Shared Nucleotide Flanks Confer Transcriptional Competency to bZip Core Motifs
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus Homo sapiens
PROVIDER: GSE111515 | GEO | 2018/07/17
REPOSITORIES: GEO
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Project description:Sequence-specific DNA binding recruits transcription factors (TFs) to the genome to regulate gene expression. Here, we perform high resolution mapping of CEBP proteins to determine how sequence dictates genomic occupancy. We demonstrate a fundamental difference between the sequence repertoire utilized by CEBPs in vivo versus the palindromic sequence preference reported by classical in vitro models, by identifying a palindromic motif at <1% of the genomic binding sites. On the native genome, CEBPs bind a diversity of related 10 bp sequences resulting from the fusion of degenerate and canonical half-sites. Altered DNA specificity of CEBPs in cells occurs through heterodimerization with other bZip TFs, and approximately 40% of CEBP-binding sites in primary human cells harbor motifs characteristic of CEBP heterodimers. In addition, we uncover an important role for sequence bias at core-motif-flanking bases for CEBPs and demonstrate that flanking bases regulate motif function across mammalian bZip TFs. Favorable flanking bases confer efficient TF occupancy and transcriptional activity, and DNA shape may explain how the flanks alter TF binding. Importantly, motif optimization within the 10-mer is strongly correlated with cell-type-independent recruitment of CEBPβ, providing key insight into how sequence sub-optimization affects genomic occupancy of widely expressed CEBPs across cell types.
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Project description:Shared Nucleotide Flanks Confer Transcriptional Competency to bZip Core Motifs
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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 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.
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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.
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Project description:To evaluate the effect of CG methylation on DNA binding of sequence-specific B-ZIP transcription factors (TFs) in a high-throughput manner, we enzymatically methylated the cytosine in the CG dinucleotide on protein binding microarrays. Using this novel technology, we show that CG methylation enhanced binding for CEBPA and CEBPB and inhibited binding for CREB, ATF4, JUN, JUND, CEBPD and CEBPG. The CEBPB|ATF4 heterodimer bound a novel motif CGAT|GCAA 10-fold better when methylated. EMSA confirmed these results. CEBPB ChIP-seq data using primary female mouse dermal fibroblasts with 50X methylome coverage for each strand indicate that the methylated sequences well-bound on the arrays are also bound in vivo. CEBPB bound 39% of the methylated canonical 10-mers ATTGC|GCAAT in the mouse genome. After ATF4 protein induction by thapsigargin which results in ER stress, CEBPB binds methylated CGAT|GCAA in vivo, recapitulating what was observed on the arrays. mRNA-seq of primary female mouse dermal fibroblasts with and without thapsigargin identified differentially expressed genes. Genes that are commonly bound by CEBPB and ATF4 to TGAT|GCAA (the best-bound 8-mer in the array) at the promoters were highly expressed and up-regulated or remained unchanged in the thapsigargin treated primary female mouse dermal fibroblasts. RNA-Seq: Examination of whole genome transcriptome profiles (RNA-seq) of primary mouse dermal fibroblasts with and without Thapsigargin treatment ChIP-Seq: Examination of transcription factor binding in dermal fibroblasts with and without Thapsigargin teratment BS-Seq: Determination of whole genome DNA methylation profiles (BS-seq) of primary mouse dermal fibroblasts
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Project description:To evaluate the effect of CG methylation on DNA binding of sequence-specific B-ZIP transcription factors (TFs) in a high-throughput manner, we enzymatically methylated the cytosine in the CG dinucleotide on protein binding microarrays. Two Agilent DNA array designs were used. One contained 40,000 features using de Bruijn sequences where each 8-mer occurs 32 times in various positions in the DNA sequence. The second contained 180,000 features with each CG containing 8-mer present three times. The first design was better for identification of binding motifs, while the second was better for quantification. Using this novel technology, we show that CG methylation enhanced binding for CEBPA and CEBPB and inhibited binding for CREB, ATF4, JUN, JUND, CEBPD and CEBPG. The CEBPB|ATF4 heterodimer bound a novel motif CGAT|GCAA 10-fold better when methylated. EMSA confirmed these results. CEBPB ChIP-seq data using primary female mouse dermal fibroblasts with 50X methylome coverage for each strand indicate that the methylated sequences well-bound on the arrays are also bound in vivo. CEBPB bound 39% of the methylated canonical 10-mers ATTGC|GCAAT in the mouse genome. After ATF4 protein induction by thapsigargin which results in ER stress, CEBPB binds methylated CGAT|GCAA in vivo, recapitulating what was observed on the arrays. This methodology can be used to identify new methylated DNA sequences preferentially bound by TF, which may be functional in vivo. To evaluate the effect of CG methylation on DNA binding of sequence-specific B-ZIP transcription factors (TFs) in a high-throughput manner, we enzymatically methylated the cytosine in the CG dinucleotide on protein binding microarrays. Two Agilent DNA array designs were used. One contained 40,000 features using de Bruijn sequences where each 8-mer occurs 32 times in various positions in the DNA sequence. The second contained 180,000 features with each CG containing 8-mer present three times. The first design was better for identification of binding motifs, while the second was better for quantification. Using this novel technology, we show that CG methylation enhanced binding for CEBPA and CEBPB and inhibited binding for CREB, ATF4, JUN, JUND, CEBPD and CEBPG. The CEBPB|ATF4 heterodimer bound a novel motif CGAT|GCAA 10-fold better when methylated. EMSA confirmed these results. CEBPB ChIP-seq data using primary female mouse dermal fibroblasts with 50X methylome coverage for each strand indicate that the methylated sequences well-bound on the arrays are also bound in vivo. CEBPB bound 39% of the methylated canonical 10-mers ATTGC|GCAAT in the mouse genome. After ATF4 protein induction by thapsigargin which results in ER stress, CEBPB binds methylated CGAT|GCAA in vivo, recapitulating what was observed on the arrays. This methodology can be used to identify new methylated DNA sequences preferentially bound by TF, which may be functional in vivo. Protein binding microarray (PBM) experiments were performed for a set of 8 mouse B-ZIP homodimers and one hetrodimer transcription factors. Briefly, the PBMs involved binding GST-tagged DNA-binding proteins to double-stranded and methylated or unmethylated 44K Agilent microarrays, containing a DeBruijn sequence design, in order to determine their sequence preferences. Details of the PBM protocol are described in Berger et al., Nature Biotechnology 2006.
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