PNAS 108:5632 (2011)
Aibin Hea,b,1, Sek Won Konga,b,c,1, Qing Maa,b, and William T. Pua,b,2
aDepartment of Cardiology and cChildren’s Hospital Informatics Program, Children’s Hospital Boston, Boston, MA 02115; bHarvard Stem Cell Institute,Harvard University, Cambridge, M
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. GATAbinding protein 4 (GATA4), NK2 transcription factor-related, locus5 (NKX2-5), T-box 5 (TBX5), serum response factor (SRF), and myocyte-enhancer factor 2A (MEF2A), here referred to as "cardiacTFs," 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 nonfermentablerelated,matrix-associated, actin-dependent regulator of hromatin,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.