Promoter-distal RNA polymerase II binding discriminates active from inactive CCAAT/ enhancer-binding protein beta binding sites.
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 TF bound sites are actively involved in transcriptional regulation. Moreover, the key genomic features that accurately discriminate between active and inactive TF binding events remain ambiguous. Recent studies have identified promoter-distal RNA polymerase II (RNAP2) binding at enhancer elements, suggesting that these interactions may serve as a marker for active regulatory sequences. Despite these correlative analyses, a thorough functional validation of these genomic co-occupancies is still lacking. To characterize 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 reporter assay, we performed cis-regulatory element sequencing (CRE-seq). We tested more than 1000 promoter-distal CCAAT/enhancer-binding protein beta (CEBPB)-bound sites in HepG2 and K562 cells, and found that CEBPB-bound sites co-occurring with RNAP2 were more likely to exhibit enhancer activity. CEBPB-bound sites further maintained substantial cell-type specificity, indicating that local DNA sequence can accurately convey cell-type-specific regulatory information. By comparing our CRE-seq results to a comprehensive set of genome annotations, we identified a variety of genomic features that are strong predictors of regulatory element activity and cell-type-specific activity. Collectively, our functional assay results indicate that RNAP2 occupancy can be used as a key genomic marker that can distinguish active from inactive TF bound sites.
HudsonAlpha Institute for Biotechnology, Huntsville, Alabama 35806, USA;
Daniel
D
Savic
HudsonAlpha Institute for Biotechnology, Huntsville, Alabama 35806, USA;
Brian S
BS
Roberts
Department of Oncological Sciences, Huntsman Cancer Institute, University of Utah, Salt Lake City, Utah 84112, USA;
Julia B
JB
Carleton
HudsonAlpha Institute for Biotechnology, Huntsville, Alabama 35806, USA;
E Christopher
EC
Partridge
Washington University at St. Louis, Center for Genome Sciences and Systems Biology, Department of Genetics, Washington University School of Medicine, St. Louis, Missouri 63108, USA.
Michael A
MA
White
Washington University at St. Louis, Center for Genome Sciences and Systems Biology, Department of Genetics, Washington University School of Medicine, St. Louis, Missouri 63108, USA.
Barak A
BA
Cohen
HudsonAlpha Institute for Biotechnology, Huntsville, Alabama 35806, USA;
Gregory M
GM
Cooper
Department of Oncological Sciences, Huntsman Cancer Institute, University of Utah, Salt Lake City, Utah 84112, USA;
Jason
J
Gertz
HudsonAlpha Institute for Biotechnology, Huntsville, Alabama 35806, USA;
Richard M
RM
Myers
D001665:Binding Sites; D022782:CCAAT-Enhancer-Binding Protein-beta / Q000378:metabolism*; D004742:Enhancer Elements, Genetic; D005786:Gene Expression Regulation; D056945:Hep G2 Cells; D006657:Histones / Q000378:metabolism; D006801:Humans; D020014:K562 Cells; D009928:Organ Specificity / Q000235:genetics; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D012319:RNA Polymerase II / Q000378:metabolism*; D020218:Response Elements; D017422:Sequence Analysis, DNA
D022782:CCAAT-Enhancer-Binding Protein-beta; D006657:Histones; D012319:RNA Polymerase II