title: Co-occupancy by multiple cardiac transcription factors identifies transcriptional enhancers active in heart.
abstract: Identification of genomic regions that control tissue-specific gene expression is currently problematic. ChIP and high-throughput sequencing (ChIP-seq) of enhancer-associated proteins such as p300 identifies some but not all enhancers active in a tissue. Here we show that co-occupancy of a chromatin region by multiple transcription factors (TFs) identifies a distinct set of enhancers. GATA-binding protein 4 (GATA4), NK2 transcription factor-related, locus 5 (NKX2-5), T-box 5 (TBX5), serum response factor (SRF), and myocyte-enhancer factor 2A (MEF2A), here referred to as "cardiac TFs," have been hypothesized to collaborate to direct cardiac gene expression. Using a modified ChIP-seq procedure, we defined chromatin occupancy by these TFs and p300 genome wide and provided unbiased support for this hypothesis. We used this principle to show that co-occupancy of a chromatin region by multiple TFs can be used to identify cardiac enhancers. Of 13 such regions tested in transient transgenic embryos, seven (54%) drove cardiac gene expression. Among these regions were three cardiac-specific enhancers of Gata4, Srf, and swItch/sucrose nonfermentable-related, matrix-associated, actin-dependent regulator of chromatin, subfamily d, member 3 (Smarcd3), an epigenetic regulator of cardiac gene expression. Multiple cardiac TFs and p300-bound regions were associated with cardiac-enriched genes and with functional annotations related to heart development. Importantly, the large majority (1,375/1,715) of loci bound by multiple cardiac TFs did not overlap loci bound by p300. Our data identify thousands of prospective cardiac regulatory sequences and indicate that multiple TF co-occupancy of a genomic region identifies developmentally relevant enhancers that are largely distinct from p300-associated enhancers.
authors.affiliation: Department of Cardiology and Children's Hospital Informatics Program, Children's Hospital Boston, Boston, MA 02115, USA.
authors.forename: Aibin
authors.initials: A
authors.lastname: He
authors.forename: Sek Won
authors.initials: SW
authors.lastname: Kong
authors.forename: Qing
authors.initials: Q
authors.lastname: Ma
authors.forename: William T
authors.initials: WT
authors.lastname: Pu
mesh_terms: D000818:Animals; D002460:Cell Line; D002843:Chromatin / Q000378:metabolism*; D047369:Chromatin Immunoprecipitation; D019295:Computational Biology; D017931:DNA Primers / Q000235:genetics; D024202:Electrophoretic Mobility Shift Assay; D004742:Enhancer Elements, Genetic / Q000235:genetics*; D020869:Gene Expression Profiling; D018507:Gene Expression Regulation, Developmental / Q000235:genetics / Q000502:physiology*; D006321:Heart / Q000196:embryology*; D016014:Linear Models; D051379:Mice; D009206:Myocardium / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism*
chemical_list: D002843:Chromatin; D017931:DNA Primers; D014157:Transcription Factors