant that we predicted and confirmed experimentally to bind cooperatively with OCT4 to the compressed motif. Ultimately, we found a strong correlation between the experimental and calculated relative cooperative-binding free energies of 12 OCT4-SOX-DNA complexes. Therefore, we validated the OCT4-SOX interfaces and demonstrated that in silico design of DNA-binding cooperativity is suitable for altering transcriptional circuitries. Copyright © 2014 Elsevier Ltd. All rights reserved.

  8. Targeting Tumor Oct4 to Deplete Prostate Tumor and Metastasis Initiating Cells

    DTIC Science & Technology

    2016-10-01

    Award Number: W81XWH-13-1-0461 TITLE: Targeting Tumor Oct4 to Deplete Prostate Tumor- and Metastasis-Initiating Cells PRINCIPAL INVESTIGATOR: Daotai...29 2016 4. TITLE AND SUBTILE Targeting Tumor Oct4 to Deplete Prostate Tumor- and Metastasis-Initiating Cells 5a. CONTRACT NUMBER 5b. GRANT NUMBER...the c-MYC oncogene. POU5F1B is a pseudogene of embryonic Oct4 (POU5F1). A recent study found that tumor Oct4 found in prostate cancer cells is due

  9. Plant nitrogen regulatory P-PII genes

    DOEpatents

    Coruzzi, Gloria M.; Lam, Hon-Ming; Hsieh, Ming-Hsiun

    2001-01-01

    The present invention generally relates to plant nitrogen regulatory PII gene (hereinafter P-PII gene), a gene involved in regulating plant nitrogen metabolism. The invention provides P-PII nucleotide sequences, expression constructs comprising said nucleotide sequences, and host cells and plants having said constructs and, optionally expressing the P-PII gene from said constructs. The invention also provides substantially pure P-PII proteins. The P-PII nucleotide sequences and constructs of the

  10. A genome-scale RNAi screen for Oct4 modulators defines a role of the Paf1 complex for embryonic stem cell identity.

    PubMed

    Ding, Li; Paszkowski-Rogacz, Maciej; Nitzsche, Anja; Slabicki, Mikolaj Michal; Heninger, Anne-Kristin; de Vries, Ingrid; Kittler, Ralf; Junqueira, Magno; Shevchenko, Andrej; Schulz, Herbert; Hubner, Norbert; Doss, Michael Xavier; Sachinidis, Agapios; Hescheler, Juergen; Iacone, Roberto; Anastassiadis, Konstantinos; Stewart, A Francis; Pisabarro, M Teresa; Caldarelli, Antonio; Poser, Ina; Theis, Mirko; Buchholz, Frank

    2009-05-08

    Pluripotent embryonic stem cells (ESCs) maintain self-renewal while ensuring a rapid response to differentiation cues. The identification of genes maintaining ESC identity is important to develop these cells for their potential therapeutic use. Here we report a genome-scale RNAi screen for a global survey of genes affecting ESC identity via alteration of Oct4 expression. Factors with the strongest effect on Oct4 expression included components of the Paf1 complex, a protein complex associated with RNA polymerase II. Using a combination of proteomics, expression profiling, and chromatin immunoprecipitation, we demonstrate that the Paf1C binds to promoters of key pluripotency genes, where it is required to maintain a transcriptionally active chromatin structure. The Paf1C is developmentally regulated and blocks ESC differentiation upon overexpression, and the knockdown in ESCs causes expression changes similar to Oct4 or Nanog depletions. We propose that the Paf1C plays an important role in maintaining ESC identity.

  11. Expression and Prognostic Value of Oct-4 in Astrocytic Brain Tumors

    PubMed Central

    Dahl Sørensen, Mia; Winther Kristensen, Bjarne

    2016-01-01

    Background Glioblastomas are the most frequent type of malignant primary brain tumor with a median overall survival less than 15 months. Therapy resistance of glioblastomas has been attributed to the presence of tumor initiating stem-like cells (TSCs). TSC-related markers have therefore been suggested to have promising potentials as prognostic markers in gliomas. Methodology/Principal Findings The aim of the present study was to investigate the expression and prognostic impact of the TSC-related marker Oct-4 in astrocytic brain tumors of increasing grade. In total 114 grade II, III and IV astrocytic brain tumors were immunohistochemically stained for Oct-4, and the fraction and intensity of Oct-4 positive cells were determined by morphometric analysis of full tumor sections. Oct-4 was expressed in all tumors, and the Oct-4 positive cell fraction increased with tumor grade (p = 0.045). There was no association between survival and Oct-4 positive cell fraction, neither when combining all tumor grades nor in analysis of individual grades. Oct-4 intensity was not associated with grade, but taking IDH1 status into account we found a tendency for high Oct-4 intensity to be associated with poor prognosis in anaplastic astrocytomas. Double immunofluorescence stainings showed co-localization in the perivascular niches of Oct-4 and two other TSC markers CD133 and nestin in glioblastomas. In some areas Oct-4 was expressed independently of CD133 and nestin. Conclusions In conclusion, high Oct-4 fraction was associated with tumor malignancy, but seemed to be without independent prognostic influence in glioblastomas. Identification of a potential prognostic value in anaplastic astrocytomas requires additional studies using larger patient cohorts. PMID:28030635

  12. Modeling gene regulatory network motifs using statecharts

    PubMed Central

    2012-01-01

    Background Gene regulatory networks are widely used by biologists to describe the interactions among genes, proteins and other components at the intra-cellular level. Recently, a great effort has been devoted to give gene regulatory networks a formal semantics based on existing computational frameworks. For this purpose, we consider Statecharts, which are a modular, hierarchical and executable formal model widely used to represent software systems. We use Statecharts for modeling small and recurring patterns of interactions in gene regulatory networks, called motifs. Results We present an improved method for modeling gene regulatory network motifs using Statecharts and we describe the successful modeling of several motifs, including those which could not be modeled or whose models could not be distinguished using the method of a previous proposal. We model motifs in an easy and intuitive way by taking advantage of the visual features of Statecharts. Our modeling approach is able to simulate some interesting temporal properties of gene regulatory network motifs: the delay in the activation and the deactivation of the "output" gene in the coherent type-1 feedforward loop, the pulse in the incoherent type-1 feedforward loop, the bistability nature of double positive and double negative feedback loops, the oscillatory behavior of the negative feedback loop, and the "lock-in" effect of positive autoregulation. Conclusions We present a Statecharts-based approach for the modeling of gene regulatory network motifs in biological systems. The basic motifs used to build more complex networks (that is, simple regulation, reciprocal regulation, feedback loop, feedforward loop, and autoregulation) can be faithfully described and their temporal dynamics can be analyzed. PMID:22536967

  13. Activation of IL6/IGFIR confers poor prognosis of HBV-related hepatocellular carcinoma through induction of OCT4/NANOG expression.

    PubMed

    Chang, Te-Sheng; Wu, Yu-Chih; Chi, Ching-Chi; Su, Wei-Chi; Chang, Pey-Jium; Lee, Kam-Fai; Tung, Tao-Hsin; Wang, Jui; Liu, Jun-Jen; Tung, Shui-Yi; Kuo, Liang-Mou; Ho, Hong-Nerng; Ling, Thai-Yen; Huang, Yen-Hua

    2015-01-01

    To unravel the role of interleukin (IL)-6 and insulin-like growth factor (IGF)-I receptor (IGFIR) in expressing stemness-related properties and to evaluate the prognostic values of pluripotent transcription factor OCT4/NANOG, and IGFIR in hepatocellular carcinoma (HCC). Serum levels of IL6 were detected using ELISA assays (n = 120). The effects of IL6/IGFI on stemness expression in HCC were examined using OCT4/NANOG promoter luciferase reporter, RNA interference, secondary sphere formation, side population, and xenograft animal models. The OCT4/NANOG protein and phospho-IGFI receptor (p-IGFIR) in tissues were detected by Western blotting (n = 8) and immunohistochemical staining (n = 85). OCT4, NANOG, and IGFIR expression levels in tissues (n = 191) were analyzed by real-time qRT-PCR and was correlated with early tumor recurrence using the Kaplan-Meier survival analysis. A high positive correlation between the expression levels of OCT4/NANOG and IGFIR/p-IGFIR in human HCC tissues was observed. The concurrent expression of OCT4/NANOG/IGFIR was mostly confined to hepatitis B virus (HBV)-related HCC (HBV-HCC) and was significantly correlated with early tumor recurrence. High serum levels of IL6 were significantly correlated with high OCT4/NANOG expression. IL6 stimulated an autocrine IGFI/IGFIR expression STAT3 dependently, which stimulated stemness-related properties in both the cell lines and the xenografted mouse tumors. The inhibition of IGFIR activation by either RNA interference or by treatment with the inhibitor picropodophyllin (PPP) significantly suppressed the IL6-induced stemness-related properties both in vitro and in vivo. The expression of pluripotency-related genes is associated with early tumor recurrence and is regulated by IL6-induced IGF/IGFIR activation, particularly in HBV-HCC. ©2014 American Association for Cancer Research.

  14. Regulatory mechanisms for floral homeotic gene expression.

    PubMed

    Liu, Zhongchi; Mara, Chloe

    2010-02-01

    Proper regulation of floral homeotic gene (or ABCE gene) expression ensures the development of floral organs in the correct number, type, and precise spatial arrangement. This review summarizes recent advances on the regulation of floral homeotic genes, highlighting the variety and the complexity of the regulatory mechanisms involved. As flower development is one of the most well characterized developmental processes in higher plants, it facilitates the discovery of novel regulatory mechanisms. To date, mechanisms for the regulation of floral homeotic genes range from transcription to post-transcription, from activators to repressors, and from microRNA- to ubiquitin-mediated post-transcriptional regulation. Region-specific activation of floral homeotic genes is dependent on the integration of a flower-specific activity provided by LEAFY (LFY) and a region- and stage-specific activating function provided by one of the LFY cofactors. Two types of regulatory loops, the feed-forward and the feedback loop, provide properly timed gene activation and subsequent maintenance and refinement in proper spatial and temporal domains of ABCE genes. Two different microRNA/target modules may have been independently recruited in different plant species to regulate C gene expression. Additionally, competition among different MADS box proteins for common interacting partners may represent a mechanism in whorl boundary demarcation. Future work using systems approaches and the development of non-model plants will provide integrated views on floral homeotic gene regulation and insights into the evolution of morphological diversity in flowering plants. Copyright 2009 Elsevier Ltd. All rights reserved.

  15. HIF-2α and Oct4 have synergistic effects on survival and myocardial repair of very small embryonic-like mesenchymal stem cells in infarcted hearts

    PubMed Central

    Zhang, Shaoheng; Zhao, Lan; Wang, Jiahong; Chen, Nannan; Yan, Jian; Pan, Xin

    2017-01-01

    Poor cell survival and limited functional benefits have restricted mesenchymal stem cell (MSC) efficacy for treating myocardial infarction (MI), suggesting that a better understanding of stem cell biology is needed. The transcription factor HIF-2α is an essential regulator of the transcriptional response to hypoxia, which can interact with embryonic stem cells (ESCs) transcription factor Oct4 and modulate its signaling. Here, we obtained very small embryonic-like mesenchymal stem cells (vselMSCs) from MI patients, which possessed the very small embryonic-like stem cells' (VSELs) morphology as well as ESCs' pluripotency. Using microarray analysis, we compared HIF-2α-regulated gene profiles in vselMSCs with ESC profiles and determined that HIF-2α coexpressed Oct4 in vselMSCs similarly to ESCs. However, this coexpression was absent in unpurified MSCs (uMSCs). Under hypoxic condition, vselMSCs exhibited stronger survival, proliferation and differentiation than uMSCs. Transplantation of vselMSCs caused greater improvement in cardiac function and heart remodeling in the infarcted rats. We further demonstrated that HIF-2α and Oct4 jointly regulate their relative downstream gene expressions, including Bcl2 and Survivin; the important pluripotent markers Nanog, Klf4, and Sox2; and Ang-1, bFGF, and VEGF, promoting angiogenesis and engraftment. Importantly, these effects were generally magnified by upregulation of HIF-2α and Oct4 induced by HIF-2α or Oct4 overexpression, and the greatest improvements were elicited after co-overexpressing HIF-2α and Oct4; overexpressing one transcription factor while silencing the other canceled this increase, and HIF-2α or Oct4 silencing abolished these effects. Together, these findings demonstrated that HIF-2α in vselMSCs cooperated with Oct4 in survival and function. The identification of the cooperation between HIF-2α and Oct4 will lead to deeper characterization of the downstream targets of this interaction in vselMSCs and will

  16. High OCT4A levels drive tumorigenicity and metastatic potential of medulloblastoma cells.

    PubMed

    da Silva, Patrícia Benites Gonçalves; Teixeira Dos Santos, Márcia Cristina; Rodini, Carolina Oliveira; Kaid, Carolini; Pereira, Márcia Cristina Leite; Furukawa, Gabriela; da Cruz, Daniel Sanzio Gimenes; Goldfeder, Mauricio Barbugiani; Rocha, Clarissa Ribeiro Reily; Rosenberg, Carla; Okamoto, Oswaldo Keith

    2017-03-21

    Medulloblastoma is a highly aggressive pediatric brain tumor, in which sporadic expression of the pluripotency factor OCT4 has been recently correlated with poor patient survival. However the contribution of specific OCT4 isoforms to tumor aggressiveness is still poorly understood. Here, we report that medulloblastoma cells stably overexpressing the OCT4A isoform displayed enhanced clonogenic, tumorsphere generation, and invasion capabilities. Moreover, in an orthotopic metastatic model of medulloblastoma, OCT4A overexpressing cells generated more developed, aggressive and infiltrative tumors, with tumor-bearing mice attaining advanced metastatic disease and shorter survival rates. Pro-oncogenic OCT4A effects were expression-level dependent and accompanied by distinct chromosomal aberrations. OCT4A overexpression in medulloblastoma cells also induced a marked differential expression of non-coding RNAs, including poorly characterized long non-coding RNAs and small nucleolar RNAs. Altogether, our findings support the relevance of pluripotency-related factors in the aggravation of medulloblastoma traits classically associated with poor clinical outcome, and underscore the prognostic and therapeutic value of OCT4A in this challenging type of pediatric brain cancer.

  17. High OCT4A levels drive tumorigenicity and metastatic potential of medulloblastoma cells

    PubMed Central

    Gonçalves da Silva, Patrícia Benites; Teixeira dos Santos, Márcia Cristina; Rodini, Carolina Oliveira; Kaid, Carolini; Leite Pereira, Márcia Cristina; Furukawa, Gabriela; Gimenes da Cruz, Daniel Sanzio; Goldfeder, Mauricio Barbugiani; Reily Rocha, Clarissa Ribeiro; Rosenberg, Carla; Okamoto, Oswaldo Keith

    2017-01-01

    Medulloblastoma is a highly aggressive pediatric brain tumor, in which sporadic expression of the pluripotency factor OCT4 has been recently correlated with poor patient survival. However the contribution of specific OCT4 isoforms to tumor aggressiveness is still poorly understood. Here, we report that medulloblastoma cells stably overexpressing the OCT4A isoform displayed enhanced clonogenic, tumorsphere generation, and invasion capabilities. Moreover, in an orthotopic metastatic model of medulloblastoma, OCT4A overexpressing cells generated more developed, aggressive and infiltrative tumors, with tumor-bearing mice attaining advanced metastatic disease and shorter survival rates. Pro-oncogenic OCT4A effects were expression-level dependent and accompanied by distinct chromosomal aberrations. OCT4A overexpression in medulloblastoma cells also induced a marked differential expression of non-coding RNAs, including poorly characterized long non-coding RNAs and small nucleolar RNAs. Altogether, our findings support the relevance of pluripotency-related factors in the aggravation of medulloblastoma traits classically associated with poor clinical outcome, and underscore the prognostic and therapeutic value of OCT4A in this challenging type of pediatric brain cancer. PMID:28186969

  18. Acquisition of pluripotency through continued environmental influence on OCT4-induced plastic human fibroblasts.

    PubMed

    Salci, Kyle R; Lee, Jung Bok; Mitchell, Ryan R; Orlando, Luca; Fiebig-Comyn, Aline; Shapovalova, Zoya; Bhatia, Mickie

    2015-07-01

    The combination of OCT4 expression and short-term exposure to reprogramming media induces a state of transcriptional plasticity in human fibroblasts, capable of responding to changes in the extracellular environment that facilitate direct cell fate conversion toward lineage specific progenitors. Here we reveal that continued exposure of OCT4-induced plastic human fibroblasts to reprogramming media (RM) is sufficient to induce pluripotency. OCT4-derived induced pluripotent stem cell (iPSC(OCT4)) colonies emerged after prolonged culture in RM, and formed independently of lineage specific progenitors. Human iPSC(OCT4) are morphologically indistinguishable from conventionally derived iPSCs and express core proteins involved in maintenance of pluripotency. iPSC(OCT4) display in vivo functional pluripotency as measured by teratoma formation consisting of the three germ layers, and are capable of targeted in vitro differentiation. Our study indicates that acquisition of pluripotency is one of multiple cell fate choices that can be facilitated through environmental stimulation of OCT4-induced plasticity, and suggests the role of other reprogramming factors to induce pluripotency can be substituted by prolonged culture of plastic fibroblasts.

  19. [Clinicopathologic features and expression of OCT4 protein in testicular diffuse large B cell lymphoma].

    PubMed

    Chen, Y P; Zhu, W F; Chen, L F; Lu, J P; He, T M; Fu, W D; Xu, C W; Chen, G

    2017-06-08

    Objective: To evaluate the expression of OCT4 and SALL4 in testicular diffuse large B-cell lymphoma (DLBCL), and the utility of an immunohistochemical (IHC) panel of OCT4, SALL4 and CD20 in the differential diagnosis of DLBCL and GCT of the testis. Methods: Eighteen cases of testicular DLBCL were selected.IHC method was used to detect the protein expression of CD20, CD3, CD5, CD10, bcl-6, MUM1, Ki-67, bcl-2, c-MYC, OCT4 and SALL4. Results: Among the 18 cases, CD20 and PAX5 were strongly and diffusely expressed in all cases, while CD21, CD3, cyclinD1, SALL4, CD117 and PLAP were all negative. CD5, bcl-2 and c-myc were expressed in 3, 16 and 8 cases, respectively. Ki-67 proliferation index ranged from 40%-95%. Bcl-2 and c-MYC were co-expressed in seven cases. Four cases were GCB-DLBCL and the remaining 14 cases were non-GCB-DLBCL, according to Hans algorithm. Nuclear OCT4 expression was present in two cases, which demonstrated moderate expression in >50% of neoplastic cells. Univariate analysis showed that clinical stage, CD5 and OCT4 expression were relevant to prognosis. Multivariate Cox regression analysis further confirmed that clinical stage, CD5 and OCT4 were independent prognostic factors in patients with testicular DLBCL. Conclusions: Care should be exercised in using OCT4 as the sole marker of germ cell differentiation in the testis. The association of OCT4 and CD5, bcl-2 co-expression raises the question of whether OCT4 expression in DLBCL may reflect more aggressive biology.

  20. Overexpression of OCT4 is associated with gefitinib resistance in non-small cell lung cancer

    PubMed Central

    Li, Bin; Yao, Zhouhong; Wan, Yunyan; Lin, Dianjie

    2016-01-01

    Epidermal growth factor receptor (EGFR)-targeted tyrosine kinase inhibitors (TKIs) have emerged as first-line drugs for non-small cell lung cancers (NSCLCs). However, the resistance to TKIs represents the key limitation for their therapeutic efficacy. We found that the difference of OCT4 expression between NSCLC and the adjacent non-tumourous tissues was statistically significant. Knockdown of OCT4 in NSCLC cells could decrease cell proliferation, and potentiate apoptosis induced by gefitinib, suggesting OCT4 may contribute to gefitinib resistance in NSCLC. PMID:27816965

  1. Latent phenotypes pervade gene regulatory circuits

    PubMed Central

    2014-01-01

    Background Latent phenotypes are non-adaptive byproducts of adaptive phenotypes. They exist in biological systems as different as promiscuous enzymes and genome-scale metabolic reaction networks, and can give rise to evolutionary adaptations and innovations. We know little about their prevalence in the gene expression phenotypes of regulatory circuits, important sources of evolutionary innovations. Results Here, we study a space of more than sixteen million three-gene model regulatory circuits, where each circuit is represented by a genotype, and has one or more functions embodied in one or more gene expression phenotypes. We find that the majority of circuits with single functions have latent expression phenotypes. Moreover, the set of circuits with a given spectrum of functions has a repertoire of latent phenotypes that is much larger than that of any one circuit. Most of this latent repertoire can be easily accessed through a series of small genetic changes that preserve a circuit’s main functions. Both circuits and gene expression phenotypes that are robust to genetic change are associated with a greater number of latent phenotypes. Conclusions Our observations suggest that latent phenotypes are pervasive in regulatory circuits, and may thus be an important source of evolutionary adaptations and innovations involving gene regulation. PMID:24884746

  2. Latent phenotypes pervade gene regulatory circuits.

    PubMed

    Payne, Joshua L; Wagner, Andreas

    2014-05-30

    Latent phenotypes are non-adaptive byproducts of adaptive phenotypes. They exist in biological systems as different as promiscuous enzymes and genome-scale metabolic reaction networks, and can give rise to evolutionary adaptations and innovations. We know little about their prevalence in the gene expression phenotypes of regulatory circuits, important sources of evolutionary innovations. Here, we study a space of more than sixteen million three-gene model regulatory circuits, where each circuit is represented by a genotype, and has one or more functions embodied in one or more gene expression phenotypes. We find that the majority of circuits with single functions have latent expression phenotypes. Moreover, the set of circuits with a given spectrum of functions has a repertoire of latent phenotypes that is much larger than that of any one circuit. Most of this latent repertoire can be easily accessed through a series of small genetic changes that preserve a circuit's main functions. Both circuits and gene expression phenotypes that are robust to genetic change are associated with a greater number of latent phenotypes. Our observations suggest that latent phenotypes are pervasive in regulatory circuits, and may thus be an important source of evolutionary adaptations and innovations involving gene regulation.

  3. Phenotypic switching in gene regulatory networks.

    PubMed

    Thomas, Philipp; Popović, Nikola; Grima, Ramon

    2014-05-13

    Noise in gene expression can lead to reversible phenotypic switching. Several experimental studies have shown that the abundance distributions of proteins in a population of isogenic cells may display multiple distinct maxima. Each of these maxima may be associated with a subpopulation of a particular phenotype, the quantification of which is important for understanding cellular decision-making. Here, we devise a methodology which allows us to quantify multimodal gene expression distributions and single-cell power spectra in gene regulatory networks. Extending the commonly used linear noise approximation, we rigorously show that, in the limit of slow promoter dynamics, these distributions can be systematically approximated as a mixture of Gaussian components in a wide class of networks. The resulting closed-form approximation provides a practical tool for studying complex nonlinear gene regulatory networks that have thus far been amenable only to stochastic simulation. We demonstrate the applicability of our approach in a number of genetic networks, uncovering previously unidentified dynamical characteristics associated with phenotypic switching. Specifically, we elucidate how the interplay of transcriptional and translational regulation can be exploited to control the multimodality of gene expression distributions in two-promoter networks. We demonstrate how phenotypic switching leads to birhythmical expression in a genetic oscillator, and to hysteresis in phenotypic induction, thus highlighting the ability of regulatory networks to retain memory.

  4. Do all roads lead to Oct4? The emerging concepts of induced pluripotency

    PubMed Central

    Radzisheuskaya, Aliaksandra; Silva, José C.R.

    2014-01-01

    Pluripotent cells have the potential to differentiate into all of the cell types of an animal. This unique cell state is governed by an interconnected network of transcription factors. Among these, Oct4 plays an essential role both in the development of pluripotent cells in the embryo and in the self-renewal of its in vitro counterpart, embryonic stem (ES) cells. Furthermore, Oct4 is one of the four Yamanaka factors and its overexpression alone can generate induced pluripotent stem (iPS) cells. Recent reports underscore Oct4 as an essential regulator of opposing cell state transitions, such as pluripotency establishment and differentiation into embryonic germ lineages. Here we discuss these recent studies and the potential mechanisms underlying these contrasting functions of Oct4. PMID:24370212

  5. Novel AKT phosphorylation sites identified in the pluripotency factors OCT4, SOX2 and KLF4.

    PubMed

    Malak, Peter N; Dannenmann, Benjamin; Hirth, Alexander; Rothfuss, Oliver C; Schulze-Osthoff, Klaus

    2015-01-01

    The four OSKM factors OCT4, SOX2, KLF4 and c-MYC are key transcription factors modulating pluripotency, self-renewal and tumorigenesis in stem cells. However, although their transcriptional targets have been extensively studied, little is known about how these factors are regulated at the posttranslational level. In this study, we established an in vitro system to identify phosphorylation patterns of the OSKM factors by AKT kinase. OCT4, SOX2, KLF4 and c-MYC were expressed in Sf9 insect cells employing the baculoviral expression system. OCT4, SOX2 and KLF4 were localized in the nucleus of insect cells, allowing their easy purification to near homogeneity upon nuclear fractionation. All transcription factors were isolated as biologically active DNA-binding proteins. Using in vitro phosphorylation and mass spectrometry-based phosphoproteome analyses several novel and known AKT phosphorylation sites could be identified in OCT4, SOX2 and KLF4.

  6. A nontranscriptional role for Oct4 in the regulation of mitotic entry

    PubMed Central

    Zhao, Rui; Deibler, Richard W.; Lerou, Paul H.; Ballabeni, Andrea; Heffner, Garrett C.; Cahan, Patrick; Unternaehrer, Juli J.; Kirschner, Marc W.; Daley, George Q.

    2014-01-01

    Rapid progression through the cell cycle and a very short G1 phase are defining characteristics of embryonic stem cells. This distinct cell cycle is driven by a positive feedback loop involving Rb inactivation and reduced oscillations of cyclins and cyclin-dependent kinase (Cdk) activity. In this setting, we inquired how ES cells avoid the potentially deleterious consequences of premature mitotic entry. We found that the pluripotency transcription factor Oct4 (octamer-binding transcription factor 4) plays an unappreciated role in the ES cell cycle by forming a complex with cyclin–Cdk1 and inhibiting Cdk1 activation. Ectopic expression of Oct4 or a mutant lacking transcriptional activity recapitulated delayed mitotic entry in HeLa cells. Reduction of Oct4 levels in ES cells accelerated G2 progression, which led to increased chromosomal missegregation and apoptosis. Our data demonstrate an unexpected nontranscriptional function of Oct4 in the regulation of mitotic entry. PMID:25324523

  7. Therapeutic gene editing: delivery and regulatory perspectives

    PubMed Central

    Shim, Gayong; Kim, Dongyoon; Park, Gyu Thae; Jin, Hyerim; Suh, Soo-Kyung; Oh, Yu-Kyoung

    2017-01-01

    Gene-editing technology is an emerging therapeutic modality for manipulating the eukaryotic genome by using target-sequence-specific engineered nucleases. Because of the exceptional advantages that gene-editing technology offers in facilitating the accurate correction of sequences in a genome, gene editing-based therapy is being aggressively developed as a next-generation therapeutic approach to treat a wide range of diseases. However, strategies for precise engineering and delivery of gene-editing nucleases, including zinc finger nucleases, transcription activator-like effector nuclease, and CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats-associated nuclease Cas9), present major obstacles to the development of gene-editing therapies, as with other gene-targeting therapeutics. Currently, viral and non-viral vectors are being studied for the delivery of these nucleases into cells in the form of DNA, mRNA, or proteins. Clinical trials are already ongoing, and in vivo studies are actively investigating the applicability of CRISPR/Cas9 techniques. However, the concept of correcting the genome poses major concerns from a regulatory perspective, especially in terms of safety. This review addresses current research trends and delivery strategies for gene editing-based therapeutics in non-clinical and clinical settings and considers the associated regulatory issues. PMID:28392568

  8. Therapeutic gene editing: delivery and regulatory perspectives.

    PubMed

    Shim, Gayong; Kim, Dongyoon; Park, Gyu Thae; Jin, Hyerim; Suh, Soo-Kyung; Oh, Yu-Kyoung

    2017-04-10

    Gene-editing technology is an emerging therapeutic modality for manipulating the eukaryotic genome by using target-sequence-specific engineered nucleases. Because of the exceptional advantages that gene-editing technology offers in facilitating the accurate correction of sequences in a genome, gene editing-based therapy is being aggressively developed as a next-generation therapeutic approach to treat a wide range of diseases. However, strategies for precise engineering and delivery of gene-editing nucleases, including zinc finger nucleases, transcription activator-like effector nuclease, and CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats-associated nuclease Cas9), present major obstacles to the development of gene-editing therapies, as with other gene-targeting therapeutics. Currently, viral and non-viral vectors are being studied for the delivery of these nucleases into cells in the form of DNA, mRNA, or proteins. Clinical trials are already ongoing, and in vivo studies are actively investigating the applicability of CRISPR/Cas9 techniques. However, the concept of correcting the genome poses major concerns from a regulatory perspective, especially in terms of safety. This review addresses current research trends and delivery strategies for gene editing-based therapeutics in non-clinical and clinical settings and considers the associated regulatory issues.

  9. Targeting Tumor Oct4 to Deplete Prostate Tumor- and Metastasis-Initiating Cells

    DTIC Science & Technology

    2015-10-01

    We hope we can validate whether tumor Oct4 can be targeted to inhibit prostate cancer progression and metastasis. In addition, we will map out the...Introduction Background: Genome -wide association studies (GWAS) have linked human chromosome 8q24.21 region with increased risk for prostatic carcinoma...al., 2013). To map the sequences or residues which are critical for the different function between POU5F1B and OCT4 would provide more clues for

  10. Identification of key player genes in gene regulatory networks.

    PubMed

    Nazarieh, Maryam; Wiese, Andreas; Will, Thorsten; Hamed, Mohamed; Helms, Volkhard

    2016-09-06

    Identifying the gene regulatory networks governing the workings and identity of cells is one of the main challenges in understanding processes such as cellular differentiation, reprogramming or cancerogenesis. One particular challenge is to identify the main drivers and master regulatory genes that control such cell fate transitions. In this work, we reformulate this problem as the optimization problems of computing a Minimum Dominating Set and a Minimum Connected Dominating Set for directed graphs. Both MDS and MCDS are applied to the well-studied gene regulatory networks of the model organisms E. coli and S. cerevisiae and to a pluripotency network for mouse embryonic stem cells. The results show that MCDS can capture most of the known key player genes identified so far in the model organisms. Moreover, this method suggests an additional small set of transcription factors as novel key players for governing the cell-specific gene regulatory network which can also be investigated with regard to diseases. To this aim, we investigated the ability of MCDS to define key drivers in breast cancer. The method identified many known drug targets as members of the MDS and MCDS. This paper proposes a new method to identify key player genes in gene regulatory networks. The Java implementation of the heuristic algorithm explained in this paper is available as a Cytoscape plugin at http://apps.cytoscape.org/apps/mcds . The SageMath programs for solving integer linear programming formulations used in the paper are available at https://github.com/maryamNazarieh/KeyRegulatoryGenes and as supplementary material.

  11. Gene regulatory networks and the underlying biology of developmental toxicity

    EPA Science Inventory

    Embryonic cells are specified by large-scale networks of functionally linked regulatory genes. Knowledge of the relevant gene regulatory networks is essential for understanding phenotypic heterogeneity that emerges from disruption of molecular functions, cellular processes or sig...

  12. Gene regulatory networks and the underlying biology of developmental toxicity

    EPA Science Inventory

    Embryonic cells are specified by large-scale networks of functionally linked regulatory genes. Knowledge of the relevant gene regulatory networks is essential for understanding phenotypic heterogeneity that emerges from disruption of molecular functions, cellular processes or sig...

  13. Dental Pulp Stem Cells Differentiation Reveals New Insights in Oct4A Dynamics

    PubMed Central

    D'Aurizio, Federica; Puppato, Elisa; Pandolfi, Maura; Beltrami, Antonio Paolo; Cesselli, Daniela; Falini, Giuseppe; Beltrami, Carlo Alberto; Curcio, Francesco

    2012-01-01

    Although the role played by the core transcription factor network, which includes c-Myc, Klf4, Nanog, and Oct4, in the maintenance of embryonic stem cell (ES) pluripotency and in the reprogramming of adult cells is well established, its persistence and function in adult stem cells are still debated. To verify its persistence and clarify the role played by these molecules in adult stem cell function, we investigated the expression pattern of embryonic and adult stem cell markers in undifferentiated and fully differentiated dental pulp stem cells (DPSC). A particular attention was devoted to the expression pattern and intracellular localization of the stemness-associated isoform A of Oct4 (Oct4A). Our data demonstrate that: Oct4, Nanog, Klf4 and c-Myc are expressed in adult stem cells and, with the exception of c-Myc, they are significantly down-regulated following differentiation. Cell differentiation was also associated with a significant reduction in the fraction of DPSC expressing the stem cell markers CD10, CD29 and CD117. Moreover, a nuclear to cytoplasm shuttling of Oct4A was identified in differentiated cells, which was associated with Oct4A phosphorylation. The present study would highlight the importance of the post-translational modifications in DPSC stemness maintenance, by which stem cells balance self-renewal versus differentiation. Understanding and controlling these mechanisms may be of great importance for stemness maintenance and stem cells clinical use, as well as for cancer research. PMID:22844522

  14. Activation of the ESC pluripotency factor OCT4 in smooth muscle cells is atheroprotective

    PubMed Central

    Cherepanova, Olga A.; Gomez, Delphine; Shankman, Laura S.; Swiatlowska, Pamela; Williams, Jason; Sarmento, Olga F.; Alencar, Gabriel F.; Hess, Daniel L.; Bevard, Melissa H.; Greene, Elizabeth S.; Murgai, Meera; Turner, Stephen D.; Geng, Yong-Jian; Bekiranov, Stefan; Connelly, Jessica J.; Tomilin, Alexey; Owens, Gary K.

    2016-01-01

    There are controversial claims that the embryonic stem cell (ESC) pluripotency factor OCT4 is activated in somatic cells, but there is no evidence it plays a functional role in these cells. Herein we demonstrate that smooth muscle cell (SMC)-specific conditional knockout of Oct4 within Apoe−/− mice resulted in increased lesion size and changes consistent with decreased plaque stability including a thinner fibrous cap, increased necrotic core, and increased intra-plaque hemorrhage. Results of SMC-lineage tracing studies showed that these changes were likely due to marked reductions in SMC number within lesions including impaired SMC migration and investment within the fibrous cap. Re-activation of Oct4 within SMCs was associated with hydroxymethylation of the Oct4 promoter and was HIF1α- and KLF4-dependent. Results provide the first direct evidence that OCT4 plays a functional role in somatic cells and highlight the importance of further investigation of possible OCT4 functions in normal and diseased somatic cells. PMID:27183216

  15. The serine 106 residue within the N-terminal transactivation domain is crucial for Oct4 function in mice.

    PubMed

    Mitani, Atsushi; Fukuda, Atsushi; Miyashita, Toshiyuki; Umezawa, Akihiro; Akutsu, Hidenori

    2017-03-07

    Pou5f1/Oct4 is a key transcription factor for the induction of pluripotency and totipotency in preimplantation mouse embryos. In mice, loss or gain of function experiments have demonstrated an important role for Oct4 in preimplantation and developmental ability. In this study, using mouse preimplantation embryos as a model for the evaluation of Oct4 function, we constructed Oct4 overexpression embryos with various mutations at the N-terminal transactivation domain. Developmental competency and molecular biological phenotypes depended on the type of mutation. The replacement of serine 106 with alanine resulted in more severe phenotypes similar to that of wild type Oct4, indicating that this alteration using alanine is negligible for Oct4 function. In contrast, we found that Oct4-specific antibodies could not recognize Oct4 protein when this residue was replaced by aspartic acid (Oct4-S106D). Oct4-S106D overexpressing embryos did not show developmental arrest and aberrant chromatin structure. Thus, these results demonstrated that the Ser-106 residue within the N-terminal transactivation domain is crucial for Oct4 function and suggested that this mutation might affect Oct4 protein conformation.

  16. Autonomous Boolean modeling of gene regulatory networks

    NASA Astrophysics Data System (ADS)

    Socolar, Joshua; Sun, Mengyang; Cheng, Xianrui

    2014-03-01

    In cases where the dynamical properties of gene regulatory networks are important, a faithful model must include three key features: a network topology; a functional response of each element to its inputs; and timing information about the transmission of signals across network links. Autonomous Boolean network (ABN) models are efficient representations of these elements and are amenable to analysis. We present an ABN model of the gene regulatory network governing cell fate specification in the early sea urchin embryo, which must generate three bands of distinct tissue types after several cell divisions, beginning from an initial condition with only two distinct cell types. Analysis of the spatial patterning problem and the dynamics of a network constructed from available experimental results reveals that a simple mechanism is at work in this case. Supported by NSF Grant DMS-10-68602

  17. Automated Identification of Core Regulatory Genes in Human Gene Regulatory Networks.

    PubMed

    Narang, Vipin; Ramli, Muhamad Azfar; Singhal, Amit; Kumar, Pavanish; de Libero, Gennaro; Poidinger, Michael; Monterola, Christopher

    2015-01-01

    Human gene regulatory networks (GRN) can be difficult to interpret due to a tangle of edges interconnecting thousands of genes. We constructed a general human GRN from extensive transcription factor and microRNA target data obtained from public databases. In a subnetwork of this GRN that is active during estrogen stimulation of MCF-7 breast cancer cells, we benchmarked automated algorithms for identifying core regulatory genes (transcription factors and microRNAs). Among these algorithms, we identified K-core decomposition, pagerank and betweenness centrality algorithms as the most effective for discovering core regulatory genes in the network evaluated based on previously known roles of these genes in MCF-7 biology as well as in their ability to explain the up or down expression status of up to 70% of the remaining genes. Finally, we validated the use of K-core algorithm for organizing the GRN in an easier to interpret layered hierarchy where more influential regulatory genes percolate towards the inner layers. The integrated human gene and miRNA network and software used in this study are provided as supplementary materials (S1 Data) accompanying this manuscript.

  18. Automated Identification of Core Regulatory Genes in Human Gene Regulatory Networks

    PubMed Central

    Singhal, Amit; Kumar, Pavanish; de Libero, Gennaro; Poidinger, Michael; Monterola, Christopher

    2015-01-01

    Human gene regulatory networks (GRN) can be difficult to interpret due to a tangle of edges interconnecting thousands of genes. We constructed a general human GRN from extensive transcription factor and microRNA target data obtained from public databases. In a subnetwork of this GRN that is active during estrogen stimulation of MCF-7 breast cancer cells, we benchmarked automated algorithms for identifying core regulatory genes (transcription factors and microRNAs). Among these algorithms, we identified K-core decomposition, pagerank and betweenness centrality algorithms as the most effective for discovering core regulatory genes in the network evaluated based on previously known roles of these genes in MCF-7 biology as well as in their ability to explain the up or down expression status of up to 70% of the remaining genes. Finally, we validated the use of K-core algorithm for organizing the GRN in an easier to interpret layered hierarchy where more influential regulatory genes percolate towards the inner layers. The integrated human gene and miRNA network and software used in this study are provided as supplementary materials (S1 Data) accompanying this manuscript. PMID:26393364

  19. Gene regulatory logic of dopaminergic neuron differentiation

    PubMed Central

    Flames, Nuria; Hobert, Oliver

    2009-01-01

    Dopamine signaling regulates a variety of complex behaviors and defects in dopaminergic neuron function or survival result in severe human pathologies, such as Parkinson's disease 1. The common denominator of all dopaminergic neurons is the expression of dopamine pathway genes, which code for a set of phylogenetically conserved proteins involved in dopamine synthesis and transport. Gene regulatory mechanisms that result in the activation of dopamine pathway genes and thereby ultimately determine the identity of dopaminergic neurons are poorly understood in any system studied to date 2. We show here that a simple cis-regulatory element, the DA motif, controls the expression of all dopamine pathway genes in all dopaminergic cell types in C. elegans. The DA motif is activated by the ETS transcription factor, AST-1. Loss of ast-1 results in the failure of all distinct dopaminergic neuronal subtypes to terminally differentiate. Ectopic expression of ast-1 is sufficient to activate the dopamine production pathway in some cellular contexts. Vertebrate dopaminergic pathway genes also contain phylogenetically conserved DA motifs that can be activated by the mouse ETS transcription factor Etv1/ER81 and a specific class of dopaminergic neurons fails to differentiate in mice lacking Etv1/ER81. Moreover, ectopic Etv1/ER81 expression induces dopaminergic fate marker expression in neuronal primary cultures. Mouse Etv1/ER81 can also functionally substitute for ast-1 in C.elegans. Our studies reveal an astoundingly simple and apparently conserved regulatory logic of dopaminergic neuron terminal differentiation and may provide new entry points into the diagnosis or therapy of conditions in which dopamine neurons are defective. PMID:19287374

  20. O-GlcNAc transferase regulates transcriptional activity of human Oct4.

    PubMed

    Constable, Sandii; Lim, Jae-Min; Vaidyanathan, Krithika; Wells, Lance

    2017-10-01

    O-linked β-N-acetylglucosamine (O-GlcNAc) is a single sugar modification found on many different classes of nuclear and cytoplasmic proteins. Addition of this modification, by the enzyme O-linked N-acetylglucosamine transferase (OGT), is dynamic and inducible. One major class of proteins modified by O-GlcNAc is transcription factors. O-GlcNAc regulates transcription factor properties through a variety of different mechanisms including localization, stability and transcriptional activation. Maintenance of embryonic stem (ES) cell pluripotency requires tight regulation of several key transcription factors, many of which are modified by O-GlcNAc. Octamer-binding protein 4 (Oct4) is one of the key transcription factors required for pluripotency of ES cells and more recently, the generation of induced pluripotent stem (iPS) cells. The action of Oct4 is modulated by the addition of several post-translational modifications, including O-GlcNAc. Previous studies in mice found a single site of O-GlcNAc addition responsible for transcriptional regulation. This study was designed to determine if this mechanism is conserved in humans. We mapped 10 novel sites of O-GlcNAc attachment on human Oct4, and confirmed a role for OGT in transcriptional activation of Oct4 at a site distinct from that found in mouse that allows distinction between different Oct4 target promoters. Additionally, we uncovered a potential new role for OGT that does not include its catalytic function. These results confirm that human Oct4 activity is being regulated by OGT by a mechanism that is distinct from mouse Oct4. © The Author 2017. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  1. VIH from the mud crab is specifically expressed in the eyestalk and potentially regulated by transactivator of Sox9/Oct4/Oct1.

    PubMed

    Liu, Chunyun; Jia, Xiwei; Zou, Zhihua; Wang, Xiaowei; Wang, Yilei; Zhang, Ziping

    2017-09-18

    Vitellogenesis-inhibiting hormone (VIH) is known to regulate ovarian maturation by suppressing the synthesis of vitellogenin (Vtg) in crustaceans, which belongs to a member of crustacean hyperglycemic hormone (CHH) family synthesized and secreted from the X-organ/sinus gland complex of eyestalks. In this study, the cDNA, genomic DNA (gDNA) and the 5'-upstream regulatory (promoter region) sequences of VIH gene were obtained by conventional PCR, genome walker and tail-PCR techniques according to our transcriptomic database of Scylla paramamosain. The full-length cDNA of SpVIH is 634 bp including 105 bp 5'UTR, 151 bp 3'UTR and 378 bp ORF that encodes a peptide of 125 amino acids. The full length gDNA of SpVIH is 790 bp containing two exons and one intron. The 5'-flanking promoter regions of SpVIH we isolated are 3070 bp from the translation initiation (ATG) and 2398 bp from the predicted transcription initiation (A), which consists of putative core promoter region and multiple potential transcription factor binding sites. SpVIH was only expressed in eyestalk. The expression level of SpVIH in eyestalk of female crab decreased gradually along with the development of ovary. As there is not cell line of crabs available, we chose the mature transfection system HEK293FT cell lines to explore the mechanism of transcription regulation of SpVIH in crabs. Sequential deletion assays using luciferase reporter gene in HEK293FT cells revealed that the possible promoter activity regions (including positive and negative transcription factors binding sites simultaneously) presented between pSpVIH-4 and pSpVIH-6. In order to further identify the crucial transcription factors binding site in this region, the site-directed mutagenesis of Sox9/Oct4/Oct1 binding site of pSpVIH-4 was created. The results demonstrated that the transcriptional activity of pSpVIH-4△ decreased significantly (p<0.05). Thus, it is reasonable to deduce that the Sox9/Oct4/Oct1 may be the essential positive

  2. Repression of Zeb1 and Hypoxia Cause Sequential MET and Induction of Aid, Oct4, and Dnmt1, Leading to Immortalization and Multipotential Reprogramming of Fibroblasts in Spheres

    PubMed Central

    Liu, Yongqing; Mukhopadhyay, Partha; Pisano, M. Michele; Lu, Xiaoqin; Huang, Li; Lu, Qingxian; Dean, Douglas C.

    2014-01-01

    Here, we demonstrate that sphere formation triggers immortalization and stable reprogramming of mouse fibroblasts. Cell contact signaling in spheres causes downregulation of the EMT transcription factor Zeb1 leading to rapid mesenchymal-to-epithelial transition. And, hypoxia within spheres together with loss of Zeb1 repression synergize to cause superinduction of Hif1a, which in turn leads to induction of the DNA demethylase Aid/Aicda, demethylation of the Oct4 promoter/enhancer and multipotency. Oct4 and Nanog expression diminish when cells are removed from the hypoxic environment of spheres and placed in monolayer culture, but the cells retain multipotential capacity, demonstrating stable reprogramming and a gene expression pattern resembling adult stem cells. Oct4 has been shown to induce Dnmt1 in mesenchymal stem cells, and we link Oct4 and Dnmt1 to silencing of cell cycle inhibitory cyclin dependent kinase inhibitors and Arf, and immortalization of the reprogrammed fibroblasts. Sphere formation then represents a novel and rapid protocol for immortalization and stable reprogramming of fibroblasts to multipotency that does not require exogenous expression of a stem cell factor or a lineage-specifying transcription factor. PMID:23554223

  3. Dual inhibiting OCT4 and AKT potently suppresses the propagation of human cancer cells

    PubMed Central

    Li, Wenxin; Zhou, Yanwen; Zhang, Xiaoqian; Yang, Ying; Dan, Songsong; Su, Tong; She, Shiqi; Dong, Weilai; Zhao, Qingwei; Jia, Jia; Yao, Hangping; Zheng, Min; Kang, Bo; Wang, Ying-Jie

    2017-01-01

    AKT serves as an epigenetic modulator that links epigenetic regulation to cell survival and proliferation while the epigenetic mediator OCT4 critically controls stem cell pluripotency and self-renewal. Emerging evidence indicated their complicated interplays in cancer cells and cancer stem cells (CSCs), and inhibiting either one may activate the other. Thus, in this study, we propose a strategy to targeting both factors simultaneously. Firstly, a combination of an OCT4-specific shRNA and the specific AKT inhibitor Akti-1/2 potently suppressed the propagation of human embryonal carcinoma cells, adherent cancer cells and stem-like cancer cells, establishing the proof-of-concept that dual inhibiting OCT4 and AKT can effectively target various cancer cells. Next, we combined Akti-1/2 with metformin, a widely-prescribed drug for treating type 2 diabetes, which was reported to down-regulate OCT4 expression. The metformin + Akti-1/2 combo significantly altered multiple signaling and epigenetic pathways, induced growth arrest and cell death of adherent and stem-like glioblastoma U87 cells, and attenuated their tumorigenicity in vivo. Taken together, we demonstrate here that simultaneously targeting an epigenetic mediator and an epigenetic modulator, by dual inhibiting OCT4 and AKT, can have significantly improved efficacies over single treatment in suppressing the propagation of CSCs as well as the entire bulk of differentiated cancer cells. PMID:28383051

  4. BMPs functionally replace Klf4 and support efficient reprogramming of mouse fibroblasts by Oct4 alone

    PubMed Central

    Chen, Jiekai; Liu, Jing; Yang, Jiaqi; Chen, You; Chen, Jing; Ni, Su; Song, Hong; Zeng, Lingwen; Ding, Ke; Pei, Duanqing

    2011-01-01

    Generation of induced pluripotent stem cells by defined factors has become a useful model to investigate the mechanism of reprogramming and cell fate determination. However, the precise mechanism of factor-based reprogramming remains unclear. Here, we show that Klf4 mainly acts at the initial phase of reprogramming to initiate mesenchymal-to-epithelial transition and can be functionally replaced by bone morphogenetic proteins (BMPs). BMPs boosted the efficiency of Oct4/Sox2-mediated reprogramming of mouse embryonic fibroblasts (MEFs) to ∼1%. BMPs also promoted single-factor Oct4-based reprogramming of MEFs and tail tibial fibroblasts. Our studies clarify the contribution of Klf4 in reprogramming and establish Oct4 as a singular setter of pluripotency in differentiated cells. PMID:21135873

  5. Coexpression of stemness factors Oct4 and Nanog predict liver resection.

    PubMed

    Yin, Xin; Li, Yi-Wei; Zhang, Bo-Heng; Ren, Zheng-Gang; Qiu, Shuang-Jian; Yi, Yong; Fan, Jia

    2012-09-01

    Oct4 and Nanog are two major transcription factors related to the stem cell self-renewal and differentiation. The aim of this study was to evaluate the correlation between these two stemness markers with recurrence, metastasis, and prognosis of hepatocellular carcinoma (HCC). Expression of Oct4 and Nanog was evaluated by immunohistochemistry in a random cohort of 228 HCC patients (cohort A), predominantly hepatitis B related, and validated in another independent cohort of 95 patients (cohort B). Survival analysis was performed by univariate and multivariate analyses. Oct4 and Nanog expression levels in 5 HCC cell lines with different metastatic potential were detected by Western blot assay and quantitative real-time PCR assay. In tissue microarrays, coexpression of Oct4 and Nanog was dramatically associated with big tumor size (P = .001) and vascular invasion (P = .02) and was an independent predictor of postoperative recurrence (hazard ratio [HR] = 1.57, 95 % confidence interval [95 % CI] 1.21-2.04, P = .01) and poor prognosis (HR = 2.20, 95 % CI 1.71-2.88, P < .001). This association was further validated in patients in cohort B. Importantly, this correlation remained significant in patients with early-stage HCC or alpha-fetoprotein (AFP) negative HCC. In addition, expression of Oct4 and Nanog increased in a concordant manner with the increase of metastatic potential in human HCC cell lines. Coexpression of stemness markers Oct4 and Nanog in HCC indicated the aggressive tumor behaviors and predicted a worse clinical outcome, which may be a useful biomarker to identify patients at high risk of postoperative recurrence.

  6. Granulosa cells and retinoic acid co-treatment enrich potential germ cells from manually selected Oct4-EGFP expressing human embryonic stem cells.

    PubMed

    Chen, Hsin-Fu; Jan, Pey-Shynan; Kuo, Hung-Chih; Wu, Fang-Chun; Lan, Chen-Wei; Huang, Mei-Chi; Chien, Chung-Liang; Ho, Hong-Nerng

    2014-09-01

    Differentiation of human embryonic stem (HES) cells to germ cells may become clinically useful in overcoming diseases related to germ-cell development. Niches were used to differentiate HES cell lines, NTU1 and H9 Oct4-enhanced green fluorescence protein (EGFP), including laminin, granulosa cell co-culture or conditioned medium, ovarian stromal cell co-culture or conditioned medium, retinoic acid, stem cell factor (SCF) and BMP4-BMP7-BMP8b treatment. Flow cytometry showed that granulosa cell co-culture (P < 0.001) or conditioned medium (P = 0.007) treatment for 14 days significantly increased the percentages of differentiated H9 Oct4-EGFP cells expressing early germ cell marker stage-specific embryonic antigen 1(SSEA1); sorted SSEA1[+] cells did not express higher levels of germ cell gene VASA and GDF9. Manually collected H9 Oct4-EGFP[+] cells expressed significantly higher levels of VASA (P = 0.005) and GDF9 (P = 0.001). H9 Oct4-EGFP[+] cells developed to ovarian follicle-like structures after culture for 28 days but with low efficiency. Unlike SCF and BMP4, retinoic acid co-treatment enhanced VASA, GDF9 and SCP3 expression. A protocol is recommended to enrich differentiated HES cells with germ-cell potential by culture with granulosa cells, conditioned medium or retinoic acid, manual selection of Oct4-EGFP[+] cells, and analysis of VASA, GDF9 expression, or both. Copyright © 2014 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

  7. Oct4 Methylation-Mediated Silencing As an Epigenetic Barrier Preventing Müller Glia Dedifferentiation in a Murine Model of Retinal Injury

    PubMed Central

    Reyes-Aguirre, Luis I.; Lamas, Monica

    2016-01-01

    Müller glia (MG) is the most abundant glial type in the vertebrate retina. Among its many functions, it is capable of responding to injury by dedifferentiating, proliferating, and differentiating into every cell types lost to damage. This regenerative ability is notoriously absent in mammals. We have previously reported that cultured mammalian MG undergoes a partial dedifferentiation, but fails to fully acquire a progenitor phenotype and differentiate into neurons. This might be explained by a mnemonic mechanism comprised by epigenetic traits, such as DNA methylation. To achieve a better understanding of this epigenetic memory, we studied the expression of pluripotency-associated genes, such as Oct4, Nanog, and Lin28, which have been reported as necessary for regeneration in fish, at early times after NMDA-induced retinal injury in a mouse experimental model. We found that although Oct4 is expressed rapidly after damage (4 hpi), it is silenced at 24 hpi. This correlates with a significant decrease in the DNA methyltransferase Dnmt3b expression, which returns to basal levels at 24 hpi. By MS-PCR, we observed a decrease in Oct4 methylation levels at 4 and 12 hpi, before returning to a fully methylated state at 24 hpi. To demonstrate that these changes are restricted to MG, we separated these cells using a GLAST antibody coupled with magnetic beads. Finally, intravitreous administration of the DNA-methyltransferase inhibitor SGI-1027 induced Oct4 expression at 24 hpi in MG. Our results suggest that mammalian MG injury-induced dedifferentiation could be restricted by DNA methylation, which rapidly silences Oct4 expression, preventing multipotency acquisition. PMID:27895551

  8. Generation of oscillating gene regulatory network motifs

    NASA Astrophysics Data System (ADS)

    van Dorp, M.; Lannoo, B.; Carlon, E.

    2013-07-01

    Using an improved version of an evolutionary algorithm originally proposed by François and Hakim [Proc. Natl. Acad. Sci. USAPNASA60027-842410.1073/pnas.0304532101 101, 580 (2004)], we generated small gene regulatory networks in which the concentration of a target protein oscillates in time. These networks may serve as candidates for oscillatory modules to be found in larger regulatory networks and protein interaction networks. The algorithm was run for 105 times to produce a large set of oscillating modules, which were systematically classified and analyzed. The robustness of the oscillations against variations of the kinetic rates was also determined, to filter out the least robust cases. Furthermore, we show that the set of evolved networks can serve as a database of models whose behavior can be compared to experimentally observed oscillations. The algorithm found three smallest (core) oscillators in which nonlinearities and number of components are minimal. Two of those are two-gene modules: the mixed feedback loop, already discussed in the literature, and an autorepressed gene coupled with a heterodimer. The third one is a single gene module which is competitively regulated by a monomer and a dimer. The evolutionary algorithm also generated larger oscillating networks, which are in part extensions of the three core modules and in part genuinely new modules. The latter includes oscillators which do not rely on feedback induced by transcription factors, but are purely of post-transcriptional type. Analysis of post-transcriptional mechanisms of oscillation may provide useful information for circadian clock research, as recent experiments showed that circadian rhythms are maintained even in the absence of transcription.

  9. Identifying genes of gene regulatory networks using formal concept analysis.

    PubMed

    Gebert, Jutta; Motameny, Susanne; Faigle, Ulrich; Forst, Christian V; Schrader, Rainer

    2008-03-01

    In order to understand the behavior of a gene regulatory network, it is essential to know the genes that belong to it. Identifying the correct members (e.g., in order to build a model) is a difficult task even for small subnetworks. Usually only few members of a network are known and one needs to guess the missing members based on experience or informed speculation. It is beneficial if one can additionally rely on experimental data to support this guess. In this work we present a new method based on formal concept analysis to detect unknown members of a gene regulatory network from gene expression time series data. We show that formal concept analysis is able to find a list of candidate genes for inclusion into a partially known basic network. This list can then be reduced by a statistical analysis so that the resulting genes interact strongly with the basic network and therefore should be included when modeling the network. The method has been applied to the DNA repair system of Mycobacterium tuberculosis. In this application, our method produces comparable results to an already existing method of component selection while it is applicable to a broader range of problems.

  10. Sox transcription factors require selective interactions with Oct4 and specific transactivation functions to mediate reprogramming.

    PubMed

    Aksoy, Irene; Jauch, Ralf; Eras, Volker; Chng, Wen-Bin Alfred; Chen, Jiaxuan; Divakar, Ushashree; Ng, Calista Keow Leng; Kolatkar, Prasanna R; Stanton, Lawrence W

    2013-12-01

    The unique ability of Sox2 to cooperate with Oct4 at selective binding sites in the genome is critical for reprogramming somatic cells into induced pluripotent stem cells (iPSCs). We have recently demonstrated that Sox17 can be converted into a reprogramming factor by alteration of a single amino acid (Sox17EK) within its DNA binding HMG domain. Here we expanded this study by introducing analogous mutations to 10 other Sox proteins and interrogated the role of N-and C-termini on the reprogramming efficiency. We found that point-mutated Sox7 and Sox17 can convert human and mouse fibroblasts into iPSCs, but Sox4, Sox5, Sox6, Sox8, Sox9, Sox11, Sox12, Sox13, and Sox18 cannot. Next we studied regions outside the HMG domain and found that the C-terminal transactivation domain of Sox17 and Sox7 enhances the potency of Sox2 in iPSC assays and confers weak reprogramming potential to the otherwise inactive Sox4EK and Sox18EK proteins. These results suggest that the glutamate (E) to lysine (K) mutation in the HMG domain is necessary but insufficient to swap the function of Sox factors. Moreover, the HMG domain alone fused to the VP16 transactivation domain is able to induce reprogramming, albeit at low efficiency. By molecular dissection of the C-terminus of Sox17, we found that the β-catenin interaction region contributes to the enhanced reprogramming efficiency of Sox17EK. To mechanistically understand the enhanced reprogramming potential of Sox17EK, we analyzed ChIP-sequencing and expression data and identified a subset of candidate genes specifically regulated by Sox17EK and not by Sox2. © AlphaMed Press.

  11. Folate Receptor Alpha Upregulates Oct4, Sox2 and Klf4 and Downregulates miR-138 and miR-let-7 in Cranial Neural Crest Cells.

    PubMed

    Mohanty, Vineet; Shah, Amar; Allender, Elise; Siddiqui, M Rizwan; Monick, Sarah; Ichi, Shunsuke; Mania-Farnell, Barbara; G McLone, David; Tomita, Tadanori; Mayanil, Chandra Shekhar

    2016-11-01

    Prenatal folic acid (FA) supplementation prevents neural tube defects. Folate receptor alpha (FRα) is critical for embryonic development, including neural crest (NC) development. Previously we showed that FRα translocates to the nucleus in response to FA, where it acts as a transcription factor. In this study, we examined if FA through interaction with FRα regulates stem cell characteristics of cranial neural crest cells (CNCCs)-critical for normal development. We hypothesized that FRα upregulates coding genes and simultaneously downregulates non-coding miRNA which targets coding genes in CNCCs. Quantitative RT-PCR and chromatin immunoprecipitation showed that FRα upregulates Oct4, Sox2, and Klf4 by binding to their cis-regulator elements-5' enhancer/promoters defined by H3K27Ac and p300 occupancy. FA via FRα downregulates miRNAs, miR-138 and miR-let-7, which target Oct4 and Trim71 (an Oct4 downstream effector), respectively. Co-immunoprecipitation data suggests that FRα interacts with the Drosha-DGCR8 complex to affect pre-miRNA processing. Transfecting anti-miR-138 or anti-miR-let-7 into non-proliferating neural crest cells (NCCs) derived from Splotch (Sp(-/-) ), restored their proliferation potential. In summary, these results suggest a novel pleiotropic role of FRα: (a) direct activation of Oct4, Sox2, and Klf4 genes; and (b) repression of biogenesis of miRNAs that target these genes or their effector molecules. Stem Cells 2016;34:2721-2732. © 2016 The Authors STEM CELLS published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.

  12. [Expression and diagnostic significance of OCT4, CD117 and CD30 in germ cell tumors].

    PubMed

    Teng, Liang-Hong; Lu, De-Hong; Xu, Qing-Zhong; Fu, Yong-Juan; Yang, Hong; He, Zhi-Li

    2005-11-01

    To study the immunohistochemical expression of OCT4, CD117 and CD30 in germ cell tumors and to assess their diagnostic value. Immunohistochemical study for OCT4 was performed on formalin-fixed, paraffin-embedded tissues of 63 cases of germ cell tumors, including seminoma (21), dysgerminoma (7), germinoma (8), embryonal carcinoma (8), yolk sac tumor (6), mature teratoma (10) and immature teratoma (3), as well as 25 cases of non-germ cell tumors, including granulosa cell tumor (8), clear cell adenocarcinoma (4), Leydig's cell tumor (5), diffuse large B-cell lymphoma (4) and malignant melanoma (4). Besides, the expression of CD117 and CD30 in all germ cell tumors was studied. All cases of seminoma and germinoma, 6/7 cases of dysgerminoma and 7/8 cases of embryonal carcinoma were positive for OCT4, with strong nuclear staining. All other germ cell tumors and non-germ cell tumors were negative for OCT4, except for 1 case of yolk sac tumor and 1 case of clear cell adenocarcinoma which showed weak staining. Positive membranous expression of CD117 was demonstrated in 19/21(90.5%) seminoma, 5/7 dysgerminoma and 7/8 germinoma. Focal weak membranous staining was also noted in 1 case of yolk sac tumor. The melanocytes in teratoma were also positive for CD117. All cases of embryonal carcinoma were negative. On the other hand, positive membranous expression of CD30 were demonstrated in 6/8 embryonal carcinoma. One case of germinoma and 1 case of yolk sac tumor showed weak cytoplasmic positivity. All cases of seminoma and dysgerminoma, 7/8 germinoma and all cases of teratoma were negative for CD30. OCT4 is a sensitive and relatively specific marker for diagnosing seminoma, dysgerminoma, germinoma and embryonal carcinoma. CD117 and CD30 immunostains, when used in combination, represent valuable tools for distinguishing embryonal carcinoma and seminoma, dysgerminoma, germinoma.

  13. Regulatory gene networks and the properties of the developmental process

    NASA Technical Reports Server (NTRS)

    Davidson, Eric H.; McClay, David R.; Hood, Leroy

    2003-01-01

    Genomic instructions for development are encoded in arrays of regulatory DNA. These specify large networks of interactions among genes producing transcription factors and signaling components. The architecture of such networks both explains and predicts developmental phenomenology. Although network analysis is yet in its early stages, some fundamental commonalities are already emerging. Two such are the use of multigenic feedback loops to ensure the progressivity of developmental regulatory states and the prevalence of repressive regulatory interactions in spatial control processes. Gene regulatory networks make it possible to explain the process of development in causal terms and eventually will enable the redesign of developmental regulatory circuitry to achieve different outcomes.

  14. Regulatory gene networks and the properties of the developmental process

    NASA Technical Reports Server (NTRS)

    Davidson, Eric H.; McClay, David R.; Hood, Leroy

    2003-01-01

    Genomic instructions for development are encoded in arrays of regulatory DNA. These specify large networks of interactions among genes producing transcription factors and signaling components. The architecture of such networks both explains and predicts developmental phenomenology. Although network analysis is yet in its early stages, some fundamental commonalities are already emerging. Two such are the use of multigenic feedback loops to ensure the progressivity of developmental regulatory states and the prevalence of repressive regulatory interactions in spatial control processes. Gene regulatory networks make it possible to explain the process of development in causal terms and eventually will enable the redesign of developmental regulatory circuitry to achieve different outcomes.

  15. A hypothesis for an embryonic origin of pluripotent Oct-4(+) stem cells in adult bone marrow and other tissues.

    PubMed

    Ratajczak, M Z; Machalinski, B; Wojakowski, W; Ratajczak, J; Kucia, M

    2007-05-01

    Accumulating evidence demonstrates that adult tissues contain a population of stem cells that express early developmental markers such as stage-specific embryonic antigen and transcription factors Oct-4 and Nanog. These are the markers characteristic for embryonic stem cells, epiblast stem cells and primordial germ cells. The presence of these stem cells in adult tissues including bone marrow, epidermis, bronchial epithelium, myocardium, pancreas and testes supports the concept that adult tissues contain some population of pluripotent stem cells that is deposited in embryogenesis during early gastrulation. In this review we will discuss these data and present a hypothesis that these cells could be direct descendants of the germ lineage. The germ lineage in order to pass genes on to the next generations creates soma and thus becomes a 'mother lineage' for all somatic cell lineages present in the adult body.

  16. Discovering Study-Specific Gene Regulatory Networks

    PubMed Central

    Bo, Valeria; Curtis, Tanya; Lysenko, Artem; Saqi, Mansoor; Swift, Stephen; Tucker, Allan

    2014-01-01

    Microarrays are commonly used in biology because of their ability to simultaneously measure thousands of genes under different conditions. Due to their structure, typically containing a high amount of variables but far fewer samples, scalable network analysis techniques are often employed. In particular, consensus approaches have been recently used that combine multiple microarray studies in order to find networks that are more robust. The purpose of this paper, however, is to combine multiple microarray studies to automatically identify subnetworks that are distinctive to specific experimental conditions rather than common to them all. To better understand key regulatory mechanisms and how they change under different conditions, we derive unique networks from multiple independent networks built using glasso which goes beyond standard correlations. This involves calculating cluster prediction accuracies to detect the most predictive genes for a specific set of conditions. We differentiate between accuracies calculated using cross-validation within a selected cluster of studies (the intra prediction accuracy) and those calculated on a set of independent studies belonging to different study clusters (inter prediction accuracy). Finally, we compare our method's results to related state-of-the art techniques. We explore how the proposed pipeline performs on both synthetic data and real data (wheat and Fusarium). Our results show that subnetworks can be identified reliably that are specific to subsets of studies and that these networks reflect key mechanisms that are fundamental to the experimental conditions in each of those subsets. PMID:25191999

  17. Regulatory considerations for translating gene therapy: a European Union perspective.

    PubMed

    Galli, Maria Cristina

    2009-11-11

    A preclinical study on a gene therapy approach for treatment of the severe muscle weakness associated with a variety of neuromuscular disorders provides a forum to discuss the translational challenges of gene therapy from a regulatory point of view. In this Perspective, the findings are considered from the view of European regulatory requirements for first clinical use.

  18. Modeling stochastic noise in gene regulatory systems.

    PubMed

    Meister, Arwen; Du, Chao; Li, Ye Henry; Wong, Wing Hung

    2014-03-01

    The Master equation is considered the gold standard for modeling the stochastic mechanisms of gene regulation in molecular detail, but it is too complex to solve exactly in most cases, so approximation and simulation methods are essential. However, there is still a lack of consensus about the best way to carry these out. To help clarify the situation, we review Master equation models of gene regulation, theoretical approximations based on an expansion method due to N.G. van Kampen and R. Kubo, and simulation algorithms due to D.T. Gillespie and P. Langevin. Expansion of the Master equation shows that for systems with a single stable steady-state, the stochastic model reduces to a deterministic model in a first-order approximation. Additional theory, also due to van Kampen, describes the asymptotic behavior of multistable systems. To support and illustrate the theory and provide further insight into the complex behavior of multistable systems, we perform a detailed simulation study comparing the various approximation and simulation methods applied to synthetic gene regulatory systems with various qualitative characteristics. The simulation studies show that for large stochastic systems with a single steady-state, deterministic models are quite accurate, since the probability distribution of the solution has a single peak tracking the deterministic trajectory whose variance is inversely proportional to the system size. In multistable stochastic systems, large fluctuations can cause individual trajectories to escape from the domain of attraction of one steady-state and be attracted to another, so the system eventually reaches a multimodal probability distribution in which all stable steady-states are represented proportional to their relative stability. However, since the escape time scales exponentially with system size, this process can take a very long time in large systems.

  19. Modeling stochastic noise in gene regulatory systems

    PubMed Central

    Meister, Arwen; Du, Chao; Li, Ye Henry; Wong, Wing Hung

    2014-01-01

    The Master equation is considered the gold standard for modeling the stochastic mechanisms of gene regulation in molecular detail, but it is too complex to solve exactly in most cases, so approximation and simulation methods are essential. However, there is still a lack of consensus about the best way to carry these out. To help clarify the situation, we review Master equation models of gene regulation, theoretical approximations based on an expansion method due to N.G. van Kampen and R. Kubo, and simulation algorithms due to D.T. Gillespie and P. Langevin. Expansion of the Master equation shows that for systems with a single stable steady-state, the stochastic model reduces to a deterministic model in a first-order approximation. Additional theory, also due to van Kampen, describes the asymptotic behavior of multistable systems. To support and illustrate the theory and provide further insight into the complex behavior of multistable systems, we perform a detailed simulation study comparing the various approximation and simulation methods applied to synthetic gene regulatory systems with various qualitative characteristics. The simulation studies show that for large stochastic systems with a single steady-state, deterministic models are quite accurate, since the probability distribution of the solution has a single peak tracking the deterministic trajectory whose variance is inversely proportional to the system size. In multistable stochastic systems, large fluctuations can cause individual trajectories to escape from the domain of attraction of one steady-state and be attracted to another, so the system eventually reaches a multimodal probability distribution in which all stable steady-states are represented proportional to their relative stability. However, since the escape time scales exponentially with system size, this process can take a very long time in large systems. PMID:25632368

  20. Caenorhabditis elegans metabolic gene regulatory networks govern the cellular economy.

    PubMed

    Watson, Emma; Walhout, Albertha J M

    2014-10-01

    Diet greatly impacts metabolism in health and disease. In response to the presence or absence of specific nutrients, metabolic gene regulatory networks sense the metabolic state of the cell and regulate metabolic flux accordingly, for instance by the transcriptional control of metabolic enzymes. Here, we discuss recent insights regarding metazoan metabolic regulatory networks using the nematode Caenorhabditis elegans as a model, including the modular organization of metabolic gene regulatory networks, the prominent impact of diet on the transcriptome and metabolome, specialized roles of nuclear hormone receptors (NHRs) in responding to dietary conditions, regulation of metabolic genes and metabolic regulators by miRNAs, and feedback between metabolic genes and their regulators.

  1. An internal regulatory element controls troponin I gene expression

    SciTech Connect

    Yutzey, K.E.; Kline, R.L.; Konieczmy, S.F. . Dept. of Biological Sciences)

    1989-04-01

    During skeletal myogenesis, approximately 20 contractile proteins and related gene products temporally accumulate as the cells fuse to form multinucleated muscle fibers. In most instances, the contractile protein genes are regulated transcriptionally, which suggests that a common molecular mechanism may coordinate the expression of this diverse and evolutionarily unrelated gene set. Recent studies have examined the muscle-specific cis-acting elements associated with numerous contractile protein genes. All of the identified regulatory elements are positioned in the 5'-flanking regions, usually within 1,500 base pairs of the transcription start site. Surprisingly, a DNA consensus sequence that is common to each contractile protein gene has not been identified. In contrast to the results of these earlier studies, the authors have found that the 5'-flanking region of the quail troponin I (TnI) gene is not sufficient to permit the normal myofiber transcriptional activation of the gene. Instead, the TnI gene utilizes a unique internal regulatory element that is responsible for the correct myofiber-specific expression pattern associated with the TnI gene. This is the first example in which a contractile protein gene has been shown to rely primarily on an internal regulatory element to elicit transcriptional activation during myogenesis. The diversity of regulatory elements associated with the contractile protein genes suggests that the temporal expression of the genes may involve individual cis-trans regulatory components specific for each gene.

  2. Lin28 regulates BMP4 and functions with Oct4 to affect ovarian tumor microenvironment

    PubMed Central

    Ma, Wei; Ma, Jing; Xu, Jie; Qiao, Chong; Branscum, Adam; Cardenas, Andres; Baron, Andre T.; Schwartz, Peter; Maihle, Nita J.; Huang, Yingqun

    2013-01-01

    Emerging evidence suggests that the tumor microenvironment plays a critical role in regulating cancer stem cells (CSCs) and tumor progression through both autocrine and paracrine signaling. Elevated production of bone morphogenetic proteins (BMPs) from human ovarian cancer cells and stroma has been shown to increase CSC proliferation and tumor growth. Here, we report that Lin28, a stem cell factor, binds to BMP4 mRNA in epithelial ovarian carcinoma cells, thereby promoting BMP4 expression at the post-transcriptional level. As co-expression of Lin28 and Oct4 (another stem cell factor) has been implicated in ovarian cancer CSCs, we also determined that high levels of Lin28 are associated with an unfavorable prognosis when co-expressed with high levels of Oct4. Together, these findings uncover a new level of regulation of BMP4 expression and imply a novel Lin28/Oct4/BMP4-mediated mechanism of regulating ovarian tumor cell growth, thus holding potential for the development of new strategies for the diagnosis and treatment of ovarian cancer. PMID:23255092

  3. Scalable topographies to support proliferation and Oct4 expression by human induced pluripotent stem cells.

    PubMed

    Reimer, Andreas; Vasilevich, Aliaksei; Hulshof, Frits; Viswanathan, Priyalakshmi; van Blitterswijk, Clemens A; de Boer, Jan; Watt, Fiona M

    2016-01-13

    It is well established that topographical features modulate cell behaviour, including cell morphology, proliferation and differentiation. To define the effects of topography on human induced pluripotent stem cells (iPSC), we plated cells on a topographical library containing over 1000 different features in medium lacking animal products (xeno-free). Using high content imaging, we determined the effect of each topography on cell proliferation and expression of the pluripotency marker Oct4 24 h after seeding. Features that maintained Oct4 expression also supported proliferation and cell-cell adhesion at 24 h, and by 4 days colonies of Oct4-positive, Sox2-positive cells had formed. Computational analysis revealed that small feature size was the most important determinant of pluripotency, followed by high wave number and high feature density. Using this information we correctly predicted whether any given topography within our library would support the pluripotent state at 24 h. This approach not only facilitates the design of substrates for optimal human iPSC expansion, but also, potentially, identification of topographies with other desirable characteristics, such as promoting differentiation.

  4. Scalable topographies to support proliferation and Oct4 expression by human induced pluripotent stem cells

    PubMed Central

    Reimer, Andreas; Vasilevich, Aliaksei; Hulshof, Frits; Viswanathan, Priyalakshmi; van Blitterswijk, Clemens A.; de Boer, Jan; Watt, Fiona M.

    2016-01-01

    It is well established that topographical features modulate cell behaviour, including cell morphology, proliferation and differentiation. To define the effects of topography on human induced pluripotent stem cells (iPSC), we plated cells on a topographical library containing over 1000 different features in medium lacking animal products (xeno-free). Using high content imaging, we determined the effect of each topography on cell proliferation and expression of the pluripotency marker Oct4 24 h after seeding. Features that maintained Oct4 expression also supported proliferation and cell-cell adhesion at 24 h, and by 4 days colonies of Oct4-positive, Sox2-positive cells had formed. Computational analysis revealed that small feature size was the most important determinant of pluripotency, followed by high wave number and high feature density. Using this information we correctly predicted whether any given topography within our library would support the pluripotent state at 24 h. This approach not only facilitates the design of substrates for optimal human iPSC expansion, but also, potentially, identification of topographies with other desirable characteristics, such as promoting differentiation. PMID:26757610

  5. Expression of OCT4A: The First Step to the Next Stage of Urothelial Bladder Cancer Progression

    PubMed Central

    Jóźwicki, Wojciech; Brożyna, Anna A.; Siekiera, Jerzy

    2014-01-01

    OCT4 (octamer-binding transcription factor) is a transcription factor responsible for maintaining the pluripotent properties of embryonic stem cells. In this paper, we present the results of studies to investigate the role of the OCT4 splicing variant in urothelial bladder cancer and the relationship between the OCT4 phenotype and the morphological parameters of tumor malignancy. Ninety patients who received a cystectomy for bladder cancer were enrolled. The expression of OCT4 protein was analyzed by immunohistochemistry. The ratio of OCT4-positive cells was the lowest in pT1 (pathological assessment (p)—tumor extent confined to mucosa (T1)) tumors and the highest in pTis (non-papillary tumor extent confined to urothelium) and pT2 (tumor extent including muscularis propria) tumors. Information about the percentage of OCT4A-positive tumor cells could facilitate choosing the treatment mode in borderline pTis–pT1 (crossing the border of the basement membrane; the first stage of progression) and pT1–pT2 (crossing the border of the muscularis propria; the second stage of progression) cases: a higher percentage of OCT4A-positive cells should support more radical therapy. A significantly higher percentage of cases with moderate OCT4 intensity was found in metastasizing (the third stage of progression) cases with >2 positive lymph nodes. The percentage of OCT4-positive cells was significantly higher for cancers with a high grade, higher non-classic differentiation number and greater aggressiveness of invasion. The differentiation, maturation and aggressiveness of tumor invasion appear to depend on the expression of the OCT4 phenotype in cancer cells, similar to the successive stages of malignancy progression in urothelial cancer. PMID:25216339

  6. Intersecting transcription networks constrain gene regulatory evolution

    PubMed Central

    Sorrells, Trevor R; Booth, Lauren N; Tuch, Brian B; Johnson, Alexander D

    2015-01-01

    Epistasis—the non-additive interactions between different genetic loci—constrains evolutionary pathways, blocking some and permitting others1–8. For biological networks such as transcription circuits, the nature of these constraints and their consequences are largely unknown. Here we describe the evolutionary pathways of a transcription network that controls the response to mating pheromone in yeasts9. A component of this network, the transcription regulator Ste12, has evolved two different modes of binding to a set of its target genes. In one group of species, Ste12 binds to specific DNA binding sites, while in another lineage it occupies DNA indirectly, relying on a second transcription regulator to recognize DNA. We show, through the construction of various possible evolutionary intermediates, that evolution of the direct mode of DNA binding was not directly accessible to the ancestor. Instead, it was contingent on a lineage-specific change to an overlapping transcription network with a different function, the specification of cell type. These results show that analyzing and predicting the evolution of cis-regulatory regions requires an understanding of their positions in overlapping networks, as this placement constrains the available evolutionary pathways. PMID:26153861

  7. Toward an orofacial gene regulatory network.

    PubMed

    Kousa, Youssef A; Schutte, Brian C

    2016-03-01

    Orofacial clefting is a common birth defect with significant morbidity. A panoply of candidate genes have been discovered through synergy of animal models and human genetics. Among these, variants in interferon regulatory factor 6 (IRF6) cause syndromic orofacial clefting and contribute risk toward isolated cleft lip and palate (1/700 live births). Rare variants in IRF6 can lead to Van der Woude syndrome (1/35,000 live births) and popliteal pterygium syndrome (1/300,000 live births). Furthermore, IRF6 regulates GRHL3 and rare variants in this downstream target can also lead to Van der Woude syndrome. In addition, a common variant (rs642961) in the IRF6 locus is found in 30% of the world's population and contributes risk for isolated orofacial clefting. Biochemical studies revealed that rs642961 abrogates one of four AP-2alpha binding sites. Like IRF6 and GRHL3, rare variants in TFAP2A can also lead to syndromic orofacial clefting with lip pits (branchio-oculo-facial syndrome). The literature suggests that AP-2alpha, IRF6 and GRHL3 are part of a pathway that is essential for lip and palate development. In addition to updating the pathways, players and pursuits, this review will highlight some of the current questions in the study of orofacial clefting.

  8. Chaotic Motifs in Gene Regulatory Networks

    PubMed Central

    Zhang, Zhaoyang; Ye, Weiming; Qian, Yu; Zheng, Zhigang; Huang, Xuhui; Hu, Gang

    2012-01-01

    Chaos should occur often in gene regulatory networks (GRNs) which have been widely described by nonlinear coupled ordinary differential equations, if their dimensions are no less than 3. It is therefore puzzling that chaos has never been reported in GRNs in nature and is also extremely rare in models of GRNs. On the other hand, the topic of motifs has attracted great attention in studying biological networks, and network motifs are suggested to be elementary building blocks that carry out some key functions in the network. In this paper, chaotic motifs (subnetworks with chaos) in GRNs are systematically investigated. The conclusion is that: (i) chaos can only appear through competitions between different oscillatory modes with rivaling intensities. Conditions required for chaotic GRNs are found to be very strict, which make chaotic GRNs extremely rare. (ii) Chaotic motifs are explored as the simplest few-node structures capable of producing chaos, and serve as the intrinsic source of chaos of random few-node GRNs. Several optimal motifs causing chaos with atypically high probability are figured out. (iii) Moreover, we discovered that a number of special oscillators can never produce chaos. These structures bring some advantages on rhythmic functions and may help us understand the robustness of diverse biological rhythms. (iv) The methods of dominant phase-advanced driving (DPAD) and DPAD time fraction are proposed to quantitatively identify chaotic motifs and to explain the origin of chaotic behaviors in GRNs. PMID:22792171

  9. Chaotic motifs in gene regulatory networks.

    PubMed

    Zhang, Zhaoyang; Ye, Weiming; Qian, Yu; Zheng, Zhigang; Huang, Xuhui; Hu, Gang

    2012-01-01

    Chaos should occur often in gene regulatory networks (GRNs) which have been widely described by nonlinear coupled ordinary differential equations, if their dimensions are no less than 3. It is therefore puzzling that chaos has never been reported in GRNs in nature and is also extremely rare in models of GRNs. On the other hand, the topic of motifs has attracted great attention in studying biological networks, and network motifs are suggested to be elementary building blocks that carry out some key functions in the network. In this paper, chaotic motifs (subnetworks with chaos) in GRNs are systematically investigated. The conclusion is that: (i) chaos can only appear through competitions between different oscillatory modes with rivaling intensities. Conditions required for chaotic GRNs are found to be very strict, which make chaotic GRNs extremely rare. (ii) Chaotic motifs are explored as the simplest few-node structures capable of producing chaos, and serve as the intrinsic source of chaos of random few-node GRNs. Several optimal motifs causing chaos with atypically high probability are figured out. (iii) Moreover, we discovered that a number of special oscillators can never produce chaos. These structures bring some advantages on rhythmic functions and may help us understand the robustness of diverse biological rhythms. (iv) The methods of dominant phase-advanced driving (DPAD) and DPAD time fraction are proposed to quantitatively identify chaotic motifs and to explain the origin of chaotic behaviors in GRNs.

  10. Transcription factor trapping by RNA in gene regulatory elements.

    PubMed

    Sigova, Alla A; Abraham, Brian J; Ji, Xiong; Molinie, Benoit; Hannett, Nancy M; Guo, Yang Eric; Jangi, Mohini; Giallourakis, Cosmas C; Sharp, Phillip A; Young, Richard A

    2015-11-20

    Transcription factors (TFs) bind specific sequences in promoter-proximal and -distal DNA elements to regulate gene transcription. RNA is transcribed from both of these DNA elements, and some DNA binding TFs bind RNA. Hence, RNA transcribed from regulatory elements may contribute to stable TF occupancy at these sites. We show that the ubiquitously expressed TF Yin-Yang 1 (YY1) binds to both gene regulatory elements and their associated RNA species across the entire genome. Reduced transcription of regulatory elements diminishes YY1 occupancy, whereas artificial tethering of RNA enhances YY1 occupancy at these elements. We propose that RNA makes a modest but important contribution to the maintenance of certain TFs at gene regulatory elements and suggest that transcription of regulatory elements produces a positive-feedback loop that contributes to the stability of gene expression programs. Copyright © 2015, American Association for the Advancement of Science.

  11. Critical regulation of miR-200/ZEB2 pathway in Oct4/Sox2-induced mesenchymal-to-epithelial transition and induced pluripotent stem cell generation.

    PubMed

    Wang, Guiying; Guo, Xudong; Hong, Wujun; Liu, Qidong; Wei, Tingyi; Lu, Chenqi; Gao, Longfei; Ye, Dan; Zhou, Yi; Chen, Jie; Wang, Jianmin; Wu, Minjuan; Liu, Houqi; Kang, Jiuhong

    2013-02-19

    Fibroblasts can be reprogrammed to induced pluripotent stem cells (iPSCs) by application of transcription factors octamer-binding protein 4 (Oct4), SRY-box containing gene 2 (Sox2), Kruppel-like factor 4 (Klf4), and c-Myelocytomatosis oncogene (c-Myc) (OSKM), but the underlying mechanisms remain unclear. Here, we report that exogenous Oct4 and Sox2 can bind at the promoter regions of mir-141/200c and mir-200a/b/429 cluster, respectively, and induce the transcription activation of miR-200 family during the OSKM-induced reprogramming. Functional suppression of miR-200s with specific inhibitors significantly represses the OSKM-caused mesenchymal-to-epithelial transition (MET, an early event in reprogramming of fibroblasts to iPSCs) and iPSC generation, whereas overexpression of miR-200s promotes the MET and iPSC generation. Mechanistic studies showed that miR-200s significantly repress the expression of zinc finger E-box binding homeobox 2 (ZEB2) through directly targeting its 3' UTR and direct inhibition of ZEB2 can mimic the effects of miR-200s on iPSC generation and MET process. Moreover, the effects of miR-200s during iPSC generation can be blocked by ZEB2 overexpression. Collectively, our findings not only reveal that members of the miR-200 family are unique mediators of the reprogramming factors Oct4/Sox2, but also demonstrate that the miR-200/ZEB2 pathway as one critical mechanism of Oct4/Sox2 to induce somatic cell reprogramming at the early stage.

  12. Curcumin induces apoptotic cell death via Oct4 inhibition and GSK-3β activation in NCCIT cells.

    PubMed

    Yun, Ji Ho; Park, Young Gyun; Lee, Kyung-Mi; Kim, Jungho; Nho, Chu Won

    2015-06-01

    Octamer-binding transcription factor 4 (Oct4) is a key regulator of pluripotent embryonic stem cell maintenance. However, increasing evidence has suggested that Oct4 is also expressed in cancer stem cells (CSCs) and is associated with tumor progression and chemoresistance. Curcumin (CUR) is a widely used cancer chemopreventive agent, and it has been used to treat several diseases including cancers. Here, we investigated whether CUR-induced apoptotic cell death by inhibiting Oct4 levels and examining molecular mechanisms in NCCIT human embryonic carcinoma cells. CUR significantly inhibited Oct4 transcription levels in a dose-dependent manner by dual luciferase experiment, also decreased mRNA and protein levels in NCCIT human embryonic carcinoma cells, which express high levels of endogenous Oct4. Interestingly, we found that CUR treatment increased apoptotic cell death including subG0/G1 contents, cleavage caspases, and pro-apoptotic protein, as confirmed with a series of loss-of-function experiments using Oct4 siRNA. Furthermore, CUR induced marked total level of glycogen synthase kinase 3 beta (GSK-3β), resulting in an increase in apoptotic cell death, was evaluated using chemical inhibitor of GSK3-3β. These data suggest that CUR induces apoptotic cell death through Oct4 inhibition and GSK-3β activation. Thus, CUR may be a useful cancer chemopreventive agent to suppress tumor progression or to improve chemoresistance by eliminating CSCs. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. Computational inference of gene regulatory networks: Approaches, limitations and opportunities.

    PubMed

    Banf, Michael; Rhee, Seung Y

    2017-01-01

    Gene regulatory networks lie at the core of cell function control. In E. coli and S. cerevisiae, the study of gene regulatory networks has led to the discovery of regulatory mechanisms responsible for the control of cell growth, differentiation and responses to environmental stimuli. In plants, computational rendering of gene regulatory networks is gaining momentum, thanks to the recent availability of high-quality genomes and transcriptomes and development of computational network inference approaches. Here, we review current techniques, challenges and trends in gene regulatory network inference and highlight challenges and opportunities for plant science. We provide plant-specific application examples to guide researchers in selecting methodologies that suit their particular research questions. Given the interdisciplinary nature of gene regulatory network inference, we tried to cater to both biologists and computer scientists to help them engage in a dialogue about concepts and caveats in network inference. Specifically, we discuss problems and opportunities in heterogeneous data integration for eukaryotic organisms and common caveats to be considered during network model evaluation. This article is part of a Special Issue entitled: Plant Gene Regulatory Mechanisms and Networks, edited by Dr. Erich Grotewold and Dr. Nathan Springer.

  14. Oct4 is required for lineage priming in the developing inner cell mass of the mouse blastocyst.

    PubMed

    Le Bin, Gloryn Chia; Muñoz-Descalzo, Silvia; Kurowski, Agata; Leitch, Harry; Lou, Xinghua; Mansfield, William; Etienne-Dumeau, Charles; Grabole, Nils; Mulas, Carla; Niwa, Hitoshi; Hadjantonakis, Anna-Katerina; Nichols, Jennifer

    2014-03-01

    The transcription factor Oct4 is required in vitro for establishment and maintenance of embryonic stem cells and for reprogramming somatic cells to pluripotency. In vivo, it prevents the ectopic differentiation of early embryos into trophoblast. Here, we further explore the role of Oct4 in blastocyst formation and specification of epiblast versus primitive endoderm lineages using conditional genetic deletion. Experiments involving mouse embryos deficient for both maternal and zygotic Oct4 suggest that it is dispensable for zygote formation, early cleavage and activation of Nanog expression. Nanog protein is significantly elevated in the presumptive inner cell mass of Oct4 null embryos, suggesting an unexpected role for Oct4 in attenuating the level of Nanog, which might be significant for priming differentiation during epiblast maturation. Induced deletion of Oct4 during the morula to blastocyst transition disrupts the ability of inner cell mass cells to adopt lineage-specific identity and acquire the molecular profile characteristic of either epiblast or primitive endoderm. Sox17, a marker of primitive endoderm, is not detected following prolonged culture of such embryos, but can be rescued by provision of exogenous FGF4. Interestingly, functional primitive endoderm can be rescued in Oct4-deficient embryos in embryonic stem cell complementation assays, but only if the host embryos are at the pre-blastocyst stage. We conclude that cell fate decisions within the inner cell mass are dependent upon Oct4 and that Oct4 is not cell-autonomously required for the differentiation of primitive endoderm derivatives, as long as an appropriate developmental environment is established.

  15. Phenotype accessibility and noise in random threshold gene regulatory networks.

    PubMed

    Pinho, Ricardo; Garcia, Victor; Feldman, Marcus W

    2014-01-01

    Evolution requires phenotypic variation in a population of organisms for selection to function. Gene regulatory processes involved in organismal development affect the phenotypic diversity of organisms. Since only a fraction of all possible phenotypes are predicted to be accessed by the end of development, organisms may evolve strategies to use environmental cues and noise-like fluctuations to produce additional phenotypic diversity, and hence to enhance the speed of adaptation. We used a generic model of organismal development --gene regulatory networks-- to investigate how different levels of noise on gene expression states (i.e. phenotypes) may affect access to new, unique phenotypes, thereby affecting phenotypic diversity. We studied additional strategies that organisms might adopt to attain larger phenotypic diversity: either by augmenting their genome or the number of gene expression states. This was done for different types of gene regulatory networks that allow for distinct levels of regulatory influence on gene expression or are more likely to give rise to stable phenotypes. We found that if gene expression is binary, increasing noise levels generally decreases phenotype accessibility for all network types studied. If more gene expression states are considered, noise can moderately enhance the speed of discovery if three or four gene expression states are allowed, and if there are enough distinct regulatory networks in the population. These results were independent of the network types analyzed, and were robust to different implementations of noise. Hence, for noise to increase the number of accessible phenotypes in gene regulatory networks, very specific conditions need to be satisfied. If the number of distinct regulatory networks involved in organismal development is large enough, and the acquisition of more genes or fine tuning of their expression states proves costly to the organism, noise can be useful in allowing access to more unique phenotypes.

  16. Phenotypic plasticity can facilitate adaptive evolution in gene regulatory circuits

    PubMed Central

    2011-01-01

    Background Many important evolutionary adaptations originate in the modification of gene regulatory circuits to produce new gene activity phenotypes. How do evolving populations sift through an astronomical number of circuits to find circuits with new adaptive phenotypes? The answer may often involve phenotypic plasticity. Phenotypic plasticity allows a genotype to produce different - alternative - phenotypes after non-genetic perturbations that include gene expression noise, environmental change, or epigenetic modification. Results We here analyze a well-studied model of gene regulatory circuits. A circuit's genotype encodes the regulatory interactions among circuit genes, and its phenotype corresponds to a stable gene activity pattern the circuit forms. For this model, we study how genotypes are arranged in genotype space, where the distance between two genotypes reflects the number of regulatory mutations that set those genotypes apart. Specifically, we address whether this arrangement favors adaptive evolution mediated by plasticity. We find that plasticity facilitates the origin of genotypes that produce a new phenotype in response to non-genetic perturbations. We also find that selection can then stabilize the new phenotype genetically, allowing it to become a circuit's dominant gene expression phenotype. These are generic properties of the circuits we study here. Conclusions Taken together, our observations suggest that phenotypic plasticity frequently facilitates the evolution of novel beneficial gene activity patterns in gene regulatory circuits. PMID:21211007

  17. Robustness and Accuracy in Sea Urchin Developmental Gene Regulatory Networks.

    PubMed

    Ben-Tabou de-Leon, Smadar

    2016-01-01

    Developmental gene regulatory networks robustly control the timely activation of regulatory and differentiation genes. The structure of these networks underlies their capacity to buffer intrinsic and extrinsic noise and maintain embryonic morphology. Here I illustrate how the use of specific architectures by the sea urchin developmental regulatory networks enables the robust control of cell fate decisions. The Wnt-βcatenin signaling pathway patterns the primary embryonic axis while the BMP signaling pathway patterns the secondary embryonic axis in the sea urchin embryo and across bilateria. Interestingly, in the sea urchin in both cases, the signaling pathway that defines the axis controls directly the expression of a set of downstream regulatory genes. I propose that this direct activation of a set of regulatory genes enables a uniform regulatory response and a clear cut cell fate decision in the endoderm and in the dorsal ectoderm. The specification of the mesodermal pigment cell lineage is activated by Delta signaling that initiates a triple positive feedback loop that locks down the pigment specification state. I propose that the use of compound positive feedback circuitry provides the endodermal cells enough time to turn off mesodermal genes and ensures correct mesoderm vs. endoderm fate decision. Thus, I argue that understanding the control properties of repeatedly used regulatory architectures illuminates their role in embryogenesis and provides possible explanations to their resistance to evolutionary change.

  18. Implications of functional similarity for gene regulatory interactions

    PubMed Central

    Glass, Kimberly; Ott, Edward; Losert, Wolfgang; Girvan, Michelle

    2012-01-01

    If one gene regulates another, those two genes are likely to be involved in many of the same biological functions. Conversely, shared biological function may be suggestive of the existence and nature of a regulatory interaction. With this in mind, we develop a measure of functional similarity between genes based on annotations made to the Gene Ontology in which the magnitude of their functional relationship is also indicative of a regulatory relationship. In contrast to other measures that have previously been used to quantify the functional similarity between genes, our measure scales the strength of any shared functional annotation by the frequency of that function's appearance across the entire set of annotations. We apply our method to both Escherichia coli and Saccharomyces cerevisiae gene annotations and find that the strength of our scaled similarity measure is more predictive of known regulatory interactions than previously published measures of functional similarity. In addition, we observe that the strength of the scaled similarity measure is correlated with the structural importance of links in the known regulatory network. By contrast, other measures of functional similarity are not indicative of any structural importance in the regulatory network. We therefore conclude that adequately adjusting for the frequency of shared biological functions is important in the construction of a functional similarity measure aimed at elucidating the existence and nature of regulatory interactions. We also compare the performance of the scaled similarity with a high-throughput method for determining regulatory interactions from gene expression data and observe that the ontology-based approach identifies a different subset of regulatory interactions compared with the gene expression approach. We show that combining predictions from the scaled similarity with those from the reconstruction algorithm leads to a significant improvement in the accuracy of the reconstructed

  19. Evolutionary conservation of regulatory elements in vertebrate Hox gene clusters.

    PubMed

    Santini, Simona; Boore, Jeffrey L; Meyer, Axel

    2003-06-01

    Comparisons of DNA sequences among evolutionarily distantly related genomes permit identification of conserved functional regions in noncoding DNA. Hox genes are highly conserved in vertebrates, occur in clusters, and are uninterrupted by other genes. We aligned (PipMaker) the nucleotide sequences of the HoxA clusters of tilapia, pufferfish, striped bass, zebrafish, horn shark, human, and mouse, which are separated by approximately 500 million years of evolution. In support of our approach, several identified putative regulatory elements known to regulate the expression of Hox genes were recovered. The majority of the newly identified putative regulatory elements contain short fragments that are almost completely conserved and are identical to known binding sites for regulatory proteins (Transfac database). The regulatory intergenic regions located between the genes that are expressed most anteriorly in the embryo are longer and apparently more evolutionarily conserved than those at the other end of Hox clusters. Different presumed regulatory sequences are retained in either the Aalpha or Abeta duplicated Hox clusters in the fish lineages. This suggests that the conserved elements are involved in different gene regulatory networks and supports the duplication-deletion-complementation model of functional divergence of duplicated genes.

  20. Evolutionary Conservation of Regulatory Elements in Vertebrate Hox Gene Clusters

    PubMed Central

    Santini, Simona; Boore, Jeffrey L.; Meyer, Axel

    2003-01-01

    Comparisons of DNA sequences among evolutionarily distantly related genomes permit identification of conserved functional regions in noncoding DNA. Hox genes are highly conserved in vertebrates, occur in clusters, and are uninterrupted by other genes. We aligned (PipMaker) the nucleotide sequences of the HoxA clusters of tilapia, pufferfish, striped bass, zebrafish, horn shark, human, and mouse, which are separated by approximately 500 million years of evolution. In support of our approach, several identified putative regulatory elements known to regulate the expression of Hox genes were recovered. The majority of the newly identified putative regulatory elements contain short fragments that are almost completely conserved and are identical to known binding sites for regulatory proteins (Transfac database). The regulatory intergenic regions located between the genes that are expressed most anteriorly in the embryo are longer and apparently more evolutionarily conserved than those at the other end of Hox clusters. Different presumed regulatory sequences are retained in either the Aα or Aβ duplicated Hox clusters in the fish lineages. This suggests that the conserved elements are involved in different gene regulatory networks and supports the duplication-deletion-complementation model of functional divergence of duplicated genes. PMID:12799348

  1. Expressions of stem cell transcription factors Nanog and Oct4 in renal cell carcinoma tissues and clinical significance.

    PubMed

    Yu, Bin; Cai, Hongzhou; Xu, Zicheng; Xu, Ting; Zou, Qing; Gu, Min

    2016-12-01

    We aimed to detect the expressions of stem cell transcription factors Nanog and Oct4 in renal cell carcinoma (RCC) tissues. Nanog and Oct4 mRNA expressions in RCC tissues significantly exceeded those in paracancerous tissues (p < 0.01 and p < 0.05), being positively correlated with histological grade (p < 0.01 and p < 0.05) and TNM stage (p < 0.05). With increasing TNM stage (p < 0.01) and lymphatic metastasis (p < 0.05), the positive expression rate of Nanog protein increased. RCC patients with low Nanog and Oct4 expressions in tumor tissues had significantly higher survival rates (p < 0.05). High Nanog and Oct4 expressions may be potential therapeutic targets.

  2. MicroRNA-145 sensitizes cervical cancer cells to low-dose irradiation by downregulating OCT4 expression

    PubMed Central

    Yan, Siqi; Li, Xiangjun; Jin, Qiao; Yuan, Jun

    2016-01-01

    Poor elucidation of the mechanisms involved in regulating the radiosensitivity of cancers prevents the extensive application of low-dose radiotherapy in clinical settings. The present study was conducted to investigate the role of microRNA-145 (miR-145) in the modulation of cervical cancer cell radiosensitivity, as well as to identify the underlying target of miR-145 during this process. Cervical cancer tera cells were initially exposed to doses of radiation between 1 and 6 Gy before the assessments of the cell viability and apoptosis rate. Irradiation at dose of 1 Gy was screened as optimum dose and used in subsequent experiments. A dual luciferase reporter assay was performed to demonstrate that octamer-binding transcription factor 4 (OCT4) is a target of miR-145 in cervical cancer. Consequently, OCT4 was suggested to be a target of miR-145, as a dual luciferase vector that was ligated to a fragment corresponding to the predicted target site of miR-145 in OCT4 3′-UTR showed an 83% reduction in fluorescence. Following exposure to 1 Gy irradiation, tera cells transfected with miR-145 mimics, which showed downregulation of OCT4 and cyclin D1, had lower cell viability and cell migration rate and higher apoptosis rate compared to non-transfected cells. However, the co-transfection of miR-145 mimics and OCT4 expression vector restored OCT4 and cyclin D1 expression levels and made no significant difference in terms of cell viability, cell migration rate and apoptosis rate. The present results indicate that miR-145 increases the radiosensitivity of cervical cancer cells by silencing OCT4, that cyclin D1 is putatively under the positive regulation of OCT4 and mediates miR-145 function. PMID:27882128

  3. A cis-Regulatory Signature for Chordate Anterior Neuroectodermal Genes

    PubMed Central

    Christiaen, Lionel; Joly, Jean-Stéphane

    2010-01-01

    One of the striking findings of comparative developmental genetics was that expression patterns of core transcription factors are extraordinarily conserved in bilaterians. However, it remains unclear whether cis-regulatory elements of their target genes also exhibit common signatures associated with conserved embryonic fields. To address this question, we focused on genes that are active in the anterior neuroectoderm and non-neural ectoderm of the ascidian Ciona intestinalis. Following the dissection of a prototypic anterior placodal enhancer, we searched all genomic conserved non-coding elements for duplicated motifs around genes showing anterior neuroectodermal expression. Strikingly, we identified an over-represented pentamer motif corresponding to the binding site of the homeodomain protein OTX, which plays a pivotal role in the anterior development of all bilaterian species. Using an in vivo reporter gene assay, we observed that 10 of 23 candidate cis-regulatory elements containing duplicated OTX motifs are active in the anterior neuroectoderm, thus showing that this cis-regulatory signature is predictive of neuroectodermal enhancers. These results show that a common cis-regulatory signature corresponding to K50-Paired homeodomain transcription factors is found in non-coding sequences flanking anterior neuroectodermal genes in chordate embryos. Thus, field-specific selector genes impose architectural constraints in the form of combinations of short tags on their target enhancers. This could account for the strong evolutionary conservation of the regulatory elements controlling field-specific selector genes responsible for body plan formation. PMID:20419150

  4. The companions: regulatory T cells and gene therapy

    PubMed Central

    Eghtesad, Saman; Morel, Penelope A; Clemens, Paula R

    2009-01-01

    Undesired immunological responses to products of therapeutic gene replacement have been obstacles to successful gene therapy. Understanding such responses of the host immune system to achieve immunological tolerance to a transferred gene product is therefore crucial. In this article, we review relevant studies of immunological responses to gene replacement therapy, the role of immunological tolerance mediated by regulatory T cells in down-regulating the unwanted immune responses, and the interrelationship of the two topics. PMID:19368560

  5. Emerging role of regulatory T cells in gene transfer.

    PubMed

    Cao, Ou; Furlan-Freguia, Christian; Arruda, Valder R; Herzog, Roland W

    2007-10-01

    Induction and maintenance of immune tolerance to therapeutic transgene products are key requirements for successful gene replacement therapies. Gene transfer may also be used to specifically induce immune tolerance and thereby augment other types of therapies. Similarly, gene therapies for treatment of autoimmune diseases are being developed in order to restore tolerance to self-antigens. Regulatory T cells have emerged as key players in many aspects of immune tolerance, and a rapidly increasing body of work documents induction and/or activation of regulatory T cells by gene transfer. Regulatory T cells may suppress antibody formation and cytotoxic T cell responses and may be critical for immune tolerance to therapeutic proteins. In this regard, CD4(+)CD25(+) regulatory T cells have been identified as important components of tolerance in several gene transfer protocols, including hepatic in vivo gene transfer. Augmentation of regulatory T cell responses should be a promising new tool to achieve tolerance and avoid immune-mediated rejection of gene therapy. During the past decade, it has become obvious that immune regulation is an important and integral component of tolerance to self-antigens and of many forms of induced tolerance. Gene therapy can only be successful if the immune system does not reject the therapeutic transgene product. Recent studies provide a rapidly growing body of evidence that regulatory T cells (T(reg)) are involved and often play a crucial role in tolerance to proteins expressed by means of gene transfer. This review seeks to provide an overview of these data and their implications for gene therapy.

  6. OCT4 controls mitotic stability and inactivates the RB tumor suppressor pathway to enhance ovarian cancer aggressiveness.

    PubMed

    Comisso, E; Scarola, M; Rosso, M; Piazza, S; Marzinotto, S; Ciani, Y; Orsaria, M; Mariuzzi, L; Schneider, C; Schoeftner, S; Benetti, R

    2017-07-27

    OCT4 (Octamer-binding transcription factor 4) is essential for embryonic stem cell self-renewal. Here we show that OCT4 increases the aggressiveness of high-grade serous ovarian cancer (HG-SOC) by inactivating the Retinoblastoma tumor suppressor pathway and enhancing mitotic stability in cancer cells. OCT4 drives the expression of Nuclear Inhibitor of Protein Phosphatase type 1 (NIPP1) and Cyclin F (CCNF) that together inhibit Protein Phosphatase 1 (PP1). This results in pRB hyper-phosphorylation, accelerated cell proliferation and increased in vitro tumorigenicity of ovarian cancer cells. In parallel, OCT4 and NIPP1/CCNF drive the expression of the central Chromosomal Passenger Complex (CPC) components, Borealin, Survivin and the mitotic kinase Aurora B, promoting the clustering of supernumerary centrosomes to increase mitotic stability. Loss of OCT4 or NIPP1/CCNF results in severe mitotic defects, multipolar spindles and supernumerary centrosomes, finally leading to the induction of apoptosis. These phenotypes were recapitulated in different cancer models indicating general relevance for human cancer. Importantly, activation of these parallel pathways leads to dramatically reduced overall survival of HG-SOC patients. Altogether, our data highlights an unprecedented role for OCT4 as central regulator of mitotic fidelity and RB tumor suppressor pathway activity. Disrupting this pathway represents a promising strategy to target an aggressive subpopulation of HG-SOC cells.

  7. The molecular and gene regulatory signature of a neuron

    PubMed Central

    Hobert, Oliver; Carrera, Inés; Stefanakis, Nikolaos

    2010-01-01

    Neuron-type specific gene batteries define the morphological and functional diversity of cell types in the nervous system. Here, we discuss the composition of neuron-type specific gene batteries and illustrate gene regulatory strategies employed by distinct organisms from C.elegans to higher vertebrates, which are instrumental in determining the unique gene expression profile and molecular composition of individual neuronal cell types. Based on principles learned from prokaryotic gene regulation, we argue that neuronal, terminal gene batteries are functionally grouped into parallel acting “regulons”. The theoretical concepts discussed here provide testable hypotheses for future experimental analysis into the exact gene regulatory mechanisms that are employed in the generation of neuronal diversity and identity. PMID:20663572

  8. Gene regulatory networks modelling using a dynamic evolutionary hybrid

    PubMed Central

    2010-01-01

    Background Inference of gene regulatory networks is a key goal in the quest for understanding fundamental cellular processes and revealing underlying relations among genes. With the availability of gene expression data, computational methods aiming at regulatory networks reconstruction are facing challenges posed by the data's high dimensionality, temporal dynamics or measurement noise. We propose an approach based on a novel multi-layer evolutionary trained neuro-fuzzy recurrent network (ENFRN) that is able to select potential regulators of target genes and describe their regulation type. Results The recurrent, self-organizing structure and evolutionary training of our network yield an optimized pool of regulatory relations, while its fuzzy nature avoids noise-related problems. Furthermore, we are able to assign scores for each regulation, highlighting the confidence in the retrieved relations. The approach was tested by applying it to several benchmark datasets of yeast, managing to acquire biologically validated relations among genes. Conclusions The results demonstrate the effectiveness of the ENFRN in retrieving biologically valid regulatory relations and providing meaningful insights for better understanding the dynamics of gene regulatory networks. The algorithms and methods described in this paper have been implemented in a Matlab toolbox and are available from: http://bioserver-1.bioacademy.gr/DataRepository/Project_ENFRN_GRN/. PMID:20298548

  9. Estimating Gene Regulatory Networks with pandaR.

    PubMed

    Schlauch, Daniel; Paulson, Joseph N; Young, Albert; Glass, Kimberly; Quackenbush, John

    2017-03-11

    PANDA (Passing Attributes betweenNetworks forData Assimilation) is a gene regulatory network inference method that begins with amodel of transcription factor-target gene interactions and usesmessage passing to update the network model given available transcriptomic and protein-protein interaction data. PANDA is used to estimate networks for each experimental group and the network models are then compared between groups to explore transcriptional processes that distinguish the groups. We present pandaR (bioconductor.org/packages/pandaR), a Bioconductor package that implements PANDA and provides a framework for exploratory data analysis on gene regulatory networks.

  10. Time-Delayed Models of Gene Regulatory Networks

    PubMed Central

    Parmar, K.; Blyuss, K. B.; Kyrychko, Y. N.; Hogan, S. J.

    2015-01-01

    We discuss different mathematical models of gene regulatory networks as relevant to the onset and development of cancer. After discussion of alternative modelling approaches, we use a paradigmatic two-gene network to focus on the role played by time delays in the dynamics of gene regulatory networks. We contrast the dynamics of the reduced model arising in the limit of fast mRNA dynamics with that of the full model. The review concludes with the discussion of some open problems. PMID:26576197

  11. Systems Approaches to Identifying Gene Regulatory Networks in Plants

    PubMed Central

    Long, Terri A.; Brady, Siobhan M.; Benfey, Philip N.

    2009-01-01

    Complex gene regulatory networks are composed of genes, noncoding RNAs, proteins, metabolites, and signaling components. The availability of genome-wide mutagenesis libraries; large-scale transcriptome, proteome, and metabalome data sets; and new high-throughput methods that uncover protein interactions underscores the need for mathematical modeling techniques that better enable scientists to synthesize these large amounts of information and to understand the properties of these biological systems. Systems biology approaches can allow researchers to move beyond a reductionist approach and to both integrate and comprehend the interactions of multiple components within these systems. Descriptive and mathematical models for gene regulatory networks can reveal emergent properties of these plant systems. This review highlights methods that researchers are using to obtain large-scale data sets, and examples of gene regulatory networks modeled with these data. Emergent properties revealed by the use of these network models and perspectives on the future of systems biology are discussed. PMID:18616425

  12. Gene regulatory network inference using out of equilibrium statistical mechanics

    PubMed Central

    Benecke, Arndt

    2008-01-01

    Spatiotemporal control of gene expression is fundamental to multicellular life. Despite prodigious efforts, the encoding of gene expression regulation in eukaryotes is not understood. Gene expression analyses nourish the hope to reverse engineer effector-target gene networks using inference techniques. Inference from noisy and circumstantial data relies on using robust models with few parameters for the underlying mechanisms. However, a systematic path to gene regulatory network reverse engineering from functional genomics data is still impeded by fundamental problems. Recently, Johannes Berg from the Theoretical Physics Institute of Cologne University has made two remarkable contributions that significantly advance the gene regulatory network inference problem. Berg, who uses gene expression data from yeast, has demonstrated a nonequilibrium regime for mRNA concentration dynamics and was able to map the gene regulatory process upon simple stochastic systems driven out of equilibrium. The impact of his demonstration is twofold, affecting both the understanding of the operational constraints under which transcription occurs and the capacity to extract relevant information from highly time-resolved expression data. Berg has used his observation to predict target genes of selected transcription factors, and thereby, in principle, demonstrated applicability of his out of equilibrium statistical mechanics approach to the gene network inference problem. PMID:19404429

  13. Gene regulatory network inference using out of equilibrium statistical mechanics.

    PubMed

    Benecke, Arndt

    2008-08-01

    Spatiotemporal control of gene expression is fundamental to multicellular life. Despite prodigious efforts, the encoding of gene expression regulation in eukaryotes is not understood. Gene expression analyses nourish the hope to reverse engineer effector-target gene networks using inference techniques. Inference from noisy and circumstantial data relies on using robust models with few parameters for the underlying mechanisms. However, a systematic path to gene regulatory network reverse engineering from functional genomics data is still impeded by fundamental problems. Recently, Johannes Berg from the Theoretical Physics Institute of Cologne University has made two remarkable contributions that significantly advance the gene regulatory network inference problem. Berg, who uses gene expression data from yeast, has demonstrated a nonequilibrium regime for mRNA concentration dynamics and was able to map the gene regulatory process upon simple stochastic systems driven out of equilibrium. The impact of his demonstration is twofold, affecting both the understanding of the operational constraints under which transcription occurs and the capacity to extract relevant information from highly time-resolved expression data. Berg has used his observation to predict target genes of selected transcription factors, and thereby, in principle, demonstrated applicability of his out of equilibrium statistical mechanics approach to the gene network inference problem.

  14. Evolutionary conservation of regulatory elements in vertebrate HOX gene clusters

    SciTech Connect

    Santini, Simona; Boore, Jeffrey L.; Meyer, Axel

    2003-12-31

    Due to their high degree of conservation, comparisons of DNA sequences among evolutionarily distantly-related genomes permit to identify functional regions in noncoding DNA. Hox genes are optimal candidate sequences for comparative genome analyses, because they are extremely conserved in vertebrates and occur in clusters. We aligned (Pipmaker) the nucleotide sequences of HoxA clusters of tilapia, pufferfish, striped bass, zebrafish, horn shark, human and mouse (over 500 million years of evolutionary distance). We identified several highly conserved intergenic sequences, likely to be important in gene regulation. Only a few of these putative regulatory elements have been previously described as being involved in the regulation of Hox genes, while several others are new elements that might have regulatory functions. The majority of these newly identified putative regulatory elements contain short fragments that are almost completely conserved and are identical to known binding sites for regulatory proteins (Transfac). The conserved intergenic regions located between the most rostrally expressed genes in the developing embryo are longer and better retained through evolution. We document that presumed regulatory sequences are retained differentially in either A or A clusters resulting from a genome duplication in the fish lineage. This observation supports both the hypothesis that the conserved elements are involved in gene regulation and the Duplication-Deletion-Complementation model.

  15. Portrait of Candida Species Biofilm Regulatory Network Genes.

    PubMed

    Araújo, Daniela; Henriques, Mariana; Silva, Sónia

    2017-01-01

    Most cases of candidiasis have been attributed to Candida albicans, but Candida glabrata, Candida parapsilosis and Candida tropicalis, designated as non-C. albicans Candida (NCAC), have been identified as frequent human pathogens. Moreover, Candida biofilms are an escalating clinical problem associated with significant rates of mortality. Biofilms have distinct developmental phases, including adhesion/colonisation, maturation and dispersal, controlled by complex regulatory networks. This review discusses recent advances regarding Candida species biofilm regulatory network genes, which are key components for candidiasis.

  16. Heterokaryon-based reprogramming of human B lymphocytes for pluripotency requires Oct4 but not Sox2.

    PubMed

    Pereira, Carlos F; Terranova, Rémi; Ryan, Natalie K; Santos, Joana; Morris, Kelly J; Cui, Wei; Merkenschlager, Matthias; Fisher, Amanda G

    2008-09-05

    Differentiated cells can be reprogrammed through the formation of heterokaryons and hybrid cells when fused with embryonic stem (ES) cells. Here, we provide evidence that conversion of human B-lymphocytes towards a multipotent state is initiated much more rapidly than previously thought, occurring in transient heterokaryons before nuclear fusion and cell division. Interestingly, reprogramming of human lymphocytes by mouse ES cells elicits the expression of a human ES-specific gene profile, in which markers of human ES cells are expressed (hSSEA4, hFGF receptors and ligands), but markers that are specific to mouse ES cells are not (e.g., Bmp4 and LIF receptor). Using genetically engineered mouse ES cells, we demonstrate that successful reprogramming of human lymphocytes is independent of Sox2, a factor thought to be required for induced pluripotent stem (iPS) cells. In contrast, there is a distinct requirement for Oct4 in the establishment but not the maintenance of the reprogrammed state. Experimental heterokaryons, therefore, offer a powerful approach to trace the contribution of individual factors to the reprogramming of human somatic cells towards a multipotent state.

  17. New POU dimer configuration mediates antagonistic control of an osteopontin preimplantation enhancer by Oct-4 and Sox-2

    PubMed Central

    Botquin, Valérie; Hess, Heike; Fuhrmann, Guy; Anastassiadis, Constantinos; Gross, Michael K.; Vriend, Gerrit; Schöler, Hans R.

    1998-01-01

    The POU transcription factor Oct-4 is expressed specifically in the germ line, pluripotent cells of the pregastrulation embryo and stem cell lines derived from the early embryo. Osteopontin (OPN) is a protein secreted by cells of the preimplantation embryo and contains a GRGDS motif that can bind to specific integrin subtypes and modulate cell adhesion/migration. We show that Oct-4 and OPN are coexpressed in the preimplantation mouse embryo and during differentiation of embryonal cell lines. Immunoprecipitation of the first intron of OPN (i-opn) from covalently fixed chromatin of embryonal stem cells by Oct-4-specific antibodies indicates that Oct-4 binds to this fragment in vivo. The i-opn fragment functions as an enhancer in cell lines that resemble cells of the preimplantation embryo. Furthermore, it contains a novel palindromic Oct factor recognition element (PORE) that is composed of an inverted pair of homeodomain-binding sites separated by exactly 5 bp (ATTTG +5 CAAAT). POU proteins can homo- and heterodimerize on the PORE in a configuration that has not been described previously. Strong transcriptional activation of the OPN element requires an intact PORE. In contrast, the canonical octamer overlapping with the downstream half of the PORE is not essential. Sox-2 is a transcription factor that contains an HMG box and is coexpressed with Oct-4 in the early mouse embryo. Sox-2 represses Oct-4 mediated activation of i-opn by way of a canonical Sox element that is located close to the PORE. Repression depends on a carboxy-terminal region of Sox-2 that is outside of the HMG box. Expression, DNA binding, and transactivation data are consistent with the hypothesis that OPN expression is regulated by Oct-4 and Sox-2 in preimplantation development. PMID:9649510

  18. Structural and mechanistic insights into nuclear transport and delivery of the critical pluripotency factor Oct4 to DNA.

    PubMed

    Okuyama, Takahide; Yamagishi, Ryosuke; Shimada, Jiro; Ikeda, Masaaki; Maruoka, Yayoi; Kaneko, Hiroki

    2017-02-17

    Oct4 is a master regulator of the induction and maintenance of cellular pluripotency, and has crucial roles in early stages of differentiation. It is the only factor that cannot be substituted by other members of the same protein family to induce pluripotency. However, although Oct4 nuclear transport and delivery to target DNA are critical events for reprogramming to pluripotency, little is known about the molecular mechanism. Oct4 is imported to the nucleus by the classical nuclear transport mechanism, which requires importin α as an adaptor to bind the nuclear localization signal (NLS). Although there are structures of complexes of the NLS of transcription factors (TFs) in complex with importin α, there are no structures available for complexes involving intact TFs. We have therefore modeled the structure of the complex of the whole Oct4 POU domain and importin α2 using protein-protein docking and molecular dynamics. The model explains how the Ebola virus VP24 protein has a negative effect on the nuclear import of STAT1 by importin α but not on Oct4, and how Nup 50 facilitates cargo release from importin α. The model demonstrates the structural differences between the Oct4 importin α bound and DNA bound crystal states. We propose that the 'expanded linker' between the two DNA-binding domains of Oct4 is an intrinsically disordered region and that its conformational changes have a key role in the recognition/binding to both DNA and importin α. Moreover, we propose that this structural change enables efficient delivery to DNA after release from importin α.

  19. Translationally-controlled tumor protein activates the transcription of Oct-4 in kidney-derived stem cells

    PubMed Central

    Jing, Ying; He, Liang-Liang; Mei, Chang-Lin

    2017-01-01

    The molecular mechanisms underlying translationally-controlled tumor protein (TCTP) in the activation of octamer-binding transcription factor 4 (Oct-4) in kidney-derived stem cells have not been characterized. The aim of the present study was to identify the transcriptional activation of Oct-4 by TCTP in kidney-derived stem cells. Homology-directed repair cDNA inserted into Fisher 344 transgenic (Tg) rats and the mouse strain 129/Svj were used for the experiments. Diphtheria toxin (DT; 10 ng/kg) injected into the Tg rats created the kidney injury, which was rapidly restored by the activation of kidney-derived stem cells. Kidney-derived stem cells were isolated from the DT-injured Tg rats using cell culture techniques. The co-expression of Oct-4 and TCTP were observed in the isolated kidney-derived stem cells. Immunoblotting and reverse transcription-polymerase chain reaction analysis of TCTP null mutant (TCTP−/−) embryos at day 9.5 (E9.5) demonstrated the absence of co-expression of Oct-4 and TCTP, but expression of paired box-2 was detected. This was in contrast with the E9.5 control embryos, which expressed all three proteins. In conclusion, the results of the present study demonstrated that TCTP activates the transcription of Oct-4 in kidney-derived stem cells, as TCTP−/− embryos exhibited knock down of TCTP and Oct-4 without disturbing the expression of Pax-2 The characteristics and functional nature of TCTP in association with Oct-4 in kidney-derived stem cells was identified. PMID:28123502

  20. Expression and significance of S100P, CD147, and OCT4 in different prostate cancer tissue TNM stages.

    PubMed

    Wang, Q; Zhang, J G; Wang, W

    2015-06-18

    The aim of this project was to investigate the expression and significance of S100P, CD147, and OCT4 in prostate cancer tissue at different TNM stages. We enrolled 54 patients with prostate cancer, 40 with benign prostatic hyperplasia, and 20 subjects with normal prostates. S100P, CD147, and OCT4 were detected by immunohistochemistry. The positive rate of S100P detection was 18.52% in prostate cancer tissues, significantly lower than in normal and benign prostate hyperplasia tissues (P ˂ 0.05). The positive expression rate of CD147 and OCT4 were 100 and 77.38% in prostate cancer tissue, respectively, both markedly higher than in normal and benign prostate hyperplasia tissue (P ˂ 0.05). The positive rate of S100P in stage V was 0, which was significantly lower than in stages I (37.50%) and II (35.71%) (P ˂ 0.05). OCT4 expression in stages III (86.67%) and V (94.12%) was higher than in stage I (37.50%). The positive rate of S100P in patients with distant metastasis was 4%, which was significantly lower than that in patients without metastases (P ˂ 0.05). In contrast, the positive rate of OCT4 in patients with distant metastasis was 92%. S100P, CD147, and OCT4 expression in prostate cancer patients with different degrees of differentiation had no significant difference (P > 0.05). Overall, our results demonstrated that S100P expression in prostate cancer tissue was significantly decreased, whereas CD147 and OCT4 expression was increased. Their expression levels were closely associated with TNM stage and distant metastasis, but were not related to the degree of differentiation.

  1. Translationally-controlled tumor protein activates the transcription of Oct-4 in kidney-derived stem cells.

    PubMed

    Jing, Ying; He, Liang-Liang; Mei, Chang-Lin

    2017-01-01

    The molecular mechanisms underlying translationally-controlled tumor protein (TCTP) in the activation of octamer-binding transcription factor 4 (Oct-4) in kidney-derived stem cells have not been characterized. The aim of the present study was to identify the transcriptional activation of Oct-4 by TCTP in kidney-derived stem cells. Homology-directed repair cDNA inserted into Fisher 344 transgenic (Tg) rats and the mouse strain 129/Svj were used for the experiments. Diphtheria toxin (DT; 10 ng/kg) injected into the Tg rats created the kidney injury, which was rapidly restored by the activation of kidney-derived stem cells. Kidney-derived stem cells were isolated from the DT-injured Tg rats using cell culture techniques. The co-expression of Oct-4 and TCTP were observed in the isolated kidney-derived stem cells. Immunoblotting and reverse transcription-polymerase chain reaction analysis of TCTP null mutant (TCTP(-)/(-)) embryos at day 9.5 (E9.5) demonstrated the absence of co-expression of Oct-4 and TCTP, but expression of paired box-2 was detected. This was in contrast with the E9.5 control embryos, which expressed all three proteins. In conclusion, the results of the present study demonstrated that TCTP activates the transcription of Oct-4 in kidney-derived stem cells, as TCTP(-)/(-) embryos exhibited knock down of TCTP and Oct-4 without disturbing the expression of Pax-2 The characteristics and functional nature of TCTP in association with Oct-4 in kidney-derived stem cells was identified.

  2. [Enzymatic regulatory processes in gene recombination].

    PubMed

    Kovarskiĭ, V A; Profir, A V

    1988-01-01

    Recombination bistability in the system of genetic regulation in pro- and eucaryots is analysed on the basis of sigmoid kinetics of regulatory enzymes. It is shown that under an increase of either exogenic factors (temperature) or endogenic factors (concentration of molecules, which activate the enzymes) of crucial values, bistability solutions for recombination frequencies are possible. Histeresic character of the dependence of this value on the external parameters is pointed out. The role of fluctuation processes in distortion of the memory effects is discussed. On the basis of monostable solutions molecular account for the empiric Plau law is given for U-shaped dependence of recombination frequency on temperature.

  3. A Maize Gene Regulatory Network for Phenolic Metabolism.

    PubMed

    Yang, Fan; Li, Wei; Jiang, Nan; Yu, Haidong; Morohashi, Kengo; Ouma, Wilberforce Zachary; Morales-Mantilla, Daniel E; Gomez-Cano, Fabio Andres; Mukundi, Eric; Prada-Salcedo, Luis Daniel; Velazquez, Roberto Alers; Valentin, Jasmin; Mejía-Guerra, Maria Katherine; Gray, John; Doseff, Andrea I; Grotewold, Erich

    2017-03-06

    The translation of the genotype into phenotype, represented for example by the expression of genes encoding enzymes required for the biosynthesis of phytochemicals that are important for interaction of plants with the environment, is largely carried out by transcription factors (TFs) that recognize specific cis-regulatory elements in the genes that they control. TFs and their target genes are organized in gene regulatory networks (GRNs), and thus uncovering GRN architecture presents an important biological challenge necessary to explain gene regulation. Linking TFs to the genes they control, central to understanding GRNs, can be carried out using gene- or TF-centered approaches. In this study, we employed a gene-centered approach utilizing the yeast one-hybrid assay to generate a network of protein-DNA interactions that participate in the transcriptional control of genes involved in the biosynthesis of maize phenolic compounds including general phenylpropanoids, lignins, and flavonoids. We identified 1100 protein-DNA interactions involving 54 phenolic gene promoters and 568 TFs. A set of 11 TFs recognized 10 or more promoters, suggesting a role in coordinating pathway gene expression. The integration of the gene-centered network with information derived from TF-centered approaches provides a foundation for a phenolics GRN characterized by interlaced feed-forward loops that link developmental regulators with biosynthetic genes.

  4. A gene regulatory network controlling the embryonic specification of endoderm.

    PubMed

    Peter, Isabelle S; Davidson, Eric H

    2011-05-29

    Specification of endoderm is the prerequisite for gut formation in the embryogenesis of bilaterian organisms. Modern lineage labelling studies have shown that in the sea urchin embryo model system, descendants of the veg1 and veg2 cell lineages produce the endoderm, and that the veg2 lineage also gives rise to mesodermal cell types. It is known that Wnt/β-catenin signalling is required for endoderm specification and Delta/Notch signalling is required for mesoderm specification. Some direct cis-regulatory targets of these signals have been found and various phenomenological patterns of gene expression have been observed in the pre-gastrular endomesoderm. However, no comprehensive, causal explanation of endoderm specification has been conceived for sea urchins, nor for any other deuterostome. Here we propose a model, on the basis of the underlying genomic control system, that provides such an explanation, built at several levels of biological organization. The hardwired core of the control system consists of the cis-regulatory apparatus of endodermal regulatory genes, which determine the relationship between the inputs to which these genes are exposed and their outputs. The architecture of the network circuitry controlling the dynamic process of endoderm specification then explains, at the system level, a sequence of developmental logic operations, which generate the biological process. The control system initiates non-interacting endodermal and mesodermal gene regulatory networks in veg2-derived cells and extinguishes the endodermal gene regulatory network in mesodermal precursors. It also generates a cross-regulatory network that specifies future anterior endoderm in veg2 descendants and institutes a distinct network specifying posterior endoderm in veg1-derived cells. The network model provides an explanatory framework that relates endoderm specification to the genomic regulatory code.

  5. Differential Role for Transcription Factor Oct4 Nucleocytoplasmic Dynamics in Somatic Cell Reprogramming and Self-renewal of Embryonic Stem Cells*

    PubMed Central

    Oka, Masahiro; Moriyama, Tetsuji; Asally, Munehiro; Kawakami, Koichi; Yoneda, Yoshihiro

    2013-01-01

    Oct4 is a member of the POU family of transcription factors and plays a critical role in both maintenance of the undifferentiated state of embryonic stem (ES) cells and in the reprogramming of somatic cells to induced pluripotent stem cells. Oct4 is imported into the nucleus where it functions as a transcription factor; however, the spatiotemporal dynamic behavior of Oct4 remains largely unknown. In the present study we show that Oct4 is a nucleocytoplasmic shuttling protein. Furthermore, although Oct4 mutants with altered nuclear import/export activity were able to maintain the self-renewal of ES cells, they displayed limited potential for cellular reprogramming. These results indicate that the intracellular localization of Oct4, which is dependent on nucleocytoplasmic shuttling, must be more strictly regulated for cellular reprogramming, suggesting that Oct4 plays differential roles in the self-renewal of ES cells and in somatic cell reprogramming. PMID:23580657

  6. Regulatory Divergence among Beta-Keratin Genes during Bird Evolution.

    PubMed

    Bhattacharjee, Maloyjo Joyraj; Yu, Chun-Ping; Lin, Jinn-Jy; Ng, Chen Siang; Wang, Tzi-Yuan; Lin, Hsin-Hung; Li, Wen-Hsiung

    2016-11-01

    Feathers, which are mainly composed of α- and β-keratins, are highly diversified, largely owing to duplication and diversification of β-keratin genes during bird evolution. However, little is known about the regulatory changes that contributed to the expressional diversification of β-keratin genes. To address this issue, we studied transcriptomes from five different parts of chicken contour and flight feathers. From these transcriptomes we inferred β-keratin enriched co-expression modules of genes and predicted transcription factors (TFs) of β-keratin genes. In total, we predicted 262 TF-target gene relationships in which 56 TFs regulate 91 β-keratin genes; we validated 14 of them by in vitro tests. A dual criterion of TF enrichment and "TF-target gene" expression correlation identified 26 TFs as the major regulators of β-keratin genes. According to our predictions, the ancestral scale and claw β-keratin genes have common and unique regulators, whereas most feather β-keratin genes show chromosome-wise regulation, distinct from scale and claw β-keratin genes. Thus, after expansion from the β-keratin gene on Chr7 to other chromosomes, which still shares a TF with scale and claw β-keratin genes, most feather β-keratin genes have recruited distinct or chromosome-specific regulators. Moreover, our data showed correlated gene expression profiles, positive or negative, between predicted TFs and their target genes over the five studied feather regions. Therefore, regulatory divergences among feather β-keratin genes have contributed to structural differences among different parts of feathers. Our study sheds light on how feather β-keratin genes have diverged in regulation from scale and claw β-keratin genes and among themselves. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  7. Gene Regulatory Evolution During Speciation in a Songbird

    PubMed Central

    Davidson, John H.; Balakrishnan, Christopher N.

    2016-01-01

    Over the last decade, tremendous progress has been made toward a comparative understanding of gene regulatory evolution. However, we know little about how gene regulation evolves in birds, and how divergent genomes interact in their hybrids. Because of the unique features of birds – female heterogamety, a highly conserved karyotype, and the slow evolution of reproductive incompatibilities – an understanding of regulatory evolution in birds is critical to a comprehensive understanding of regulatory evolution and its implications for speciation. Using a novel complement of analyses of replicated RNA-seq libraries, we demonstrate abundant divergence in brain gene expression between zebra finch (Taeniopygia guttata) subspecies. By comparing parental populations and their F1 hybrids, we also show that gene misexpression is relatively rare among brain-expressed transcripts in male birds. If this pattern is consistent across tissues and sexes, it may partially explain the slow buildup of postzygotic reproductive isolation observed in birds relative to other taxa. Although we expected that the action of genetic drift on the island-dwelling zebra finch subspecies would be manifested in a higher rate of trans regulatory divergence, we found that most divergence was in cis regulation, following a pattern commonly observed in other taxa. Thus, our study highlights both unique and shared features of avian regulatory evolution. PMID:26976438

  8. Regulatory links between imprinted genes: evolutionary predictions and consequences.

    PubMed

    Patten, Manus M; Cowley, Michael; Oakey, Rebecca J; Feil, Robert

    2016-02-10

    Genomic imprinting is essential for development and growth and plays diverse roles in physiology and behaviour. Imprinted genes have traditionally been studied in isolation or in clusters with respect to cis-acting modes of gene regulation, both from a mechanistic and evolutionary point of view. Recent studies in mammals, however, reveal that imprinted genes are often co-regulated and are part of a gene network involved in the control of cellular proliferation and differentiation. Moreover, a subset of imprinted genes acts in trans on the expression of other imprinted genes. Numerous studies have modulated levels of imprinted gene expression to explore phenotypic and gene regulatory consequences. Increasingly, the applied genome-wide approaches highlight how perturbation of one imprinted gene may affect other maternally or paternally expressed genes. Here, we discuss these novel findings and consider evolutionary theories that offer a rationale for such intricate interactions among imprinted genes. An evolutionary view of these trans-regulatory effects provides a novel interpretation of the logic of gene networks within species and has implications for the origin of reproductive isolation between species.

  9. Regulatory links between imprinted genes: evolutionary predictions and consequences

    PubMed Central

    Patten, Manus M.; Cowley, Michael; Oakey, Rebecca J.; Feil, Robert

    2016-01-01

    Genomic imprinting is essential for development and growth and plays diverse roles in physiology and behaviour. Imprinted genes have traditionally been studied in isolation or in clusters with respect to cis-acting modes of gene regulation, both from a mechanistic and evolutionary point of view. Recent studies in mammals, however, reveal that imprinted genes are often co-regulated and are part of a gene network involved in the control of cellular proliferation and differentiation. Moreover, a subset of imprinted genes acts in trans on the expression of other imprinted genes. Numerous studies have modulated levels of imprinted gene expression to explore phenotypic and gene regulatory consequences. Increasingly, the applied genome-wide approaches highlight how perturbation of one imprinted gene may affect other maternally or paternally expressed genes. Here, we discuss these novel findings and consider evolutionary theories that offer a rationale for such intricate interactions among imprinted genes. An evolutionary view of these trans-regulatory effects provides a novel interpretation of the logic of gene networks within species and has implications for the origin of reproductive isolation between species. PMID:26842569

  10. Dynamics of gene regulatory networks with cell division cycle

    NASA Astrophysics Data System (ADS)

    Chen, Luonan; Wang, Ruiqi; Kobayashi, Tetsuya J.; Aihara, Kazuyuki

    2004-07-01

    This paper focuses on modeling and analyzing the nonlinear dynamics of gene regulatory networks with the consideration of a cell division cycle with duplication process of DNA , in particular for switches and oscillators of synthetic networks. We derive two models that may correspond to the eukaryotic and prokaryotic cells, respectively. A biologically plausible three-gene model ( lac,tetR , and cI ) and a repressilator as switch and oscillator examples are used to illustrate our theoretical results. We show that the cell cycle may play a significant role in gene regulation due to the nonlinear dynamics of a gene regulatory network although gene expressions are usually tightly controlled by transcriptional factors.

  11. Gene regulatory networks elucidating huanglongbing disease mechanisms.

    PubMed

    Martinelli, Federico; Reagan, Russell L; Uratsu, Sandra L; Phu, My L; Albrecht, Ute; Zhao, Weixiang; Davis, Cristina E; Bowman, Kim D; Dandekar, Abhaya M

    2013-01-01

    Next-generation sequencing was exploited to gain deeper insight into the response to infection by Candidatus liberibacter asiaticus (CaLas), especially the immune disregulation and metabolic dysfunction caused by source-sink disruption. Previous fruit transcriptome data were compared with additional RNA-Seq data in three tissues: immature fruit, and young and mature leaves. Four categories of orchard trees were studied: symptomatic, asymptomatic, apparently healthy, and healthy. Principal component analysis found distinct expression patterns between immature and mature fruits and leaf samples for all four categories of trees. A predicted protein - protein interaction network identified HLB-regulated genes for sugar transporters playing key roles in the overall plant responses. Gene set and pathway enrichment analyses highlight the role of sucrose and starch metabolism in disease symptom development in all tissues. HLB-regulated genes (glucose-phosphate-transporter, invertase, starch-related genes) would likely determine the source-sink relationship disruption. In infected leaves, transcriptomic changes were observed for light reactions genes (downregulation), sucrose metabolism (upregulation), and starch biosynthesis (upregulation). In parallel, symptomatic fruits over-expressed genes involved in photosynthesis, sucrose and raffinose metabolism, and downregulated starch biosynthesis. We visualized gene networks between tissues inducing a source-sink shift. CaLas alters the hormone crosstalk, resulting in weak and ineffective tissue-specific plant immune responses necessary for bacterial clearance. Accordingly, expression of WRKYs (including WRKY70) was higher in fruits than in leaves. Systemic acquired responses were inadequately activated in young leaves, generally considered the sites where most new infections occur.

  12. Over-expression of Oct4 and Sox2 transcription factors enhances differentiation of human umbilical cord blood cells in vivo

    SciTech Connect

    Guseva, Daria; Rizvanov, Albert A.; Salafutdinov, Ilnur I.; Kudryashova, Nezhdana V.; Palotás, András; Islamov, Rustem R.

    2014-09-05

    Highlights: • Gene and cell-based therapies comprise innovative aspects of regenerative medicine. • Genetically modified hUCB-MCs enhanced differentiation of cells in a mouse model of ALS. • Stem cells successfully transformed into micro-glial and endothelial lines in spinal cords. • Over-expressing oct4 and sox2 also induced production of neural marker PGP9.5. • Formation of new nerve cells, secreting trophic factors and neo-vascularisation could improve symptoms in ALS. - Abstract: Gene and cell-based therapies comprise innovative aspects of regenerative medicine. Even though stem cells represent a highly potential therapeutic strategy, their wide-spread exploitation is marred by ethical concerns, potential for malignant transformation and a plethora of other technical issues, largely restricting their use to experimental studies. Utilizing genetically modified human umbilical cord blood mono-nuclear cells (hUCB-MCs), this communication reports enhanced differentiation of transplants in a mouse model of amyotrophic lateral sclerosis (ALS). Over-expressing Oct4 and Sox2 induced production of neural marker PGP9.5, as well as transformation of hUCB-MCs into micro-glial and endothelial lines in ALS spinal cords. In addition to producing new nerve cells, providing degenerated areas with trophic factors and neo-vascularisation might prevent and even reverse progressive loss of moto-neurons and skeletal muscle paralysis.

  13. CpG oligodeoxyribonucleotide 7909 enhances radiosensitivity via downregulating Oct-4 expression in radioresistant lung cancer cells

    PubMed Central

    Xing, Na; Qiao, Tiankui; Zhuang, Xibing; Yuan, Sujuan; Zhang, Qi; Xu, Guoxiong

    2015-01-01

    Radiotherapy is a powerful cure for local advanced non-small cell lung cancer. However, radioresistance and tumor relapse still occur in a high proportion of patients. Octamer-4 (Oct-4), a transcription factor of the POU family, plays a key role in maintaining chemoradioresistant properties and regulating cancer progression. In this study, we demonstrated that Oct-4 expression was significantly increased in radioresistant H460 (H460R) cell line. CpG oligodeoxyribonucleotide (CpG-ODN) 7909 sensitized H460R cells when combined with irradiation treatment. The clonogenic capacity was significantly decreased, and the values of D0 and Dq were lower than those of irradiation alone group. The sensitive enhancement ratio (SER) of D0 was 1.224. This combined treatment led to a dramatic reduction in Oct-4 expression in a dose-dependent manner and also showed increased percentage of cells in the radiosensitive G2/M phase relative to either treatment alone. These results identified that Oct-4 was involved in radioresistance. CpG-ODN 7909 could enhance radiosensitivity partly through downregulating Oct-4 expression in radioresistant lung cancer cells. PMID:26109868

  14. CpG oligodeoxyribonucleotide 7909 enhances radiosensitivity via downregulating Oct-4 expression in radioresistant lung cancer cells.

    PubMed

    Xing, Na; Qiao, Tiankui; Zhuang, Xibing; Yuan, Sujuan; Zhang, Qi; Xu, Guoxiong

    2015-01-01

    Radiotherapy is a powerful cure for local advanced non-small cell lung cancer. However, radioresistance and tumor relapse still occur in a high proportion of patients. Octamer-4 (Oct-4), a transcription factor of the POU family, plays a key role in maintaining chemoradioresistant properties and regulating cancer progression. In this study, we demonstrated that Oct-4 expression was significantly increased in radioresistant H460 (H460R) cell line. CpG oligodeoxyribonucleotide (CpG-ODN) 7909 sensitized H460R cells when combined with irradiation treatment. The clonogenic capacity was significantly decreased, and the values of D0 and Dq were lower than those of irradiation alone group. The sensitive enhancement ratio (SER) of D0 was 1.224. This combined treatment led to a dramatic reduction in Oct-4 expression in a dose-dependent manner and also showed increased percentage of cells in the radiosensitive G2/M phase relative to either treatment alone. These results identified that Oct-4 was involved in radioresistance. CpG-ODN 7909 could enhance radiosensitivity partly through downregulating Oct-4 expression in radioresistant lung cancer cells.

  15. Evolutionary conservation of the eumetazoan gene regulatory landscape

    PubMed Central

    Schwaiger, Michaela; Schönauer, Anna; Rendeiro, André F.; Pribitzer, Carina; Schauer, Alexandra; Gilles, Anna F.; Schinko, Johannes B.; Renfer, Eduard; Fredman, David; Technau, Ulrich

    2014-01-01

    Despite considerable differences in morphology and complexity of body plans among animals, a great part of the gene set is shared among Bilateria and their basally branching sister group, the Cnidaria. This suggests that the common ancestor of eumetazoans already had a highly complex gene repertoire. At present it is therefore unclear how morphological diversification is encoded in the genome. Here we address the possibility that differences in gene regulation could contribute to the large morphological divergence between cnidarians and bilaterians. To this end, we generated the first genome-wide map of gene regulatory elements in a nonbilaterian animal, the sea anemone Nematostella vectensis. Using chromatin immunoprecipitation followed by deep sequencing of five chromatin modifications and a transcriptional cofactor, we identified over 5000 enhancers in the Nematostella genome and could validate 75% of the tested enhancers in vivo. We found that in Nematostella, but not in yeast, enhancers are characterized by the same combination of histone modifications as in bilaterians, and these enhancers preferentially target developmental regulatory genes. Surprisingly, the distribution and abundance of gene regulatory elements relative to these genes are shared between Nematostella and bilaterian model organisms. Our results suggest that complex gene regulation originated at least 600 million yr ago, predating the common ancestor of eumetazoans. PMID:24642862

  16. Dynamic integration of splicing within gene regulatory pathways

    PubMed Central

    Braunschweig, Ulrich; Gueroussov, Serge; Plocik, Alex; Graveley, Brenton R.; Blencowe, Benjamin J.

    2013-01-01

    Precursor mRNA splicing is one of the most highly regulated processes in metazoan species. In addition to generating vast repertoires of RNAs and proteins, splicing has a profound impact on other gene regulatory layers, including mRNA transcription, turnover, transport and translation. Conversely, factors regulating chromatin and transcription complexes impact the splicing process. This extensive cross-talk between gene regulatory layers takes advantage of dynamic spatial, physical and temporal organizational properties of the cell nucleus, and further emphasizes the importance of developing a multidimensional understanding of splicing control. PMID:23498935

  17. Variable neighborhood search for reverse engineering of gene regulatory networks.

    PubMed

    Nicholson, Charles; Goodwin, Leslie; Clark, Corey

    2017-01-01

    A new search heuristic, Divided Neighborhood Exploration Search, designed to be used with inference algorithms such as Bayesian networks to improve on the reverse engineering of gene regulatory networks is presented. The approach systematically moves through the search space to find topologies representative of gene regulatory networks that are more likely to explain microarray data. In empirical testing it is demonstrated that the novel method is superior to the widely employed greedy search techniques in both the quality of the inferred networks and computational time.

  18. Learning gene regulatory networks from next generation sequencing data.

    PubMed

    Jia, Bochao; Xu, Suwa; Xiao, Guanghua; Lamba, Vishal; Liang, Faming

    2017-03-10

    In recent years, next generation sequencing (NGS) has gradually replaced microarray as the major platform in measuring gene expressions. Compared to microarray, NGS has many advantages, such as less noise and higher throughput. However, the discreteness of NGS data also challenges the existing statistical methodology. In particular, there still lacks an appropriate statistical method for reconstructing gene regulatory networks using NGS data in the literature. The existing local Poisson graphical model method is not consistent and can only infer certain local structures of the network. In this article, we propose a random effect model-based transformation to continuize NGS data and then we transform the continuized data to Gaussian via a semiparametric transformation and apply an equivalent partial correlation selection method to reconstruct gene regulatory networks. The proposed method is consistent. The numerical results indicate that the proposed method can lead to much more accurate inference of gene regulatory networks than the local Poisson graphical model and other existing methods. The proposed data-continuized transformation fills the theoretical gap for how to transform discrete data to continuous data and facilitates NGS data analysis. The proposed data-continuized transformation also makes it feasible to integrate different types of data, such as microarray and RNA-seq data, in reconstruction of gene regulatory networks.

  19. Follicular progesterone concentrations and messenger RNA expression of MATER and OCT-4 in immature bovine oocytes as predictors of developmental competence.

    PubMed

    Urrego, R; Herrera-Puerta, E; Chavarria, N A; Camargo, O; Wrenzycki, C; Rodriguez-Osorio, N

    2015-04-15

    The ability of bovine embryos to develop to the blastocyst stage and to implant and generate healthy offspring depends greatly on the competence of the oocyte. Oocyte competence is attributed to its close communication with the follicular environment and to its capacity to synthesize and store substantial amounts of messenger RNA. Higher developmental competence of bovine oocytes has been associated with both the expression of a cohort of developmental genes and the concentration of sex steroids in the follicular fluid. The aim of this study was to identify differences in the expression of FST in cumulus cells and OCT-4 and MATER in oocytes and the influence of the follicular progesterone and follicular estrogen concentration on the competence of bovine oocytes retrieved 30 minutes or 4 hours after slaughter. Cumulus-oocyte complexes (COCs) were left in postmortem ovaries for 30 minutes (group I) or 4 hours (group II) at 30 °C. Aspirated oocytes were then subjected to IVM, IVF, and IVC or were evaluated for MATER and OCT-4 messenger RNA abundance by quantitative real-time polymerase chain reaction. Total RNA was isolated from pools of 100 oocytes for each experimental replicate. Progesterone and estradiol concentration in follicular fluid was evaluated by immunoassay using an IMMULITE 2000 analyzer. Three repeats of in vitro embryo production were performed with a total of 455 (group I) and 470 (group II) COCs. There were no significant differences between the cleavage rates (72 hours postinsemination [hpi]) between both groups (63.5% vs. 69.1%). However, blastocyst (168 hpi) and hatching (216 hpi) rates were higher (P < 0.05) in group II compared with those of group I (21.3% vs. 30.7% and 27.6% vs. 51.5%, respectively). Group II oocytes exhibited the highest MATER and OCT-4 abundance (P < 0.05). Follicular estradiol concentration was not different between both the groups, whereas the progesterone concentration was lower (P ≤ 0.05) in group II follicles. These

  20. [Regulatory functions of Pax gene family in Drosophila development].

    PubMed

    Li, Li; Yang, Yang; Xue, Lei

    2010-02-01

    The Pax gene family encodes a group of important transcription factors that have been evolutionary conserved from Drosophila to human. Pax genes play pivotal roles in regulating diverse signal transduction pathways and organogenesis during embryonic development through modulating cell proliferation and self-renewal, embryonic precursor cell migration, and the coordination of specific differentiation programs. Ten members of the Pax gene family, which perform crucial regulatory functions during embryonic and postembryonic development, have been identified in Drosophila. In this report, we described the protein structures, expression patterns, and main functions of Drosophila Pax genes.

  1. The incorporation of epigenetics in artificial gene regulatory networks.

    PubMed

    Turner, Alexander P; Lones, Michael A; Fuente, Luis A; Stepney, Susan; Caves, Leo S D; Tyrrell, Andy M

    2013-05-01

    Artificial gene regulatory networks are computational models that draw inspiration from biological networks of gene regulation. Since their inception they have been used to infer knowledge about gene regulation and as methods of computation. These computational models have been shown to possess properties typically found in the biological world, such as robustness and self organisation. Recently, it has become apparent that epigenetic mechanisms play an important role in gene regulation. This paper describes a new model, the Artificial Epigenetic Regulatory Network (AERN) which builds upon existing models by adding an epigenetic control layer. Our results demonstrate that AERNs are more adept at controlling multiple opposing trajectories when applied to a chaos control task within a conservative dynamical system, suggesting that AERNs are an interesting area for further investigation.

  2. Charting gene regulatory networks: strategies, challenges and perspectives

    PubMed Central

    2004-01-01

    One of the foremost challenges in the post-genomic era will be to chart the gene regulatory networks of cells, including aspects such as genome annotation, identification of cis-regulatory elements and transcription factors, information on protein–DNA and protein–protein interactions, and data mining and integration. Some of these broad sets of data have already been assembled for building networks of gene regulation. Even though these datasets are still far from comprehensive, and the approach faces many important and difficult challenges, some strategies have begun to make connections between disparate regulatory events and to foster new hypotheses. In this article we review several different genomics and proteomics technologies, and present bioinformatics methods for exploring these data in order to make novel discoveries. PMID:15080794

  3. Transient Downregulation of Nanog and Oct4 Induced by DETA/NO Exposure in Mouse Embryonic Stem Cells Leads to Mesodermal/Endodermal Lineage Differentiation.

    PubMed

    Mora-Castilla, Sergio; Tejedo, Juan R; Tapia-Limonchi, Rafael; Díaz, Irene; Hitos, Ana B; Cahuana, Gladys M; Hmadcha, Abdelkrim; Martín, Franz; Soria, Bernat; Bedoya, Francisco J

    2014-01-01

    The function of pluripotency genes in differentiation is a matter of investigation. We report here that Nanog and Oct4 are reexpressed in two mouse embryonic stem cell (mESC) lines following exposure to the differentiating agent DETA/NO. Both cell lines express a battery of both endoderm and mesoderm markers following induction of differentiation with DETA/NO-based protocols. Confocal analysis of cells undergoing directed differentiation shows that the majority of cells expressing Nanog express also endoderm genes such as Gata4 and FoxA2 (75.4% and 96.2%, resp.). Simultaneously, mRNA of mesodermal markers Flk1 and Mef2c are also regulated by the treatment. Acetylated histone H3 occupancy at the promoter of Nanog is involved in the process of reexpression. Furthermore, Nanog binding to the promoter of Brachyury leads to repression of this gene, thus disrupting mesendoderm transition.

  4. A gene regulatory network armature for T-lymphocyte specification

    SciTech Connect

    Fung, Elizabeth-sharon

    2008-01-01

    Choice of a T-lymphoid fate by hematopoietic progenitor cells depends on sustained Notch-Delta signaling combined with tightly-regulated activities of multiple transcription factors. To dissect the regulatory network connections that mediate this process, we have used high-resolution analysis of regulatory gene expression trajectories from the beginning to the end of specification; tests of the short-term Notchdependence of these gene expression changes; and perturbation analyses of the effects of overexpression of two essential transcription factors, namely PU.l and GATA-3. Quantitative expression measurements of >50 transcription factor and marker genes have been used to derive the principal components of regulatory change through which T-cell precursors progress from primitive multipotency to T-lineage commitment. Distinct parts of the path reveal separate contributions of Notch signaling, GATA-3 activity, and downregulation of PU.l. Using BioTapestry, the results have been assembled into a draft gene regulatory network for the specification of T-cell precursors and the choice of T as opposed to myeloid dendritic or mast-cell fates. This network also accommodates effects of E proteins and mutual repression circuits of Gfil against Egr-2 and of TCF-l against PU.l as proposed elsewhere, but requires additional functions that remain unidentified. Distinctive features of this network structure include the intense dose-dependence of GATA-3 effects; the gene-specific modulation of PU.l activity based on Notch activity; the lack of direct opposition between PU.l and GATA-3; and the need for a distinct, late-acting repressive function or functions to extinguish stem and progenitor-derived regulatory gene expression.

  5. Angiotensin II-regulated transcription regulatory genes in adrenal steroidogenesis.

    PubMed

    Romero, Damian G; Gomez-Sanchez, Elise P; Gomez-Sanchez, Celso E

    2010-11-29

    Transcription regulatory genes are crucial modulators of cell physiology and metabolism whose intracellular levels are tightly controlled in response to extracellular stimuli. We previously reported a set of 29 transcription regulatory genes modulated by angiotensin II in H295R human adrenocortical cells and their roles in regulating the expression of the last and unique enzymes of the glucocorticoid and mineralocorticoid biosynthetic pathways, 11β-hydroxylase and aldosterone synthase, respectively, using gene expression reporter assays. To study the effect of this set of transcription regulatory genes on adrenal steroidogenesis, H295R cells were transfected by high-efficiency nucleofection and aldosterone and cortisol were measured in cell culture supernatants under basal and angiotensin II-stimulated conditions. BCL11B, BHLHB2, CITED2, ELL2, HMGA1, MAFF, NFIL3, PER1, SERTAD1, and VDR significantly stimulated aldosterone secretion, while EGR1, FOSB, and ZFP295 decreased aldosterone secretion. BTG2, HMGA1, MITF, NR4A1, and ZFP295 significantly increased cortisol secretion, while BCL11B, NFIL3, PER1, and SIX2 decreased cortisol secretion. We also report the effect of some of these regulators on the expression of endogenous aldosterone synthase and 11β-hydroxylase under basal and angiotensin II-stimulated conditions. In summary, this study reports for the first time the effects of a set of angiotensin II-modulated transcription regulatory genes on aldosterone and cortisol secretion and the expression levels of the last and unique enzymes of the mineralocorticoid and glucocorticoid biosynthetic pathways. Abnormal regulation of mineralocorticoid or glucocorticoid secretion is involved in several pathophysiological conditions. These transcription regulatory genes may be involved in adrenal steroidogenesis pathologies; thus they merit additional study as potential candidates for therapeutic intervention.

  6. Combinatorial Gene Regulatory Functions Underlie Ultraconserved Elements in Drosophila

    PubMed Central

    Warnefors, Maria; Hartmann, Britta; Thomsen, Stefan; Alonso, Claudio R.

    2016-01-01

    Ultraconserved elements (UCEs) are discrete genomic elements conserved across large evolutionary distances. Although UCEs have been linked to multiple facets of mammalian gene regulation their extreme evolutionary conservation remains largely unexplained. Here, we apply a computational approach to investigate this question in Drosophila, exploring the molecular functions of more than 1,500 UCEs shared across the genomes of 12 Drosophila species. Our data indicate that Drosophila UCEs are hubs for gene regulatory functions and suggest that UCE sequence invariance originates from their combinatorial roles in gene control. We also note that the gene regulatory roles of intronic and intergenic UCEs (iUCEs) are distinct from those found in exonic UCEs (eUCEs). In iUCEs, transcription factor (TF) and epigenetic factor binding data strongly support iUCE roles in transcriptional and epigenetic regulation. In contrast, analyses of eUCEs indicate that they are two orders of magnitude more likely than the expected to simultaneously include protein-coding sequence, TF-binding sites, splice sites, and RNA editing sites but have reduced roles in transcriptional or epigenetic regulation. Furthermore, we use a Drosophila cell culture system and transgenic Drosophila embryos to validate the notion of UCE combinatorial regulatory roles using an eUCE within the Hox gene Ultrabithorax and show that its protein-coding region also contains alternative splicing regulatory information. Taken together our experiments indicate that UCEs emerge as a result of combinatorial gene regulatory roles and highlight common features in mammalian and insect UCEs implying that similar processes might underlie ultraconservation in diverse animal taxa. PMID:27247329

  7. Combinatorial Gene Regulatory Functions Underlie Ultraconserved Elements in Drosophila.

    PubMed

    Warnefors, Maria; Hartmann, Britta; Thomsen, Stefan; Alonso, Claudio R

    2016-09-01

    Ultraconserved elements (UCEs) are discrete genomic elements conserved across large evolutionary distances. Although UCEs have been linked to multiple facets of mammalian gene regulation their extreme evolutionary conservation remains largely unexplained. Here, we apply a computational approach to investigate this question in Drosophila, exploring the molecular functions of more than 1,500 UCEs shared across the genomes of 12 Drosophila species. Our data indicate that Drosophila UCEs are hubs for gene regulatory functions and suggest that UCE sequence invariance originates from their combinatorial roles in gene control. We also note that the gene regulatory roles of intronic and intergenic UCEs (iUCEs) are distinct from those found in exonic UCEs (eUCEs). In iUCEs, transcription factor (TF) and epigenetic factor binding data strongly support iUCE roles in transcriptional and epigenetic regulation. In contrast, analyses of eUCEs indicate that they are two orders of magnitude more likely than the expected to simultaneously include protein-coding sequence, TF-binding sites, splice sites, and RNA editing sites but have reduced roles in transcriptional or epigenetic regulation. Furthermore, we use a Drosophila cell culture system and transgenic Drosophila embryos to validate the notion of UCE combinatorial regulatory roles using an eUCE within the Hox gene Ultrabithorax and show that its protein-coding region also contains alternative splicing regulatory information. Taken together our experiments indicate that UCEs emerge as a result of combinatorial gene regulatory roles and highlight common features in mammalian and insect UCEs implying that similar processes might underlie ultraconservation in diverse animal taxa.

  8. Oct-4 Expression Maintained Cancer Stem-Like Properties in Lung Cancer-Derived CD133-Positive Cells

    PubMed Central

    Tsai, Tung-Hu; How, Chorng-Kuang; Wang, Chien-Ying; Hung, Shih-Chieh; Chang, Yuh-Lih; Tsai, Ming-Long; Lee, Yi-Yen; Ku, Hung-Hai; Chiou, Shih-Hwa

    2008-01-01

    CD133 (prominin-1), a 5-transmembrane glycoprotein, has recently been considered to be an important marker that represents the subset population of cancer stem-like cells. Herein we report the isolation of CD133-positive cells (LC-CD133+) and CD133-negative cells (LC-CD133−) from tissue samples of ten patients with non-small cell lung cancer (LC) and five LC cell lines. LC-CD133+ displayed higher Oct-4 expressions with the ability to self-renew and may represent a reservoir with proliferative potential for generating lung cancer cells. Furthermore, LC-CD133+, unlike LC-CD133−, highly co-expressed the multiple drug-resistant marker ABCG2 and showed significant resistance to chemotherapy agents (i.e., cisplatin, etoposide, doxorubicin, and paclitaxel) and radiotherapy. The treatment of Oct-4 siRNA with lentiviral vector can specifically block the capability of LC-CD133+ to form spheres and can further facilitate LC-CD133+ to differentiate into LC-CD133−. In addition, knock-down of Oct-4 expression in LC-CD133+ can significantly inhibit the abilities of tumor invasion and colony formation, and increase apoptotic activities of caspase 3 and poly (ADP-ribose) polymerase (PARP). Finally, in vitro and in vivo studies further confirm that the treatment effect of chemoradiotherapy for LC-CD133+ can be improved by the treatment of Oct-4 siRNA. In conclusion, we demonstrated that Oct-4 expression plays a crucial role in maintaining the self-renewing, cancer stem-like, and chemoradioresistant properties of LC-CD133+. Future research is warranted regarding the up-regulated expression of Oct-4 in LC-CD133+ and malignant lung cancer. PMID:18612434

  9. Efficient Reverse-Engineering of a Developmental Gene Regulatory Network

    PubMed Central

    Cicin-Sain, Damjan; Ashyraliyev, Maksat; Jaeger, Johannes

    2012-01-01

    Understanding the complex regulatory networks underlying development and evolution of multi-cellular organisms is a major problem in biology. Computational models can be used as tools to extract the regulatory structure and dynamics of such networks from gene expression data. This approach is called reverse engineering. It has been successfully applied to many gene networks in various biological systems. However, to reconstitute the structure and non-linear dynamics of a developmental gene network in its spatial context remains a considerable challenge. Here, we address this challenge using a case study: the gap gene network involved in segment determination during early development of Drosophila melanogaster. A major problem for reverse-engineering pattern-forming networks is the significant amount of time and effort required to acquire and quantify spatial gene expression data. We have developed a simplified data processing pipeline that considerably increases the throughput of the method, but results in data of reduced accuracy compared to those previously used for gap gene network inference. We demonstrate that we can infer the correct network structure using our reduced data set, and investigate minimal data requirements for successful reverse engineering. Our results show that timing and position of expression domain boundaries are the crucial features for determining regulatory network structure from data, while it is less important to precisely measure expression levels. Based on this, we define minimal data requirements for gap gene network inference. Our results demonstrate the feasibility of reverse-engineering with much reduced experimental effort. This enables more widespread use of the method in different developmental contexts and organisms. Such systematic application of data-driven models to real-world networks has enormous potential. Only the quantitative investigation of a large number of developmental gene regulatory networks will allow us to

  10. Gene regulatory networks in the early ascidian embryo.

    PubMed

    Satou, Yutaka; Satoh, Nori; Imai, Kaoru S

    2009-04-01

    Ascidians, or sea squirts, are tunicates that diverged from the vertebrate lineage early in the chordate evolution. The compact and simple organization of the ascidian genome makes this organism an ideal model system for analyzing gene regulatory networks in embryonic development. Embryos contain relatively few cells and gene activities by individual cells have been determined. Here we review and discuss advances in our understanding of the ascidian embryogenesis emerging from genomic expression studies and analyses at the single cell level.

  11. Efficient reverse-engineering of a developmental gene regulatory network.

    PubMed

    Crombach, Anton; Wotton, Karl R; Cicin-Sain, Damjan; Ashyraliyev, Maksat; Jaeger, Johannes

    2012-01-01

    Understanding the complex regulatory networks underlying development and evolution of multi-cellular organisms is a major problem in biology. Computational models can be used as tools to extract the regulatory structure and dynamics of such networks from gene expression data. This approach is called reverse engineering. It has been successfully applied to many gene networks in various biological systems. However, to reconstitute the structure and non-linear dynamics of a developmental gene network in its spatial context remains a considerable challenge. Here, we address this challenge using a case study: the gap gene network involved in segment determination during early development of Drosophila melanogaster. A major problem for reverse-engineering pattern-forming networks is the significant amount of time and effort required to acquire and quantify spatial gene expression data. We have developed a simplified data processing pipeline that considerably increases the throughput of the method, but results in data of reduced accuracy compared to those previously used for gap gene network inference. We demonstrate that we can infer the correct network structure using our reduced data set, and investigate minimal data requirements for successful reverse engineering. Our results show that timing and position of expression domain boundaries are the crucial features for determining regulatory network structure from data, while it is less important to precisely measure expression levels. Based on this, we define minimal data requirements for gap gene network inference. Our results demonstrate the feasibility of reverse-engineering with much reduced experimental effort. This enables more widespread use of the method in different developmental contexts and organisms. Such systematic application of data-driven models to real-world networks has enormous potential. Only the quantitative investigation of a large number of developmental gene regulatory networks will allow us to

  12. Inferring transcription factor collaborations in gene regulatory networks

    PubMed Central

    2014-01-01

    Background Living cells are realized by complex gene expression programs that are moderated by regulatory proteins called transcription factors (TFs). The TFs control the differential expression of target genes in the context of transcriptional regulatory networks (TRNs), either individually or in groups. Deciphering the mechanisms of how the TFs control the expression of target genes is a challenging task, especially when multiple TFs collaboratively participate in the transcriptional regulation. Results We model the underlying regulatory interactions in terms of the directions (activation or repression) and their logical roles (necessary and/or sufficient) with a modified association rule mining approach, called mTRIM. The experiment on Yeast discovered 670 regulatory interactions, in which multiple TFs express their functions on common target genes collaboratively. The evaluation on yeast genetic interactions, TF knockouts and a synthetic dataset shows that our algorithm is significantly better than the existing ones. Conclusions mTRIM is a novel method to infer TF collaborations in transcriptional regulation networks. mTRIM is available at http://www.msu.edu/~jinchen/mTRIM. PMID:24565025

  13. Compartmentalized gene regulatory network of the pathogenic fungus Fusarium graminearum

    USDA-ARS?s Scientific Manuscript database

    Head blight caused by Fusarium graminearum (Fg) is a major limiting factor of wheat production with both yield loss and mycotoxin contamination. Here we report a model for global Fg gene regulatory networks (GRNs) inferred from a large collection of transcriptomic data using a machine-learning appro...

  14. Second order optimization for the inference of gene regulatory pathways.

    PubMed

    Das, Mouli; Murthy, Chivukula A; De, Rajat K

    2014-02-01

    With the increasing availability of experimental data on gene interactions, modeling of gene regulatory pathways has gained special attention. Gradient descent algorithms have been widely used for regression and classification applications. Unfortunately, results obtained after training a model by gradient descent are often highly variable. In this paper, we present a new second order learning rule based on the Newton's method for inferring optimal gene regulatory pathways. Unlike the gradient descent method, the proposed optimization rule is independent of the learning parameter. The flow vectors are estimated based on biomass conservation. A set of constraints is formulated incorporating weighting coefficients. The method calculates the maximal expression of the target gene starting from a given initial gene through these weighting coefficients. Our algorithm has been benchmarked and validated on certain types of functions and on some gene regulatory networks, gathered from literature. The proposed method has been found to perform better than the gradient descent learning. Extensive performance comparison with the extreme pathway analysis method has underlined the effectiveness of our proposed methodology.

  15. TALEN/CRISPR-Mediated eGFP Knock-In Add-On at the OCT4 Locus Does Not Impact Differentiation of Human Embryonic Stem Cells towards Endoderm

    PubMed Central

    Krentz, Nicole A. J.; Nian, Cuilan; Lynn, Francis C.

    2014-01-01

    Human embryonic stem cells (hESCs) have great promise as a source of unlimited transplantable cells for regenerative medicine. However, current progress on producing the desired cell type for disease treatment has been limited due to an insufficient understanding of the developmental processes that govern their differentiation, as well as a paucity of tools to systematically study differentiation in the lab. In order to overcome these limitations, cell-type reporter hESC lines will be required. Here we outline two strategies using Transcription Activator Like Effector Nucleases (TALENs) and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-Associated protein (Cas) to create OCT4-eGFP knock-in add-on hESC lines. Thirty-one and forty-seven percent of clones were correctly modified using the TALEN and CRISPR-Cas9 systems, respectively. Further analysis of three correctly targeted clones demonstrated that the insertion of eGFP in-frame with OCT4 neither significantly impacted expression from the wild type allele nor did the fusion protein have a dramatically different biological stability. Importantly, the OCT4-eGFP fusion was easily detected using microscopy, flow cytometry and western blotting. The OCT4 reporter lines remained equally competent at producing CXCR4+ definitive endoderm that expressed a panel of endodermal genes. Moreover, the genomic modification did not impact the formation of NKX6.1+/SOX9+ pancreatic progenitor cells following directed differentiation. In conclusion, these findings demonstrate for the first time that CRISPR-Cas9 can be used to modify OCT4 and highlight the feasibility of creating cell-type specific reporter hESC lines utilizing genome-editing tools that facilitate homologous recombination. PMID:25474420

  16. Molecular characterization of a maize regulatory gene

    SciTech Connect

    Wessler, S.R.

    1991-12-01

    Based on initial bombardment studies we have previously concluded that promoter diversity was responsible for the diversity of naturally occurring R alleles. During this period we have found that R is controlled at the level of translation initiation and intron 1 is alternatively spliced. The experiments described in Sections 1 and 2 sought to quantify these effects and to determine whether they contribute to the tissue specific expression of select R alleles. This study was done because very little is understood about the post-transcriptional regulation of plant genes. Section 3 and 4 describe experiments designed to identify important structural components of the R protein.

  17. Function does not follow form in gene regulatory circuits

    PubMed Central

    Payne, Joshua L.; Wagner, Andreas

    2015-01-01

    Gene regulatory circuits are to the cell what arithmetic logic units are to the chip: fundamental components of information processing that map an input onto an output. Gene regulatory circuits come in many different forms, distinct structural configurations that determine who regulates whom. Studies that have focused on the gene expression patterns (functions) of circuits with a given structure (form) have examined just a few structures or gene expression patterns. Here, we use a computational model to exhaustively characterize the gene expression patterns of nearly 17 million three-gene circuits in order to systematically explore the relationship between circuit form and function. Three main conclusions emerge. First, function does not follow form. A circuit of any one structure can have between twelve and nearly thirty thousand distinct gene expression patterns. Second, and conversely, form does not follow function. Most gene expression patterns can be realized by more than one circuit structure. And third, multifunctionality severely constrains circuit form. The number of circuit structures able to drive multiple gene expression patterns decreases rapidly with the number of these patterns. These results indicate that it is generally not possible to infer circuit function from circuit form, or vice versa. PMID:26290154

  18. Expression of TAT recombinant Oct4, Sox2, Lin28, and Nanog proteins from baculovirus-infected Sf9 insect cells.

    PubMed

    Pan, Chuanying; Jia, Wenchao; Lu, Baisong; Bishop, Colin E

    2015-02-10

    Somatic cell reprogramming has generated enormous interest, following the first report of generation of induced pluripotent stem cells (iPSCs) from mouse fibroblasts, but the integration of viral transgenes into the genome is unlikely to be accepted. Given these safety considerations, a method for virus-free transient gene expression from suspension-adapted Sf9 insect cells was developed. Here, we expressed transactivator of transcription (TAT)-fused proteins, Sox2, Oct4, Lin28, and Nanog in Sf9 cells using the baculovirus expression vector system (BEVS). The molecular weights of the TAT-Sox2, TAT-Oct4, TAT-Lin28, and TAT-Nanog fusion proteins were 36kD, 40kD, 24kD, and 36kD, respectively. Further investigation indicated that most of the recombinant proteins remained in the nuclei of the Sf9 insect cells and were therefore unavailable for purification and cellular reprogramming. Once this problem has been solved, it seems likely that protein expressed from baculovirus-infected Sf9 insect cells will be the method of choice for cellular reprogramming.

  19. Two-stage induced differentiation of OCT4+/Nanog+ stem-like cells in lung adenocarcinoma.

    PubMed

    Li, Rong; Huang, Jinsu; Ma, Meili; Lou, Yuqing; Zhang, Yanwei; Wu, Lixia; Chang, David W; Zhao, Picheng; Dong, Qianggang; Wu, Xifeng; Han, Baohui

    2016-10-18

    Stem-like cells in solid tumors are purported to contribute to cancer development and poor treatment outcome. The abilities to self-renew, differentiate, and resist anticancer therapies are hallmarks of these rare cells, and steering them into lineage commitment may be one strategy to curb cancer development or progression. Vitamin D is a prohormone that can alter cell growth and differentiation and may induce the differentiation cancer stem-like cells. In this study, octamer-binding transcription factor 4 (OCT4)-positive/Nanog homeobox (Nanog)- positive lung adenocarcinoma stem-like cells (LACSCs) were enriched from spheroid cultured SPC-A1 cells and differentiated by a two-stage induction (TSI) method, which involved knockdown of hypoxia-inducible factor 1-alpha (HIF1α) expression (first stage) followed by sequential induction with 1alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3, VD3) and suberoylanilide hydroxamic acid (SAHA) treatment (second stage). The results showed the HIF1α-knockdowned cells displayed diminished cell invasion and clonogenic activities. Moreover, the TSI cells highly expressed tumor suppressor protein p63 (P63) and forkhead box J1 (FOXJ1) and lost stem cell characteristics, including absent expression of OCT4 and Nanog. These cells regained sensitivity to cisplatin in vitro while losing tumorigenic capacity and decreased tumor cell proliferation in vivo. Our results suggest that induced transdifferentiation of LACSCs by vitamin D and SAHA may become novel therapeutic avenue to alter tumor cell phenotypes and improve patient outcome.The development and progression of lung cancer may involve rare population of stem-like cells that have the ability to grow, differentiate, and resist drug treatment. However, current therapeutic strategies have mostly focused on tumor characteristics and neglected the potential source of cells that may contribute to poor clinical outcome. We generated lung adenocarcinoma stem-like cells from spheroid culture and

  20. Two-stage induced differentiation of OCT4+/Nanog+ stem-like cells in lung adenocarcinoma

    PubMed Central

    Ma, Meili; Lou, Yuqing; Zhang, Yanwei; Wu, Lixia; Chang, David W.; Zhao, Picheng; Dong, Qianggang; Wu, Xifeng; Han, Baohui

    2016-01-01

    Stem-like cells in solid tumors are purported to contribute to cancer development and poor treatment outcome. The abilities to self-renew, differentiate, and resist anticancer therapies are hallmarks of these rare cells, and steering them into lineage commitment may be one strategy to curb cancer development or progression. Vitamin D is a prohormone that can alter cell growth and differentiation and may induce the differentiation cancer stem-like cells. In this study, octamer-binding transcription factor 4 (OCT4)-positive/Nanog homeobox (Nanog)- positive lung adenocarcinoma stem-like cells (LACSCs) were enriched from spheroid cultured SPC-A1 cells and differentiated by a two-stage induction (TSI) method, which involved knockdown of hypoxia-inducible factor 1-alpha (HIF1α) expression (first stage) followed by sequential induction with 1alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3, VD3) and suberoylanilide hydroxamic acid (SAHA) treatment (second stage). The results showed the HIF1α-knockdowned cells displayed diminished cell invasion and clonogenic activities. Moreover, the TSI cells highly expressed tumor suppressor protein p63 (P63) and forkhead box J1 (FOXJ1) and lost stem cell characteristics, including absent expression of OCT4 and Nanog. These cells regained sensitivity to cisplatin in vitro while losing tumorigenic capacity and decreased tumor cell proliferation in vivo. Our results suggest that induced transdifferentiation of LACSCs by vitamin D and SAHA may become novel therapeutic avenue to alter tumor cell phenotypes and improve patient outcome. SIGNIFICANCE STATEMENT The development and progression of lung cancer may involve rare population of stem-like cells that have the ability to grow, differentiate, and resist drug treatment. However, current therapeutic strategies have mostly focused on tumor characteristics and neglected the potential source of cells that may contribute to poor clinical outcome. We generated lung adenocarcinoma stem-like cells from

  1. Inferring slowly-changing dynamic gene-regulatory networks

    PubMed Central

    2015-01-01

    Dynamic gene-regulatory networks are complex since the interaction patterns between their components mean that it is impossible to study parts of the network in separation. This holistic character of gene-regulatory networks poses a real challenge to any type of modelling. Graphical models are a class of models that connect the network with a conditional independence relationships between random variables. By interpreting these random variables as gene activities and the conditional independence relationships as functional non-relatedness, graphical models have been used to describe gene-regulatory networks. Whereas the literature has been focused on static networks, most time-course experiments are designed in order to tease out temporal changes in the underlying network. It is typically reasonable to assume that changes in genomic networks are few, because biological systems tend to be stable. We introduce a new model for estimating slow changes in dynamic gene-regulatory networks, which is suitable for high-dimensional data, e.g. time-course microarray data. Our aim is to estimate a dynamically changing genomic network based on temporal activity measurements of the genes in the network. Our method is based on the penalized likelihood with ℓ1-norm, that penalizes conditional dependencies between genes as well as differences between conditional independence elements across time points. We also present a heuristic search strategy to find optimal tuning parameters. We re-write the penalized maximum likelihood problem into a standard convex optimization problem subject to linear equality constraints. We show that our method performs well in simulation studies. Finally, we apply the proposed model to a time-course T-cell dataset. PMID:25917062

  2. Topological origin of global attractors in gene regulatory networks

    NASA Astrophysics Data System (ADS)

    Zhang, YunJun; Ouyang, Qi; Geng, Zhi

    2015-02-01

    Fixed-point attractors with global stability manifest themselves in a number of gene regulatory networks. This property indicates the stability of regulatory networks against small state perturbations and is closely related to other complex dynamics. In this paper, we aim to reveal the core modules in regulatory networks that determine their global attractors and the relationship between these core modules and other motifs. This work has been done via three steps. Firstly, inspired by the signal transmission in the regulation process, we extract the model of chain-like network from regulation networks. We propose a module of "ideal transmission chain (ITC)", which is proved sufficient and necessary (under certain condition) to form a global fixed-point in the context of chain-like network. Secondly, by examining two well-studied regulatory networks (i.e., the cell-cycle regulatory networks of Budding yeast and Fission yeast), we identify the ideal modules in true regulation networks and demonstrate that the modules have a superior contribution to network stability (quantified by the relative size of the biggest attraction basin). Thirdly, in these two regulation networks, we find that the double negative feedback loops, which are the key motifs of forming bistability in regulation, are connected to these core modules with high network stability. These results have shed new light on the connection between the topological feature and the dynamic property of regulatory networks.

  3. Gap Gene Regulatory Dynamics Evolve along a Genotype Network

    PubMed Central

    Crombach, Anton; Wotton, Karl R.; Jiménez-Guri, Eva; Jaeger, Johannes

    2016-01-01

    Developmental gene networks implement the dynamic regulatory mechanisms that pattern and shape the organism. Over evolutionary time, the wiring of these networks changes, yet the patterning outcome is often preserved, a phenomenon known as “system drift.” System drift is illustrated by the gap gene network—involved in segmental patterning—in dipteran insects. In the classic model organism Drosophila melanogaster and the nonmodel scuttle fly Megaselia abdita, early activation and placement of gap gene expression domains show significant quantitative differences, yet the final patterning output of the system is essentially identical in both species. In this detailed modeling analysis of system drift, we use gene circuits which are fit to quantitative gap gene expression data in M. abdita and compare them with an equivalent set of models from D. melanogaster. The results of this comparative analysis show precisely how compensatory regulatory mechanisms achieve equivalent final patterns in both species. We discuss the larger implications of the work in terms of “genotype networks” and the ways in which the structure of regulatory networks can influence patterns of evolutionary change (evolvability). PMID:26796549

  4. Heart morphogenesis gene regulatory networks revealed by temporal expression analysis.

    PubMed

    Hill, Jonathon T; Demarest, Bradley; Gorsi, Bushra; Smith, Megan; Yost, H Joseph

    2017-10-01

    During embryogenesis the heart forms as a linear tube that then undergoes multiple simultaneous morphogenetic events to obtain its mature shape. To understand the gene regulatory networks (GRNs) driving this phase of heart development, during which many congenital heart disease malformations likely arise, we conducted an RNA-seq timecourse in zebrafish from 30 hpf to 72 hpf and identified 5861 genes with altered expression. We clustered the genes by temporal expression pattern, identified transcription factor binding motifs enriched in each cluster, and generated a model GRN for the major gene batteries in heart morphogenesis. This approach predicted hundreds of regulatory interactions and found batteries enriched in specific cell and tissue types, indicating that the approach can be used to narrow the search for novel genetic markers and regulatory interactions. Subsequent analyses confirmed the GRN using two mutants, Tbx5 and nkx2-5, and identified sets of duplicated zebrafish genes that do not show temporal subfunctionalization. This dataset provides an essential resource for future studies on the genetic/epigenetic pathways implicated in congenital heart defects and the mechanisms of cardiac transcriptional regulation. © 2017. Published by The Company of Biologists Ltd.

  5. How difficult is inference of mammalian causal gene regulatory networks?

    PubMed

    Djordjevic, Djordje; Yang, Andrian; Zadoorian, Armella; Rungrugeecharoen, Kevin; Ho, Joshua W K

    2014-01-01

    Gene regulatory networks (GRNs) play a central role in systems biology, especially in the study of mammalian organ development. One key question remains largely unanswered: Is it possible to infer mammalian causal GRNs using observable gene co-expression patterns alone? We assembled two mouse GRN datasets (embryonic tooth and heart) and matching microarray gene expression profiles to systematically investigate the difficulties of mammalian causal GRN inference. The GRNs were assembled based on > 2,000 pieces of experimental genetic perturbation evidence from manually reading > 150 primary research articles. Each piece of perturbation evidence records the qualitative change of the expression of one gene following knock-down or over-expression of another gene. Our data have thorough annotation of tissue types and embryonic stages, as well as the type of regulation (activation, inhibition and no effect), which uniquely allows us to estimate both sensitivity and specificity of the inference of tissue specific causal GRN edges. Using these unprecedented datasets, we found that gene co-expression does not reliably distinguish true positive from false positive interactions, making inference of GRN in mammalian development very difficult. Nonetheless, if we have expression profiling data from genetic or molecular perturbation experiments, such as gene knock-out or signalling stimulation, it is possible to use the set of differentially expressed genes to recover causal regulatory relationships with good sensitivity and specificity. Our result supports the importance of using perturbation experimental data in causal network reconstruction. Furthermore, we showed that causal gene regulatory relationship can be highly cell type or developmental stage specific, suggesting the importance of employing expression profiles from homogeneous cell populations. This study provides essential datasets and empirical evidence to guide the development of new GRN inference methods for

  6. Modularity and evolutionary constraints in a baculovirus gene regulatory network

    PubMed Central

    2013-01-01

    Background The structure of regulatory networks remains an open question in our understanding of complex biological systems. Interactions during complete viral life cycles present unique opportunities to understand how host-parasite network take shape and behave. The Anticarsia gemmatalis multiple nucleopolyhedrovirus (AgMNPV) is a large double-stranded DNA virus, whose genome may encode for 152 open reading frames (ORFs). Here we present the analysis of the ordered cascade of the AgMNPV gene expression. Results We observed an earlier onset of the expression than previously reported for other baculoviruses, especially for genes involved in DNA replication. Most ORFs were expressed at higher levels in a more permissive host cell line. Genes with more than one copy in the genome had distinct expression profiles, which could indicate the acquisition of new functionalities. The transcription gene regulatory network (GRN) for 149 ORFs had a modular topology comprising five communities of highly interconnected nodes that separated key genes that are functionally related on different communities, possibly maximizing redundancy and GRN robustness by compartmentalization of important functions. Core conserved functions showed expression synchronicity, distinct GRN features and significantly less genetic diversity, consistent with evolutionary constraints imposed in key elements of biological systems. This reduced genetic diversity also had a positive correlation with the importance of the gene in our estimated GRN, supporting a relationship between phylogenetic data of baculovirus genes and network features inferred from expression data. We also observed that gene arrangement in overlapping transcripts was conserved among related baculoviruses, suggesting a principle of genome organization. Conclusions Albeit with a reduced number of nodes (149), the AgMNPV GRN had a topology and key characteristics similar to those observed in complex cellular organisms, which indicates

  7. Interrogating the topological robustness of gene regulatory circuits by randomization

    PubMed Central

    Levine, Herbert; Onuchic, Jose N.

    2017-01-01

    One of the most important roles of cells is performing their cellular tasks properly for survival. Cells usually achieve robust functionality, for example, cell-fate decision-making and signal transduction, through multiple layers of regulation involving many genes. Despite the combinatorial complexity of gene regulation, its quantitative behavior has been typically studied on the basis of experimentally verified core gene regulatory circuitry, composed of a small set of important elements. It is still unclear how such a core circuit operates in the presence of many other regulatory molecules and in a crowded and noisy cellular environment. Here we report a new computational method, named random circuit perturbation (RACIPE), for interrogating the robust dynamical behavior of a gene regulatory circuit even without accurate measurements of circuit kinetic parameters. RACIPE generates an ensemble of random kinetic models corresponding to a fixed circuit topology, and utilizes statistical tools to identify generic properties of the circuit. By applying RACIPE to simple toggle-switch-like motifs, we observed that the stable states of all models converge to experimentally observed gene state clusters even when the parameters are strongly perturbed. RACIPE was further applied to a proposed 22-gene network of the Epithelial-to-Mesenchymal Transition (EMT), from which we identified four experimentally observed gene states, including the states that are associated with two different types of hybrid Epithelial/Mesenchymal phenotypes. Our results suggest that dynamics of a gene circuit is mainly determined by its topology, not by detailed circuit parameters. Our work provides a theoretical foundation for circuit-based systems biology modeling. We anticipate RACIPE to be a powerful tool to predict and decode circuit design principles in an unbiased manner, and to quantitatively evaluate the robustness and heterogeneity of gene expression. PMID:28362798

  8. Dynamic Gene Regulatory Networks Drive Hematopoietic Specification and Differentiation

    PubMed Central

    Goode, Debbie K.; Obier, Nadine; Vijayabaskar, M.S.; Lie-A-Ling, Michael; Lilly, Andrew J.; Hannah, Rebecca; Lichtinger, Monika; Batta, Kiran; Florkowska, Magdalena; Patel, Rahima; Challinor, Mairi; Wallace, Kirstie; Gilmour, Jane; Assi, Salam A.; Cauchy, Pierre; Hoogenkamp, Maarten; Westhead, David R.; Lacaud, Georges; Kouskoff, Valerie; Göttgens, Berthold; Bonifer, Constanze

    2016-01-01

    Summary Metazoan development involves the successive activation and silencing of specific gene expression programs and is driven by tissue-specific transcription factors programming the chromatin landscape. To understand how this process executes an entire developmental pathway, we generated global gene expression, chromatin accessibility, histone modification, and transcription factor binding data from purified embryonic stem cell-derived cells representing six sequential stages of hematopoietic specification and differentiation. Our data reveal the nature of regulatory elements driving differential gene expression and inform how transcription factor binding impacts on promoter activity. We present a dynamic core regulatory network model for hematopoietic specification and demonstrate its utility for the design of reprogramming experiments. Functional studies motivated by our genome-wide data uncovered a stage-specific role for TEAD/YAP factors in mammalian hematopoietic specification. Our study presents a powerful resource for studying hematopoiesis and demonstrates how such data advance our understanding of mammalian development. PMID:26923725

  9. Constraint and Contingency in Multifunctional Gene Regulatory Circuits

    PubMed Central

    Payne, Joshua L.; Wagner, Andreas

    2013-01-01

    Gene regulatory circuits drive the development, physiology, and behavior of organisms from bacteria to humans. The phenotypes or functions of such circuits are embodied in the gene expression patterns they form. Regulatory circuits are typically multifunctional, forming distinct gene expression patterns in different embryonic stages, tissues, or physiological states. Any one circuit with a single function can be realized by many different regulatory genotypes. Multifunctionality presumably constrains this number, but we do not know to what extent. We here exhaustively characterize a genotype space harboring millions of model regulatory circuits and all their possible functions. As a circuit's number of functions increases, the number of genotypes with a given number of functions decreases exponentially but can remain very large for a modest number of functions. However, the sets of circuits that can form any one set of functions becomes increasingly fragmented. As a result, historical contingency becomes widespread in circuits with many functions. Whether a circuit can acquire an additional function in the course of its evolution becomes increasingly dependent on the function it already has. Circuits with many functions also become increasingly brittle and sensitive to mutation. These observations are generic properties of a broad class of circuits and independent of any one circuit genotype or phenotype. PMID:23762020

  10. Constraint and contingency in multifunctional gene regulatory circuits.

    PubMed

    Payne, Joshua L; Wagner, Andreas

    2013-01-01

    Gene regulatory circuits drive the development, physiology, and behavior of organisms from bacteria to humans. The phenotypes or functions of such circuits are embodied in the gene expression patterns they form. Regulatory circuits are typically multifunctional, forming distinct gene expression patterns in different embryonic stages, tissues, or physiological states. Any one circuit with a single function can be realized by many different regulatory genotypes. Multifunctionality presumably constrains this number, but we do not know to what extent. We here exhaustively characterize a genotype space harboring millions of model regulatory circuits and all their possible functions. As a circuit's number of functions increases, the number of genotypes with a given number of functions decreases exponentially but can remain very large for a modest number of functions. However, the sets of circuits that can form any one set of functions becomes increasingly fragmented. As a result, historical contingency becomes widespread in circuits with many functions. Whether a circuit can acquire an additional function in the course of its evolution becomes increasingly dependent on the function it already has. Circuits with many functions also become increasingly brittle and sensitive to mutation. These observations are generic properties of a broad class of circuits and independent of any one circuit genotype or phenotype.

  11. Selecting and Weighting Data for Building Consensus Gene Regulatory Networks

    NASA Astrophysics Data System (ADS)

    Steele, Emma; Tucker, Allan

    Microarrays are the major source of data for gene expression activity, allowing the expression of thousands of genes to be measured simultaneously. Gene regulatory networks (GRNs) describe how the expression level of genes affect the expression of the other genes. Modelling GRNs from expression data is a topic of great interest in current bioinformatics research. Previously, we took advantage of publicly available gene expression datasets generated by similar biological studies by drawing together a richer and/or broader collection of data in order to produce GRN models that are more robust, have greater confidence and place less reliance on a single dataset. In this paper a new approach, Weighted Consensus Bayesian Networks, introduces the use of weights in order to place more influence on certain input networks or remove the least reliable networks from the input with encouraging results on both synthetic data and real world yeast microarray datasets.

  12. Genomic imprinting-an epigenetic gene-regulatory model.

    PubMed

    Koerner, Martha V; Barlow, Denise P

    2010-04-01

    Epigenetic mechanisms (Box 1) are considered to play major gene-regulatory roles in development, differentiation and disease. However, the relative importance of epigenetics in defining the mammalian transcriptome in normal and disease states is unknown. The mammalian genome contains only a few model systems where epigenetic gene regulation has been shown to play a major role in transcriptional control. These model systems are important not only to investigate the biological function of known epigenetic modifications but also to identify new and unexpected epigenetic mechanisms in the mammalian genome. Here we review recent progress in understanding how epigenetic mechanisms control imprinted gene expression.

  13. Additive Functions in Boolean Models of Gene Regulatory Network Modules

    PubMed Central

    Darabos, Christian; Di Cunto, Ferdinando; Tomassini, Marco; Moore, Jason H.; Provero, Paolo; Giacobini, Mario

    2011-01-01

    Gene-on-gene regulations are key components of every living organism. Dynamical abstract models of genetic regulatory networks help explain the genome's evolvability and robustness. These properties can be attributed to the structural topology of the graph formed by genes, as vertices, and regulatory interactions, as edges. Moreover, the actual gene interaction of each gene is believed to play a key role in the stability of the structure. With advances in biology, some effort was deployed to develop update functions in Boolean models that include recent knowledge. We combine real-life gene interaction networks with novel update functions in a Boolean model. We use two sub-networks of biological organisms, the yeast cell-cycle and the mouse embryonic stem cell, as topological support for our system. On these structures, we substitute the original random update functions by a novel threshold-based dynamic function in which the promoting and repressing effect of each interaction is considered. We use a third real-life regulatory network, along with its inferred Boolean update functions to validate the proposed update function. Results of this validation hint to increased biological plausibility of the threshold-based function. To investigate the dynamical behavior of this new model, we visualized the phase transition between order and chaos into the critical regime using Derrida plots. We complement the qualitative nature of Derrida plots with an alternative measure, the criticality distance, that also allows to discriminate between regimes in a quantitative way. Simulation on both real-life genetic regulatory networks show that there exists a set of parameters that allows the systems to operate in the critical region. This new model includes experimentally derived biological information and recent discoveries, which makes it potentially useful to guide experimental research. The update function confers additional realism to the model, while reducing the complexity

  14. Additive functions in boolean models of gene regulatory network modules.

    PubMed

    Darabos, Christian; Di Cunto, Ferdinando; Tomassini, Marco; Moore, Jason H; Provero, Paolo; Giacobini, Mario

    2011-01-01

    Gene-on-gene regulations are key components of every living organism. Dynamical abstract models of genetic regulatory networks help explain the genome's evolvability and robustness. These properties can be attributed to the structural topology of the graph formed by genes, as vertices, and regulatory interactions, as edges. Moreover, the actual gene interaction of each gene is believed to play a key role in the stability of the structure. With advances in biology, some effort was deployed to develop update functions in boolean models that include recent knowledge. We combine real-life gene interaction networks with novel update functions in a boolean model. We use two sub-networks of biological organisms, the yeast cell-cycle and the mouse embryonic stem cell, as topological support for our system. On these structures, we substitute the original random update functions by a novel threshold-based dynamic function in which the promoting and repressing effect of each interaction is considered. We use a third real-life regulatory network, along with its inferred boolean update functions to validate the proposed update function. Results of this validation hint to increased biological plausibility of the threshold-based function. To investigate the dynamical behavior of this new model, we visualized the phase transition between order and chaos into the critical regime using Derrida plots. We complement the qualitative nature of Derrida plots with an alternative measure, the criticality distance, that also allows to discriminate between regimes in a quantitative way. Simulation on both real-life genetic regulatory networks show that there exists a set of parameters that allows the systems to operate in the critical region. This new model includes experimentally derived biological information and recent discoveries, which makes it potentially useful to guide experimental research. The update function confers additional realism to the model, while reducing the complexity

  15. Effects of Four Different Regulatory Mechanisms on the Dynamics of Gene Regulatory Cascades

    PubMed Central

    Hansen, Sabine; Krishna, Sandeep; Semsey, Szabolcs; Lo Svenningsen, Sine

    2015-01-01

    Gene regulatory cascades (GRCs) are common motifs in cellular molecular networks. A given logical function in these cascades, such as the repression of the activity of a transcription factor, can be implemented by a number of different regulatory mechanisms. The potential consequences for the dynamic performance of the GRC of choosing one mechanism over another have not been analysed systematically. Here, we report the construction of a synthetic GRC in Escherichia coli, which allows us for the first time to directly compare and contrast the dynamics of four different regulatory mechanisms, affecting the transcription, translation, stability, or activity of a transcriptional repressor. We developed a biologically motivated mathematical model which is sufficient to reproduce the response dynamics determined by experimental measurements. Using the model, we explored the potential response dynamics that the constructed GRC can perform. We conclude that dynamic differences between regulatory mechanisms at an individual step in a GRC are often concealed in the overall performance of the GRC, and suggest that the presence of a given regulatory mechanism in a certain network environment does not necessarily mean that it represents a single optimal evolutionary solution. PMID:26184971

  16. Aberrant expression of KPNA2 is associated with a poor prognosis and contributes to OCT4 nuclear transportation in bladder cancer

    PubMed Central

    Zhou, Jingcheng; Dong, Daoquan; Cheng, Ran; Wang, Yan; Jiang, Shuqi; Zhu, Yuhong; Fan, Longlong; Mao, Xiangming; Gui, Yaoting; Li, Zesong; Li, Xianxin; Shi, Bentao

    2016-01-01

    Recent studies show that Karyopherin alpha 2 (KPNA2) is up-regulated in quite a number of cancers and associated with poor prognosis. Here, we found that expression levels of KPNA2 and OCT4 are up-regulated in bladder cancer tissues and significantly associated with primary tumor stage and bladder cancer patients' poorer prognosis. Our data also showed decreased cell proliferation and migration rates of bladder cancer cell lines when the expression of KPNA2 and OCT4 was silenced. Meanwhile, cell apoptosis rate was increased. Furthermore, Co-IP and immunofluorescence assay showed the KPNA2 interacts with OCT4 and inhibits OCT4 nuclear transportation when KPNA2 was silenced. Thus, we confirmed that up-regulated KPNA2 and OCT4 expression is a common feature of bladder cancer that is correlated with increased aggressive tumor behavior. Also, we propose that KPNA2 regulates the process of OCT4 nuclear transportation in bladder cancer. PMID:27611951

  17. Identification of cancer-related genes and motifs in the human gene regulatory network.

    PubMed

    Carson, Matthew B; Gu, Jianlei; Yu, Guangjun; Lu, Hui

    2015-08-01

    The authors investigated the regulatory network motifs and corresponding motif positions of cancer-related genes. First, they mapped disease-related genes to a transcription factor regulatory network. Next, they calculated statistically significant motifs and subsequently identified positions within these motifs that were enriched in cancer-related genes. Potential mechanisms of these motifs and positions are discussed. These results could be used to identify other disease- and cancer-related genes and could also suggest mechanisms for how these genes relate to co-occurring diseases.

  18. Mapping gene regulatory circuitry of Pax6 during neurogenesis

    PubMed Central

    Thakurela, Sudhir; Tiwari, Neha; Schick, Sandra; Garding, Angela; Ivanek, Robert; Berninger, Benedikt; Tiwari, Vijay K

    2016-01-01

    Pax6 is a highly conserved transcription factor among vertebrates and is important in various aspects of the central nervous system development. However, the gene regulatory circuitry of Pax6 underlying these functions remains elusive. We find that Pax6 targets a large number of promoters in neural progenitors cells. Intriguingly, many of these sites are also bound by another progenitor factor, Sox2, which cooperates with Pax6 in gene regulation. A combinatorial analysis of Pax6-binding data set with transcriptome changes in Pax6-deficient neural progenitors reveals a dual role for Pax6, in which it activates the neuronal (ectodermal) genes while concurrently represses the mesodermal and endodermal genes, thereby ensuring the unidirectionality of lineage commitment towards neuronal differentiation. Furthermore, Pax6 is critical for inducing activity of transcription factors that elicit neurogenesis and repress others that promote non-neuronal lineages. In addition to many established downstream effectors, Pax6 directly binds and activates a number of genes that are specifically expressed in neural progenitors but have not been previously implicated in neurogenesis. The in utero knockdown of one such gene, Ift74, during brain development impairs polarity and migration of newborn neurons. These findings demonstrate new aspects of the gene regulatory circuitry of Pax6, revealing how it functions to control neuronal development at multiple levels to ensure unidirectionality and proper execution of the neurogenic program. PMID:27462442

  19. Crosstalks between Raf-kinase inhibitor protein and cancer stem cell transcription factors (Oct4, KLF4, Sox2, Nanog).

    PubMed

    Lee, SoHyun; Wottrich, Stephanie; Bonavida, Benjamin

    2017-04-01

    Raf-kinase inhibitor protein has been reported to inhibit both the Raf/mitogen extracellular signal-regulated kinase/extracellular signal-regulated kinase and nuclear factor kappa-light-chain of activated B cells pathways. It has also been reported in cancers that Raf-kinase inhibitor protein behaves as a metastatic suppressor as well as a chemo-immunosensitizing factor to drug/immune-mediated apoptosis. The majority of cancers exhibit low or no levels of Raf-kinase inhibitor protein. Hence, the activities of Raf-kinase inhibitor protein contrast, in part, to those mediated by several cancer stem cell transcription factors for their roles in resistance and metastasis. In this review, the existence of crosstalks in the signaling pathways between Raf-kinase inhibitor protein and several cancer stem cell transcription factors (Oct4, KLF4, Sox2 and Nanog) was assembled. Oct4 is induced by Lin28, and Raf-kinase inhibitor protein inhibits the microRNA binding protein Lin28. The expression of Raf-kinase inhibitor protein inversely correlates with the expression of Oct4. KLF4 does not interact directly with Raf-kinase inhibitor protein, but rather interacts indirectly via Raf-kinase inhibitor protein's regulation of the Oct4/Sox2/KLF4 complex through the mitogen-activated protein kinase pathway. The mechanism by which Raf-kinase inhibitor protein inhibits Sox2 is via the inhibition of the mitogen-activated protein kinase pathway by Raf-kinase inhibitor protein. Thus, Raf-kinase inhibitor protein's relationship with Sox2 is via its regulation of Oct4. Inhibition of extracellular signal-regulated kinase by Raf-kinase inhibitor protein results in the upregulation of Nanog. The inhibition of Oct4 by Raf-kinase inhibitor protein results in the failure of the heterodimer formation of Oct4 and Sox2 that is necessary to bind to the Nanog promoter for the transcription of Nanog. The findings revealed that there exists a direct correlation between the expression of Raf

  20. Modeling stochasticity and robustness in gene regulatory networks

    PubMed Central

    Garg, Abhishek; Mohanram, Kartik; Di Cara, Alessandro; De Micheli, Giovanni; Xenarios, Ioannis

    2009-01-01

    Motivation: Understanding gene regulation in biological processes and modeling the robustness of underlying regulatory networks is an important problem that is currently being addressed by computational systems biologists. Lately, there has been a renewed interest in Boolean modeling techniques for gene regulatory networks (GRNs). However, due to their deterministic nature, it is often difficult to identify whether these modeling approaches are robust to the addition of stochastic noise that is widespread in gene regulatory processes. Stochasticity in Boolean models of GRNs has been addressed relatively sparingly in the past, mainly by flipping the expression of genes between different expression levels with a predefined probability. This stochasticity in nodes (SIN) model leads to over representation of noise in GRNs and hence non-correspondence with biological observations. Results: In this article, we introduce the stochasticity in functions (SIF) model for simulating stochasticity in Boolean models of GRNs. By providing biological motivation behind the use of the SIF model and applying it to the T-helper and T-cell activation networks, we show that the SIF model provides more biologically robust results than the existing SIN model of stochasticity in GRNs. Availability: Algorithms are made available under our Boolean modeling toolbox, GenYsis. The software binaries can be downloaded from http://si2.epfl.ch/∼garg/genysis.html. Contact: abhishek.garg@epfl.ch PMID:19477975

  1. Modeling stochasticity and robustness in gene regulatory networks.

    PubMed

    Garg, Abhishek; Mohanram, Kartik; Di Cara, Alessandro; De Micheli, Giovanni; Xenarios, Ioannis

    2009-06-15

    Understanding gene regulation in biological processes and modeling the robustness of underlying regulatory networks is an important problem that is currently being addressed by computational systems biologists. Lately, there has been a renewed interest in Boolean modeling techniques for gene regulatory networks (GRNs). However, due to their deterministic nature, it is often difficult to identify whether these modeling approaches are robust to the addition of stochastic noise that is widespread in gene regulatory processes. Stochasticity in Boolean models of GRNs has been addressed relatively sparingly in the past, mainly by flipping the expression of genes between different expression levels with a predefined probability. This stochasticity in nodes (SIN) model leads to over representation of noise in GRNs and hence non-correspondence with biological observations. In this article, we introduce the stochasticity in functions (SIF) model for simulating stochasticity in Boolean models of GRNs. By providing biological motivation behind the use of the SIF model and applying it to the T-helper and T-cell activation networks, we show that the SIF model provides more biologically robust results than the existing SIN model of stochasticity in GRNs. Algorithms are made available under our Boolean modeling toolbox, GenYsis. The software binaries can be downloaded from http://si2.epfl.ch/ approximately garg/genysis.html.

  2. EXAMINE: a computational approach to reconstructing gene regulatory networks.

    PubMed

    Deng, Xutao; Geng, Huimin; Ali, Hesham

    2005-08-01

    Reverse-engineering of gene networks using linear models often results in an underdetermined system because of excessive unknown parameters. In addition, the practical utility of linear models has remained unclear. We address these problems by developing an improved method, EXpression Array MINing Engine (EXAMINE), to infer gene regulatory networks from time-series gene expression data sets. EXAMINE takes advantage of sparse graph theory to overcome the excessive-parameter problem with an adaptive-connectivity model and fitting algorithm. EXAMINE also guarantees that the most parsimonious network structure will be found with its incremental adaptive fitting process. Compared to previous linear models, where a fully connected model is used, EXAMINE reduces the number of parameters by O(N), thereby increasing the chance of recovering the underlying regulatory network. The fitting algorithm increments the connectivity during the fitting process until a satisfactory fit is obtained. We performed a systematic study to explore the data mining ability of linear models. A guideline for using linear models is provided: If the system is small (3-20 elements), more than 90% of the regulation pathways can be determined correctly. For a large-scale system, either clustering is needed or it is necessary to integrate information in addition to expression profile. Coupled with the clustering method, we applied EXAMINE to rat central nervous system development (CNS) data with 112 genes. We were able to efficiently generate regulatory networks with statistically significant pathways that have been predicted previously.

  3. Dynamic Gene Regulatory Networks of Human Myeloid Differentiation.

    PubMed

    Ramirez, Ricardo N; El-Ali, Nicole C; Mager, Mikayla Anne; Wyman, Dana; Conesa, Ana; Mortazavi, Ali

    2017-03-27

    The reconstruction of gene regulatory networks underlying cell differentiation from high-throughput gene expression and chromatin data remains a challenge. Here, we derive dynamic gene regulatory networks for human myeloid differentiation using a 5-day time series of RNA-seq and ATAC-seq data. We profile HL-60 promyelocytes differentiating into macrophages, neutrophils, monocytes, and monocyte-derived macrophages. We find a rapid response in the expression of key transcription factors and lineage markers that only regulate a subset of their targets at a given time, which is followed by chromatin accessibility changes that occur later along with further gene expression changes. We observe differences between promyelocyte- and monocyte-derived macrophages at both the transcriptional and chromatin landscape level, despite using the same differentiation stimulus, which suggest that the path taken by cells in the differentiation landscape defines their end cell state. More generally, our approach of combining neighboring time points and replicates to achieve greater sequencing depth can efficiently infer footprint-based regulatory networks from long series data.

  4. Expression of regulatory nif genes in Rhodobacter capsulatus.

    PubMed Central

    Hübner, P; Willison, J C; Vignais, P M; Bickle, T A

    1991-01-01

    Translational fusions of the Escherichia coli lacZ gene to Rhodobacter capsulatus nif genes were constructed in order to determine the regulatory circuit of nif gene expression in R. capsulatus, a free-living photosynthetic diazotroph. The expression of nifH, nifA (copies I and II), and nifR4 was measured in different regulatory mutant strains under different physiological conditions. The expression of nifH and nifR4 (the analog of ntrA in Klebsiella pneumoniae) depends on the NIFR1/R2 system (the analog of the ntr system in K. pneumoniae), on NIFA, and on NIFR4. The expression of both copies of nifA is regulated by the NIFR1/R2 system and is modulated by the N source of the medium under anaerobic photosynthetic growth conditions. In the presence of ammonia or oxygen, moderate expression of nifA was detectable, whereas nifH and nifR4 were not expressed under these conditions. The implications for the regulatory circuit of nif gene expression in R. capsulatus are discussed and compared with the situation in K. pneumoniae, another free-living diazotroph. PMID:1902215

  5. Characterization of nif regulatory genes in Rhodopseudomonas capsulata using lac gene fusions.

    PubMed

    Kranz, R G; Haselkorn, R

    1985-01-01

    Translational fusions of the Escherichia coli lacZYA operon to Rhodopseudomonas capsulata nif genes were obtained by using mini-MudII1734 [Castilho et al., J. Bacteriol. 158 (1984) 488-495] inserts into cloned fragments of R. capsulata DNA. A lac fusion to the nifH gene, which encodes dinitrogenase reductase, was used to classify Nif- mutations occurring in regulatory genes. Nine mutations were unable to activate nifHDK transcription. The nine mutations define four nif regulatory genes. Three of these genes are located on the same R. capsulata 8.4-kb EcoRI fragment. Each is transcribed independently. One of these (complementing mutant J61) is partially homologous with the ntrC gene of Escherichia coli, based on Southern hybridization. The fourth nif regulatory gene (complementing mutants LJ1, AH1 and AH3) is unlinked to the others. Lac fusions to all four regulatory genes were constructed. Each regulatory gene is weakly expressed compared to derepressed nifH and partially repressed in the presence of ammonia.

  6. Quiescent Oct4(+) Neural Stem Cells (NSCs) Repopulate Ablated Glial Fibrillary Acidic Protein(+) NSCs in the Adult Mouse Brain.

    PubMed

    Reeve, Rachel L; Yammine, Samantha Z; Morshead, Cindi M; van der Kooy, Derek

    2017-09-01

    Adult primitive neural stem cells (pNSCs) are a rare population of glial fibrillary acidic protein (GFAP)(-) Oct4(+) cells in the mouse forebrain subependymal zone bordering the lateral ventricles that give rise to clonal neurospheres in leukemia inhibitory factor in vitro. pNSC neurospheres can be passaged to self-renew or give rise to GFAP(+) NSCs that form neurospheres in epidermal growth factor and fibroblast growth factor 2, which we collectively refer to as definitive NSCs (dNSCs). Label retention experiments using doxycycline-inducible histone-2B (H2B)-green fluorescent protein (GFP) mice and several chase periods of up to 1 year quantified the adult pNSC cell cycle time as 3-5 months. We hypothesized that while pNSCs are not very proliferative at baseline, they may exist as a reserve pool of NSCs in case of injury. To test this function of pNSCs, we obtained conditional Oct4 knockout mice, Oct4(fl/fl) ;Sox1(Cre) (Oct4(CKO) ), which do not yield adult pNSC-derived neurospheres. When we ablated the progeny of pNSCs, namely all GFAP(+) dNSCs, in these Oct4(CKO) mice, we found that dNSCs did not recover as they do in wild-type mice, suggesting that pNSCs are necessary for dNSC repopulation. Returning to the H2B-GFP mice, we observed that the cytosine β-d-arabinofuranoside ablation of proliferating cells including dNSCs-induced quiescent pNSCs to proliferate and significantly dilute their H2B-GFP label. In conclusion, we demonstrate that pNSCs are the most quiescent stem cells in the adult brain reported to date and that their lineage position upstream of GFAP(+) dNSCs allows them to repopulate a depleted neural lineage. Stem Cells 2017;35:2071-2082. © 2017 AlphaMed Press.

  7. Gene therapy for cancer: regulatory considerations for approval

    PubMed Central

    Husain, S R; Han, J; Au, P; Shannon, K; Puri, R K

    2015-01-01

    The rapidly changing field of gene therapy promises a number of innovative treatments for cancer patients. Advances in genetic modification of cancer and immune cells and the use of oncolytic viruses and bacteria have led to numerous clinical trials for cancer therapy, with several progressing to late-stage product development. At the time of this writing, no gene therapy product has been approved by the United States Food and Drug Administration (FDA). Some of the key scientific and regulatory issues include understanding of gene transfer vector biology, safety of vectors in vitro and in animal models, optimum gene transfer, long-term persistence or integration in the host, shedding of a virus and ability to maintain transgene expression in vivo for a desired period of time. Because of the biological complexity of these products, the FDA encourages a flexible, data-driven approach for preclinical safety testing programs. The clinical trial design should be based on the unique features of gene therapy products, and should ensure the safety of enrolled subjects. This article focuses on regulatory considerations for gene therapy product development and also discusses guidance documents that have been published by the FDA. PMID:26584531

  8. Regulatory sequences of duck hepatitis B virus C gene transcription.

    PubMed Central

    Schneider, R; Will, H

    1991-01-01

    The regulatory elements involved in transcription of the C gene of duck hepatitis B virus (DHBV) were investigated. Several DHBV DNA fragments were assayed for C gene promoter, enhancer, and silencer activity by using a chloramphenicol acetyltransferase (CAT) reporter gene and transfection of established liver and nonliver cell lines. A major transcript initiating at nucleotide positions 2532 and 2533 and three minor transcripts initiating at positions 2453/2454 and 2461 were identified in cells containing these constructs. These positions correspond to the 5' end of the C mRNA and were close to that of the pre-C mRNAs, respectively, found in infected livers. The pre-C mRNAs were only detected when sequences located between the initiation sites of the pre-C and C mRNAs were deleted. These sequences downregulated, in an orientation-independent fashion, a heterologous promoter and were found to contain a consensus motif common to negative transcriptional regulatory elements previously characterized in other cellular and viral genes. C gene promoter activity was only observed in highly differentiated liver cells and was dependent on a short DHBV DNA fragment containing an enhancer core consensus motif. These data indicate that transcription of the DHBV C gene is regulated by positive, negative, and differentiation factor-responsive elements. Images PMID:1920612

  9. Gene therapy for cancer: regulatory considerations for approval.

    PubMed

    Husain, S R; Han, J; Au, P; Shannon, K; Puri, R K

    2015-12-01

    The rapidly changing field of gene therapy promises a number of innovative treatments for cancer patients. Advances in genetic modification of cancer and immune cells and the use of oncolytic viruses and bacteria have led to numerous clinical trials for cancer therapy, with several progressing to late-stage product development. At the time of this writing, no gene therapy product has been approved by the United States Food and Drug Administration (FDA). Some of the key scientific and regulatory issues include understanding of gene transfer vector biology, safety of vectors in vitro and in animal models, optimum gene transfer, long-term persistence or integration in the host, shedding of a virus and ability to maintain transgene expression in vivo for a desired period of time. Because of the biological complexity of these products, the FDA encourages a flexible, data-driven approach for preclinical safety testing programs. The clinical trial design should be based on the unique features of gene therapy products, and should ensure the safety of enrolled subjects. This article focuses on regulatory considerations for gene therapy product development and also discusses guidance documents that have been published by the FDA.

  10. Beyond antioxidant genes in the ancient NRF2 regulatory network

    PubMed Central

    Lacher, Sarah E.; Lee, Joslynn S.; Wang, Xuting; Campbell, Michelle R.; Bell, Douglas A.; Slattery, Matthew

    2016-01-01

    NRF2, a basic leucine zipper transcription factor encoded by the gene NFE2L2, is a master regulator of the transcriptional response to oxidative stress. NRF2 is structurally and functionally conserved from insects to humans, and it heterodimerizes with the small MAF transcription factors to bind a consensus DNA sequence (the antioxidant response element, or ARE) and regulate gene expression. We have used genome-wide chromatin immunoprecipitation (ChIP-seq) and gene expression data to identify direct NRF2 target genes in Drosophila and humans. These data have allowed us to construct the deeply conserved ancient NRF2 regulatory network – target genes that are conserved from Drosophila to human. The ancient network consists of canonical antioxidant genes, as well as genes related to proteasomal pathways, metabolism, and a number of less expected genes. We have also used enhancer reporter assays and electrophoretic mobility shift assays to confirm NRF2-mediated regulation of ARE (antioxidant response element) activity at a number of these novel target genes. Interestingly, the ancient network also highlights a prominent negative feedback loop; this, combined with the finding that and NRF2-mediated regulatory output is tightly linked to the quality of the ARE it is targeting, suggests that precise regulation of nuclear NRF2 concentration is necessary to achieve proper quantitative regulation of distinct gene sets. Together, these findings highlight the importance of balance in the NRF2-ARE pathway, and indicate that NRF2-mediated regulation of xenobiotic metabolism, glucose metabolism, and proteostasis have been central to this pathway since its inception. PMID:26163000

  11. Establishing neural crest identity: a gene regulatory recipe

    PubMed Central

    Simões-Costa, Marcos; Bronner, Marianne E.

    2015-01-01

    The neural crest is a stem/progenitor cell population that contributes to a wide variety of derivatives, including sensory and autonomic ganglia, cartilage and bone of the face and pigment cells of the skin. Unique to vertebrate embryos, it has served as an excellent model system for the study of cell behavior and identity owing to its multipotency, motility and ability to form a broad array of cell types. Neural crest development is thought to be controlled by a suite of transcriptional and epigenetic inputs arranged hierarchically in a gene regulatory network. Here, we examine neural crest development from a gene regulatory perspective and discuss how the underlying genetic circuitry results in the features that define this unique cell population. PMID:25564621

  12. Statistical ensemble of gene regulatory networks of macrophage differentiation.

    PubMed

    Castiglione, Filippo; Tieri, Paolo; Palma, Alessandro; Jarrah, Abdul Salam

    2016-12-22

    Macrophages cover a major role in the immune system, being the most plastic cell yielding several key immune functions. Here we derived a minimalistic gene regulatory network model for the differentiation of macrophages into the two phenotypes M1 (pro-) and M2 (anti-inflammatory). To test the model, we simulated a large number of such networks as in a statistical ensemble. In other words, to enable the inter-cellular crosstalk required to obtain an immune activation in which the macrophage plays its role, the simulated networks are not taken in isolation but combined with other cellular agents, thus setting up a discrete minimalistic model of the immune system at the microscopic/intracellular (i.e., genetic regulation) and mesoscopic/intercellular scale. We show that within the mesoscopic level description of cellular interaction and cooperation, the gene regulatory logic is coherent and contributes to the overall dynamics of the ensembles that shows, statistically, the expected behaviour.

  13. Transient exposure to proteins SOX2, Oct-4, and NANOG immortalizes exhausted tumor-infiltrating CTLs

    SciTech Connect

    Bhadurihauck, Anjuli; Li, Lei; Li, Qianqian; Wang, Jianjun; Xiao, Zhengguo

    2016-05-13

    Adoptive cell transfer therapy (ACT) is one of the most promising immunotherapies against cancer, using tumor-infiltrating lymphocytes (TILs) expanded in vitro. Tumor-infiltrating cytotoxic T lymphocytes (TICTLs) play a prominent role in cancer control. TILs terminally differentiate in response to immunosuppressive environments within tumors, and thus are slow to expand and challenging to maintain both in vitro and in patients. To reverse this exhaustion, we utilize a nuclear protein delivery system that exposes TICTLs to the SOX2, Oct-4, and NANOG (SON) proteins. Unlike activated naïve CTLs (effector CTLs), TICTLs respond favorably to SON treatment, exhibiting steady proliferation and extended survivability independent of cytokine and antigen stimulation. Though TICTLs treated with SON (STICTLs) still express T cell receptors as well as other critical downstream components, they are unresponsive to antigen challenge, suggesting that SON treatment regresses TICTLs into a state similar to that of an early double negative T cell. Our findings indicate the TICTL response to SON proteins is unique when compared to effector CTLs, suggesting TICTLs may be sensitive to regulation by other lineage-specific transcription factors and opening a promising new avenue into cancer immunotherapy. To our knowledge, this is the first report on lineage reprogramming of TILs using protein stem cell transcription factors delivered directly to the nucleus. -- Highlights: •TICTLs are sensitive to reprogramming by proteins of stem cell transcription factors, but effector CTLs were not. •TICTLs are regressed back to an early double negative T cell stage. •TCR signaling is deregulated by these transcription factors.

  14. Analyzing stationary states of gene regulatory network using petri nets.

    PubMed

    Gambin, Anna; Lasota, Sławomir; Rutkowski, Michał

    2006-01-01

    We introduce and formally define the notion of a stationary state for Petri nets. We also propose a fully automatic method for finding such states. The procedure makes use of the Presburger arithmetic to describe all the stationary states. Finally we apply this novel approach to find stationary states of a gene regulatory network describing the flower morphogenesis of A. thaliana. This shows that the proposed method can be successfully applied in the study of biological systems.

  15. Analyzing stationary States of gene regulatory network using petri nets.

    PubMed

    Gambin, Anna; Lasota, Sławomir; Rutkowski, Michał

    2011-01-01

    We introduce and formally define the notion of a stationary state for Petri nets. We also propose a fully automatic method for finding such states. The procedure makes use of the Presburger arithmetic to describe all the stationary states. Finally we apply this novel approach to find stationary states of a gene regulatory network describing the flower morphogenesis of A. thaliana. This shows that the proposed method can be successfully applied in the study of biological systems.

  16. Modeling stochasticity and variability in gene regulatory networks.

    PubMed

    Murrugarra, David; Veliz-Cuba, Alan; Aguilar, Boris; Arat, Seda; Laubenbacher, Reinhard

    2012-06-06

    Modeling stochasticity in gene regulatory networks is an important and complex problem in molecular systems biology. To elucidate intrinsic noise, several modeling strategies such as the Gillespie algorithm have been used successfully. This article contributes an approach as an alternative to these classical settings. Within the discrete paradigm, where genes, proteins, and other molecular components of gene regulatory networks are modeled as discrete variables and are assigned as logical rules describing their regulation through interactions with other components. Stochasticity is modeled at the biological function level under the assumption that even if the expression levels of the input nodes of an update rule guarantee activation or degradation there is a probability that the process will not occur due to stochastic effects. This approach allows a finer analysis of discrete models and provides a natural setup for cell population simulations to study cell-to-cell variability. We applied our methods to two of the most studied regulatory networks, the outcome of lambda phage infection of bacteria and the p53-mdm2 complex.

  17. Modeling stochasticity and variability in gene regulatory networks

    PubMed Central

    2012-01-01

    Modeling stochasticity in gene regulatory networks is an important and complex problem in molecular systems biology. To elucidate intrinsic noise, several modeling strategies such as the Gillespie algorithm have been used successfully. This article contributes an approach as an alternative to these classical settings. Within the discrete paradigm, where genes, proteins, and other molecular components of gene regulatory networks are modeled as discrete variables and are assigned as logical rules describing their regulation through interactions with other components. Stochasticity is modeled at the biological function level under the assumption that even if the expression levels of the input nodes of an update rule guarantee activation or degradation there is a probability that the process will not occur due to stochastic effects. This approach allows a finer analysis of discrete models and provides a natural setup for cell population simulations to study cell-to-cell variability. We applied our methods to two of the most studied regulatory networks, the outcome of lambda phage infection of bacteria and the p53-mdm2 complex. PMID:22673395

  18. Transcriptional Targeting in the Airway Using Novel Gene Regulatory Elements

    PubMed Central

    Burnight, Erin R.; Wang, Guoshun; McCray, Paul B.

    2012-01-01

    The delivery of cystic fibrosis transmembrane conductance regulator (CFTR) to airway epithelia is a goal of many gene therapy strategies to treat cystic fibrosis. Because the native regulatory elements of the CFTR are not well characterized, the development of vectors with heterologous promoters of varying strengths and specificity would aid in our selection of optimal reagents for the appropriate expression of the vector-delivered CFTR gene. Here we contrasted the performance of several novel gene-regulatory elements. Based on airway expression analysis, we selected putative regulatory elements from BPIFA1 and WDR65 to investigate. In addition, we selected a human CFTR promoter region (∼ 2 kb upstream of the human CFTR transcription start site) to study. Using feline immunodeficiency virus vectors containing the candidate elements driving firefly luciferase, we transduced murine nasal epithelia in vivo. Luciferase expression persisted for 30 weeks, which was the duration of the experiment. Furthermore, when the nasal epithelium was ablated using the detergent polidocanol, the mice showed a transient loss of luciferase expression that returned 2 weeks after administration, suggesting that our vectors transduced a progenitor cell population. Importantly, the hWDR65 element drove sufficient CFTR expression to correct the anion transport defect in CFTR-null epithelia. These results will guide the development of optimal vectors for sufficient, sustained CFTR expression in airway epithelia. PMID:22447971

  19. Gene regulatory elements of the cardiac conduction system.

    PubMed

    van Duijvenboden, Karel; Ruijter, Jan M; Christoffels, Vincent M

    2014-01-01

    The coordinated contraction of the heart relies on the generation and conduction of the electrical impulse. Aberrations of the function of the cardiac conduction system have been associated with various arrhythmogenic disorders and increased risk of sudden cardiac death. The genetics underlying conduction system function have been investigated using functional studies and genome-wide association studies. Both methods point towards the involvement of ion channel genes and the transcription factors that govern their activity. A large fraction of disease- and trait-associated sequence variants lie within non-coding sequences, enriched with epigenetic marks indicative of regulatory DNA. Although sequence conservation as a result of functional constraint has been a useful property to identify transcriptional enhancers, this identification process has been advanced through the development of techniques such as ChIP-seq and chromatin conformation capture technologies. The role of variation in gene regulatory elements in the cardiac conduction system has recently been demonstrated by studies on enhancers of SCN5A/SCN10A and TBX5. In both studies, a region harbouring a functionally implicated single-nucleotide polymorphism was shown to drive reproducible cardiac expression in a reporter gene assay. Furthermore, the risk variant of the allele abrogated enhancer function in both cases. Functional studies on regulatory DNA will likely receive a boost through recent developments in genome modification technologies.

  20. Phase transitions in the evolution of gene regulatory networks

    NASA Astrophysics Data System (ADS)

    Skanata, Antun; Kussell, Edo

    The role of gene regulatory networks is to respond to environmental conditions and optimize growth of the cell. A typical example is found in bacteria, where metabolic genes are activated in response to nutrient availability, and are subsequently turned off to conserve energy when their specific substrates are depleted. However, in fluctuating environmental conditions, regulatory networks could experience strong evolutionary pressures not only to turn the right genes on and off, but also to respond optimally under a wide spectrum of fluctuation timescales. The outcome of evolution is predicted by the long-term growth rate, which differentiates between optimal strategies. Here we present an analytic computation of the long-term growth rate in randomly fluctuating environments, by using mean-field and higher order expansion in the environmental history. We find that optimal strategies correspond to distinct regions in the phase space of fluctuations, separated by first and second order phase transitions. The statistics of environmental randomness are shown to dictate the possible evolutionary modes, which either change the structure of the regulatory network abruptly, or gradually modify and tune the interactions between its components.

  1. Functional studies of regulatory genes in the sea urchin embryo.

    PubMed

    Cavalieri, Vincenzo; Di Bernardo, Maria; Spinelli, Giovanni

    2009-01-01

    Sea urchin embryos are characterized by an extremely simple mode of development, rapid cleavage, high transparency, and well-defined cell lineage. Although they are not suitable for genetic studies, other approaches are successfully used to unravel mechanisms and molecules involved in cell fate specification and morphogenesis. Microinjection is the elective method to study gene function in sea urchin embryos. It is used to deliver precise amounts of DNA, RNA, oligonucleotides, peptides, or antibodies into the eggs or even into blastomeres. Here we describe microinjection as it is currently applied in our laboratory and show how it has been used in gene perturbation analyses and dissection of cis-regulatory DNA elements.

  2. Functional Studies of Regulatory Genes in the Sea Urchin Embryo

    NASA Astrophysics Data System (ADS)

    Cavalieri, Vincenzo; Bernardo, Maria Di; Spinelli, Giovanni

    Sea urchin embryos are characterized by an extremely simple mode of development, rapid cleavage, high transparency, and well-defined cell lineage. Although they are not suitable for genetic studies, other approaches are successfully used to unravel mechanisms and molecules involved in cell fate specification and morphogenesis. Microinjection is the elective method to study gene function in sea urchin embryos. It is used to deliver precise amounts of DNA, RNA, oligonucleotides, peptides, or antibodies into the eggs or even into blastomeres. Here we describe microinjection as it is currently applied in our laboratory and show how it has been used in gene perturbation analyses and dissection of cis-regulatory DNA elements.

  3. Roles of lignin biosynthesis and regulatory genes in plant development.

    PubMed

    Yoon, Jinmi; Choi, Heebak; An, Gynheung

    2015-11-01

    Lignin is an important factor affecting agricultural traits, biofuel production, and the pulping industry. Most lignin biosynthesis genes and their regulatory genes are expressed mainly in the vascular bundles of stems and leaves, preferentially in tissues undergoing lignification. Other genes are poorly expressed during normal stages of development, but are strongly induced by abiotic or biotic stresses. Some are expressed in non-lignifying tissues such as the shoot apical meristem. Alterations in lignin levels affect plant development. Suppression of lignin biosynthesis genes causes abnormal phenotypes such as collapsed xylem, bending stems, and growth retardation. The loss of expression by genes that function early in the lignin biosynthesis pathway results in more severe developmental phenotypes when compared with plants that have mutations in later genes. Defective lignin deposition is also associated with phenotypes of seed shattering or brittle culm. MYB and NAC transcriptional factors function as switches, and some homeobox proteins negatively control lignin biosynthesis genes. Ectopic deposition caused by overexpression of lignin biosynthesis genes or master switch genes induces curly leaf formation and dwarfism.

  4. Roles of lignin biosynthesis and regulatory genes in plant development

    PubMed Central

    Yoon, Jinmi; Choi, Heebak

    2015-01-01

    Abstract Lignin is an important factor affecting agricultural traits, biofuel production, and the pulping industry. Most lignin biosynthesis genes and their regulatory genes are expressed mainly in the vascular bundles of stems and leaves, preferentially in tissues undergoing lignification. Other genes are poorly expressed during normal stages of development, but are strongly induced by abiotic or biotic stresses. Some are expressed in non‐lignifying tissues such as the shoot apical meristem. Alterations in lignin levels affect plant development. Suppression of lignin biosynthesis genes causes abnormal phenotypes such as collapsed xylem, bending stems, and growth retardation. The loss of expression by genes that function early in the lignin biosynthesis pathway results in more severe developmental phenotypes when compared with plants that have mutations in later genes. Defective lignin deposition is also associated with phenotypes of seed shattering or brittle culm. MYB and NAC transcriptional factors function as switches, and some homeobox proteins negatively control lignin biosynthesis genes. Ectopic deposition caused by overexpression of lignin biosynthesis genes or master switch genes induces curly leaf formation and dwarfism. PMID:26297385

  5. Using gene expression programming to infer gene regulatory networks from time-series data.

    PubMed

    Zhang, Yongqing; Pu, Yifei; Zhang, Haisen; Su, Yabo; Zhang, Lifang; Zhou, Jiliu

    2013-12-01

    Gene regulatory networks inference is currently a topic under heavy research in the systems biology field. In this paper, gene regulatory networks are inferred via evolutionary model based on time-series microarray data. A non-linear differential equation model is adopted. Gene expression programming (GEP) is applied to identify the structure of the model and least mean square (LMS) is used to optimize the parameters in ordinary differential equations (ODEs). The proposed work has been first verified by synthetic data with noise-free and noisy time-series data, respectively, and then its effectiveness is confirmed by three real time-series expression datasets. Finally, a gene regulatory network was constructed with 12 Yeast genes. Experimental results demonstrate that our model can improve the prediction accuracy of microarray time-series data effectively. Copyright © 2013 Elsevier Ltd. All rights reserved.

  6. Downregulation of transcription factor Oct4 induces an epithelial-to-mesenchymal transition via enhancement of Ca{sup 2+} influx in breast cancer cells

    SciTech Connect

    Hu, Jiajia; Qin, Kunhua; Zhang, Yan; Gong, Junbo; Li, Na; Lv, Dan; Xiang, Rong; Tan, Xiaoyue

    2011-08-12

    Highlights: {yields} We examine the role of Oct4 in metastasis in cultured MCF-7 cells. {yields} The down regulation of Oct4 induces EMT and increases the capability of migration and invasion in MCF-7 cells. {yields} TGF-{beta}1 inhibits Oct4 expression in both time- and dose-dependent manners. {yields} The EMT induced by TGF-{beta}1 or down regulation of Oct4 could be abrogated by inhibitor of SOCE. {yields} The down regulation of STIM1 (one of the major components of the CRAC channel) alleviates the EMT induce by Oct4 silencing down. -- Abstract: The stem cell-related transcription factor Oct4 regulates tumor proliferation and apoptosis, but its role in tumor migration and invasion is still undefined. Here, we compared Oct4 expression in MCF-7 and MDA-MB-231 cells, two breast cancer cell lines with similar epithelial origins, but distinct invasive and metastatic characteristics. We found MCF-7 cells to express very high levels of Oct4, while no obvious expression was detected in MDA-MB-231 cells. We then downregulated Oct4 expression using small interfering RNA (siRNA) to explore its effects on migration and invasion. Transwell assays showed that silencing Oct4 in MCF-7 cells improved their migration and invasion capabilities. Reverse-transcriptase PCR and western blots showed that E-cadherin expression decreased, and {alpha}-smooth muscle actin expression increased with Oct4 downregulation, which suggests that epithelial-to-mesenchymal transition (EMT) occurred. A potent EMT stimulus, TGF-{beta}1, significantly inhibited Oct4 expression in both dose- and time course-dependent manners. Silencing Oct4 also upregulated expression of two major components of store-operated Ca{sup 2+} entry channels (SOCs), STIM1 and Orai1, and enhanced SOC-directed Ca{sup 2+} influx. Silencing STIM1 blocked the Ca{sup 2+} influx and rescued the EMT initiated by Oct4 downregulation. In conclusion, silencing Oct4 promotes invasion and metastasis in breast cancer cells by inducing EMT

  7. Comparison of evolutionary algorithms in gene regulatory network model inference

    PubMed Central

    2010-01-01

    Background The evolution of high throughput technologies that measure gene expression levels has created a data base for inferring GRNs (a process also known as reverse engineering of GRNs). However, the nature of these data has made this process very difficult. At the moment, several methods of discovering qualitative causal relationships between genes with high accuracy from microarray data exist, but large scale quantitative analysis on real biological datasets cannot be performed, to date, as existing approaches are not suitable for real microarray data which are noisy and insufficient. Results This paper performs an analysis of several existing evolutionary algorithms for quantitative gene regulatory network modelling. The aim is to present the techniques used and offer a comprehensive comparison of approaches, under a common framework. Algorithms are applied to both synthetic and real gene expression data from DNA microarrays, and ability to reproduce biological behaviour, scalability and robustness to noise are assessed and compared. Conclusions Presented is a comparison framework for assessment of evolutionary algorithms, used to infer gene regulatory networks. Promising methods are identified and a platform for development of appropriate model formalisms is established. PMID:20105328

  8. Comparison of evolutionary algorithms in gene regulatory network model inference.

    PubMed

    Sîrbu, Alina; Ruskin, Heather J; Crane, Martin

    2010-01-27

    The evolution of high throughput technologies that measure gene expression levels has created a data base for inferring GRNs (a process also known as reverse engineering of GRNs). However, the nature of these data has made this process very difficult. At the moment, several methods of discovering qualitative causal relationships between genes with high accuracy from microarray data exist, but large scale quantitative analysis on real biological datasets cannot be performed, to date, as existing approaches are not suitable for real microarray data which are noisy and insufficient. This paper performs an analysis of several existing evolutionary algorithms for quantitative gene regulatory network modelling. The aim is to present the techniques used and offer a comprehensive comparison of approaches, under a common framework. Algorithms are applied to both synthetic and real gene expression data from DNA microarrays, and ability to reproduce biological behaviour, scalability and robustness to noise are assessed and compared. Presented is a comparison framework for assessment of evolutionary algorithms, used to infer gene regulatory networks. Promising methods are identified and a platform for development of appropriate model formalisms is established.

  9. Expression of OCT4 and SALL4 in Diffuse Large B-cell Lymphoma: An Analysis of 145 Consecutive Cases and Testicular Lymphomas.

    PubMed

    Williams, Andrew S; Shawwa, Allam; Merrimen, Jennifer; Dakin Haché, Kelly

    2016-07-01

    OCT4 and SALL4 are transcription factors within a complex network that functions to maintain pluripotency in primitive stem cells and germ cells. Nuclear expression of OCT4 is widely cited as sensitive and specific for primary and metastatic germ cell tumors and is commonly used in the diagnosis of central nervous system (CNS) germinomas. Studies have failed to systematically examine the expression of OCT4 or SALL4 in diffuse large B-cell lymphoma (DLBCL), although this entity enters the morphologic differential diagnosis of some germ cell tumors. A retrospective review was conducted on 145 consecutive cases of DLBCL and testicular lymphoma to evaluate the prevalence of OCT4 and SALL4 expression. Nuclear OCT4 expression was present in 2/11 (18%) testicular DLBCLs and 6/134 (4.5%) nontesticular DLBCLs. Most OCT4 cases demonstrated moderate to strong expression in >50% of neoplastic cells. Rare, weak nuclear SALL4 expression was detected in only 3 nontesticular DLBCLs. Within the extratesticular DLBCL group, 2/6 (33%) primary CNS DLBCLs expressed nuclear OCT4. In addition, OCT4 DLBCL showed an overall predilection toward non-germinal center B-cell phenotype (7/8; 88%) and had a higher than expected rate of CD5 coexpression (4/8, 50%). These results are cautionary against using OCT4 as a sole marker of germ cell differentiation in testicular and extratesticular sites, especially in the CNS. The apparent associations of OCT4 expression with primary CNS DLBCL, non-germinal center B-cell phenotype, and CD5 coexpression raise the question of whether OCT4 expression in DLBCL may reflect more aggressive biology.

  10. Efficiently finding regulatory elements using correlation with gene expression.

    PubMed

    Bannai, Hideo; Inenaga, Shunsuke; Shinohara, Ayumi; Takeda, Masayuki; Miyano, Satoru

    2004-06-01

    We present an efficient algorithm for detecting putative regulatory elements in the upstream DNA sequences of genes, using gene expression information obtained from microarray experiments. Based on a generalized suffix tree, our algorithm looks for motif patterns whose appearance in the upstream region is most correlated with the expression levels of the genes. We are able to find the optimal pattern, in time linear in the total length of the upstream sequences. We implement and apply our algorithm to publicly available microarray gene expression data, and show that our method is able to discover biologically significant motifs, including various motifs which have been reported previously using the same data set. We further discuss applications for which the efficiency of the method is essential, as well as possible extensions to our algorithm.

  11. An Arabidopsis gene regulatory network for secondary cell wall synthesis

    SciTech Connect

    Taylor-Teeples, M.; Lin, L.; de Lucas, M.; Turco, G.; Toal, T. W.; Gaudinier, A.; Young, N. F.; Trabucco, G. M.; Veling, M. T.; Lamothe, R.; Handakumbura, P. P.; Xiong, G.; Wang, C.; Corwin, J.; Tsoukalas, A.; Zhang, L.; Ware, D.; Pauly, M.; Kliebenstein, D. J.; Dehesh, K.; Tagkopoulos, I.; Breton, G.; Pruneda-Paz, J. L.; Ahnert, S. E.; Kay, S. A.; Hazen, S. P.; Brady, S. M.

    2014-12-24

    The plant cell wall is an important factor for determining cell shape, function and response to the environment. Secondary cell walls, such as those found in xylem, are composed of cellulose, hemicelluloses and lignin and account for the bulk of plant biomass. The coordination between transcriptional regulation of synthesis for each polymer is complex and vital to cell function. A regulatory hierarchy of developmental switches has been proposed, although the full complement of regulators remains unknown. In this paper, we present a protein–DNA network between Arabidopsis thaliana transcription factors and secondary cell wall metabolic genes with gene expression regulated by a series of feed-forward loops. This model allowed us to develop and validate new hypotheses about secondary wall gene regulation under abiotic stress. Distinct stresses are able to perturb targeted genes to potentially promote functional adaptation. Finally, these interactions will serve as a foundation for understanding the regulation of a complex, integral plant component.

  12. Using shRNA experiments to validate gene regulatory networks.

    PubMed

    Olsen, Catharina; Fleming, Kathleen; Prendergast, Niall; Rubio, Renee; Emmert-Streib, Frank; Bontempi, Gianluca; Quackenbush, John; Haibe-Kains, Benjamin

    2015-06-01

    Quantitative validation of gene regulatory networks (GRNs) inferred from observational expression data is a difficult task usually involving time intensive and costly laboratory experiments. We were able to show that gene knock-down experiments can be used to quantitatively assess the quality of large-scale GRNs via a purely data-driven approach (Olsen et al. 2014). Our new validation framework also enables the statistical comparison of multiple network inference techniques, which was a long-standing challenge in the field. In this Data in Brief we detail the contents and quality controls for the gene expression data (available from NCBI Gene Expression Omnibus repository with accession number GSE53091) associated with our study published in Genomics (Olsen et al. 2014). We also provide R code to access the data and reproduce the analysis presented in this article.

  13. Identification of cis-regulatory mutations generating de novo edges in personalized cancer gene regulatory networks.

    PubMed

    Kalender Atak, Zeynep; Imrichova, Hana; Svetlichnyy, Dmitry; Hulselmans, Gert; Christiaens, Valerie; Reumers, Joke; Ceulemans, Hugo; Aerts, Stein

    2017-08-30

    The identification of functional non-coding mutations is a key challenge in the field of genomics. Here we introduce μ-cisTarget to filter, annotate and prioritize cis-regulatory mutations based on their putative effect on the underlying "personal" gene regulatory network. We validated μ-cisTarget by re-analyzing the TAL1 and LMO1 enhancer mutations in T-ALL, and the TERT promoter mutation in melanoma. Next, we re-sequenced the full genomes of ten cancer cell lines and used matched transcriptome data and motif discovery to identify master regulators with de novo binding sites that result in the up-regulation of nearby oncogenic drivers. μ-cisTarget is available from http://mucistarget.aertslab.org .

  14. Inference of Gene Regulatory Network Based on Local Bayesian Networks.

    PubMed

    Liu, Fei; Zhang, Shao-Wu; Guo, Wei-Feng; Wei, Ze-Gang; Chen, Luonan

    2016-08-01

    The inference of gene regulatory networks (GRNs) from expression data can mine the direct regulations among genes and gain deep insights into biological processes at a network level. During past decades, numerous computational approaches have been introduced for inferring the GRNs. However, many of them still suffer from various problems, e.g., Bayesian network (BN) methods cannot handle large-scale networks due to their high computational complexity, while information theory-based methods cannot identify the directions of regulatory interactions and also suffer from false positive/negative problems. To overcome the limitations, in this work we present a novel algorithm, namely local Bayesian network (LBN), to infer GRNs from gene expression data by using the network decomposition strategy and false-positive edge elimination scheme. Specifically, LBN algorithm first uses conditional mutual information (CMI) to construct an initial network or GRN, which is decomposed into a number of local networks or GRNs. Then, BN method is employed to generate a series of local BNs by selecting the k-nearest neighbors of each gene as its candidate regulatory genes, which significantly reduces the exponential search space from all possible GRN structures. Integrating these local BNs forms a tentative network or GRN by performing CMI, which reduces redundant regulations in the GRN and thus alleviates the false positive problem. The final network or GRN can be obtained by iteratively performing CMI and local BN on the tentative network. In the iterative process, the false or redundant regulations are gradually removed. When tested on the benchmark GRN datasets from DREAM challenge as well as the SOS DNA repair network in E.coli, our results suggest that LBN outperforms other state-of-the-art methods (ARACNE, GENIE3 and NARROMI) significantly, with more accurate and robust performance. In particular, the decomposition strategy with local Bayesian networks not only effectively reduce

  15. Regulatory Features for Odorant Receptor Genes in the Mouse Genome.

    PubMed

    Degl'Innocenti, Andrea; D'Errico, Anna

    2017-01-01

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

  16. Regulatory Features for Odorant Receptor Genes in the Mouse Genome

    PubMed Central

    Degl’Innocenti, Andrea; D’Errico, Anna

    2017-01-01

    The odorant receptor genes, seven transmembrane receptor genes constituting the vastest mammalian gene multifamily, are expressed monogenically and monoallelicaly in each sensory neuron in the olfactory epithelium. This characteristic, often referred to as the one neuron–one receptor rule, is driven by mostly uncharacterized molecular dynamics, generally named odorant receptor gene choice. Much attention has been paid by the scientific community to the identification of sequences regulating the expression of odorant receptor genes within their loci, where related genes are usually arranged in genomic clusters. A number of studies identified transcription factor binding sites on odorant receptor promoter sequences. Similar binding sites were also found on a number of enhancers that regulate in cis their transcription, but have been proposed to form interchromosomal networks. Odorant receptor gene choice seems to occur via the local removal of strongly repressive epigenetic markings, put in place during the maturation of the sensory neuron on each odorant receptor locus. Here we review the fast-changing state of art for the study of regulatory features for odorant receptor genes. PMID:28270833

  17. Helicobacter pylori upregulates Nanog and Oct4 via Wnt/β-catenin signaling pathway to promote cancer stem cell-like properties in human gastric cancer.

    PubMed

    Yong, Xin; Tang, Bo; Xiao, Yu-Feng; Xie, Rui; Qin, Yong; Luo, Gang; Hu, Chang-Jiang; Dong, Hui; Yang, Shi-Ming

    2016-05-01

    Helicobacter pylori (H. pylori) infection is considered a major risk factor for gastric cancer. CagA behaves as a major bacterial oncoprotein playing a key role in H. pylori-induced tumorigenesis. Cancer stem cells (CSCs) are believed to possess the ability to initiate tumorigenesis and promote progression. Although studies have suggested that cancer cells can exhibit CSC-like properties in the tumor microenvironment, it remains unclear whether H. pylori infection could induce the emergence of CSC-like properties in gastric cancer cells and, the underlying mechanism. Here, gastric cancer cells were co-cultured with a CagA-positive H. pylori strain or a CagA isogenic mutant strain. We found that H. pylori-infected gastric cancer cells exhibited CSC-like properties, including an increased expression of CSC specific surface markers CD44 and Lgr5, as well as that of Nanog, Oct4 and c-myc, which are known pluripotency genes, and an increased capacity for self-renewal, whereas these properties were not observed in the CagA isogenic mutant strain-infected cells. Further studies revealed that H. pylori activated Wnt/β-catenin signaling pathway in a CagA-dependent manner and that the activation of this pathway was dependent upon CagA-positive H. pylori-mediated phosphorylation of β-catenin at the C-terminal Ser675 and Ser552 residues in a c-met- and/or Akt-dependent manner. We further demonstrated that this activation was responsible for H. pylori-induced CSC-like properties. Moreover, we found the promoter activity of Nanog and Oct4 were upregulated, and β-catenin was observed to bind to these promoters during H. pylori infection, while a Wnt/β-catenin inhibitor suppressed promoter activity and binding. Taken together, these results suggest that H. pylori upregulates Nanog and Oct4 via Wnt/β-catenin signaling pathway to promote CSC-like properties in gastric cancer cells.

  18. Gene regulatory mechanisms governing energy metabolism during cardiac hypertrophic growth.

    PubMed

    Lehman, John J; Kelly, Daniel P

    2002-04-01

    Studies in a variety of mammalian species, including humans, have demonstrated a reduction in fatty acid oxidation (FAO) and increased glucose utilization in pathologic cardiac hypertrophy, consistent with reinduction of the fetal energy metabolic program. This review describes results of recent molecular studies aimed at delineating the gene regulatory events which facilitate myocardial energy substrate switches during hypertrophic growth of the heart. Studies aimed at the characterization of transcriptional control mechanisms governing FAO enzyme gene expression in the cardiac myocyte have defined a central role for the fatty acid-activated nuclear receptor peroxisome proliferator-activated receptor alpha (PPAR(alpha)). Cardiac FAO enzyme gene expression was shown to be coordinately downregulated in murine models of ventricular pressure overload, consistent with the energy substrate switch away from fatty acid utilization in the hypertrophied heart. Nuclear protein levels of PPAR(alpha) decline in the ventricle in response to pressure overload, while several Sp and nuclear receptor transcription factors are induced to fetal levels, consistent with their binding to DNA as transcriptional repressors of rate-limiting FAO enzyme genes with hypertrophy. Knowledge of key components of this transcriptional regulatory pathway will allow for the development of genetic engineering strategies in mice that will modulate fatty acid oxidative flux and assist in defining whether energy metabolic derangements play a primary role in the development of pathologic cardiac hypertrophy and eventual progression to heart failure.

  19. Regulatory myosin light-chain genes of Caenorhabditis elegans.

    PubMed Central

    Cummins, C; Anderson, P

    1988-01-01

    We have cloned and analyzed the Caenorhabditis elegans regulatory myosin light-chain genes. C. elegans contains two such genes, which we have designated mlc-1 and mlc-2. The two genes are separated by 2.6 kilobases and are divergently transcribed. We determined the complete nucleotide sequences of both mlc-1 and mlc-2. A single, conservative amino acid substitution distinguishes the sequences of the two proteins. The C. elegans proteins are strongly homologous to regulatory myosin light chains of Drosophila melanogaster and vertebrates and weakly homologous to a superfamily of eucaryotic calcium-binding proteins. Both mlc-1 and mlc-2 encode abundant mRNAs. We mapped the 5' termini of these transcripts by using primer extension sequencing of mRNA templates. mlc-1 mRNAs initiate within conserved hexanucleotides at two different positions, located at -28 and -38 relative to the start of translation. The 5' terminus of mlc-2 mRNA is not encoded in the 4.8-kilobase genomic region upstream of mlc-2. Rather, mlc-2 mRNA contains at its 5' end a short, untranslated leader sequence that is identical to the trans-spliced leader sequence of three C. elegans actin genes. Images PMID:3244358

  20. Fused Regression for Multi-source Gene Regulatory Network Inference

    PubMed Central

    Lam, Kari Y.; Westrick, Zachary M.; Müller, Christian L.; Christiaen, Lionel; Bonneau, Richard

    2016-01-01

    Understanding gene regulatory networks is critical to understanding cellular differentiation and response to external stimuli. Methods for global network inference have been developed and applied to a variety of species. Most approaches consider the problem of network inference independently in each species, despite evidence that gene regulation can be conserved even in distantly related species. Further, network inference is often confined to single data-types (single platforms) and single cell types. We introduce a method for multi-source network inference that allows simultaneous estimation of gene regulatory networks in multiple species or biological processes through the introduction of priors based on known gene relationships such as orthology incorporated using fused regression. This approach improves network inference performance even when orthology mapping and conservation are incomplete. We refine this method by presenting an algorithm that extracts the true conserved subnetwork from a larger set of potentially conserved interactions and demonstrate the utility of our method in cross species network inference. Last, we demonstrate our method’s utility in learning from data collected on different experimental platforms. PMID:27923054

  1. SRY and OCT4 Are Required for the Acquisition of Cancer Stem Cell-Like Properties and Are Potential Differentiation Therapy Targets.

    PubMed

    Murakami, Shigekazu; Ninomiya, Wataru; Sakamoto, Erina; Shibata, Tatsuhiro; Akiyama, Hirotada; Tashiro, Fumio

    2015-09-01

    The acquisition of stemness is a hallmark of aggressive human hepatocellular carcinoma (hHCC). The stem cell marker OCT4 is frequently expressed in HCCs, and its expression correlates with those of putative cancer stem cell (CSC) markers and CSC properties. Here, we describe a novel mechanism of CSC maintenance by SRY through OCT4. We previously reported that Sry is involved in tumor malignancy in rodent HCCs. However, the oncogenic function of SRY in hHCCs is poorly understood. Ectopic expression of SRY increased multiple stem cell factors, including OCT4 and CD13. The OCT4 promoter contained SRY-binding sites that were directly activated by SRY. In HCC-derived cells, SRY knockdown decreased OCT4 expression and cancer stem-like phenotypes such as self-renewal, chemoresistance, and tumorigenicity. Conversely, OCT4 and SRY overexpression promoted cancer stem-like phenotypes. OCT4 knockdown in SRY clones downregulated the self-renewal capacity and chemoresistance. These data suggest that SRY is involved in the maintenance of cancer stem-like characteristics through OCT4. Moreover, CSCs of HCC-derived cells differentiated into Tuj1-positive neuron-like cells by retinoic acid. Noteworthily, SRY was highly expressed in some hHCC patients. Taken together, our findings imply a novel therapeutic strategy against CSCs of hHCCs.

  2. Regulatory Oversight of Cell and Gene Therapy Products in Canada.

    PubMed

    Ridgway, Anthony; Agbanyo, Francisca; Wang, Jian; Rosu-Myles, Michael

    2015-01-01

    Health Canada regulates gene therapy products and many cell therapy products as biological drugs under the Canadian Food and Drugs Act and its attendant regulations. Cellular products that meet certain criteria, including minimal manipulation and homologous use, may be subjected to a standards-based approach under the Safety of Human Cells, Tissues and Organs for Transplantation Regulations. The manufacture and clinical testing of cell and gene therapy products (CGTPs) presents many challenges beyond those for protein biologics. Cells cannot be subjected to pathogen removal or inactivation procedures and must frequently be administered shortly after final formulation. Viral vector design and manufacturing control are critically important to overall product quality and linked to safety and efficacy in patients through concerns such as replication competence, vector integration, and vector shedding. In addition, for many CGTPs, the value of nonclinical studies is largely limited to providing proof of concept, and the first meaningful data relating to appropriate dosing, safety parameters, and validity of surrogate or true determinants of efficacy must come from carefully designed clinical trials in patients. Addressing these numerous challenges requires application of various risk mitigation strategies and meeting regulatory expectations specifically adapted to the product types. Regulatory cooperation and harmonisation at an international level are essential for progress in the development and commercialisation of these products. However, particularly in the area of cell therapy, new regulatory paradigms may be needed to harness the benefits of clinical progress in situations where the resources and motivation to pursue a typical drug product approval pathway may be lacking.

  3. Resolution of gene regulatory conflicts caused by combinations of antibiotics

    PubMed Central

    Bollenbach, Tobias; Kishony, Roy

    2011-01-01

    SUMMARY Regulatory conflicts occur when two signals which individually trigger opposite cellular responses are present simultaneously. Here, we investigate regulatory conflicts in the bacterial response to antibiotic combinations. We use an Escherichia coli promoter-GFP library to study the transcriptional response of many promoters to either additive or antagonistic drug pairs at fine two-dimensional resolution of drug concentration. Surprisingly, we find that this dataset can be characterized as a linear sum of only two principal components. Component one, accounting for over 70% of the response, represents the response to growth inhibition by the drugs. Component two describes how regulatory conflicts are resolved. For the additive drug pair, conflicts are resolved by linearly interpolating the single drug responses, while for the antagonistic drug pair, the growth-limiting drug dominates the response. Importantly, for a given drug pair, the same conflict resolution strategy applies to almost all genes. These results provide a recipe for predicting gene expression responses to antibiotic combinations. PMID:21596308

  4. The gene regulatory network for breast cancer: integrated regulatory landscape of cancer hallmarks.

    PubMed

    Emmert-Streib, Frank; de Matos Simoes, Ricardo; Mullan, Paul; Haibe-Kains, Benjamin; Dehmer, Matthias

    2014-01-01

    In this study, we infer the breast cancer gene regulatory network from gene expression data. This network is obtained from the application of the BC3Net inference algorithm to a large-scale gene expression data set consisting of 351 patient samples. In order to elucidate the functional relevance of the inferred network, we are performing a Gene Ontology (GO) analysis for its structural components. Our analysis reveals that most significant GO-terms we find for the breast cancer network represent functional modules of biological processes that are described by known cancer hallmarks, including translation, immune response, cell cycle, organelle fission, mitosis, cell adhesion, RNA processing, RNA splicing and response to wounding. Furthermore, by using a curated list of census cancer genes, we find an enrichment in these functional modules. Finally, we study cooperative effects of chromosomes based on information of interacting genes in the beast cancer network. We find that chromosome 21 is most coactive with other chromosomes. To our knowledge this is the first study investigating the genome-scale breast cancer network.

  5. Regulatory aspects for translating gene therapy research into the clinic.

    PubMed

    Laurencot, Carolyn M; Ruppel, Sheryl

    2009-01-01

    Gene therapy products are highly regulated, therefore moving a promising candidate from the laboratory into the clinic can present unique challenges. Success can only be achieved by proper planning and communication within the clinical development team, as well as consultation with the regulatory scientists who will eventually review the clinical plan. Regulators should not be considered as obstacles but rather as collaborators whose advice can significantly expedite the product development. Sound scientific data is required and reviewed by the regulatory agencies to determine whether the potential benefit to the patient population outweighs the risk. Therefore, compliance with Good Manufacturing Practice (GMP) and Good Laboratory Practice (GLP) principles to ensure quality, safety, purity, and potency of the product, and to establish "proof of concept" for efficacy, and for safety information, respectively, is essential. The design and conduct of the clinical trial must adhere to Good Clinical Practice (GCP) principals. The clinical protocol should contain adequate rationale, supported by nonclinical data, to justify the starting dose and regimen, and adequate safety monitoring based on the patient population and the anticipated toxicities. Proper review and approval of gene therapy clinical studies by numerous committees, and regulatory agencies before and throughout the study allows for ongoing risk assessment of these novel and innovative products. The ethical conduct of clinical trials must be a priority for all clinical investigators and sponsors. As history has shown us, only a few fatal mistakes can dramatically alter the regulation of investigational products for all individuals involved in gene therapy clinical research, and further delay the advancement of gene therapy to licensed medicinal products.

  6. Signaling and Gene Regulatory Networks in Mammalian Lens Development.

    PubMed

    Cvekl, Ales; Zhang, Xin

    2017-10-01

    Ocular lens development represents an advantageous system in which to study regulatory mechanisms governing cell fate decisions, extracellular signaling, cell and tissue organization, and the underlying gene regulatory networks. Spatiotemporally regulated domains of BMP, FGF, and other signaling molecules in late gastrula-early neurula stage embryos generate the border region between the neural plate and non-neural ectoderm from which multiple cell types, including lens progenitor cells, emerge and undergo initial tissue formation. Extracellular signaling and DNA-binding transcription factors govern lens and optic cup morphogenesis. Pax6, c-Maf, Hsf4, Prox1, Sox1, and a few additional factors regulate the expression of the lens structural proteins, the crystallins. Extensive crosstalk between a diverse array of signaling pathways controls the complexity and order of lens morphogenetic processes and lens transparency. Copyright © 2017 Elsevier Ltd. All rights reserved.

  7. Autonomous Boolean modelling of developmental gene regulatory networks

    PubMed Central

    Cheng, Xianrui; Sun, Mengyang; Socolar, Joshua E. S.

    2013-01-01

    During early embryonic development, a network of regulatory interactions among genes dynamically determines a pattern of differentiated tissues. We show that important timing information associated with the interactions can be faithfully represented in autonomous Boolean models in which binary variables representing expression levels are updated in continuous time, and that such models can provide a direct insight into features that are difficult to extract from ordinary differential equation (ODE) models. As an application, we model the experimentally well-studied network controlling fly body segmentation. The Boolean model successfully generates the patterns formed in normal and genetically perturbed fly embryos, permits the derivation of constraints on the time delay parameters, clarifies the logic associated with different ODE parameter sets and provides a platform for studying connectivity and robustness in parameter space. By elucidating the role of regulatory time delays in pattern formation, the results suggest new types of experimental measurements in early embryonic development. PMID:23034351

  8. Optimal finite horizon control in gene regulatory networks

    NASA Astrophysics Data System (ADS)

    Liu, Qiuli

    2013-06-01

    As a paradigm for modeling gene regulatory networks, probabilistic Boolean networks (PBNs) form a subclass of Markov genetic regulatory networks. To date, many different stochastic optimal control approaches have been developed to find therapeutic intervention strategies for PBNs. A PBN is essentially a collection of constituent Boolean networks via a probability structure. Most of the existing works assume that the probability structure for Boolean networks selection is known. Such an assumption cannot be satisfied in practice since the presence of noise prevents the probability structure from being accurately determined. In this paper, we treat a case in which we lack the governing probability structure for Boolean network selection. Specifically, in the framework of PBNs, the theory of finite horizon Markov decision process is employed to find optimal constituent Boolean networks with respect to the defined objective functions. In order to illustrate the validity of our proposed approach, an example is also displayed.

  9. Expression of Ki-67, Oct-4, γ-tubulin and α-tubulin in human tooth development.

    PubMed

    Kero, Darko; Novakovic, Josip; Vukojevic, Katarina; Petricevic, Josko; Kalibovic Govorko, Danijela; Biocina-Lukenda, Dolores; Saraga-Babic, Mirna

    2014-11-01

    To analyze factors controlling cell proliferation and differentiation, and appearance of primary cilia during the cap and bell stages of incisor or/and canine human enamel organs. Qualitative and quantitative analysis of proliferating Ki-67 positive cells and expression of γ-tubulin, α-tubulin and Oct-4 was immunohistochemically analyzed in the cap an bell stages of 10 developing human incisor and canine germs, 8-21 weeks old. During the analyzed period, ratio of Ki-67 positive cells changed in outer enamel epithelium from 48.86% to 24.52%, in inner enamel epithelium increased from 56.11% to 60.06% and then dropped to 44.24%. While in dental papilla proliferation first increased from 46.26% to 55.45%, and then dropped to 22.08%, a constant decrease of proliferation characterized enamel reticulum (from 46.26% to 15.49%). Strong cytoplasmic Oct-4 expression characterized epithelial parts of enamel organ, particularly the differentiating ameloblasts. During further development, Oct-4 expression shifted to both nuclear and cytoplasmic expression in mesenchymal tooth components. Primary cilia characterized most of the cells in developing enamel organ. While non-ciliated (proliferating) cells mainly contained two centrioles (γ-tubulin), the primary cilia (α-tubulin) were arising from basal bodies (γ-tubulin) of non-proliferating cells. We suggest that increase in cell proliferation enables growth of enamel organ, while its selective decrease leads to disintegration of some tooth parts. Drop of proliferation coincided with initiation of ameloblast and odontoblast differentiation. Additionally, cell differentiation was accompanied by increased expression of Oct-4 and probably by signalling via primary cilia, both regulating processes of cell proliferation and differentiation. Copyright © 2014 Elsevier Ltd. All rights reserved.

  10. Innovation and robustness in complex regulatory gene networks

    PubMed Central

    Ciliberti, S.; Martin, O. C.; Wagner, A.

    2007-01-01

    The history of life involves countless evolutionary innovations, a steady stream of ingenuity that has been flowing for more than 3 billion years. Very little is known about the principles of biological organization that allow such innovation. Here, we examine these principles for evolutionary innovation in gene expression patterns. To this end, we study a model for the transcriptional regulation networks that are at the heart of embryonic development. A genotype corresponds to a regulatory network of a given topology, and a phenotype corresponds to a steady-state gene expression pattern. Networks with the same phenotype form a connected graph in genotype space, where two networks are immediate neighbors if they differ by one regulatory interaction. We show that an evolutionary search on this graph can reach genotypes that are as different from each other as if they were chosen at random in genotype space, allowing evolutionary access to different kinds of innovation while staying close to a viable phenotype. Thus, although robustness to mutations may hinder innovation in the short term, we conclude that long-term innovation in gene expression patterns can only emerge in the presence of the robustness caused by connected genotype graphs. PMID:17690244

  11. A Provisional Gene Regulatory Atlas for Mouse Heart Development

    PubMed Central

    Chen, Hailin; VanBuren, Vincent

    2014-01-01

    Congenital Heart Disease (CHD) is one of the most common birth defects. Elucidating the molecular mechanisms underlying normal cardiac development is an important step towards early identification of abnormalities during the developmental program and towards the creation of early intervention strategies. We developed a novel computational strategy for leveraging high-content data sets, including a large selection of microarray data associated with mouse cardiac development, mouse genome sequence, ChIP-seq data of selected mouse transcription factors and Y2H data of mouse protein-protein interactions, to infer the active transcriptional regulatory network of mouse cardiac development. We identified phase-specific expression activity for 765 overlapping gene co-expression modules that were defined for obtained cardiac lineage microarray data. For each co-expression module, we identified the phase of cardiac development where gene expression for that module was higher than other phases. Co-expression modules were found to be consistent with biological pathway knowledge in Wikipathways, and met expectations for enrichment of pathways involved in heart lineage development. Over 359,000 transcription factor-target relationships were inferred by analyzing the promoter sequences within each gene module for overrepresentation against the JASPAR database of Transcription Factor Binding Site (TFBS) motifs. The provisional regulatory network will provide a framework of studying the genetic basis of CHD. PMID:24421884

  12. Reverse Engineering of Genome-wide Gene Regulatory Networks from Gene Expression Data.

    PubMed

    Liu, Zhi-Ping

    2015-02-01

    Transcriptional regulation plays vital roles in many fundamental biological processes. Reverse engineering of genome-wide regulatory networks from high-throughput transcriptomic data provides a promising way to characterize the global scenario of regulatory relationships between regulators and their targets. In this review, we summarize and categorize the main frameworks and methods currently available for inferring transcriptional regulatory networks from microarray gene expression profiling data. We overview each of strategies and introduce representative methods respectively. Their assumptions, advantages, shortcomings, and possible improvements and extensions are also clarified and commented.

  13. Reverse Engineering of Genome-wide Gene Regulatory Networks from Gene Expression Data

    PubMed Central

    Liu, Zhi-Ping

    2015-01-01

    Transcriptional regulation plays vital roles in many fundamental biological processes. Reverse engineering of genome-wide regulatory networks from high-throughput transcriptomic data provides a promising way to characterize the global scenario of regulatory relationships between regulators and their targets. In this review, we summarize and categorize the main frameworks and methods currently available for inferring transcriptional regulatory networks from microarray gene expression profiling data. We overview each of strategies and introduce representative methods respectively. Their assumptions, advantages, shortcomings, and possible improvements and extensions are also clarified and commented. PMID:25937810

  14. Identifying gene regulatory network rewiring using latent differential graphical models

    PubMed Central

    Tian, Dechao; Gu, Quanquan; Ma, Jian

    2016-01-01

    Gene regulatory networks (GRNs) are highly dynamic among different tissue types. Identifying tissue-specific gene regulation is critically important to understand gene function in a particular cellular context. Graphical models have been used to estimate GRN from gene expression data to distinguish direct interactions from indirect associations. However, most existing methods estimate GRN for a specific cell/tissue type or in a tissue-naive way, or do not specifically focus on network rewiring between different tissues. Here, we describe a new method called Latent Differential Graphical Model (LDGM). The motivation of our method is to estimate the differential network between two tissue types directly without inferring the network for individual tissues, which has the advantage of utilizing much smaller sample size to achieve reliable differential network estimation. Our simulation results demonstrated that LDGM consistently outperforms other Gaussian graphical model based methods. We further evaluated LDGM by applying to the brain and blood gene expression data from the GTEx consortium. We also applied LDGM to identify network rewiring between cancer subtypes using the TCGA breast cancer samples. Our results suggest that LDGM is an effective method to infer differential network using high-throughput gene expression data to identify GRN dynamics among different cellular conditions. PMID:27378774

  15. Engineering nucleases for gene targeting: safety and regulatory considerations.

    PubMed

    Pauwels, Katia; Podevin, Nancy; Breyer, Didier; Carroll, Dana; Herman, Philippe

    2014-01-25

    Nuclease-based gene targeting (NBGT) represents a significant breakthrough in targeted genome editing since it is applicable from single-celled protozoa to human, including several species of economic importance. Along with the fast progress in NBGT and the increasing availability of customized nucleases, more data are available about off-target effects associated with the use of this approach. We discuss how NBGT may offer a new perspective for genetic modification, we address some aspects crucial for a safety improvement of the corresponding techniques and we also briefly relate the use of NBGT applications and products to the regulatory oversight.

  16. Gene regulatory networks governing haematopoietic stem cell development and identity.

    PubMed

    Pimanda, John E; Göttgens, Berthold

    2010-01-01

    Development can be viewed as a dynamic progression through regulatory states which characterise the various cell types within a given differentiation cascade. To understand the progression of regulatory states that define the origin and subsequent development of haematopoietic stem cells, the first imperative is to understand the ontogeny of haematopoiesis. We are fortunate that the ontogeny of blood development is one of the best characterized mammalian developmental systems. However, the field is still in its infancy with regard to the reconstruction of gene regulatory networks and their interactions with cell signalling cascades that drive a mesodermal progenitor to adopt the identity of a haematopoietic stem cell and beyond. Nevertheless, a framework to dissect these networks and comprehend the logic of its circuitry does exist and although they may not as yet be available, a sense for the tools that will be required to achieve this aim is also emerging. In this review we cover the fundamentals of network architecture, methods used to reconstruct networks, current knowledge of haematopoietic and related transcriptional networks, current challenges and future outlook.

  17. Selection for distinct gene expression properties favours the evolution of mutational robustness in gene regulatory networks.

    PubMed

    Espinosa-Soto, C

    2016-11-01

    Mutational robustness is a genotype's tendency to keep a phenotypic trait with little and few changes in the face of mutations. Mutational robustness is both ubiquitous and evolutionarily important as it affects in different ways the probability that new phenotypic variation arises. Understanding the origins of robustness is specially relevant for systems of development that are phylogenetically widespread and that construct phenotypic traits with a strong impact on fitness. Gene regulatory networks are examples of this class of systems. They comprise sets of genes that, through cross-regulation, build the gene activity patterns that define cellular responses, different tissues or distinct cell types. Several empirical observations, such as a greater robustness of wild-type phenotypes, suggest that stabilizing selection underlies the evolution of mutational robustness. However, the role of selection in the evolution of robustness is still under debate. Computer simulations of the dynamics and evolution of gene regulatory networks have shown that selection for any gene activity pattern that is steady and self-sustaining is sufficient to promote the evolution of mutational robustness. Here, I generalize this scenario using a computational model to show that selection for different aspects of a gene activity phenotype increases mutational robustness. Mutational robustness evolves even when selection favours properties that conflict with the stationarity of a gene activity pattern. The results that I present support an important role for stabilizing selection in the evolution of robustness in gene regulatory networks.

  18. Regulatory dynamics of synthetic gene networks with positive feedback.

    PubMed

    Maeda, Yusuke T; Sano, Masaki

    2006-06-16

    Biological processes are governed by complex networks ranging from gene regulation to signal transduction. Positive feedback is a key element in such networks. The regulation enables cells to adopt multiple internal expression states in response to a single external input signal. However, past works lacked a dynamical aspect of this system. To address the dynamical property of the positive feedback system, we employ synthetic gene circuits in Escherichia coli to measure the rise-time of both the no-feedback system and the positive feedback system. We show that the kinetics of gene expression is slowed down if the gene regulatory system includes positive feedback. We also report that the transition of gene switching behaviors from the hysteretic one to the graded one occurs. A mathematical model based on the chemical reactions shows that the response delay is an inherited property of the positive feedback system. Furthermore, with the aid of the phase diagram, we demonstrate the decline of the feedback activation causes the transition of switching behaviors. Our findings provide a further understanding of a positive feedback system in a living cell from a dynamical point of view.

  19. Using synthetic biology to study gene regulatory evolution.

    PubMed

    Crocker, Justin; Ilsley, Garth R

    2017-09-29

    Transcriptional enhancers specify the precise time, level, and location of gene expression. Disentangling and characterizing the components of enhancer activity in multicellular eukaryotic development has proven challenging because enhancers contain activator and repressor binding sites for multiple factors that each exert nuanced, context-dependent control of enhancer activity. Recent advances in synthetic biology provide an almost unlimited ability to create and modify regulatory elements and networks, offering unprecedented power to study gene regulation. Here we review several studies demonstrating the utility of synthetic biology for studying enhancer function during development and evolution. These studies clearly show that synthetic biology can provide a way to reverse-engineer and reengineer transcriptional regulation in animal genomes with enormous potential for understanding evolution. Copyright © 2017 Elsevier Ltd. All rights reserved.

  20. Reverse engineering of gene regulatory networks: a comparative study.

    PubMed

    Hache, Hendrik; Lehrach, Hans; Herwig, Ralf

    2009-01-01

    Reverse engineering of gene regulatory networks has been an intensively studied topic in bioinformatics since it constitutes an intermediate step from explorative to causative gene expression analysis. Many methods have been proposed through recent years leading to a wide range of mathematical approaches. In practice, different mathematical approaches will generate different resulting network structures, thus, it is very important for users to assess the performance of these algorithms. We have conducted a comparative study with six different reverse engineering methods, including relevance networks, neural networks, and Bayesian networks. Our approach consists of the generation of defined benchmark data, the analysis of these data with the different methods, and the assessment of algorithmic performances by statistical analyses. Performance was judged by network size and noise levels. The results of the comparative study highlight the neural network approach as best performing method among those under study.

  1. Diverse Gene Expression in Human Regulatory T Cell Subsets Uncovers Connection between Regulatory T Cell Genes and Suppressive Function.

    PubMed

    Hua, Jing; Davis, Scott P; Hill, Jonathan A; Yamagata, Tetsuya

    2015-10-15

    Regulatory T (Treg) cells have a critical role in the control of immunity, and their diverse subpopulations may allow adaptation to different types of immune responses. In this study, we analyzed human Treg cell subpopulations in the peripheral blood by performing genome-wide expression profiling of 40 Treg cell subsets from healthy donors. We found that the human peripheral blood Treg cell population is comprised of five major genomic subgroups, represented by 16 tractable subsets with a particular cell surface phenotype. These subsets possess a range of suppressive function and cytokine secretion and can exert a genomic footprint on target effector T (Teff) cells. Correlation analysis of variability in gene expression in the subsets identified several cell surface molecules associated with Treg suppressive function, and pharmacological interrogation revealed a set of genes having causative effect. The five genomic subgroups of Treg cells imposed a preserved pattern of gene expression on Teff cells, with a varying degree of genes being suppressed or induced. Notably, there was a cluster of genes induced by Treg cells that bolstered an autoinhibitory effect in Teff cells, and this induction appears to be governed by a different set of genes than ones involved in counteracting Teff activation. Our work shows an example of exploiting the diversity within human Treg cell subpopulations to dissect Treg cell biology.

  2. Genomic Aberrations Frequently Alter Chromatin Regulatory Genes in Chordoma

    PubMed Central

    Wang, Lu; Zehir, Ahmet; Nafa, Khedoudja; Zhou, Nengyi; Berger, Michael F.; Casanova, Jacklyn; Sadowska, Justyna; Lu, Chao; Allis, C. David; Gounder, Mrinal; Chandhanayingyong, Chandhanarat; Ladanyi, Marc; Boland, Patrick J; Hameed, Meera

    2016-01-01

    Chordoma is a rare primary bone neoplasm that is resistant to standard chemotherapies. Despite aggressive surgical management, local recurrence and metastasis is not uncommon. To identify the specific genetic aberrations that play key roles in chordoma pathogenesis, we utilized a genome-wide high-resolution SNP-array and next generation sequencing (NGS)-based molecular profiling platform to study 24 patient samples with typical histopathologic features of chordoma. Matching normal tissues were available for 16 samples. SNP-array analysis revealed nonrandom copy number losses across the genome, frequently involving 3, 9p, 1p, 14, 10, and 13. In contrast, copy number gain is uncommon in chordomas. Two minimum deleted regions were observed on 3p within a ~8 Mb segment at 3p21.1–p21.31, which overlaps SETD2, BAP1 and PBRM1. The minimum deleted region on 9p was mapped to CDKN2A locus at 9p21.3, and homozygous deletion of CDKN2A was detected in 5/22 chordomas (~23%). NGS-based molecular profiling demonstrated an extremely low level of mutation rate in chordomas, with an average of 0.5 mutations per sample for the 16 cases with matched normal. When the mutated genes were grouped based on molecular functions, many of the mutation events (~40%) were found in chromatin regulatory genes. The combined copy number and mutation profiling revealed that SETD2 is the single gene affected most frequently in chordomas, either by deletion or by mutations. Our study demonstrated that chordoma belongs to the C-class (copy number changes) tumors whose oncogenic signature is non-random multiple copy number losses across the genome and genomic aberrations frequently alter chromatin regulatory genes. PMID:27072194

  3. Exogenous Oct4 in combination with valproic acid increased neural progenitor markers: an approach for enhancing the repair potential of the brain.

    PubMed

    Dehghan, Samaneh; Asadi, Sareh; Hajikaram, Maryam; Soleimani, Masoud; Mowla, Seyed Javad; Fathollahi, Yaghoub; Ahmadiani, Abolhassan; Javan, Mohammad

    2015-02-01

    Attempts are aimed to introduce new approaches toward enhancing the brain's potential for repair in neurodegenerative diseases and traumatic injuries. Here we report an increased expression of pluripotency and progenitor markers within the brain following pretreatment with valproic acid (VPA) and in vivo transfection of inducible Oct4-expressing viral particles. Systemic administration of VPA was performed for one week prior to an intracerebroventricular injection of the Oct4-expressing vector into the right side of the brain. Oct4 expression was induced by doxycycline from day 1 post-transfection for an additional week. Real time-PCR and immunohistofluorescence were used for evaluation of marker expression. Real time-PCR analyses of samples collected from the area of transfection within the injected-lateral ventricle revealed increased expression of some stem cell and progenitor markers, which included endogenous Oct4, Nanog, Klf4, c-Myc, Pax6 and Sox1. Expressions of Oct4, SSEA1 and Nanog were further confirmed by immunohistofluorescence. The increased neural progenitor and pluripotency markers due to Oct4 overexpression did not lead to teratoma formation during a 100day follow-up. Our findings suggest that the application of Oct4 as a reprogramming factor in conjunction with VPA, an epigenetic modifier, might be a potential strategy for increasing the brain's capability to repair itself. Copyright © 2014 Elsevier Inc. All rights reserved.

  4. Toxin-mediated gene regulatory mechanism in Staphylococcus aureus

    PubMed Central

    Joo, Hwang-Soo; Otto, Michael

    2016-01-01

    The dangerous human pathogen Staphylococcus aureus relies heavily on toxins to cause disease, but toxin production can put a strong burden on the bacteria’s energy balance. Thus, controlling the synthesis of proteins solely needed in times of toxin production represents a way for the bacteria to avoid wasting energy. One hypothetical manner to accomplish this sort of regulation is by gene regulatory functions of the toxins themselves. There have been several reports about gene regulation by toxins in S. aureus, but these were never verified on the molecular level. In our study published in MBio [Joo et al., 7(5). pii: e01579-16], we show that phenol-soluble modulins (PSMs), important peptide toxins of S. aureus, release a repressor from the promoter of the operon encoding the toxin export system, thereby enabling toxin secretion. This study describes the first molecular regulatory mechanism exerted by an S. aureus toxin, setting a paradigmatic example of how S. aureus toxins may influence cell functions to adjust them to times of toxin production.

  5. Neurogenic gene regulatory pathways in the sea urchin embryo

    PubMed Central

    Wei, Zheng; Angerer, Lynne M.; Angerer, Robert C.

    2016-01-01

    During embryogenesis the sea urchin early pluteus larva differentiates 40-50 neurons marked by expression of the pan-neural marker synaptotagmin B (SynB) that are distributed along the ciliary band, in the apical plate and pharyngeal endoderm, and 4-6 serotonergic neurons that are confined to the apical plate. Development of all neurons has been shown to depend on the function of Six3. Using a combination of molecular screens and tests of gene function by morpholino-mediated knockdown, we identified SoxC and Brn1/2/4, which function sequentially in the neurogenic regulatory pathway and are also required for the differentiation of all neurons. Misexpression of Brn1/2/4 at low dose caused an increase in the number of serotonin-expressing cells and at higher dose converted most of the embryo to a neurogenic epithelial sphere expressing the Hnf6 ciliary band marker. A third factor, Z167, was shown to work downstream of the Six3 and SoxC core factors and to define a branch specific for the differentiation of serotonergic neurons. These results provide a framework for building a gene regulatory network for neurogenesis in the sea urchin embryo. PMID:26657764

  6. Gene Regulatory Networks in Cardiac Conduction System Development

    PubMed Central

    Munshi, Nikhil V.

    2014-01-01

    The cardiac conduction system is a specialized tract of myocardial cells responsible for maintaining normal cardiac rhythm. Given its critical role in coordinating cardiac performance, a detailed analysis of the molecular mechanisms underlying conduction system formation should inform our understanding of arrhythmia pathophysiology and affect the development of novel therapeutic strategies. Historically, the ability to distinguish cells of the conduction system from neighboring working myocytes presented a major technical challenge for performing comprehensive mechanistic studies. Early lineage tracing experiments suggested that conduction cells derive from cardiomyocyte precursors, and these claims have been substantiated by using more contemporary approaches. However, regional specialization of conduction cells adds an additional layer of complexity to this system, and it appears that different components of the conduction system utilize unique modes of developmental formation. The identification of numerous transcription factors and their downstream target genes involved in regional differentiation of the conduction system has provided insight into how lineage commitment is achieved. Furthermore, by adopting cutting-edge genetic techniques in combination with sophisticated phenotyping capabilities, investigators have made substantial progress in delineating the regulatory networks that orchestrate conduction system formation and their role in cardiac rhythm and physiology. This review describes the connectivity of these gene regulatory networks in cardiac conduction system development and discusses how they provide a foundation for understanding normal and pathological human cardiac rhythms. PMID:22628576

  7. Neurogenic gene regulatory pathways in the sea urchin embryo.

    PubMed

    Wei, Zheng; Angerer, Lynne M; Angerer, Robert C

    2016-01-15

    During embryogenesis the sea urchin early pluteus larva differentiates 40-50 neurons marked by expression of the pan-neural marker synaptotagmin B (SynB) that are distributed along the ciliary band, in the apical plate and pharyngeal endoderm, and 4-6 serotonergic neurons that are confined to the apical plate. Development of all neurons has been shown to depend on the function of Six3. Using a combination of molecular screens and tests of gene function by morpholino-mediated knockdown, we identified SoxC and Brn1/2/4, which function sequentially in the neurogenic regulatory pathway and are also required for the differentiation of all neurons. Misexpression of Brn1/2/4 at low dose caused an increase in the number of serotonin-expressing cells and at higher dose converted most of the embryo to a neurogenic epithelial sphere expressing the Hnf6 ciliary band marker. A third factor, Z167, was shown to work downstream of the Six3 and SoxC core factors and to define a branch specific for the differentiation of serotonergic neurons. These results provide a framework for building a gene regulatory network for neurogenesis in the sea urchin embryo. © 2016. Published by The Company of Biologists Ltd.

  8. Deduced products of C4-dicarboxylate transport regulatory genes of Rhizobium leguminosarum are homologous to nitrogen regulatory gene products.

    PubMed Central

    Ronson, C W; Astwood, P M; Nixon, B T; Ausubel, F M

    1987-01-01

    We have sequenced two genes dctB and dctD required for the activation of the C4-dicarboxylate transport structural gene dctA in free-living Rhizobium leguminosarum. The hydropathic profile of the dctB gene product (DctB) suggested that its N-terminal region may be located in the periplasm and its C-terminal region in the cytoplasm. The C-terminal region of DctB was strongly conserved with similar regions of the products of several regulatory genes that may act as environmental sensors, including ntrB, envZ, virA, phoR, cpxA, and phoM. The N-terminal domains of the products of several regulatory genes thought to be transcriptional activators, including ntrC, ompR, virG, phoB and sfrA. In addition, the central and C-terminal regions of DctD were strongly conserved with the products of ntrC and nifA, transcriptional activators that require the alternate sigma factor rpoN (ntrA) as co-activator. The central region of DctD also contained a potential ATP-binding domain. These results are consistent with recent results that show that rpoN product is required for dctA activation, and suggest that DctB plus DctD-mediated transcriptional activation of dctA may be mechanistically similar to NtrB plus NtrC-mediated activation of glnA in E. coli. PMID:3671068

  9. Contrasting Frequencies and Effects of cis- and trans-Regulatory Mutations Affecting Gene Expression

    PubMed Central

    Metzger, Brian P. H.; Duveau, Fabien; Yuan, David C.; Tryban, Stephen; Yang, Bing; Wittkopp, Patricia J.

    2016-01-01

    Heritable differences in gene expression are caused by mutations in DNA sequences encoding cis-regulatory elements and trans-regulatory factors. These two classes of regulatory change differ in their relative contributions to expression differences in natural populations because of the combined effects of mutation and natural selection. Here, we investigate how new mutations create the regulatory variation upon which natural selection acts by quantifying the frequencies and effects of hundreds of new cis- and trans-acting mutations altering activity of the TDH3 promoter in the yeast Saccharomyces cerevisiae in the absence of natural selection. We find that cis-regulatory mutations have larger effects on expression than trans-regulatory mutations and that while trans-regulatory mutations are more common overall, cis- and trans-regulatory changes in expression are equally abundant when only the largest changes in expression are considered. In addition, we find that cis-regulatory mutations are skewed toward decreased expression while trans-regulatory mutations are skewed toward increased expression. We also measure the effects of cis- and trans-regulatory mutations on the variability in gene expression among genetically identical cells, a property of gene expression known as expression noise, finding that trans-regulatory mutations are much more likely to decrease expression noise than cis-regulatory mutations. Because new mutations are the raw material upon which natural selection acts, these differences in the frequencies and effects of cis- and trans-regulatory mutations should be considered in models of regulatory evolution. PMID:26782996

  10. Graphlet Based Metrics for the Comparison of Gene Regulatory Networks

    PubMed Central

    Martin, Alberto J. M.; Dominguez, Calixto; Contreras-Riquelme, Sebastián; Holmes, David S.; Perez-Acle, Tomas

    2016-01-01

    Understanding the control of gene expression remains one of the main challenges in the post-genomic era. Accordingly, a plethora of methods exists to identify variations in gene expression levels. These variations underlay almost all relevant biological phenomena, including disease and adaptation to environmental conditions. However, computational tools to identify how regulation changes are scarce. Regulation of gene expression is usually depicted in the form of a gene regulatory network (GRN). Structural changes in a GRN over time and conditions represent variations in the regulation of gene expression. Like other biological networks, GRNs are composed of basic building blocks called graphlets. As a consequence, two new metrics based on graphlets are proposed in this work: REConstruction Rate (REC) and REC Graphlet Degree (RGD). REC determines the rate of graphlet similarity between different states of a network and RGD identifies the subset of nodes with the highest topological variation. In other words, RGD discerns how th GRN was rewired. REC and RGD were used to compare the local structure of nodes in condition-specific GRNs obtained from gene expression data of Escherichia coli, forming biofilms and cultured in suspension. According to our results, most of the network local structure remains unaltered in the two compared conditions. Nevertheless, changes reported by RGD necessarily imply that a different cohort of regulators (i.e. transcription factors (TFs)) appear on the scene, shedding light on how the regulation of gene expression occurs when E. coli transits from suspension to biofilm. Consequently, we propose that both metrics REC and RGD should be adopted as a quantitative approach to conduct differential analyses of GRNs. A tool that implements both metrics is available as an on-line web server (http://dlab.cl/loto). PMID:27695050

  11. Graphlet Based Metrics for the Comparison of Gene Regulatory Networks.

    PubMed

    Martin, Alberto J M; Dominguez, Calixto; Contreras-Riquelme, Sebastián; Holmes, David S; Perez-Acle, Tomas

    2016-01-01

    Understanding the control of gene expression remains one of the main challenges in the post-genomic era. Accordingly, a plethora of methods exists to identify variations in gene expression levels. These variations underlay almost all relevant biological phenomena, including disease and adaptation to environmental conditions. However, computational tools to identify how regulation changes are scarce. Regulation of gene expression is usually depicted in the form of a gene regulatory network (GRN). Structural changes in a GRN over time and conditions represent variations in the regulation of gene expression. Like other biological networks, GRNs are composed of basic building blocks called graphlets. As a consequence, two new metrics based on graphlets are proposed in this work: REConstruction Rate (REC) and REC Graphlet Degree (RGD). REC determines the rate of graphlet similarity between different states of a network and RGD identifies the subset of nodes with the highest topological variation. In other words, RGD discerns how th GRN was rewired. REC and RGD were used to compare the local structure of nodes in condition-specific GRNs obtained from gene expression data of Escherichia coli, forming biofilms and cultured in suspension. According to our results, most of the network local structure remains unaltered in the two compared conditions. Nevertheless, changes reported by RGD necessarily imply that a different cohort of regulators (i.e. transcription factors (TFs)) appear on the scene, shedding light on how the regulation of gene expression occurs when E. coli transits from suspension to biofilm. Consequently, we propose that both metrics REC and RGD should be adopted as a quantitative approach to conduct differential analyses of GRNs. A tool that implements both metrics is available as an on-line web server (http://dlab.cl/loto).

  12. Evolutionary and Topological Properties of Genes and Community Structures in Human Gene Regulatory Networks.

    PubMed

    Szedlak, Anthony; Smith, Nicholas; Liu, Li; Paternostro, Giovanni; Piermarocchi, Carlo

    2016-06-01

    The diverse, specialized genes present in today's lifeforms evolved from a common core of ancient, elementary genes. However, these genes did not evolve individually: gene expression is controlled by a complex network of interactions, and alterations in one gene may drive reciprocal changes in its proteins' binding partners. Like many complex networks, these gene regulatory networks (GRNs) are composed of communities, or clusters of genes with relatively high connectivity. A deep understanding of the relationship between the evolutionary history of single genes and the topological properties of the underlying GRN is integral to evolutionary genetics. Here, we show that the topological properties of an acute myeloid leukemia GRN and a general human GRN are strongly coupled with its genes' evolutionary properties. Slowly evolving ("cold"), old genes tend to interact with each other, as do rapidly evolving ("hot"), young genes. This naturally causes genes to segregate into community structures with relatively homogeneous evolutionary histories. We argue that gene duplication placed old, cold genes and communities at the center of the networks, and young, hot genes and communities at the periphery. We demonstrate this with single-node centrality measures and two new measures of efficiency, the set efficiency and the interset efficiency. We conclude that these methods for studying the relationships between a GRN's community structures and its genes' evolutionary properties provide new perspectives for understanding evolutionary genetics.

  13. Implications of Developmental Gene Regulatory Networks Inside and Outside Developmental Biology.

    PubMed

    Peter, Isabelle S; Davidson, Eric H

    2016-01-01

    The insight that the genomic control of developmental process is encoded in the form of gene regulatory networks has profound impacts on many areas of modern bioscience. Most importantly, it affects developmental biology itself, as it means that a causal understanding of development requires knowledge of the architecture of regulatory network interactions. Furthermore, it follows that functional changes in developmental gene regulatory networks have to be considered as a primary mechanism for evolutionary process. We here discuss some of the recent advances in gene regulatory network biology and how they have affected our current understanding of development, evolution, and regulatory genomics.

  14. Enhancing gene regulatory network inference through data integration with markov random fields

    DOE PAGES

    Banf, Michael; Rhee, Seung Y.

    2017-02-01

    Here, a gene regulatory network links transcription factors to their target genes and represents a map of transcriptional regulation. Much progress has been made in deciphering gene regulatory networks computationally. However, gene regulatory network inference for most eukaryotic organisms remain challenging. To improve the accuracy of gene regulatory network inference and facilitate candidate selection for experimentation, we developed an algorithm called GRACE (Gene Regulatory network inference ACcuracy Enhancement). GRACE exploits biological a priori and heterogeneous data integration to generate high- confidence network predictions for eukaryotic organisms using Markov Random Fields in a semi-supervised fashion. GRACE uses a novel optimization schememore » to integrate regulatory evidence and biological relevance. It is particularly suited for model learning with sparse regulatory gold standard data. We show GRACE’s potential to produce high confidence regulatory networks compared to state of the art approaches using Drosophila melanogaster and Arabidopsis thaliana data. In an A. thaliana developmental gene regulatory network, GRACE recovers cell cycle related regulatory mechanisms and further hypothesizes several novel regulatory links, including a putative control mechanism of vascular structure formation due to modifications in cell proliferation.« less

  15. Enhancing gene regulatory network inference through data integration with markov random fields

    PubMed Central

    Banf, Michael; Rhee, Seung Y.

    2017-01-01

    A gene regulatory network links transcription factors to their target genes and represents a map of transcriptional regulation. Much progress has been made in deciphering gene regulatory networks computationally. However, gene regulatory network inference for most eukaryotic organisms remain challenging. To improve the accuracy of gene regulatory network inference and facilitate candidate selection for experimentation, we developed an algorithm called GRACE (Gene Regulatory network inference ACcuracy Enhancement). GRACE exploits biological a priori and heterogeneous data integration to generate high- confidence network predictions for eukaryotic organisms using Markov Random Fields in a semi-supervised fashion. GRACE uses a novel optimization scheme to integrate regulatory evidence and biological relevance. It is particularly suited for model learning with sparse regulatory gold standard data. We show GRACE’s potential to produce high confidence regulatory networks compared to state of the art approaches using Drosophila melanogaster and Arabidopsis thaliana data. In an A. thaliana developmental gene regulatory network, GRACE recovers cell cycle related regulatory mechanisms and further hypothesizes several novel regulatory links, including a putative control mechanism of vascular structure formation due to modifications in cell proliferation. PMID:28145456

  16. Enhancing gene regulatory network inference through data integration with markov random fields.

    PubMed

    Banf, Michael; Rhee, Seung Y

    2017-02-01

    A gene regulatory network links transcription factors to their target genes and represents a map of transcriptional regulation. Much progress has been made in deciphering gene regulatory networks computationally. However, gene regulatory network inference for most eukaryotic organisms remain challenging. To improve the accuracy of gene regulatory network inference and facilitate candidate selection for experimentation, we developed an algorithm called GRACE (Gene Regulatory network inference ACcuracy Enhancement). GRACE exploits biological a priori and heterogeneous data integration to generate high- confidence network predictions for eukaryotic organisms using Markov Random Fields in a semi-supervised fashion. GRACE uses a novel optimization scheme to integrate regulatory evidence and biological relevance. It is particularly suited for model learning with sparse regulatory gold standard data. We show GRACE's potential to produce high confidence regulatory networks compared to state of the art approaches using Drosophila melanogaster and Arabidopsis thaliana data. In an A. thaliana developmental gene regulatory network, GRACE recovers cell cycle related regulatory mechanisms and further hypothesizes several novel regulatory links, including a putative control mechanism of vascular structure formation due to modifications in cell proliferation.

  17. An Arabidopsis gene regulatory network for secondary cell wall synthesis

    DOE PAGES

    Taylor-Teeples, M.; Lin, L.; de Lucas, M.; ...

    2014-12-24

    The plant cell wall is an important factor for determining cell shape, function and response to the environment. Secondary cell walls, such as those found in xylem, are composed of cellulose, hemicelluloses and lignin and account for the bulk of plant biomass. The coordination between transcriptional regulation of synthesis for each polymer is complex and vital to cell function. A regulatory hierarchy of developmental switches has been proposed, although the full complement of regulators remains unknown. In this paper, we present a protein–DNA network between Arabidopsis thaliana transcription factors and secondary cell wall metabolic genes with gene expression regulated bymore » a series of feed-forward loops. This model allowed us to develop and validate new hypotheses about secondary wall gene regulation under abiotic stress. Distinct stresses are able to perturb targeted genes to potentially promote functional adaptation. Finally, these interactions will serve as a foundation for understanding the regulation of a complex, integral plant component.« less

  18. Identification of a gene regulatory network associated with prion replication

    PubMed Central

    Marbiah, Masue M; Harvey, Anna; West, Billy T; Louzolo, Anais; Banerjee, Priya; Alden, Jack; Grigoriadis, Anita; Hummerich, Holger; Kan, Ho-Man; Cai, Ying; Bloom, George S; Jat, Parmjit; Collinge, John; Klöhn, Peter-Christian

    2014-01-01

    Prions consist of aggregates of abnormal conformers of the cellular prion protein (PrPC). They propagate by recruiting host-encoded PrPC although the critical interacting proteins and the reasons for the differences in susceptibility of distinct cell lines and populations are unknown. We derived a lineage of cell lines with markedly differing susceptibilities, unexplained by PrPC expression differences, to identify such factors. Transcriptome analysis of prion-resistant revertants, isolated from highly susceptible cells, revealed a gene expression signature associated with susceptibility and modulated by differentiation. Several of these genes encode proteins with a role in extracellular matrix (ECM) remodelling, a compartment in which disease-related PrP is deposited. Silencing nine of these genes significantly increased susceptibility. Silencing of Papss2 led to undersulphated heparan sulphate and increased PrPC deposition at the ECM, concomitantly with increased prion propagation. Moreover, inhibition of fibronectin 1 binding to integrin α8 by RGD peptide inhibited metalloproteinases (MMP)-2/9 whilst increasing prion propagation. In summary, we have identified a gene regulatory network associated with prion propagation at the ECM and governed by the cellular differentiation state. PMID:24843046

  19. Construction of gene regulatory networks using biclustering and bayesian networks

    PubMed Central

    2011-01-01

    Background Understanding gene interactions in complex living systems can be seen as the ultimate goal of the systems biology revolution. Hence, to elucidate disease ontology fully and to reduce the cost of drug development, gene regulatory networks (GRNs) have to be constructed. During the last decade, many GRN inference algorithms based on genome-wide data have been developed to unravel the complexity of gene regulation. Time series transcriptomic data measured by genome-wide DNA microarrays are traditionally used for GRN modelling. One of the major problems with microarrays is that a dataset consists of relatively few time points with respect to the large number of genes. Dimensionality is one of the interesting problems in GRN modelling. Results In this paper, we develop a biclustering function enrichment analysis toolbox (BicAT-plus) to study the effect of biclustering in reducing data dimensions. The network generated from our system was validated via available interaction databases and was compared with previous methods. The results revealed the performance of our proposed method. Conclusions Because of the sparse nature of GRNs, the results of biclustering techniques differ significantly from those of previous methods. PMID:22018164

  20. Gene regulatory networks and the role of robustness and stochasticity in the control of gene expression

    PubMed Central

    MacNeil, Lesley T.; Walhout, Albertha J.M.

    2011-01-01

    In any given cell, thousands of genes are expressed and work in concert to ensure the cell's function, fitness, and survival. Each gene, in turn, must be expressed at the proper time and in the proper amounts to ensure the appropriate functional outcome. The regulation and expression of some genes are highly robust; their expression is controlled by invariable expression programs. For instance, developmental gene expression is extremely similar in a given cell type from one individual to another. The expression of other genes is more variable: Their levels are noisy and are different from cell to cell and from individual to individual. This can be highly beneficial in physiological responses to outside cues and stresses. Recent advances have enabled the analysis of differential gene expression at a systems level. Gene regulatory networks (GRNs) involving interactions between large numbers of genes and their regulators have been mapped onto graphic diagrams that are used to visualize the regulatory relationships. The further characterization of GRNs has already uncovered global principles of gene regulation. Together with synthetic network biology, such studies are starting to provide insights into the transcriptional mechanisms that cause robust versus stochastic gene expression and their relationships to phenotypic robustness and variability. Here, we discuss GRNs and their topological properties in relation to transcriptional and phenotypic outputs in development and organismal physiology. PMID:21324878

  1. Nitrogen fixation specific regulatory genes of Klebsiella pneumoniae and Rhizobium meliloti share homology with the general nitrogen regulatory gene ntrC of K. pneumoniae.

    PubMed Central

    Buikema, W J; Szeto, W W; Lemley, P V; Orme-Johnson, W H; Ausubel, F M

    1985-01-01

    We have determined the complete nucleotide sequences of three functionally related nitrogen assimilation regulatory genes from Klebsiella pneumoniae and Rhizobium meliloti. These genes are: 1) The K. pneumoniae general nitrogen assimilation regulatory gene ntrC (formerly called glnG), 2) the K. pneumoniae nif-specific regulatory gene nifA, and 3) an R. meliloti nif-specific regulatory gene that appears to be functionally analogous to the K. pneumoniae nifA gene. In addition to the DNA sequence data, gel-purified K. pneumoniae nifA protein was used to determine the amino acid composition of the nifA protein. The K. pneumoniae ntrC and nifA genes code for proteins of 52,259 and 53,319 d respectively. The R. meliloti nifA gene codes for a 59,968 d protein. A central region within each polypeptide, consisting of approximately 200 amino acids, is between 52% and 58% conserved among the three proteins. Neither the amino termini nor the carboxy termini show any conserved sequences. Together with data that shows that the three regulatory proteins activate promoters that share a common consensus sequence in the -10 (5'-TTGCA-3') and -23 (5'-CTGG-3') regions, the sequence data presented here suggest a common evolutionary origin for the three regulatory genes. Images PMID:2989799

  2. Dose response relationship in anti-stress gene regulatory networks.

    PubMed

    Zhang, Qiang; Andersen, Melvin E

    2007-03-02

    To maintain a stable intracellular environment, cells utilize complex and specialized defense systems against a variety of external perturbations, such as electrophilic stress, heat shock, and hypoxia, etc. Irrespective of the type of stress, many adaptive mechanisms contributing to cellular homeostasis appear to operate through gene regulatory networks that are organized into negative feedback loops. In general, the degree of deviation of the controlled variables, such as electrophiles, misfolded proteins, and O2, is first detected by specialized sensor molecules, then the signal is transduced to specific transcription factors. Transcription factors can regulate the expression of a suite of anti-stress genes, many of which encode enzymes functioning to counteract the perturbed variables. The objective of this study was to explore, using control theory and computational approaches, the theoretical basis that underlies the steady-state dose response relationship between cellular stressors and intracellular biochemical species (controlled variables, transcription factors, and gene products) in these gene regulatory networks. Our work indicated that the shape of dose response curves (linear, superlinear, or sublinear) depends on changes in the specific values of local response coefficients (gains) distributed in the feedback loop. Multimerization of anti-stress enzymes and transcription factors into homodimers, homotrimers, or even higher-order multimers, play a significant role in maintaining robust homeostasis. Moreover, our simulation noted that dose response curves for the controlled variables can transition sequentially through four distinct phases as stressor level increases: initial superlinear with lesser control, superlinear more highly controlled, linear uncontrolled, and sublinear catastrophic. Each phase relies on specific gain-changing events that come into play as stressor level increases. The low-dose region is intrinsically nonlinear, and depending on

  3. The influence of assortativity on the robustness and evolvability of gene regulatory networks upon gene birth

    PubMed Central

    Pechenick, Dov A.; Moore, Jason H.; Payne, Joshua L.

    2013-01-01

    Gene regulatory networks (GRNs) represent the interactions between genes and gene products, which drive the gene expression patterns that produce cellular phenotypes. GRNs display a number of characteristics that are beneficial for the development and evolution of organisms. For example, they are often robust to genetic perturbation, such as mutations in regulatory regions or loss of gene function. Simultaneously, GRNs are often evolvable as these genetic perturbations are occasionally exploited to innovate novel regulatory programs. Several topological properties, such as degree distribution, are known to influence the robustness and evolvability of GRNs. Assortativity, which measures the propensity of nodes of similar connectivity to connect to one another, is a separate topological property that has recently been shown to influence the robustness of GRNs to point mutations in cis-regulatory regions. However, it remains to be seen how assortativity may influence the robustness and evolvability of GRNs to other forms of genetic perturbation, such as gene birth via duplication or de novo origination. Here, we employ a computational model of genetic regulation to investigate whether the assortativity of a GRN influences its robustness and evolvability upon gene birth. We find that the robustness of a GRN generally increases with increasing assortativity, while its evolvability generally decreases. However, the rate of change in robustness outpaces that of evolvability, resulting in an increased proportion of assortative GRNs that are simultaneously robust and evolvable. By providing a mechanistic explanation for these observations, this work extends our understanding of how the assortativity of a GRN influences its robustness and evolvability upon gene birth. PMID:23542384

  4. Gene regulatory network clustering for graph layout based on microarray gene expression data.

    PubMed

    Kojima, Kaname; Imoto, Seiya; Nagasaki, Masao; Miyano, Satoru

    2010-01-01

    We propose a statistical model realizing simultaneous estimation of gene regulatory network and gene module identification from time series gene expression data from microarray experiments. Under the assumption that genes in the same module are densely connected, the proposed method detects gene modules based on the variational Bayesian technique. The model can also incorporate existing biological prior knowledge such as protein subcellular localization. We apply the proposed model to the time series data from a synthetically generated network and verified the effectiveness of the proposed model. The proposed model is also applied the time series microarray data from HeLa cell. Detected gene module information gives the great help on drawing the estimated gene network.

  5. Evolutionary and Topological Properties of Genes and Community Structures in Human Gene Regulatory Networks

    PubMed Central

    Szedlak, Anthony; Smith, Nicholas; Liu, Li; Paternostro, Giovanni; Piermarocchi, Carlo

    2016-01-01

    The diverse, specialized genes present in today’s lifeforms evolved from a common core of ancient, elementary genes. However, these genes did not evolve individually: gene expression is controlled by a complex network of interactions, and alterations in one gene may drive reciprocal changes in its proteins’ binding partners. Like many complex networks, these gene regulatory networks (GRNs) are composed of communities, or clusters of genes with relatively high connectivity. A deep understanding of the relationship between the evolutionary history of single genes and the topological properties of the underlying GRN is integral to evolutionary genetics. Here, we show that the topological properties of an acute myeloid leukemia GRN and a general human GRN are strongly coupled with its genes’ evolutionary properties. Slowly evolving (“cold”), old genes tend to interact with each other, as do rapidly evolving (“hot”), young genes. This naturally causes genes to segregate into community structures with relatively homogeneous evolutionary histories. We argue that gene duplication placed old, cold genes and communities at the center of the networks, and young, hot genes and communities at the periphery. We demonstrate this with single-node centrality measures and two new measures of efficiency, the set efficiency and the interset efficiency. We conclude that these methods for studying the relationships between a GRN’s community structures and its genes’ evolutionary properties provide new perspectives for understanding evolutionary genetics. PMID:27359334

  6. Topological effects of data incompleteness of gene regulatory networks

    PubMed Central

    2012-01-01

    Background The topological analysis of biological networks has been a prolific topic in network science during the last decade. A persistent problem with this approach is the inherent uncertainty and noisy nature of the data. One of the cases in which this situation is more marked is that of transcriptional regulatory networks (TRNs) in bacteria. The datasets are incomplete because regulatory pathways associated to a relevant fraction of bacterial genes remain unknown. Furthermore, direction, strengths and signs of the links are sometimes unknown or simply overlooked. Finally, the experimental approaches to infer the regulations are highly heterogeneous, in a way that induces the appearance of systematic experimental-topological correlations. And yet, the quality of the available data increases constantly. Results In this work we capitalize on these advances to point out the influence of data (in)completeness and quality on some classical results on topological analysis of TRNs, specially regarding modularity at different levels. Conclusions In doing so, we identify the most relevant factors affecting the validity of previous findings, highlighting important caveats to future prokaryotic TRNs topological analysis. PMID:22920968

  7. Stability Depends on Positive Autoregulation in Boolean Gene Regulatory Networks

    PubMed Central

    Pinho, Ricardo; Garcia, Victor; Irimia, Manuel; Feldman, Marcus W.

    2014-01-01

    Network motifs have been identified as building blocks of regulatory networks, including gene regulatory networks (GRNs). The most basic motif, autoregulation, has been associated with bistability (when positive) and with homeostasis and robustness to noise (when negative), but its general importance in network behavior is poorly understood. Moreover, how specific autoregulatory motifs are selected during evolution and how this relates to robustness is largely unknown. Here, we used a class of GRN models, Boolean networks, to investigate the relationship between autoregulation and network stability and robustness under various conditions. We ran evolutionary simulation experiments for different models of selection, including mutation and recombination. Each generation simulated the development of a population of organisms modeled by GRNs. We found that stability and robustness positively correlate with autoregulation; in all investigated scenarios, stable networks had mostly positive autoregulation. Assuming biological networks correspond to stable networks, these results suggest that biological networks should often be dominated by positive autoregulatory loops. This seems to be the case for most studied eukaryotic transcription factor networks, including those in yeast, flies and mammals. PMID:25375153

  8. Stochastic S-system modeling of gene regulatory network.

    PubMed

    Chowdhury, Ahsan Raja; Chetty, Madhu; Evans, Rob

    2015-10-01

    Microarray gene expression data can provide insights into biological processes at a system-wide level and is commonly used for reverse engineering gene regulatory networks (GRN). Due to the amalgamation of noise from different sources, microarray expression profiles become inherently noisy leading to significant impact on the GRN reconstruction process. Microarray replicates (both biological and technical), generated to increase the reliability of data obtained under noisy conditions, have limited influence in enhancing the accuracy of reconstruction . Therefore, instead of the conventional GRN modeling approaches which are deterministic, stochastic techniques are becoming increasingly necessary for inferring GRN from noisy microarray data. In this paper, we propose a new stochastic GRN model by investigating incorporation of various standard noise measurements in the deterministic S-system model. Experimental evaluations performed for varying sizes of synthetic network, representing different stochastic processes, demonstrate the effect of noise on the accuracy of genetic network modeling and the significance of stochastic modeling for GRN reconstruction . The proposed stochastic model is subsequently applied to infer the regulations among genes in two real life networks: (1) the well-studied IRMA network, a real-life in-vivo synthetic network constructed within the Saccharomyces cerevisiae yeast, and (2) the SOS DNA repair network in Escherichia coli.

  9. Identifying sleep regulatory genes using a Drosophila model of insomnia

    PubMed Central

    Seugnet, Laurent; Suzuki, Yasuko; Thimgan, Matthew; Donlea, Jeff; Gimbel, Sarah I.; Gottschalk, Laura; Duntley, Steve P.; Shaw, Paul J.

    2009-01-01

    Although it is widely accepted that sleep must serve an essential biological function, little is known about molecules that underlie sleep regulation. Given that insomnia is a common sleep disorder that disrupts the ability to initiate and maintain restorative sleep, a better understanding of its molecular underpinning may provide crucial insights into sleep regulatory processes. Thus, we created a line of flies using laboratory selection that share traits with human insomnia. After 60 generations insomnia-like (ins-l) flies sleep 60 min a day, exhibit difficulty initiating sleep, difficulty maintaining sleep, and show evidence of daytime cognitive impairment. ins-l flies are also hyperactive and hyper responsive to environmental perturbations. In addition they have difficulty maintaining their balance, have elevated levels of dopamine, are short-lived and show increased levels of triglycerides, cholesterol, and free fatty acids. While their core molecular clock remains intact, ins-l flies lose their ability to sleep when placed into constant darkness. Whole genome profiling identified genes that are modified in ins-l flies. Among those differentially expressed transcripts genes involved in metabolism, neuronal activity, and sensory perception constituted over-represented categories. We demonstrate that two of these genes are upregulated in human subjects following acute sleep deprivation. Together these data indicate that the ins-l flies are a useful tool that can be used to identify molecules important for sleep regulation and may provide insights into both the causes and long-term consequences of insomnia. PMID:19494137

  10. Complex Dynamic Behavior in Simple Gene Regulatory Networks

    NASA Astrophysics Data System (ADS)

    Santillán Zerón, Moisés

    2007-02-01

    Knowing the complete genome of a given species is just a piece of the puzzle. To fully unveil the systems behavior of an organism, an organ, or even a single cell, we need to understand the underlying gene regulatory dynamics. Given the complexity of the whole system, the ultimate goal is unattainable for the moment. But perhaps, by analyzing the most simple genetic systems, we may be able to develop the mathematical techniques and procedures required to tackle more complex genetic networks in the near future. In the present work, the techniques for developing mathematical models of simple bacterial gene networks, like the tryptophan and lactose operons are introduced. Despite all of the underlying assumptions, such models can provide valuable information regarding gene regulation dynamics. Here, we pay special attention to robustness as an emergent property. These notes are organized as follows. In the first section, the long historical relation between mathematics, physics, and biology is briefly reviewed. Recently, the multidisciplinary work in biology has received great attention in the form of systems biology. The main concepts of this novel science are discussed in the second section. A very slim introduction to the essential concepts of molecular biology is given in the third section. In the fourth section, a brief introduction to chemical kinetics is presented. Finally, in the fifth section, a mathematical model for the lactose operon is developed and analyzed..

  11. Algebraic model checking for Boolean gene regulatory networks.

    PubMed

    Tran, Quoc-Nam

    2011-01-01

    We present a computational method in which modular and Groebner bases (GB) computation in Boolean rings are used for solving problems in Boolean gene regulatory networks (BN). In contrast to other known algebraic approaches, the degree of intermediate polynomials during the calculation of Groebner bases using our method will never grow resulting in a significant improvement in running time and memory space consumption. We also show how calculation in temporal logic for model checking can be done by means of our direct and efficient Groebner basis computation in Boolean rings. We present our experimental results in finding attractors and control strategies of Boolean networks to illustrate our theoretical arguments. The results are promising. Our algebraic approach is more efficient than the state-of-the-art model checker NuSMV on BNs. More importantly, our approach finds all solutions for the BN problems.

  12. Modeling gene regulatory networks: A network simplification algorithm

    NASA Astrophysics Data System (ADS)

    Ferreira, Luiz Henrique O.; de Castro, Maria Clicia S.; da Silva, Fabricio A. B.

    2016-12-01

    Boolean networks have been used for some time to model Gene Regulatory Networks (GRNs), which describe cell functions. Those models can help biologists to make predictions, prognosis and even specialized treatment when some disturb on the GRN lead to a sick condition. However, the amount of information related to a GRN can be huge, making the task of inferring its boolean network representation quite a challenge. The method shown here takes into account information about the interactome to build a network, where each node represents a protein, and uses the entropy of each node as a key to reduce the size of the network, allowing the further inferring process to focus only on the main protein hubs, the ones with most potential to interfere in overall network behavior.

  13. ERIC DAVIDSON: STEPS TO A GENE REGULATORY NETWORK FOR DEVELOPMENT

    PubMed Central

    Rothenberg, Ellen V.

    2016-01-01

    Eric Harris Davidson was a unique and creative intellectual force who grappled with the diversity of developmental processes used by animal embryos and wrestled them into an intelligible set of principles, then spent his life translating these process elements into molecularly definable terms through the architecture of gene regulatory networks. He took speculative risks in his theoretical writing but ran a highly organized, rigorous experimental program that yielded an unprecedentedly full characterization of a developing organism. His writings created logical order and a framework for mechanism from the complex phenomena at the heart of advanced multicellular organism development. This is a reminiscence of intellectual currents in his work as observed by the author through the last 30–35 years of Davidson’s life. PMID:26825392

  14. Transactivation of anthocyanin biosynthetic genes following transfer of B regulatory genes into maize tissues.

    PubMed Central

    Goff, S A; Klein, T M; Roth, B A; Fromm, M E; Cone, K C; Radicella, J P; Chandler, V L

    1990-01-01

    The C1, B and R genes regulating the maize anthocyanin biosynthetic pathway encode tissue-specific regulatory proteins with similarities to transcriptional activators. The C1 and R regulatory genes are usually responsible for pigmentation of seed tissues, and the B-Peru allele of B, but not the B-I allele, can substitute for R function in the seed. In this study, members of the B family of regulatory genes were delivered to intact maize tissues by high velocity microprojectiles. In colorless r aleurones or embryos, the introduction of the B-Peru genomic clone or the expressed cDNAs of B-Peru or B-I resulted in anthocyanin-producing cells. Luciferase produced from the Bronze1 anthocyanin structural gene promoter was induced 100-fold when co-introduced with the expressed B-Peru or B-I cDNAs. This quantitative transactivation assay demonstrates that the proteins encoded by these two B alleles are equally able to transactivate the Bronze1 promoter. Analogous results were obtained using embryogenic callus cells. These observations suggest that one major contribution towards tissue-specific anthocyanin synthesis controlled by the various alleles of the B and R genes is the differential expression of functionally similar proteins. Images Fig. 2. PMID:2369901

  15. Reverse engineering of gene regulatory network using restricted gene expression programming.

    PubMed

    Yang, Bin; Liu, Sanrong; Zhang, Wei

    2016-10-01

    Inference of gene regulatory networks has been becoming a major area of interest in the field of systems biology over the past decade. In this paper, we present a novel representation of S-system model, named restricted gene expression programming (RGEP), to infer gene regulatory network. A new hybrid evolutionary algorithm based on structure-based evolutionary algorithm and cuckoo search (CS) is proposed to optimize the architecture and corresponding parameters of model, respectively. Two synthetic benchmark datasets and one real biological dataset from SOS DNA repair network in E. coli are used to test the validity of our method. Experimental results demonstrate that our proposed method performs better than previously proposed popular methods.

  16. Integrated module and gene-specific regulatory inference implicates upstream signaling networks.

    PubMed

    Roy, Sushmita; Lagree, Stephen; Hou, Zhonggang; Thomson, James A; Stewart, Ron; Gasch, Audrey P

    2013-01-01

    Regulatory networks that control gene expression are important in diverse biological contexts including stress response and development. Each gene's regulatory program is determined by module-level regulation (e.g. co-regulation via the same signaling system), as well as gene-specific determinants that can fine-tune expression. We present a novel approach, Modular regulatory network learning with per gene information (MERLIN), that infers regulatory programs for individual genes while probabilistically constraining these programs to reveal module-level organization of regulatory networks. Using edge-, regulator- and module-based comparisons of simulated networks of known ground truth, we find MERLIN reconstructs regulatory programs of individual genes as well or better than existing approaches of network reconstruction, while additionally identifying modular organization of the regulatory networks. We use MERLIN to dissect global transcriptional behavior in two biological contexts: yeast stress response and human embryonic stem cell differentiation. Regulatory modules inferred by MERLIN capture co-regulatory relationships between signaling proteins and downstream transcription factors thereby revealing the upstream signaling systems controlling transcriptional responses. The inferred networks are enriched for regulators with genetic or physical interactions, supporting the inference, and identify modules of functionally related genes bound by the same transcriptional regulators. Our method combines the strengths of per-gene and per-module methods to reveal new insights into transcriptional regulation in stress and development.

  17. Integrated Module and Gene-Specific Regulatory Inference Implicates Upstream Signaling Networks

    PubMed Central

    Roy, Sushmita; Lagree, Stephen; Hou, Zhonggang; Thomson, James A.; Stewart, Ron; Gasch, Audrey P.

    2013-01-01

    Regulatory networks that control gene expression are important in diverse biological contexts including stress response and development. Each gene's regulatory program is determined by module-level regulation (e.g. co-regulation via the same signaling system), as well as gene-specific determinants that can fine-tune expression. We present a novel approach, Modular regulatory network learning with per gene information (MERLIN), that infers regulatory programs for individual genes while probabilistically constraining these programs to reveal module-level organization of regulatory networks. Using edge-, regulator- and module-based comparisons of simulated networks of known ground truth, we find MERLIN reconstructs regulatory programs of individual genes as well or better than existing approaches of network reconstruction, while additionally identifying modular organization of the regulatory networks. We use MERLIN to dissect global transcriptional behavior in two biological contexts: yeast stress response and human embryonic stem cell differentiation. Regulatory modules inferred by MERLIN capture co-regulatory relationships between signaling proteins and downstream transcription factors thereby revealing the upstream signaling systems controlling transcriptional responses. The inferred networks are enriched for regulators with genetic or physical interactions, supporting the inference, and identify modules of functionally related genes bound by the same transcriptional regulators. Our method combines the strengths of per-gene and per-module methods to reveal new insights into transcriptional regulation in stress and development. PMID:24146602

  18. SOX-2, but not Oct4, is highly expressed in early-stage endometrial adenocarcinoma and is related to tumour grading.

    PubMed

    Pityński, Kazimierz; Banas, Tomasz; Pietrus, Milosz; Milian-Ciesielska, Katarzyna; Ludwin, Artur; Okon, Krzysztof

    2015-01-01

    Expression of SOX-2 and Oct4 as markers for the identification of cancer stem cells (CSCs) has been revealed in several malignancies. In this study, the co-expression of SOX-2 and Oct4 and their correlation with clinicopathological features of endometrial adenocarcinomas (EACs) was investigated. SOX-2 and Oct4 expression was assessed by immunohistochemistry in 27 (39.13%) stage IA and in 42 (60.87%) stage IB International Federation of Gynaecology and Obstetrics (FIGO) EACs and related to the clinicopathological features of patients. The expression of SOX-2 was confirmed in 62/69 tumour specimens compared to Oct4 expression in 46/69 specimens (P = 0.015) and no difference in median staining intensity between SOX-2 and Oct-4 was observed. The highest median SOX-2 expression was found in high-grade (G3) EAC samples compared to moderate-grade (G2) EAC specimens (P = 0.020) and low-grade (G1) specimens (P = 0.008), while no differences in median Oct4 expression in EAC samples according to grading were present. In G3 specimens, significantly higher median SOX-2 expression was noted compared to Oct4 (P = 0.002). SOX-2 and Oct4 co-expression was observed only in G1 EAC (R: 0.51; P = 0.031). Age of EAC diagnosis was positively correlated with SOX-2 expression (b = 0.193; R(2) = 10.83%; P = 0.003) but not to age of menarche, menopause, parity or body mass index. There is no need to use SOX-2 expression as a poor outcome predictor in stage I EAC, and SOX-2 expression should be analysed in more advanced stages.

  19. Dynamical analysis of regulatory interactions in the gap gene system of Drosophila melanogaster.

    PubMed Central

    Jaeger, Johannes; Blagov, Maxim; Kosman, David; Kozlov, Konstantin N; Manu; Myasnikova, Ekaterina; Surkova, Svetlana; Vanario-Alonso, Carlos E; Samsonova, Maria; Sharp, David H; Reinitz, John

    2004-01-01

    Genetic studies have revealed that segment determination in Drosophila melanogaster is based on hierarchical regulatory interactions among maternal coordinate and zygotic segmentation genes. The gap gene system constitutes the most upstream zygotic layer of this regulatory hierarchy, responsible for the initial interpretation of positional information encoded by maternal gradients. We present a detailed analysis of regulatory interactions involved in gap gene regulation based on gap gene circuits, which are mathematical gene network models used to infer regulatory interactions from quantitative gene expression data. Our models reproduce gap gene expression at high accuracy and temporal resolution. Regulatory interactions found in gap gene circuits provide consistent and sufficient mechanisms for gap gene expression, which largely agree with mechanisms previously inferred from qualitative studies of mutant gene expression patterns. Our models predict activation of Kr by Cad and clarify several other regulatory interactions. Our analysis suggests a central role for repressive feedback loops between complementary gap genes. We observe that repressive interactions among overlapping gap genes show anteroposterior asymmetry with posterior dominance. Finally, our models suggest a correlation between timing of gap domain boundary formation and regulatory contributions from the terminal maternal system. PMID:15342511

  20. Regulatory component analysis: a semi-blind extraction approach to infer gene regulatory networks with imperfect biological knowledge

    PubMed Central

    Wang, Chen; Xuan, Jianhua; Shih, Ie-Ming; Clarke, Robert; Wang, Yue

    2011-01-01

    With the advent of high-throughput biotechnology capable of monitoring genomic signals, it becomes increasingly promising to understand molecular cellular mechanisms through systems biology approaches. One of the active research topics in systems biology is to infer gene transcriptional regulatory networks using various genomic data; this inference problem can be formulated as a linear model with latent signals associated with some regulatory proteins called transcription factors (TFs). As common statistical assumptions may not hold for genomic signals, typical latent variable algorithms such as independent component analysis (ICA) are incapable to reveal underlying true regulatory signals. Liao et al. [1] proposed to perform inference using an approach named network component analysis (NCA), the optimization of which is achieved by a least-squares fitting approach with biological knowledge constraints. However, the incompleteness of biological knowledge and its inconsistency with gene expression data are not considered in the original NCA solution, which could greatly affect the inference accuracy. To overcome these limitations, we propose a linear extraction scheme, namely regulatory component analysis (RCA), to infer underlying regulatory signals even with partial biological knowledge. Numerical simulations show a significant improvement of our proposed RCA over NCA, not only when signal-to-noise-ratio (SNR) is low, but also when the given biological knowledge is incomplete and inconsistent to gene expression data. Furthermore, real biological experiments on E. coli are performed for regulatory network inference in comparison with several typical linear latent variable methods, which again demonstrates the effectiveness and improved performance of the proposed algorithm. PMID:22685363

  1. Regulation of photoreceptor gene transcription via a highly conserved transcriptional regulatory element by vsx gene products

    PubMed Central

    Pan, Yi; Comiskey, Daniel F.; Kelly, Lisa E.; Chandler, Dawn S.

    2016-01-01

    Purpose The photoreceptor conserved element-1 (PCE-1) sequence is found in the transcriptional regulatory regions of many genes expressed in photoreceptors. The retinal homeobox (Rx or Rax) gene product functions by binding to PCE-1 sites. However, other transcriptional regulators have also been reported to bind to PCE-1. One of these, vsx2, is expressed in retinal progenitor and bipolar cells. The purpose of this study is to identify Xenopus laevis vsx gene products and characterize vsx gene product expression and function with respect to the PCE-1 site. Methods X. laevis vsx gene products were amplified with PCR. Expression patterns were determined with in situ hybridization using whole or sectioned X. laevis embryos and digoxigenin- or fluorescein-labeled antisense riboprobes. DNA binding characteristics of the vsx gene products were analyzed with electrophoretic mobility shift assays (EMSAs) using in vitro translated proteins and radiolabeled oligonucleotide probes. Gene transactivation assays were performed using luciferase-based reporters and in vitro transcribed effector gene products, injected into X. laevis embryos. Results We identified one vsx1 and two vsx2 gene products. The two vsx2 gene products are generated by alternate mRNA splicing. We verified that these gene products are expressed in the developing retina and that expression resolves into distinct cell types in the mature retina. Finally, we found that vsx gene products can bind the PCE-1 site in vitro and that the two vsx2 isoforms have different gene transactivation activities. Conclusions vsx gene products are expressed in the developing and mature neural retina. vsx gene products can bind the PCE-1 site in vitro and influence the expression of a rhodopsin promoter-luciferase reporter gene. The two isoforms of vsx have different gene transactivation activities in this reporter gene system. PMID:28003732

  2. Genome-wide network of regulatory genes for construction of a chordate embryo.

    PubMed

    Shoguchi, Eiichi; Hamaguchi, Makoto; Satoh, Nori

    2008-04-15

    Animal development is controlled by gene regulation networks that are composed of sequence-specific transcription factors (TF) and cell signaling molecules (ST). Although housekeeping genes have been reported to show clustering in the animal genomes, whether the genes comprising a given regulatory network are physically clustered on a chromosome is uncertain. We examined this question in the present study. Ascidians are the closest living relatives of vertebrates, and their tadpole-type larva represents the basic body plan of chordates. The Ciona intestinalis genome contains 390 core TF genes and 119 major ST genes. Previous gene disruption assays led to the formulation of a basic chordate embryonic blueprint, based on over 3000 genetic interactions among 79 zygotic regulatory genes. Here, we mapped the regulatory genes, including all 79 regulatory genes, on the 14 pairs of Ciona chromosomes by fluorescent in situ hybridization (FISH). Chromosomal localization of upstream and downstream regulatory genes demonstrates that the components of coherent developmental gene networks are evenly distributed over the 14 chromosomes. Thus, this study provides the first comprehensive evidence that the physical clustering of regulatory genes, or their target genes, is not relevant for the genome-wide control of gene expression during development.

  3. A Structural Investigation into Oct4 Regulation by Orphan Nuclear Receptors, Germ Cell Nuclear Factor (GCNF), and Liver Receptor Homolog-1 (LRH-1).

    PubMed

    Weikum, Emily R; Tuntland, Micheal L; Murphy, Michael N; Ortlund, Eric A

    2016-12-04

    Oct4 is a transcription factor required for maintaining pluripotency and self-renewal in stem cells. Prior to differentiation, Oct4 must be silenced to allow for the development of the three germ layers in the developing embryo. This fine-tuning is controlled by the nuclear receptors (NRs), liver receptor homolog-1 (LRH-1) and germ cell nuclear factor (GCNF). Liver receptor homolog-1 is responsible for driving the expression of Oct4 where GCNF represses its expression upon differentiation. Both receptors bind to a DR0 motif located within the Oct4 promoter. Here, we present the first structure of mouse GCNF DNA-binding domain in complex with the Oct4 DR0. The overall structure revealed two molecules bound in a head-to-tail fashion on opposite sides of the DNA. Additionally, we solved the structure of the human LRH-1 DNA-binding domain bound to the same element. We explore the structural elements that govern Oct4 recognition by these two NRs. Copyright © 2016 Elsevier Ltd. All rights reserved.

  4. Unraveling gene regulatory networks from time-resolved gene expression data -- a measures comparison study

    PubMed Central

    2011-01-01

    Background Inferring regulatory interactions between genes from transcriptomics time-resolved data, yielding reverse engineered gene regulatory networks, is of paramount importance to systems biology and bioinformatics studies. Accurate methods to address this problem can ultimately provide a deeper insight into the complexity, behavior, and functions of the underlying biological systems. However, the large number of interacting genes coupled with short and often noisy time-resolved read-outs of the system renders the reverse engineering a challenging task. Therefore, the development and assessment of methods which are computationally efficient, robust against noise, applicable to short time series data, and preferably capable of reconstructing the directionality of the regulatory interactions remains a pressing research problem with valuable applications. Results Here we perform the largest systematic analysis of a set of similarity measures and scoring schemes within the scope of the relevance network approach which are commonly used for gene regulatory network reconstruction from time series data. In addition, we define and analyze several novel measures and schemes which are particularly suitable for short transcriptomics time series. We also compare the considered 21 measures and 6 scoring schemes according to their ability to correctly reconstruct such networks from short time series data by calculating summary statistics based on the corresponding specificity and sensitivity. Our results demonstrate that rank and symbol based measures have the highest performance in inferring regulatory interactions. In addition, the proposed scoring scheme by asymmetric weighting has shown to be valuable in reducing the number of false positive interactions. On the other hand, Granger causality as well as information-theoretic measures, frequently used in inference of regulatory networks, show low performance on the short time series analyzed in this study. Conclusions Our

  5. Evolution of the CNS myelin gene regulatory program.

    PubMed

    Li, Huiliang; Richardson, William D

    2016-06-15

    Myelin is a specialized subcellular structure that evolved uniquely in vertebrates. A myelinated axon conducts action potentials many times faster than an unmyelinated axon of the same diameter; for the same conduction speed, the unmyelinated axon would need a much larger diameter and volume than its myelinated counterpart. Hence myelin speeds information transfer and saves space, allowing the evolution of a powerful yet portable brain. Myelination in the central nervous system (CNS) is controlled by a gene regulatory program that features a number of master transcriptional regulators including Olig1, Olig2 and Myrf. Olig family genes evolved from a single ancestral gene in non-chordates. Olig2, which executes multiple functions with regard to oligodendrocyte identity and development in vertebrates, might have evolved functional versatility through post-translational modification, especially phosphorylation, as illustrated by its evolutionarily conserved serine/threonine phospho-acceptor sites and its accumulation of serine residues during more recent stages of vertebrate evolution. Olig1, derived from a duplicated copy of Olig2 in early bony fish, is involved in oligodendrocyte development and is critical to remyelination in bony vertebrates, but is lost in birds. The origin of Myrf orthologs might be the result of DNA integration between an invading phage or bacterium and an early protist, producing a fusion protein capable of self-cleavage and DNA binding. Myrf seems to have adopted new functions in early vertebrates - initiation of the CNS myelination program as well as the maintenance of mature oligodendrocyte identity and myelin structure - by developing new ways to interact with DNA motifs specific to myelin genes. This article is part of a Special Issue entitled SI: Myelin Evolution. Copyright © 2015 Elsevier B.V. All rights reserved.

  6. BCIP: a gene-centered platform for identifying potential regulatory genes in breast cancer

    PubMed Central

    Wu, Jiaqi; Hu, Shuofeng; Chen, Yaowen; Li, Zongcheng; Zhang, Jian; Yuan, Hanyu; Shi, Qiang; Shao, Ningsheng; Ying, Xiaomin

    2017-01-01

    Breast cancer is a disease with high heterogeneity. Many issues on tumorigenesis and progression are still elusive. It is critical to identify genes that play important roles in the progression of tumors, especially for tumors with poor prognosis such as basal-like breast cancer and tumors in very young women. To facilitate the identification of potential regulatory or driver genes, we present the Breast Cancer Integrative Platform (BCIP, http://omics.bmi.ac.cn/bcancer/). BCIP maintains multi-omics data selected with strict quality control and processed with uniform normalization methods, including gene expression profiles from 9,005 tumor and 376 normal tissue samples, copy number variation information from 3,035 tumor samples, microRNA-target interactions, co-expressed genes, KEGG pathways, and mammary tissue-specific gene functional networks. This platform provides a user-friendly interface integrating comprehensive and flexible analysis tools on differential gene expression, copy number variation, and survival analysis. The prominent characteristic of BCIP is that users can perform analysis by customizing subgroups with single or combined clinical features, including subtypes, histological grades, pathologic stages, metastasis status, lymph node status, ER/PR/HER2 status, TP53 mutation status, menopause status, age, tumor size, therapy responses, and prognosis. BCIP will help to identify regulatory or driver genes and candidate biomarkers for further research in breast cancer. PMID:28327601

  7. Coordinated regulation of biosynthetic and regulatory genes coincides with anthocyanin accumulation in developing eggplant fruit

    USDA-ARS?s Scientific Manuscript database

    Violet to black pigmentation of eggplant (Solanum melongena) fruit is attributed to anthocyanin accumulation. Model systems support the interaction of biosynthetic and regulatory genes for anthocyanin biosynthesis. Anthocyanin structural gene transcription requires the expression of at least one m...

  8. Stochastic models and numerical algorithms for a class of regulatory gene networks.

    PubMed

    Fournier, Thomas; Gabriel, Jean-Pierre; Pasquier, Jerôme; Mazza, Christian; Galbete, José; Mermod, Nicolas

    2009-08-01

    Regulatory gene networks contain generic modules, like those involving feedback loops, which are essential for the regulation of many biological functions (Guido et al. in Nature 439:856-860, 2006). We consider a class of self-regulated genes which are the building blocks of many regulatory gene networks, and study the steady-state distribution of the associated Gillespie algorithm by providing efficient numerical algorithms. We also study a regulatory gene network of interest in gene therapy, using mean-field models with time delays. Convergence of the related time-nonhomogeneous Markov chain is established for a class of linear catalytic networks with feedback loops.

  9. Framework for engineering finite state machines in gene regulatory networks.

    PubMed

    Oishi, Kevin; Klavins, Eric

    2014-09-19

    Finite state machines are fundamental computing devices at the core of many models of computation. In biology, finite state machines are commonly used as models of development in multicellular organisms. However, it remains unclear to what extent cells can remember state, how they can transition from one state to another reliably, and whether the existing parts available to the synthetic biologist are sufficient to implement specified finite state machines in living cells. Furthermore, how complex multicellular behaviors can be realized by multiple cells coordinating their states with signaling, growth, and division is not well understood. Here, we describe a method by which any finite state machine can be built using nothing more than a suitably engineered network of readily available repressing transcription factors. In particular, we show the mathematical equivalence of finite state machines with a Boolean model of gene regulatory networks. We describe how such networks can be realized with a small class of promoters and transcription factors. To demonstrate the effectiveness of our approach, we show that the behavior of the coarse grained ideal Boolean network model approximates a fine grained delay differential equation model of gene expression. Finally, we explore a framework for the design of more complex systems via an example, synthetic bacterial microcolony edge detection, that illustrates how finite state machines could be used together with cell signaling to construct novel multicellular behaviors.

  10. GeRNet: a gene regulatory network tool.

    PubMed

    Dussaut, J S; Gallo, C A; Cravero, F; Martínez, M J; Carballido, J A; Ponzoni, I

    2017-08-30

    Gene regulatory networks (GRNs) are crucial in every process of life since they govern the majority of the molecular processes. Therefore, the task of assembling these networks is highly important. In particular, the so called model-free approaches have an advantage modeling the complexities of dynamic molecular networks, since most of the gene networks are hard to be mapped with accuracy by any other mathematical model. A highly abstract model-free approach, called rule-based approach, offers several advantages performing data-driven analysis; such as the requirement of the least amount of data. They also have an important ability to perform inferences: its simplicity allows the inference of large size models with a higher speed of analysis. However, regarding these techniques, the reconstruction of the relational structure of the network is partial, hence incomplete, for an effective biological analysis. This situation motivated us to explore the possibility of hybridizing with other approaches, such as biclustering techniques. This led to incorporate a biclustering tool that finds new relations between the nodes of the GRN. In this work we present a new software, called GeRNeT that integrates the algorithms of GRNCOP2 and BiHEA along a set of tools for interactive visualization, statistical analysis and ontological enrichment of the resulting GRNs. In this regard, results associated with Alzheimer disease datasets are presented that show the usefulness of integrating both bioinformatics tools. Copyright © 2017 Elsevier B.V. All rights reserved.

  11. Improving gene regulatory network inference using network topology information.

    PubMed

    Nair, Ajay; Chetty, Madhu; Wangikar, Pramod P

    2015-09-01

    Inferring the gene regulatory network (GRN) structure from data is an important problem in computational biology. However, it is a computationally complex problem and approximate methods such as heuristic search techniques, restriction of the maximum-number-of-parents (maxP) for a gene, or an optimal search under special conditions are required. The limitations of a heuristic search are well known but literature on the detailed analysis of the widely used maxP technique is lacking. The optimal search methods require large computational time. We report the theoretical analysis and experimental results of the strengths and limitations of the maxP technique. Further, using an optimal search method, we combine the strengths of the maxP technique and the known GRN topology to propose two novel algorithms. These algorithms are implemented in a Bayesian network framework and tested on biological, realistic, and in silico networks of different sizes and topologies. They overcome the limitations of the maxP technique and show superior computational speed when compared to the current optimal search algorithms.

  12. Automated large-scale control of gene regulatory networks.

    PubMed

    Tan, Mehmet; Alhajj, Reda; Polat, Faruk

    2010-04-01

    Controlling gene regulatory networks (GRNs) is an important and hard problem. As it is the case in all control problems, the curse of dimensionality is the main issue in real applications. It is possible that hundreds of genes may regulate one biological activity in an organism; this implies a huge state space, even in the case of Boolean models. This is also evident in the literature that shows that only models of small portions of the genome could be used in control applications. In this paper, we empower our framework for controlling GRNs by eliminating the need for expert knowledge to specify some crucial threshold that is necessary for producing effective results. Our framework is characterized by applying the factored Markov decision problem (FMDP) method to the control problem of GRNs. The FMDP is a suitable framework for large state spaces as it represents the probability distribution of state transitions using compact models so that more space and time efficient algorithms could be devised for solving control problems. We successfully mapped the GRN control problem to an FMDP and propose a model reduction algorithm that helps find approximate solutions for large networks by using existing FMDP solvers. The test results reported in this paper demonstrate the efficiency and effectiveness of the proposed approach.

  13. Amylase and chitinase genes in Streptomyces lividans are regulated by reg1, a pleiotropic regulatory gene.

    PubMed Central

    Nguyen, J; Francou, F; Virolle, M J; Guérineau, M

    1997-01-01

    A regulatory gene, reg1, was identified in Streptomyces lividans. It encodes a 345-amino-acid protein (Reg1) which contains a helix-turn-helix DNA-binding motif in the N-terminal region. Reg1 exhibits similarity with the LacI/GalR family members over the entire sequence. It displays 95% identity with MalR (the repressor of malE in S. coelicolor), 65% identity with ORF-Sl (a putative regulatory gene of alpha-amylase of S. limosus), and 31% identity with CcpA (the carbon catabolite repressor in Bacillus subtilis). In S. lividans, the chromosomal disruption of reg1 affected the expression of several genes. The production of alpha-amylases of S. lividans and that of the alpha-amylase of S. limosus in S. lividans were enhanced in the reg1 mutant strains and relieved of carbon catabolite repression. As a result, the transcription level of the alpha-amylase of S. limosus was noticeably increased in the reg1 mutant strain. Moreover, the induction of chitinase production in S. lividans was relieved of carbon catabolite repression by glucose in the reg1 mutant strain, while the induction by chitin was lost. Therefore, reg1 can be regarded as a pleiotropic regulatory gene in S. lividans. PMID:9335287

  14. An algebra-based method for inferring gene regulatory networks.

    PubMed

    Vera-Licona, Paola; Jarrah, Abdul; Garcia-Puente, Luis David; McGee, John; Laubenbacher, Reinhard

    2014-03-26

    The inference of gene regulatory networks (GRNs) from experimental observations is at the heart of systems biology. This includes the inference of both the network topology and its dynamics. While there are many algorithms available to infer the network topology from experimental data, less emphasis has been placed on methods that infer network dynamics. Furthermore, since the network inference problem is typically underdetermined, it is essential to have the option of incorporating into the inference process, prior knowledge about the network, along with an effective description of the search space of dynamic models. Finally, it is also important to have an understanding of how a given inference method is affected by experimental and other noise in the data used. This paper contains a novel inference algorithm using the algebraic framework of Boolean polynomial dynamical systems (BPDS), meeting all these requirements. The algorithm takes as input time series data, including those from network perturbations, such as knock-out mutant strains and RNAi experiments. It allows for the incorporation of prior biological knowledge while being robust to significant levels of noise in the data used for inference. It uses an evolutionary algorithm for local optimization with an encoding of the mathematical models as BPDS. The BPDS framework allows an effective representation of the search space for algebraic dynamic models that improves computational performance. The algorithm is validated with both simulated and experimental microarray expression profile data. Robustness to noise is tested using a published mathematical model of the segment polarity gene network in Drosophila melanogaster. Benchmarking of the algorithm is done by comparison with a spectrum of state-of-the-art network inference methods on data from the synthetic IRMA network to demonstrate that our method has good precision and recall for the network reconstruction task, while also predicting several of the

  15. An algebra-based method for inferring gene regulatory networks

    PubMed Central

    2014-01-01

    Background The inference of gene regulatory networks (GRNs) from experimental observations is at the heart of systems biology. This includes the inference of both the network topology and its dynamics. While there are many algorithms available to infer the network topology from experimental data, less emphasis has been placed on methods that infer network dynamics. Furthermore, since the network inference problem is typically underdetermined, it is essential to have the option of incorporating into the inference process, prior knowledge about the network, along with an effective description of the search space of dynamic models. Finally, it is also important to have an understanding of how a given inference method is affected by experimental and other noise in the data used. Results This paper contains a novel inference algorithm using the algebraic framework of Boolean polynomial dynamical systems (BPDS), meeting all these requirements. The algorithm takes as input time series data, including those from network perturbations, such as knock-out mutant strains and RNAi experiments. It allows for the incorporation of prior biological knowledge while being robust to significant levels of noise in the data used for inference. It uses an evolutionary algorithm for local optimization with an encoding of the mathematical models as BPDS. The BPDS framework allows an effective representation of the search space for algebraic dynamic models that improves computational performance. The algorithm is validated with both simulated and experimental microarray expression profile data. Robustness to noise is tested using a published mathematical model of the segment polarity gene network in Drosophila melanogaster. Benchmarking of the algorithm is done by comparison with a spectrum of state-of-the-art network inference methods on data from the synthetic IRMA network to demonstrate that our method has good precision and recall for the network reconstruction task, while also

  16. The impact of gene expression variation on the robustness and evolvability of a developmental gene regulatory network.

    PubMed

    Garfield, David A; Runcie, Daniel E; Babbitt, Courtney C; Haygood, Ralph; Nielsen, William J; Wray, Gregory A

    2013-10-01

    Regulatory interactions buffer development against genetic and environmental perturbations, but adaptation requires phenotypes to change. We investigated the relationship between robustness and evolvability within the gene regulatory network underlying development of the larval skeleton in the sea urchin Strongylocentrotus purpuratus. We find extensive variation in gene expression in this network throughout development in a natural population, some of which has a heritable genetic basis. Switch-like regulatory interactions predominate during early development, buffer expression variation, and may promote the accumulation of cryptic genetic variation affecting early stages. Regulatory interactions during later development are typically more sensitive (linear), allowing variation in expression to affect downstream target genes. Variation in skeletal morphology is associated primarily with expression variation of a few, primarily structural, genes at terminal positions within the network. These results indicate that the position and properties of gene interactions within a network can have important evolutionary consequences independent of their immediate regulatory role.

  17. The Impact of Gene Expression Variation on the Robustness and Evolvability of a Developmental Gene Regulatory Network

    PubMed Central

    Garfield, David A.; Runcie, Daniel E.; Babbitt, Courtney C.; Haygood, Ralph; Nielsen, William J.; Wray, Gregory A.

    2013-01-01

    Regulatory interactions buffer development against genetic and environmental perturbations, but adaptation requires phenotypes to change. We investigated the relationship between robustness and evolvability within the gene regulatory network underlying development of the larval skeleton in the sea urchin Strongylocentrotus purpuratus. We find extensive variation in gene expression in this network throughout development in a natural population, some of which has a heritable genetic basis. Switch-like regulatory interactions predominate during early development, buffer expression variation, and may promote the accumulation of cryptic genetic variation affecting early stages. Regulatory interactions during later development are typically more sensitive (linear), allowing variation in expression to affect downstream target genes. Variation in skeletal morphology is associated primarily with expression variation of a few, primarily structural, genes at terminal positions within the network. These results indicate that the position and properties of gene interactions within a network can have important evolutionary consequences independent of their immediate regulatory role. PMID:24204211

  18. Inferring orthologous gene regulatory networks using interspecies data fusion

    PubMed Central

    Penfold, Christopher A.; Millar, Jonathan B. A.; Wild, David L.

    2015-01-01

    Motivation: The ability to jointly learn gene regulatory networks (GRNs) in, or leverage GRNs between related species would allow the vast amount of legacy data obtained in model organisms to inform the GRNs of more complex, or economically or medically relevant counterparts. Examples include transferring information from Arabidopsis thaliana into related crop species for food security purposes, or from mice into humans for medical applications. Here we develop two related Bayesian approaches to network inference that allow GRNs to be jointly inferred in, or leveraged between, several related species: in one framework, network information is directly propagated between species; in the second hierarchical approach, network information is propagated via an unobserved ‘hypernetwork’. In both frameworks, information about network similarity is captured via graph kernels, with the networks additionally informed by species-specific time series gene expression data, when available, using Gaussian processes to model the dynamics of gene expression. Results: Results on in silico benchmarks demonstrate that joint inference, and leveraging of known networks between species, offers better accuracy than standalone inference. The direct propagation of network information via the non-hierarchical framework is more appropriate when there are relatively few species, while the hierarchical approach is better suited when there are many species. Both methods are robust to small amounts of mislabelling of orthologues. Finally, the use of Saccharomyces cerevisiae data and networks to inform inference of networks in the budding yeast Schizosaccharomyces pombe predicts a novel role in cell cycle regulation for Gas1 (SPAC19B12.02c), a 1,3-beta-glucanosyltransferase. Availability and implementation: MATLAB code is available from http://go.warwick.ac.uk/systemsbiology/software/. Contact: d.l.wild@warwick.ac.uk Supplementary information: Supplementary data are available at Bioinformatics

  19. A new approach for modelling gene regulatory networks using fuzzy petri nets.

    PubMed

    Hamed, Raed I; Ahson, S I; Parveen, R

    2010-02-04

    Gene Regulatory Networks are models of genes and gene interactions at the expression level. The advent of microarray technology has challenged computer scientists to develop better algorithms for modeling the underlying regulatory relationship in between the genes. Fuzzy system has an ability to search microarray datasets for activator/repressor regulatory relationship. In this paper, we present a fuzzy reasoning model based on the Fuzzy Petri Net. The model considers the regulatory triplets by means of predicting changes in expression level of the target based on input expression level. This method eliminates possible false predictions from the classical fuzzy model thereby allowing a wider search space for inferring regulatory relationship. Through formalization of fuzzy reasoning, we propose an approach to construct a rulebased reasoning system. The experimental results show the proposed approach is feasible and acceptable to predict changes in expression level of the target gene.

  20. Regulatory elements of Caenorhabditis elegans ribosomal protein genes

    PubMed Central

    2012-01-01

    Background Ribosomal protein genes (RPGs) are essential, tightly regulated, and highly expressed during embryonic development and cell growth. Even though their protein sequences are strongly conserved, their mechanism of regulation is not conserved across yeast, Drosophila, and vertebrates. A recent investigation of genomic sequences conserved across both nematode species and associated with different gene groups indicated the existence of several elements in the upstream regions of C. elegans RPGs, providing a new insight regarding the regulation of these genes in C. elegans. Results In this study, we performed an in-depth examination of C. elegans RPG regulation and found nine highly conserved motifs in the upstream regions of C. elegans RPGs using the motif discovery algorithm DME. Four motifs were partially similar to transcription factor binding sites from C. elegans, Drosophila, yeast, and human. One pair of these motifs was found to co-occur in the upstream regions of 250 transcripts including 22 RPGs. The distance between the two motifs displayed a complex frequency pattern that was related to their relative orientation. We tested the impact of three of these motifs on the expression of rpl-2 using a series of reporter gene constructs and showed that all three motifs are necessary to maintain the high natural expression level of this gene. One of the motifs was similar to the binding site of an orthologue of POP-1, and we showed that RNAi knockdown of pop-1 impacts the expression of rpl-2. We further determined the transcription start site of rpl-2 by 5’ RACE and found that the motifs lie 40–90 bases upstream of the start site. We also found evidence that a noncoding RNA, contained within the outron of rpl-2, is co-transcribed with rpl-2 and cleaved during trans-splicing. Conclusions Our results indicate that C. elegans RPGs are regulated by a complex novel series of regulatory elements that is evolutionarily distinct from those of all other species

  1. A review on the computational approaches for gene regulatory network construction.

    PubMed

    Chai, Lian En; Loh, Swee Kuan; Low, Swee Thing; Mohamad, Mohd Saberi; Deris, Safaai; Zakaria, Zalmiyah

    2014-05-01

    Many biological research areas such as drug design require gene regulatory networks to provide clear insight and understanding of the cellular process in living cells. This is because interactions among the genes and their products play an important role in many molecular processes. A gene regulatory network can act as a blueprint for the researchers to observe the relationships among genes. Due to its importance, several computational approaches have been proposed to infer gene regulatory networks from gene expression data. In this review, six inference approaches are discussed: Boolean network, probabilistic Boolean network, ordinary differential equation, neural network, Bayesian network, and dynamic Bayesian network. These approaches are discussed in terms of introduction, methodology and recent applications of these approaches in gene regulatory network construction. These approaches are also compared in the discussion section. Furthermore, the strengths and weaknesses of these computational approaches are described. Copyright © 2014 Elsevier Ltd. All rights reserved.

  2. Equine umbilical cord blood contains a population of stem cells that express Oct4 and differentiate into mesodermal and endodermal cell types.

    PubMed

    Reed, Sarah A; Johnson, Sally E

    2008-05-01

    Mesenchymal stem cells (MSCs) offer promise as therapeutic aids in the repair of tendon, ligament, and bone damage suffered by sport horses. The objective of the study was to identify and characterize stem-like cells from newborn foal umbilical cord blood (UCB). UCB was collected and MSC isolated using human reagents. The cells exhibit a fibroblast-like morphology and express the stem cell markers Oct4, SSEA-1, Tra1-60 and Tra1-81. Culture of the cells in tissue-specific differentiation media leads to the formation of cell types characteristic of mesodermal and endodermal origins. Chondrogenic differentiation reveals proteoglycan and glycosaminoglycan synthesis as measured histochemically and Sox9 and collagen 2A1 gene transcription. Osteocytes capable of mineral deposition, osteonectin and Runx2 transcription were evident. Hepatogenic cells formed from UCBs express albumin and cytokeratin 18. Multinucleated myofibers that express desmin were observed indicating partial differentiation into mature muscle cells. Interestingly, conventional human protocols for UCB differentiation into adipocytes were unsuccessful in foal UCB and adult horse adipose-derived MSC. These results demonstrate that equine UCB can be induced to form multiple cell types that underlie their value for regenerative medicine in injured horses. In addition, this work suggests that subtle differences exist between equine and human UCB stem cells. (c) 2007 Wiley-Liss, Inc.

  3. Lin28B/Let-7 Regulates Expression of Oct4 and Sox2 and Reprograms Oral Squamous Cell Carcinoma Cells to a Stem-like State.

    PubMed

    Chien, Chian-Shiu; Wang, Mong-Lien; Chu, Pen-Yuan; Chang, Yuh-Lih; Liu, Wei-Hsiu; Yu, Cheng-Chia; Lan, Yuan-Tzu; Huang, Pin-I; Lee, Yi-Yen; Chen, Yi-Wei; Lo, Wen-Liang; Chiou, Shih-Hwa

    2015-06-15

    Lin28, a key factor for cellular reprogramming and generation of induced pluripotent stem cell (iPSC), makes a critical contribution to tumorigenicity by suppressing Let-7. However, it is unclear whether Lin28 is involved in regulating cancer stem-like cells (CSC), including in oral squamous carcinoma cells (OSCC). In this study, we demonstrate a correlation between high levels of Lin28B, Oct4, and Sox2, and a high percentage of CD44(+)ALDH1(+) CSC in OSCC. Ectopic Lin28B expression in CD44(-)ALDH1(-)/OSCC cells was sufficient to enhance Oct4/Sox2 expression and CSC properties, whereas Let7 co-overexpression effectively reversed these phenomena. We identified ARID3B and HMGA2 as downstream effectors of Lin28B/Let7 signaling in regulating endogenous Oct4 and Sox2 expression. Let7 targeted the 3' untranslated region of ARID3B and HMGA2 and suppressed their expression, whereas ARID3B and HMGA2 increased the transcription of Oct4 and Sox2, respectively, through promoter binding. Chromatin immunoprecipitation assays revealed a direct association between ARID3B and a specific ARID3B-binding sequence in the Oct4 promoter. Notably, by modulating Oct4/Sox2 expression, the Lin28B-Let7 pathway not only regulated stemness properties in OSCC but also determined the efficiency by which normal human oral keratinocytes could be reprogrammed to iPSC. Clinically, a Lin28B(high)-Let7(low) expression pattern was highly correlated with high levels of ARID3B, HMGA2, OCT4, and SOX2 expression in OSCC specimens. Taken together, our results show how Lin28B/Let7 regulates key cancer stem-like properties in oral squamous cancers.

  4. The effect of steroid hormones on the mRNA expression of oct4 and sox2 in uterine tissue of the ovariectomized mice model of menopause

    PubMed Central

    Davoudi, Marzieh; Zavareh, Saeed; Ghorbanian, Mohammad Taghi; Paylakhi, Seyed Hassan; Mohebbi, Seyed Reza

    2016-01-01

    Background: The uterus is a dynamic tissue responding to hormonal changes during reproductive cycles. As such, uterine stem cells have been studied in recent years. Transcription factors oct4 and sox2 are critical for effective maintenance of pluripotent cell identity. Objective: The present research evaluated the mRNA expression of oct4 and sox2 in the uterine tissues of ovariectomized mice treated with steroid hormones. Materials and Methods: In this experimental study, adult virgin female mice were ovariectomized and treated with estradiol 17β (E2), progesterone (P4), and a combination of E2 and P4 (E2 & P4) for 5 days. Uterine tissues were removed, and immunofluorescent (IF) staining and quantitative real-time PCR of oct4 and sox2 markers were performed. Results: IF showed oct4 and sox2 expression in the uterine endometrium and myometrium among all groups. The mRNA expression of oct4 (p=0.022) and sox2 (p=0.042) in the E2-treated group significantly were decreased compared to that in the control group. By contrast, the mRNA expression of oct4 and sox2 in the P4 (p=0.641 and 0.489 respectively) and E2 & P4-treated groups (p=0.267 and 0.264 respectively) did not show significant differences compared to the control group. Conclusion: The results indicate ovarian steroid hormones change the expression of oct4 and sox2 in the mice uterine tissues, which suggest the involvement of steroid hormonal regulation in uterine stem cells. PMID:27525332

  5. Discovery of time-delayed gene regulatory networks based on temporal gene expression profiling

    PubMed Central

    Li, Xia; Rao, Shaoqi; Jiang, Wei; Li, Chuanxing; Xiao, Yun; Guo, Zheng; Zhang, Qingpu; Wang, Lihong; Du, Lei; Li, Jing; Li, Li; Zhang, Tianwen; Wang, Qing K

    2006-01-01

    Background It is one of the ultimate goals for modern biological research to fully elucidate the intricate interplays and the regulations of the molecular determinants that propel and characterize the progression of versatile life phenomena, to name a few, cell cycling, developmental biology, aging, and the progressive and recurrent pathogenesis of complex diseases. The vast amount of large-scale and genome-wide time-resolved data is becoming increasing available, which provides the golden opportunity to unravel the challenging reverse-engineering problem of time-delayed gene regulatory networks. Results In particular, this methodological paper aims to reconstruct regulatory networks from temporal gene expression data by using delayed correlations between genes, i.e., pairwise overlaps of expression levels shifted in time relative each other. We have thus developed a novel model-free computational toolbox termed TdGRN (Time-delayed Gene Regulatory Network) to address the underlying regulations of genes that can span any unit(s) of time intervals. This bioinformatics toolbox has provided a unified approach to uncovering time trends of gene regulations through decision analysis of the newly designed time-delayed gene expression matrix. We have applied the proposed method to yeast cell cycling and human HeLa cell cycling and have discovered most of the underlying time-delayed regulations that are supported by multiple lines of experimental evidence and that are remarkably consistent with the current knowledge on phase characteristics for the cell cyclings. Conclusion We established a usable and powerful model-free approach to dissecting high-order dynamic trends of gene-gene interactions. We have carefully validated the proposed algorithm by applying it to two publicly available cell cycling datasets. In addition to uncovering the time trends of gene regulations for cell cycling, this unified approach can also be used to study the complex gene regulations related to

  6. Applying Attractor Dynamics to Infer Gene Regulatory Interactions Involved in Cellular Differentiation.

    PubMed

    Ghaffarizadeh, Ahmadreza; Podgorski, Gregory J; Flann, Nicholas S

    2017-02-27

    The dynamics of gene regulatory networks (GRNs) guide cellular differentiation. Determining the ways regulatory genes control expression of their targets is essential to understand and control cellular differentiation. The way a regulatory gene controls its target can be expressed as a gene regulatory function. Manual derivation of these regulatory functions is slow, error-prone and difficult to update as new information arises. Automating this process is a significant challenge and the subject of intensive effort. This work presents a novel approach to discovering biologically plausible gene regulatory interactions that control cellular differentiation. This method integrates known cell type expression data, genetic interactions, and knowledge of the effects of gene knockouts to determine likely GRN regulatory functions. We employ a genetic algorithm to search for candidate GRNs that use a set of transcription factors that control differentiation within a lineage. Nested canalyzing functions are used to constrain the search space to biologically plausible networks. The method identifies an ensemble of GRNs whose dynamics reproduce the gene expression pattern for each cell type within a particular lineage. The method's effectiveness was tested by inferring consensus GRNs for myeloid and pancreatic cell differentiation and comparing the predicted gene regulatory interactions to manually derived interactions. We identified many regulatory interactions reported in the literature and also found differences from published reports. These discrepancies suggest areas for biological studies of myeloid and pancreatic differentiation. We also performed a study that used defined synthetic networks to evaluate the accuracy of the automated search method and found that the search algorithm was able to discover the regulatory interactions in these defined networks with high accuracy. We suggest that the GRN functions derived from the methods described here can be used to fill

  7. Gene regulatory networks and their applications: understanding biological and medical problems in terms of networks

    PubMed Central

    Emmert-Streib, Frank; Dehmer, Matthias; Haibe-Kains, Benjamin

    2014-01-01

    In recent years gene regulatory networks (GRNs) have attracted a lot of interest and many methods have been introduced for their statistical inference from gene expression data. However, despite their popularity, GRNs are widely misunderstood. For this reason, we provide in this paper a general discussion and perspective of gene regulatory networks. Specifically, we discuss their meaning, the consistency among different network inference methods, ensemble methods, the assessment of GRNs, the estimated number of existing GRNs and their usage in different application domains. Furthermore, we discuss open questions and necessary steps in order to utilize gene regulatory networks in a clinical context and for personalized medicine. PMID:25364745

  8. Large-scale time-lapse microscopy of Oct4 expression in human embryonic stem cell colonies.

    PubMed

    Bhadriraju, Kiran; Halter, Michael; Amelot, Julien; Bajcsy, Peter; Chalfoun, Joe; Vandecreme, Antoine; Mallon, Barbara S; Park, Kye-Yoon; Sista, Subhash; Elliott, John T; Plant, Anne L

    2016-07-01

    Identification and quantification of the characteristics of stem cell preparations is critical for understanding stem cell biology and for the development and manufacturing of stem cell based therapies. We have developed image analysis and visualization software that allows effective use of time-lapse microscopy to provide spatial and dynamic information from large numbers of human embryonic stem cell colonies. To achieve statistically relevant sampling, we examined >680 colonies from 3 different preparations of cells over 5days each, generating a total experimental dataset of 0.9 terabyte (TB). The 0.5 Giga-pixel images at each time point were represented by multi-resolution pyramids and visualized using the Deep Zoom Javascript library extended to support viewing Giga-pixel images over time and extracting data on individual colonies. We present a methodology that enables quantification of variations in nominally-identical preparations and between colonies, correlation of colony characteristics with Oct4 expression, and identification of rare events.

  9. Clinical characteristics and prognosis of acute myeloid leukemia associated with DNA-methylation regulatory gene mutations

    PubMed Central

    Ryotokuji, Takeshi; Yamaguchi, Hiroki; Ueki, Toshimitsu; Usuki, Kensuke; Kurosawa, Saiko; Kobayashi, Yutaka; Kawata, Eri; Tajika, Kenji; Gomi, Seiji; Kanda, Junya; Kobayashi, Anna; Omori, Ikuko; Marumo, Atsushi; Fujiwara, Yusuke; Yui, Shunsuke; Terada, Kazuki; Fukunaga, Keiko; Hirakawa, Tsuneaki; Arai, Kunihito; Kitano, Tomoaki; Kosaka, Fumiko; Tamai, Hayato; Nakayama, Kazutaka; Wakita, Satoshi; Fukuda, Takahiro; Inokuchi, Koiti

    2016-01-01

    In recent years, it has been reported that the frequency of DNA-methylation regulatory gene mutations – mutations of the genes that regulate gene expression through DNA methylation – is high in acute myeloid leukemia. The objective of the present study was to elucidate the clinical characteristics and prognosis of acute myeloid leukemia with associated DNA-methylation regulatory gene mutation. We studied 308 patients with acute myeloid leukemia. DNA-methylation regulatory gene mutations were observed in 135 of the 308 cases (43.8%). Acute myeloid leukemia associated with a DNA-methylation regulatory gene mutation was more frequent in older patients (P<0.0001) and in patients with intermediate cytogenetic risk (P<0.0001) accompanied by a high white blood cell count (P=0.0032). DNA-methylation regulatory gene mutation was an unfavorable prognostic factor for overall survival in the whole cohort (P=0.0018), in patients aged ≤70 years, in patients with intermediate cytogenetic risk, and in FLT3-ITD-negative patients (P=0.0409). Among the patients with DNA-methylation regulatory gene mutations, 26.7% were found to have two or more such mutations and prognosis worsened with increasing number of mutations. In multivariate analysis DNA-methylation regulatory gene mutation was an independent unfavorable prognostic factor for overall survival (P=0.0424). However, patients with a DNA-methylation regulatory gene mutation who underwent allogeneic stem cell transplantation in first remission had a significantly better prognosis than those who did not undergo such transplantation (P=0.0254). Our study establishes that DNA-methylation regulatory gene mutation is an important unfavorable prognostic factor in acute myeloid leukemia. PMID:27247325

  10. Efficient computation of minimal perturbation sets in gene regulatory networks

    PubMed Central

    Garg, Abhishek; Mohanram, Kartik; Di Cara, Alessandro; Degueurce, Gwendoline; Ibberson, Mark; Dorier, Julien; Xenarios, Ioannis

    2013-01-01

    In the last few decades, technological and experimental advancements have enabled a more precise understanding of the mode of action of drugs with respect to human cell signaling pathways and have positively influenced the design of new drug compounds. However, as the design of compounds has become increasingly target-specific, the overall effects of a drug on adjacent cellular signaling pathways remain difficult to predict because of the complexity of the interactions involved. Off-target effects of drugs are known to influence their efficacy and safety. Similarly, drugs which are more target-specific also suffer from lack of efficacy because their scope might be too limited in the context of cellular signaling. Even in situations where the signaling pathways targeted by a drug are known, the presence of point mutations in some of the components of the pathways can render a therapy ineffective in a considerable target subpopulation. Some of these issues can be addressed by predicting Minimal Intervention Sets (MIS) of elements of the signaling pathways that when perturbed give rise to a pre-defined cellular phenotype. These minimal gene perturbation sets can then be further used to screen a library of drug compounds in order to discover effective drug therapies. This manuscript describes algorithms that can be used to discover MIS in a gene regulatory network that can lead to a defined cellular phenotype. Algorithms are implemented in our Boolean modeling toolbox, GenYsis. The software binaries of GenYsis are available for download from http://www.vital-it.ch/software/genYsis/. PMID:24391592

  11. Regulatory and Structural Genes for Lysozymes of Mice

    PubMed Central

    Hammer, Michael F.; Wilson, Allan C.

    1987-01-01

    The molecular and genetic basis of large differences in the concentration of P lysozyme in the small intestine has been investigated by crossing inbred strains of two species of house mouse (genus Mus). The concentration of P in domesticus is about 130-fold higher than in castaneus . An autosomal genetic element determining the concentration of P has been identified and named the P lysozyme regulator, Lzp-r . The level of P in interspecific hybrids (domesticus x castaneus) as well as in certain classes of backcross progeny is intermediate relative to parental levels, which shows that the two alleles of Lzp-r are inherited additively. There are two forms of P lysozyme in the intestine of the interspecific hybrid—one having the heat stability of domesticus P, the other being more stable and presumably the product of the castaneus P locus. These two forms occur in equal amounts, and it appears that Lzp-r acts in trans. The linkage of Lzp-r to three structural genes (Lzp-s, Lzm-s1, and Lzm-s2), one specifying P lysozyme and two specifying M lysozymes, was shown by electrophoretic analysis of backcrosses involving domesticus and castaneus and also domesticus and spretus . The role of regulatory mutations in evolution is discussed in light of these results. PMID:3569879

  12. Treatment of agarose-agarose RENCA macrobeads with docetaxel selects for OCT4(+) cells with tumor-initiating capability.

    PubMed

    Gazda, Lawrence S; Martis, Prithy C; Laramore, Melissa A; Bautista, Melissa A; Dudley, Atira; Vinerean, Horatiu V; Smith, Barry H

    2013-12-01

    The cancer stem cell (CSC) theory depicts such cells as having the capacity to produce both identical CSCs (symmetrical division) and tumor-amplifying daughter cells (asymmetric division). CSCs are thought to reside in niches similar to those of normal stem cells as described for neural, intestinal, and epidermal tissue, are resistant to chemotherapy, and are responsible for tumor recurrence. We recently described the niche-like nature of mouse renal adenocarcinoma (RENCA) cells following encapsulation in agarose macrobeads. In this paper we tested the hypothesis that encapsulated RENCA colonies function as an in vitro model of a CSC niche and that the majority of cells would undergo chemotherapy-induced death, followed by tumor recurrence. After exposure to docetaxel (5 µg/ml), 50% of cells were lost one week post-treatment while only one or two cells remained in each colony by 6 weeks. Surviving cells expressed OCT4 and reformed tumors at 16 weeks post-treatment. Docetaxel-resistant cells also grew as monolayers in cell culture (16-17 weeks post-exposure) or as primary tumors following transplantation to Balb/c mice (6 of 10 mice) or NOD.CB17-Prkdc(scid)/J mice (9 of 9 mice; 10 weeks post-transplantation or 28 weeks post-exposure). These data support the hypothesis that a rare subpopulation of OCT4(+) cells are resistant to docetaxel and these cells are sufficient for tumor recurrence. The reported methodology can be used to obtain purified populations of tumor-initiating cells, to screen for anti-tumor-initiating cell agents, and to investigate the in vitro correlate of a CSC niche, especially as it relates to chemo-resistance and tumor recurrence.

  13. Perturbation analysis analyzed - Mathematical modeling of intact and perturbed gene regulatory circuits for animal development

    PubMed Central

    de-Leon, Smadar Ben-Tabou

    2010-01-01

    Gene regulatory networks for animal development are the underlying mechanisms controlling cell fate specification and differentiation. The architecture of gene regulatory circuits determines their information processing properties and their developmental function. It is a major task to derive realistic network models from exceedingly advanced high throughput experimental data. Here we use mathematical modeling to study the dynamics of gene regulatory circuits to advance the ability to infer regulatory connections and logic function from experimental data. This study is guided by experimental methodologies that are commonly used to study gene regulatory networks that control cell fate specification. We study the effect of a perturbation of an input on the level of its downstream genes and compare between the cis-regulatory execution of OR and AND logics. Circuits that initiate gene activation and circuits that lock on the expression of genes are analyzed. The model improves our ability to analyze experimental data and construct from it the network topology. The model also illuminates information processing properties of gene regulatory circuits for animal development. PMID:20599898

  14. A Novel Regulatory Gene, Tri10, Controls Trichothecene Toxin Production and Gene Expression

    PubMed Central

    Tag, Andrew G.; Garifullina, Gulnara F.; Peplow, Andrew W.; Ake, Charles; Phillips, T. D.; Hohn, Thomas M.; Beremand, Marian N.

    2001-01-01

    We report here the characterization of Tri10, a novel regulatory gene within the trichothecene gene cluster. Comparison of Tri10 genomic and mRNA sequences revealed that removal of a single 77-bp intron provided a 1,260-bp open reading frame, encoding a 420-amino-acid protein. Disruption of Tri10 in Fusarium sporotrichioides abolished T-2 toxin production and dramatically decreased the transcript accumulation for four trichothecene genes (Tri4, Tri5, Tri6, and Tri101) and an apparent farnesyl pyrophosphate synthetase (Fpps) gene. Conversely, homologous integration of a disruption vector by a single upstream crossover event significantly increased T-2 toxin production and elevated the transcript accumulation of the trichothecene genes and Fpps. Further analysis revealed that disruption of Tri10, and to a greater extent the disruption of Tri6, increased sensitivity to T-2 toxin under certain growth conditions. Although Tri10 is conserved in Fusarium graminearum and Fusarium sambucinum and clearly plays a central role in regulating trichothecene gene expression, it does not show any significant matches to proteins of known or predicted function or to motifs except a single transmembrane domain. We suggest a model in which Tri10 acts upstream of the cluster-encoded transcription factor TRI6 and is necessary for full expression of both the other trichothecene genes and the genes for the primary metabolic pathway that precedes the trichothecene biosynthetic pathway, as well as for wild-type levels of trichothecene self-protection. We further suggest the presence of a regulatory loop where Tri6 is not required for the transcription of Tri10 but is required to limit the expression of Tri10. PMID:11679358

  15. Creating and validating cis-regulatory maps of tissue-specific gene expression regulation

    PubMed Central

    O'Connor, Timothy R.; Bailey, Timothy L.

    2014-01-01

    Predicting which genomic regions control the transcription of a given gene is a challenge. We present a novel computational approach for creating and validating maps that associate genomic regions (cis-regulatory modules–CRMs) with genes. The method infers regulatory relationships that explain gene expression observed in a test tissue using widely available genomic data for ‘other’ tissues. To predict the regulatory targets of a CRM, we use cross-tissue correlation between histone modifications present at the CRM and expression at genes within 1 Mbp of it. To validate cis-regulatory maps, we show that they yield more accurate models of gene expression than carefully constructed control maps. These gene expression models predict observed gene expression from transcription factor binding in the CRMs linked to that gene. We show that our maps are able to identify long-range regulatory interactions and improve substantially over maps linking genes and CRMs based on either the control maps or a ‘nearest neighbor’ heuristic. Our results also show that it is essential to include CRMs predicted in multiple tissues during map-building, that H3K27ac is the most informative histone modification, and that CAGE is the most informative measure of gene expression for creating cis-regulatory maps. PMID:25200088

  16. Creating and validating cis-regulatory maps of tissue-specific gene expression regulation.

    PubMed

    O'Connor, Timothy R; Bailey, Timothy L

    2014-01-01

    Predicting which genomic regions control the transcription of a given gene is a challenge. We present a novel computational approach for creating and validating maps that associate genomic regions (cis-regulatory modules-CRMs) with genes. The method infers regulatory relationships that explain gene expression observed in a test tissue using widely available genomic data for 'other' tissues. To predict the regulatory targets of a CRM, we use cross-tissue correlation between histone modifications present at the CRM and expression at genes within 1 Mbp of it. To validate cis-regulatory maps, we show that they yield more accurate models of gene expression than carefully constructed control maps. These gene expression models predict observed gene expression from transcription factor binding in the CRMs linked to that gene. We show that our maps are able to identify long-range regulatory interactions and improve substantially over maps linking genes and CRMs based on either the control maps or a 'nearest neighbor' heuristic. Our results also show that it is essential to include CRMs predicted in multiple tissues during map-building, that H3K27ac is the most informative histone modification, and that CAGE is the most informative measure of gene expression for creating cis-regulatory maps.

  17. Anomaly detection in gene expression via stochastic models of gene regulatory networks.

    PubMed

    Kim, Haseong; Gelenbe, Erol

    2009-12-03

    The steady-state behaviour of gene regulatory networks (GRNs) can provide crucial evidence for detecting disease-causing genes. However, monitoring the dynamics of GRNs is particularly difficult because biological data only reflects a snapshot of the dynamical behaviour of the living organism. Also most GRN data and methods are used to provide limited structural inferences. In this study, the theory of stochastic GRNs, derived from G-Networks, is applied to GRNs in order to monitor their steady-state behaviours. This approach is applied to a simulation dataset which is generated by using the stochastic gene expression model, and observe that the G-Network properly detects the abnormally expressed genes in the simulation study. In the analysis of real data concerning the cell cycle microarray of budding yeast, our approach finds that the steady-state probability of CLB2 is lower than that of other agents, while most of the genes have similar steady-state probabilities. These results lead to the conclusion that the key regulatory genes of the cell cycle can be expressed in the absence of CLB type cyclines, which was also the conclusion of the original microarray experiment study. G-networks provide an efficient way to monitor steady-state of GRNs. Our method produces more reliable results then the conventional t-test in detecting differentially expressed genes. Also G-networks are successfully applied to the yeast GRNs. This study will be the base of further GRN dynamics studies cooperated with conventional GRN inference algorithms.

  18. The Death-inducer Obliterator 1 (Dido1) Gene Regulates Embryonic Stem Cell Self-renewal*

    PubMed Central

    Liu, Yinyin; Kim, Hyeung; Liang, Jiancong; Lu, Weisi; Ouyang, Bin; Liu, Dan; Songyang, Zhou

    2014-01-01

    The regulatory network of factors that center on master transcription factors such as Oct4, Nanog, and Sox2 help maintain embryonic stem (ES) cells and ensure their pluripotency. The target genes of these master transcription factors define the ES cell transcriptional landscape. In this study, we report our findings that Dido1, a target of canonical transcription factors such as Oct4, Sox2, and Nanog, plays an important role in regulating ES cell maintenance. We found that depletion of Dido1 in mouse ES cells led to differentiation, and ectopic expression of Dido1 inhibited differentiation induced by leukemia inhibitory factor withdrawal. We further demonstrated that whereas Nanog and Oct4 could occupy the Dido1 locus and promote its transcription, Dido1 could also target to the loci of pluripotency factors such as Nanog and Oct4 and positively regulate their expression. Through this feedback and feedforward loop, Dido1 is able to regulate self-renewal of mouse ES cells PMID:24347171

  19. A spatially dynamic cohort of regulatory genes in the endomesodermal gene network of the sea urchin embryo.

    PubMed

    Smith, Joel; Kraemer, Ebba; Liu, Hongdau; Theodoris, Christina; Davidson, Eric

    2008-01-15

    A gene regulatory network subcircuit comprising the otx, wnt8, and blimp1 genes accounts for a moving torus of gene expression that sweeps concentrically across the vegetal domain of the sea urchin embryo. Here we confirm by mutation the inputs into the blimp1cis-regulatory module predicted by network analysis. Its essential design feature is that it includes both activation and autorepression sites. The wnt8 gene is functionally linked into the subcircuit in that cells receiving this ligand generate a beta-catenin/Tcf input required for blimp1 expression, while the wnt8 gene in turn requires a Blimp1 input. Their torus-like spatial expression patterns and gene regulatory analysis indicate that the genes even-skipped and hox11/13b are also entrained by this subcircuit. We verify the cis-regulatory inputs of even-skipped predicted by network analysis. These include activation by beta-catenin/Tcf and Blimp1, repression within the torus by Hox11/13b, and repression outside the torus by Tcf in the absence of Wnt8 signal input. Thus even-skipped and hox11/13b, along with blimp1 and wnt8, are members of a cohort of torus genes with similar regulatory inputs and similar, though slightly out-of-phase, expression patterns, which reflect differences in cis-regulatory design.

  20. Regulatory Divergence between Parental Alleles Determines Gene Expression Patterns in Hybrids

    PubMed Central

    Combes, Marie-Christine; Hueber, Yann; Dereeper, Alexis; Rialle, Stéphanie; Herrera, Juan-Carlos; Lashermes, Philippe

    2015-01-01

    Both hybridization and allopolyploidization generate novel phenotypes by conciliating divergent genomes and regulatory networks in the same cellular context. To understand the rewiring of gene expression in hybrids, the total expression of 21,025 genes and the allele-specific expression of over 11,000 genes were quantified in interspecific hybrids and their parental species, Coffea canephora and Coffea eugenioides using RNA-seq technology. Between parental species, cis- and trans-regulatory divergences affected around 32% and 35% of analyzed genes, respectively, with nearly 17% of them showing both. The relative importance of trans-regulatory divergences between both species could be related to their low genetic divergence and perennial habit. In hybrids, among divergently expressed genes between parental species and hybrids, 77% was expressed like one parent (expression level dominance), including 65% like C. eugenioides. Gene expression was shown to result from the expression of both alleles affected by intertwined parental trans-regulatory factors. A strong impact of C. eugenioides trans-regulatory factors on the upregulation of C. canephora alleles was revealed. The gene expression patterns appeared determined by complex combinations of cis- and trans-reg