GABPα and CREB1 binding to double nucleotide polymorphisms of their consensus motifs and cooperative binding to the ETS-CRE motif (ACCGGAAGTGACGTCA).
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PROVIDER: PRJNA516929 | ENA |
REPOSITORIES: ENA
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Project description:Previously, cooperative binding to DNA between the bZIP domain of CREB1 and the ETS domain of GABPα was observed for the composite ETS-CRE motif (A0C1C2G3G4A5A6G7T8G9A10C11G12T13C14A15). Single nucleotide polymorphisms (SNPs) at the beginning and end of the ETS motif (ACCGGAAGT) increased cooperative binding. Here, we use a microarray containing all double nucleotide polymorphisms (DNPs) of the ETS-CRE motif to explore GABPα and CREB1 binding to their canonical motifs and their cooperative binding to the ETS-CRE motif. For GABPα, binding to SNPs predicted binding the DNPs. In contrast, CREB1 binding to DNPs showed cooperativity. Similar results were observed for other ETS and bZIP family members. Cooperative binding between GABPα and CREB1 was typically weaker than expected except for DNPs containing A7 and SNPs at the beginning of the ETS motif.
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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:Previously, cooperative binding of the bZIP domain of CREB1 and the ETS domain of GABPα was observed for the composite DNA ETS ⇔ CRE motif (A 0 C 1 C 2 G 3 G 4 A 5 A 6 G 7 T 8 G 9 A 10 C 11 G 12 T 13 C 14 A 15 ). Single nucleotide polymorphisms (SNPs) at the beginning and end of the ETS motif (ACCGGAAGT) increased cooperative binding. Here, we use an Agilent microarray of 60-mers containing all double nucleotide polymorphisms (DNPs) of the ETS ⇔ CRE motif to explore GABPα and CREB1 binding to their individual motifs and their cooperative binding. For GABPα, all DNPs were bound as if each SNP acted independently. In contrast, CREB1 binding to some DNPs was stronger or weaker than expected, depending on the locations of each SNP. CREB1 binding to DNPs where both SNPs were in the same half site, T 8 G 9 A 10 or T 13 C 14 A 15 , was greater than expected, indicating that an additional SNP cannot destroy binding as much as expected, suggesting that an individual SNP is enough to abolish sequence-specific DNA binding of a single bZIP monomer. If a DNP contains SNPs in each half site, binding is weaker than expected. Similar results were observed for additional ETS and bZIP family members. Cooperative binding between GABPα and CREB1 to the ETS ⇔ CRE motif was weaker than expected except for DNPs containing A 7 and SNPs at the beginning of the ETS motif.
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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
2008-10-01 | GSE12126 | GEO
Project description:The bZIP homodimers CEBPB and CREB1 bind DNA containing methylated cytosines differently. CREB1 binds stronger to the C/EBP half-site GCAA when the cytosine is methylated. For CEBPB, methylation of the same cytosine does not affect DNA binding. The X-ray structure of CREB1 binding the half site GTCA identifies an alanine in the DNA binding region interacting with the methyl group of T, structurally analogous to the methyl group of methylated C. This alanine is replaced with a valine in CEBPB. To explore the contribution of this amino acid to binding with methylated cytosine of the GCAA half-site, we made the reciprocal mutants CEBPB(V285A) and CREB1(A297V) and used protein binding microarrays (PBM) to examine binding to four types of double-stranded DNA (dsDNA): 1) DNA with cytosine in both strands (DNA(C|C)), 2) DNA with 5-methylcytosine (M) in one strand and cytosine in the second strand (DNA(M|C)), 3) DNA with 5-hydroxymethylcytosine (H) in one strand and cytosine in the second strand (DNA(H|C)), and 4) DNA with both cytosines in all CG dinucleotides contain- ing 5mC (DNA(5mCG)). When binding to DNA(C|C), CEBPB (V285A) preferentially binds the CRE consensus motif (TGACGTCA), similar to CREB1. The reciprocal mutant, CREB1(A297V) binds DNA with some similarity to CEBPB with strongest binding to the methylated PAR site 8-mer TTACGTAA. These data demonstrate that V285 residue inhibits CEBPB binding to methylated cytosine of the GCAA half-site.
2019-03-01 | GSE112018 | GEO
Project description:E2F in vivo binding specificity: comparison of consensus vs. non-consensus binding sites
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Project description:The bZIP homodimers CEBPB and CREB1 bind DNA containing methylated cytosines differently. CREB1 binds stronger to the C/EBP half-site GCAA when the cytosine is methylated. For CEBPB, methylation of the same cytosine does not affect DNA binding. The X-ray structure of CREB1 binding the half site GTCA identifies an alanine in the DNA binding region interacting with the methyl group of T, structurally analogous to the methyl group of methylated C. This alanine is replaced with a valine in CEBPB. To explore the contribution of this amino acid to binding with methylated cytosine of the GCAA half-site, we made the reciprocal mutants CEBPB(V285A) and CREB1(A297V) and used protein binding microarrays (PBM) to examine binding to four types of double-stranded DNA (dsDNA): 1) DNA with cytosine in both strands (DNA(C|C)), 2) DNA with 5-methylcytosine (M) in one strand and cytosine in the second strand (DNA(M|C)), 3) DNA with 5-hydroxymethylcytosine (H) in one strand and cytosine in the second strand (DNA(H|C)), and 4) DNA with both cytosines in all CG dinucleotides containing 5mC (DNA(5mCG)). When binding to DNA(C|C), CEBPB (V285A) preferentially binds the CRE consensus motif (TGACGTCA), similar to CREB1. The reciprocal mutant, CREB1(A297V) binds DNA with some similarity to CEBPB with strongest binding to the methylated PAR site 8-mer TTACGTAA. These data demonstrate that V285 residue inhibits CEBPB binding to methylated cytosine of the GCAA half-site.
2019-03-01 | GSE112019 | GEO
Project description:The complexities of DNA recognition by transcription factors with multiple Cys2-His2 zinc fingers (C2H2-ZFs) remain poorly studied. We previously reported a mutation (R1092W) in the C2H2-ZF transcription factor Zfp335 which led to selective loss of binding at a subset of targets, although the basis for this was unclear. We show that Zfp335 binds DNA and drives transcription via recognition of two distinct consensus motifs by separate ZF clusters, and identify the specific motif interaction disrupted by R1092W. Our work presents Zfp335 as a model for understanding how C2H2-ZF TFs may utilize multiple recognition motifs to control gene expression.
2016-07-11 | GSE83116 | GEO
Project description:To achieve proper spatiotemporal control of gene expression, transcription factors cooperatively assemble onto specific DNA sequences. The ETS domain protein monomer of GABP? and the B-ZIP domain protein dimer of CREB1 cooperatively bind DNA only when the ETS ((C)/GCGGAA GT: ) and CRE ( GT: GACGTCAC) motifs overlap precisely, producing the ETS?CRE motif ((C)/GCGGAA GT: GACGTCAC). We designed a Protein Binding Microarray (PBM) with 60-bp DNAs containing four identical sectors, each with 177,440 features that explore the cooperative interactions between GABP? and CREB1 upon binding the ETS?CRE motif. The DNA sequences include all 15-mers of the form (C)/GCGGA--CG-, the ETS?CRE motif, and all single nucleotide polymorphisms (SNPs), and occurrences in the human and mouse genomes. CREB1 enhanced GABP? binding to the canonical ETS?CRE motif CCGGAAGT two-fold, and up to 23-fold for several SNPs at the beginning and end of the ETS motif, which is suggestive of two separate and distinct allosteric mechanisms of cooperative binding. We show that the ETS-CRE array data can be used to identify regions likely cooperatively bound by GABP? and CREB1 in vivo, and demonstrate their ability to identify human genetic variants that might inhibit cooperative binding.
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Project description:In human and mouse stem cells and brain, 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) can occur outside of CG dinucleotides. Using protein binding microarrays (PBMs) containing 60-mer DNA probes, we evaluated the effect of 5mC and 5hmC on one DNA strand on the double-stranded DNA binding of the mouse B-ZIP transcription factors (TFs) CREB1, ATF1, and JUND. 5mC inhibited CREB1 binding to the canonical CRE half-site |GTCA, but increased binding to the C/EBP half-site |GCAA. 5hmC inhibited CREB1 binding to all 8-mers except TGAT|GCAA, where binding is enhanced. We observed similar DNA binding patterns with the closely related TF: ATF1. In contrast, both 5mC and 5hmC inhibited binding of JUND. These results identify new DNA sequences that are well-bound by CREB1 and ATF1 only when they contain 5mC or 5hmC. Analysis of two x-ray structures examines the consequences of 5mC and 5hmC on DNA binding by CREB and FOS|JUN.
2016-10-19 | GSE88897 | GEO