E2F in vivo binding specificity: comparison of consensus vs. non-consensus binding sites
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PROVIDER: PRJNA113485 | ENA |
REPOSITORIES: ENA
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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: Not available
| S-EPMC2577861 | biostudies-literature
Project description:Structural insights into the DNA-binding specificity of E2F family transcription factors
| PRJEB8671 | ENA
Project description:The E2F family of transcription factors is typically described as binding the family consensus sequence TTTSSCGC, were S is G or C. Analysis of ChIP-seq experiments, however, reveals that this consensus sequence is found in only 10% of ChIP-seq peaks, suggesting that the mechanism for E2F sequence recognition cannot be explained using previous assumptions. In order to better understand E2F sequence specificity, we performed high-throughput Universal Protein Binding Microarray experiments to obtain the relative binding affinity for every possible 8-mer, as well a large number of bound and unbound probes intheir native genomic sequence context. Our results show that while the consensus sequence is bound with relatively high affinity, numerous other 8-mers, many distinctly different from the consensus motif, are bound with similar or greater affinity. These data suggest that the mechanism for E2F sequence specificity is likely complex, and cannot readily be explained through a simple consensus sequence. Because of this, complex regression models were created using the bound and unbound probe binding affinities, and were able to predict binding in vivo, where the consensus sequence and varoius E2F PWMs were not.
2014-11-04 | GSE61854 | GEO
Project description:ChIP-seq for the strongest cell cycle regulator transcription factors in Drosophila Melanogaster S2 cells. These assays have been used to validate the direct transcriptional targets of the same transcription factors investigated in RNA-seq (E-MTAB-1364) and Affymetrix microarray experiments (E-MTAB-453). ChIP-seq assays have been done with tagged fusion proteins (for example, since we dont have functional E2f antibodies against endogenous E2f , we are transfecting v5-tagged-E2f-ORF to S2 cells and then use antibodies against v5 to detect the signal from E2f binding). If the ChIP-seq has been done with tagged fusion proteins (such as v5-tagged-E2f-ORF), the protein expression has been induced with CuSO4 treatment 48h prior to cell crosslinking & lysis. Our fusion protein constructs are driven by metallothionein promoter, which is induced by CuSO4. E-MTAB-1648, E-MTAB-1364 and E-MTAB-453 are all data from: Bonke M, et al. (2013) Transcriptional networks controlling the cell cycle. G3 (Bethesda) 3, 75-90, PMID: 23316440.
2013-08-01 | E-MTAB-1648 | biostudies-arrayexpress
Project description:Somatic mutations are highly enriched at transcription factor (TF) binding sites, with the strongest trend being observed for ultraviolet light (UV)-induced mutations in melanomas. One of the main mechanisms proposed for this hyper-mutation pattern is the inefficient repair of UV lesions within TF-binding sites, caused by competition between TFs bound to these lesions and the DNA repair proteins that must recognize the lesions to initiate repair. However, TF binding to UV-irradiated DNA is poorly characterized, and it is unclear whether TFs maintain specificity for their DNA sites after UV exposure. We developed UV-Bind, a high-throughput approach to investigate the impact of UV irradiation on protein-DNA binding specificity. We applied UV-Bind to ten TFs from eight structural families, and found that UV lesions significantly altered the DNA-binding preferences of all TFs tested. The main effect was a decrease in binding specificity, but the precise effects and their magnitude differ across factors. Importantly, we found that despite the overall reduction in DNA-binding specificity in the presence of UV lesions, TFs can still compete with repair proteins for lesion recognition, in a manner consistent with their specificity for UV-irradiated DNA. In addition, for a subset of TFs we identified a surprising but reproducible effect at certain non-consensus DNA sequences, where UV irradiation leads to a high increase in the level of TF binding. These changes in DNA-binding specificity after UV irradiation, at both consensus and non-consensus sites, have important implications for the regulatory and mutagenic roles of TFs in the cell.
2023-01-20 | GSE223022 | GEO
Project description:Somatic mutations are highly enriched at transcription factor (TF) binding sites, with the strongest trend being observed for ultraviolet light (UV)-induced mutations in melanomas. One of the main mechanisms proposed for this hyper-mutation pattern is the inefficient repair of UV lesions within TF-binding sites, caused by competition between TFs bound to these lesions and the DNA repair proteins that must recognize the lesions to initiate repair. However, TF binding to UV-irradiated DNA is poorly characterized, and it is unclear whether TFs maintain specificity for their DNA sites after UV exposure. We developed UV-Bind, a high-throughput approach to investigate the impact of UV irradiation on protein-DNA binding specificity. We applied UV-Bind to ten TFs from eight structural families, and found that UV lesions significantly altered the DNA-binding preferences of all TFs tested. The main effect was a decrease in binding specificity, but the precise effects and their magnitude differ across factors. Importantly, we found that despite the overall reduction in DNA-binding specificity in the presence of UV lesions, TFs can still compete with repair proteins for lesion recognition, in a manner consistent with their specificity for UV-irradiated DNA. In addition, for a subset of TFs we identified a surprising but reproducible effect at certain non-consensus DNA sequences, where UV irradiation leads to a high increase in the level of TF binding. These changes in DNA-binding specificity after UV irradiation, at both consensus and non-consensus sites, have important implications for the regulatory and mutagenic roles of TFs in the cell.
2023-01-20 | GSE223019 | GEO
Project description:Somatic mutations are highly enriched at transcription factor (TF) binding sites, with the strongest trend being observed for ultraviolet light (UV)-induced mutations in melanomas. One of the main mechanisms proposed for this hyper-mutation pattern is the inefficient repair of UV lesions within TF-binding sites, caused by competition between TFs bound to these lesions and the DNA repair proteins that must recognize the lesions to initiate repair. However, TF binding to UV-irradiated DNA is poorly characterized, and it is unclear whether TFs maintain specificity for their DNA sites after UV exposure. We developed UV-Bind, a high-throughput approach to investigate the impact of UV irradiation on protein-DNA binding specificity. We applied UV-Bind to ten TFs from eight structural families, and found that UV lesions significantly altered the DNA-binding preferences of all TFs tested. The main effect was a decrease in binding specificity, but the precise effects and their magnitude differ across factors. Importantly, we found that despite the overall reduction in DNA-binding specificity in the presence of UV lesions, TFs can still compete with repair proteins for lesion recognition, in a manner consistent with their specificity for UV-irradiated DNA. In addition, for a subset of TFs we identified a surprising but reproducible effect at certain non-consensus DNA sequences, where UV irradiation leads to a high increase in the level of TF binding. These changes in DNA-binding specificity after UV irradiation, at both consensus and non-consensus sites, have important implications for the regulatory and mutagenic roles of TFs in the cell.
2023-01-20 | GSE223017 | GEO
Project description:Somatic mutations are highly enriched at transcription factor (TF) binding sites, with the strongest trend being observed for ultraviolet light (UV)-induced mutations in melanomas. One of the main mechanisms proposed for this hyper-mutation pattern is the inefficient repair of UV lesions within TF-binding sites, caused by competition between TFs bound to these lesions and the DNA repair proteins that must recognize the lesions to initiate repair. However, TF binding to UV-irradiated DNA is poorly characterized, and it is unclear whether TFs maintain specificity for their DNA sites after UV exposure. We developed UV-Bind, a high-throughput approach to investigate the impact of UV irradiation on protein-DNA binding specificity. We applied UV-Bind to ten TFs from eight structural families, and found that UV lesions significantly altered the DNA-binding preferences of all TFs tested. The main effect was a decrease in binding specificity, but the precise effects and their magnitude differ across factors. Importantly, we found that despite the overall reduction in DNA-binding specificity in the presence of UV lesions, TFs can still compete with repair proteins for lesion recognition, in a manner consistent with their specificity for UV-irradiated DNA. In addition, for a subset of TFs we identified a surprising but reproducible effect at certain non-consensus DNA sequences, where UV irradiation leads to a high increase in the level of TF binding. These changes in DNA-binding specificity after UV irradiation, at both consensus and non-consensus sites, have important implications for the regulatory and mutagenic roles of TFs in the cell.
2023-01-20 | GSE223015 | GEO