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Promoter-distal RNA polymerase II binding discriminates active from inactive CCAAT/enhancer-binding protein beta binding sites

ABSTRACT: Transcription factors (TFs) bind to thousands of DNA sequences in mammalian genomes, but most of these binding events appear to have no direct effect on gene expression. It is unclear why only a subset of transcription factor bound sites are actively involved in transcriptional regulation, nor are the key genomic features known that discriminate between active and inactive TF binding events. Recent studies have identified promoter-distal RNA polymerase II (RNAP2) binding at enhancer elements, suggesting that these interactions may serve as a potential marker for active regulatory sequences. Despite these correlative analyses, a thorough functional validation of these genomic co-occupancies is still lacking. To characterize systematically the gene regulatory activity of DNA sequences underlying promoter-distal TF binding events that co-occur with RNAP2 and TF sites devoid of RNAP2 occupancy using a functional assay, we performed cis-regulatory element sequencing (CRE-seq) reporter assays. We tested more than 1,000 promoter-distal CCAAT/enhancer-binding protein beta (CEBPB) bound sites in two human cell types, HepG2 and K562. We found that CEBPB bound sites that co-occur with RNAP2 were more likely to exhibit enhancer activity in our reporter assay and these active sequences display significantly stronger RNAP2 read enrichment and local enhancer RNA (eRNA) activity compared to inactive sites. CEBPB-bound sites further maintained substantial cell type specificity, indicating that DNA sequence information at such sites can accurately convey cell type-specific regulatory information, supporting a strong role for local DNA sequence in distinguishing active from inactive TF-bound loci. By comparing our CRE-seq results to a comprehensive set of over 1,000 genome annotations, we identified a variety of genomic features that are strong predictors of regulatory element activity as well as cell type-specific activity. Collectively, our functional assay results indicate that RNAP2 occupancy can be used as a key genomic marker that can discriminate active versus inactive transcription factor bound sites.

ORGANISM(S): Homo sapiens

PROVIDER: GSE73183 | GEO | 2015/10/14

SECONDARY ACCESSION(S): PRJNA296098

REPOSITORIES: GEO

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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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Promoter-distal RNA polymerase II binding discriminates active from inactive CCAAT/enhancer-binding protein beta binding sites

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