A ChIP-chip analysis of E2F family in normal and tumor cells reveals interchangeable roles of E2F family members
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE8716 | GEO | 2007/08/22
SECONDARY ACCESSION(S): PRJNA101959
REPOSITORIES: GEO
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Project description:Using ChIP-chip assays, we examined the binding patterns of three members of E2F family in five different cell types. Keywords: ChIP-chip We used ENCODE arrays to determine the loaction of E2F family, then identify their binding motifs in vivo. We used promoter arrays to assess the relationship of E2F family in five different cells including tumor and normal cells.
2007-08-22 | E-GEOD-8716 | biostudies-arrayexpress
Project description:Using ChIP-chip assays (employing ENCODE arrays and core promoter arrays), we examined the binding patterns of three members of the E2F family in five cell types. We determined that most E2F1, E2F4, and E2F6 binding sites are located within 2 kb of a transcription start site, in both normal and tumor cells. In fact, the majority of promoters that are active (as defined by TAF1 or POLR2A binding) in GM06990 B lymphocytes and Ntera2 carcinoma cells were also bound by an E2F. This very close relationship between E2F binding sites and binding sites for general transcription factors in both normal and tumor cells suggests that a chromatin-bound E2F may be a signpost for active transcription initiation complexes. In general, we found that several E2Fs bind to a given promoter and that there is only modest cell type specificity of the E2F family. Thus, it is difficult to assess the role of any particular E2F in transcriptional regulation, due to extreme redundancy of target promoters. However, Ntera2 carcinoma cells were exceptional in that a large set of promoters were bound by E2F6, but not by E2F1 or E2F4. It has been proposed that E2F6 contributes to gene silencing by recruiting enzymes involved in methylating histone H3. To test this hypothesis, we created Ntera2 cell lines harboring shRNAs to E2F6. We found that reduction of E2F6 only induced minimal alteration of the transcriptome of Ntera2 transcriptome. Our results support the concept of functional redundancy in the E2F family and suggest that E2F6 is not critical for histone methylation.
| S-EPMC2045138 | biostudies-literature
Project description:Mutations in cancer are due in part to DNA cytosine deamination by APOBEC3B (A3B). While low in healthy tissues, A3B expression and activity are elevated in tumors and further increase in metastases. However, molecular mechanisms responsible for A3B transcriptional regulation are poorly understood. Here, we address whether the RB/E2F pathway, which is often dysregulated in breast cancer, is a molecular trigger of A3B overexpression. First, an A3B promoter-driven luciferase reporter was used to demonstrate reporter activation by disruption of only one out of five predicted E2F binding sites. Second, A3B-luciferase reporter activation was also triggered by expressing the BK polyomavirus T-antigen, which inactivates RB and thereby alleviates repression of E2F regulated genes. Importantly, A3B-luciferase reporter induction by BK polyomavirus T-antigen could not be further increased by mutating the functional E2F binding site. Third, both CRISPR disruption and targeted base substitutions in the endogenous E2F binding site caused strong A3B upregulation and confirmed importance of this regulatory element. Fourth, proteomics experiments showed that members of the DREAM and PRC1.6-complexes including multiple E2F family members are able to bind to wild-type but not E2F mutant promoter sequences. Finally, a combination of genetic and biochemical studies implicated E2F4 and E2F6 in endogenous A3B gene repression. Altogether, our studies demonstrate that A3B expression is suppressed in normal cells by the RB/E2F axis and that viral or mutational disruption of this pathway causes overexpression of this DNA deaminase in cancer and contributes mutational fuel to tumor evolution.
2020-10-19 | PXD020473 | Pride
Project description:E2F is a heterogenous transcription factor and its role in cell cycle control results from the integrated activities of many different E2F family members. Unlike mammalian cells, that have a large number of E2F-related genes, the Drosophila genome encodes just two E2F genes, de2f1 and de2f2. Here we show that de2f1 and de2f2 provide different elements of E2F regulation and that they have opposing functions during Drosophila development. dE2F1 and dE2F2 both heterodimerize with dDP and bind to the promoters of E2F-regulated genes in vivo. dE2F1 is a potent activator of transcription, and the loss of de2f1 results in the reduced expression of E2F-regulated genes. In contrast, dE2F2 represses the transcription of E2F reporters and the loss of de2f2 function results in increased and expanded patterns of gene expression. The loss of de2f1 function has previously been reported to compromise cell proliferation. de2f1 mutant embryos have reduced expression of E2F-regulated genes, low levels of DNA synthesis, and hatch to give slow-growing larvae. We find that these defects are due in large part to the unchecked activity of dE2F2, since they can be suppressed by mutation of de2f2. Examination of eye discs from de2f1; de2f2 double-mutant animals reveals that relatively normal patterns of DNA synthesis can occur in the absence of both E2F proteins. This study shows how repressor and activator E2Fs are used to pattern transcription and how the net effect of E2F on cell proliferation results from the interplay between two types of E2F complexes that have antagonistic functions.
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Project description:Most E2F-binding sites repress transcription through the recruitment of Retinoblasoma (RB) family members until the end of the G1 cell-cycle phase. Although the MYB promoter contains an E2F-binding site, its transcription is activated shortly after the exit from quiescence, before RB family members inactivation, by unknown mechanisms. We had previously uncovered a nuclear factor distinct from E2F, Myb-sp, whose DNA-binding site overlapped the E2F element and had hypothesized that this factor might overcome the transcriptional repression of MYB by E2F-RB family members. We have purified Myb-sp and discovered that Myc-associated zinc finger proteins (MAZ) are major components. We show that various MAZ isoforms are present in Myb-sp and activate transcription via the MYB-E2F element. Moreover, while forced RB or p130 expression repressed the activity of a luciferase reporter driven by the MYB-E2F element, co-expression of MAZ proteins not only reverted repression, but also activated transcription. Finally, we show that MAZ binds the MYB promoter in vivo, that its binding site is critical for MYB transactivation, and that MAZ knockdown inhibits MYB expression during the exit from quiescence. Together, these data indicate that MAZ is essential to bypass MYB promoter repression by RB family members and to induce MYB expression.
2017-10-04 | PXD006857 | Pride
Project description:Recent genome-scale ChIP-chip studies of transcription factors have shown that a low percentage of experimentally determined binding sites contain the consensus motif for the immunoprecipitated factor. In most cases, differences between in vivo target sites that contain or lack a consensus motif have not been explored. We have previously shown that most sites to which E2F family members are bound in vivo do not contain E2F consensus motifs. The main purpose of this study was to develop an understanding of how E2F binding specificity is achieved in vivo. In particular, we have addressed how E2F family members are recruited to core promoter regions that lack a consensus motif and are excluded from other regions that contain a consensus motif. Using promoter and ENCODE arrays, we have shown that the predominant factors specifying whether E2F is recruited to an in vivo binding site are a) the site must be in a core promoter and b) the promoter region must be utilized as a promoter by the transcriptional machinery in that particular cell type. We have tested three models for recruitment of E2F to core promoters lacking a consensus site, including a) indirect recruitment, b) looping to the core promoter mediated by an E2F bound to a distal consensus motif, and c) assisted binding of E2F to a site that weakly resembles an E2F consensus motif within the core promoter. To test these models, we developed a new in vivo assay, termed eChIP, which allows analysis of transcription factor binding to isolated promoter fragments. Our findings suggest that in vivo a) the presence of a consensus motif is not sufficient to recruit E2Fs, b) E2Fs can bind to isolated regions that lack a consensus motif, and c) binding can require regions other than the best match to the E2F PWM in the core promoter. Keywords: E2F, ChIP-chip, transcription factor binding, consensus motifs 37 ChIP-chip arrays (of these, 14 array sets are biological duplicates). 22 samples are included in this series, the rest can be found in supplementary info to the following papers: Xu 2007, Jin 2006, Komashko 2008
2008-10-01 | E-GEOD-12126 | biostudies-arrayexpress
Project description:Recent genome-scale ChIP-chip studies of transcription factors have shown that a low percentage of experimentally determined binding sites contain the consensus motif for the immunoprecipitated factor. In most cases, differences between in vivo target sites that contain or lack a consensus motif have not been explored. We have previously shown that most sites to which E2F family members are bound in vivo do not contain E2F consensus motifs. The main purpose of this study was to develop an understanding of how E2F binding specificity is achieved in vivo. In particular, we have addressed how E2F family members are recruited to core promoter regions that lack a consensus motif and are excluded from other regions that contain a consensus motif. Using promoter and ENCODE arrays, we have shown that the predominant factors specifying whether E2F is recruited to an in vivo binding site are a) the site must be in a core promoter and b) the promoter region must be utilized as a promoter by the transcriptional machinery in that particular cell type. We have tested three models for recruitment of E2F to core promoters lacking a consensus site, including a) indirect recruitment, b) looping to the core promoter mediated by an E2F bound to a distal consensus motif, and c) assisted binding of E2F to a site that weakly resembles an E2F consensus motif within the core promoter. To test these models, we developed a new in vivo assay, termed eChIP, which allows analysis of transcription factor binding to isolated promoter fragments. Our findings suggest that in vivo a) the presence of a consensus motif is not sufficient to recruit E2Fs, b) E2Fs can bind to isolated regions that lack a consensus motif, and c) binding can require regions other than the best match to the E2F PWM in the core promoter. Keywords: E2F, ChIP-chip, transcription factor binding, consensus motifs
2008-10-01 | GSE12126 | GEO
Project description:The Rb/E2F tumor suppressor regulates gene expression to control the onset and timing of differentiation in many different cell types during development. This critical function makes Rb/E2F a frequent target for inactivation in tumors of diverse tissue origin. However, the mechanisms by which Rb/E2F governs tissue-specific gene regulation in vivo are poorly understood. We have determined the genome-wide binding profiles for components of the C. elegans Rb/E2F transcriptional regulatory pathway in the germ line, the intestine, and broadly in somatic tissues. Rb/E2F exhibits highly tissue-specific regulation, with unique sets of target genes that are activated or repressed depending on whether cells are in a progenitor (germ line) or terminally differentiated (somatic) state. In particular, LIN-35 binding is impaired in the germ line relative to the soma. Moreover, in the intestine, LIN-35 binds independently of EFL-1/DPL-1 at broad intergenic sites at which the heterochromatin protein HPL-2 also binds. Finally, recently defined âHOTâ sites, which are non-specifically bound by many unrelated factors, appear to be restricted primarily to somatic tissues, suggesting that germ line chromatin is in a state that globally restricts promiscuous association of regulatory factors. In sum, these tissue-specific binding profiles led to significant mechanistic insights into tissue-specific properties of Rb/E2F function in C. elegans that are likely relevant to other organisms. Staged C. elegans worms carrying different transgenes expressing GFP-transcription factors under different promoters were treated to ChIP-seq in biological replicate. A total of eleven different transgenic lines were analyzed. Three transcription factors (LIN-35, EFL-1 and DPL-1) were each tagged with GFP and expressed under the pie-1 promoter (germline), the ges-1 promoter (intestine), or their endogenous promoter (somatic expression), for a total of nine lines. In addition, LIN-35:GFP was expressed under the mex-5 promoter (germline), and HPL-2 was expressed under the ges-1 promoter, for an additional two lines. All strains with germline expression were analyzed as young adults, while the intestine or somatic expressed lines were analyzed as L1s. For each replicate of each transgenic line at the appropriate stage, anti-GFP was used to immunoprecipitate the tagged transcription factor, and the factor-enriched chromatin was subjected to Illumina sequencing. As a control, for every ChIP sample, the input (without immunoprecipitation) was also sequenced.
2012-06-26 | E-GEOD-30246 | biostudies-arrayexpress
Project description:Members of the GATA protein family play important roles in lineage specification and transdifferentiation. Previous reports show that some members of GATA protein family also can induce pluripotency in somatic cells by substituting for the key pluripotency-associated factor Oct4. However, the mechanism that links the lineage specifying cues and activation of pluripotency remains elusive. Here, we report that all GATA family members can substitute for Oct4 to induce pluripotency. We found that all members of the GATA family can inhibit elevated ectodermal-lineage genes, which is consistent with previous reports that a balance of different lineage-specifying forces is important for restoration of pluripotency. A conserved DNA-binding domain in the C-terminal zinc finger is critical for the GATA family to induce pluripotency. Using RNA-seq and ChIP-seq we identified that the pluripotency-related gene Sall4 is a direct target of GATA family members during reprogramming, serving as a bridge linking the lineage-specific GATA family to the pluripotency circuit. Thus, the GATA family is the first family of proteins wherein all members can function as inducers of the reprogramming process that can substitute for Oct4. Our results suggest that the roles of the GATA family in reprogramming have been greatly underestimated, and that the GATA family may serve as a general mediator for cell fate conversion.
2014-12-02 | GSE57849 | GEO
Project description:Five members of the Arabidopsis thaliana NF-YA gene family are strongly induced by several stress conditions via transcriptional and miR169-related posttranscriptional mechanisms. These transcription factors participate in gene regulation via two different mechanisms, one depending on binding to the CCAAT-box in the promoter of regulated genes and the other, independent of the CCAAT-box, in which NF-YA prevents the interaction of the NF-YB/YC heterodimer with transcription factors.
2012-04-25 | GSE36092 | GEO