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.

Fig. 1.

In vivo TF binding motifs. De novo motif discovery of in vivo motifs by MEME and Weeder using the top 500 peaks of ChIP-seq data. All MEME E-values were less than 10⁻⁶⁰. For TBX5, high GC-content peaks were excluded. Motifs found by de novo discovery were compared with available consensus and optimal in vitro motifs from JASPAR, UniPROBE, or the indicated reference. Dashed boxes highlight differences between in vivo and in vitro motifs.

Fig. 2.

Expansion of the cardiac TF interaction network by motif enrichment analysis. (A) Heat map showing statistical enrichment of selected JASPAR and TRANSFAC motifs among top 500 peaks bound by cardiac TF. A heat map of all analyzed motifs is shown in SI Appendix, Fig. S5. (B) TEAD1 ChIP-qPCR assay of cardiac TF peaks containing predicted TEAD1 motifs. Fold enrichment indicates TEAD1 compared with IgG1 ChIP and normalized to Actin-β (Actb) intronic control. Filled bars indicate greater than twofold enrichment (dotted line). Actn4, actinin α4; Afap1, actin filament-associated protein 1; Cap2, adenylate cyclase-associated protein, 2; Cdh1, cadherin 1 type 1; Cdh2, cadherin 2; Chd2, chromodomain helicase DNA binding protein 2; Col4a3, collagen type IV, α3; Fgf12, fibroblast growth factor 12; Galnt2, UDP-N-acetyl-α-d-galactosamine:polypeptide N-acetylgalactosaminyltransferase 2; Myst4, MYST histone acetyltransferase (monocytic leukemia) 4; Rbpms, RNA binding protein gene with multiple splicing; Scn10a, sodium channel, voltage-gated type X α subunit; Tcf3, transcription factor 3.

Fig. 3.

Genomic regions co-occupied by multiple cardiac TFs direct cardiac gene expression. (A) Genes with a higher cardiac enrichment score (cardiac expression/average expression in other tissues) were associated more frequently with MTLs. (B) Enhancer activity in vitro. Enhancers cloned upstream of hsp68-lacZ were transfected into neonatal rat cardiomyocytes or cardiac fibroblasts along with pGL3-luc. Ratio of LacZ activity in neonatal rat ventricular myocytes (NRVM) to fibroblasts was plotted after normalization to luciferase activity. n = 3. *P < 0.05; **P < 0.01; ***P < 0.001. NS, not significant. (C) Enhancer–hsp68–lacZ constructs were used to generate E10.5 transgenic embryos. Representative Xgal-stained embryos are shown. Numbers indicate embryos with cardiac expression over the total PCR⁺ embryos. Arrowheads indicate myocardial expression. Black arrows indicate activity in endocardium and endocardial cushions. PE, proepicardium; SHF, second heart field. (White scale bars: 400 μm; black scale bars: 200 μm.)

Fig. 4.

GATA4 and TBX5 binding sites are required for Smarcd3 −1497 activity. (A) Validation of GATA4 occupancy of Smarcd3 −1497 by ChIP-qPCR from mouse heart at the indicated developmental stages. E, embryonic; P, postnatal. (B) Activity of Smarcd3 −1497 enhancers containing mutation of GATA4 (G4m), TBX5 (T5m), or both motifs indicated in SI Appendix, Fig. S6D. Arrow indicates residual activity in outflow tract. Yellow arrowhead indicates loss of activity in cardiac chambers. Numbers indicate Xgal⁺ and PCR⁺ embryos. (Scale bars: 500 μm.)

Fig. 5.

MultiTF and p300 binding mark distinct sets of enhancers. (A) p300 frequently co-occupied genomic loci with cardiac TF, most notably GATA4. (B) Genes with higher cardiac enrichment scores were associated more frequently with p300. (C) The preponderance of multiTF and p300 enhancers did not overlap. (D) MultiTF and p300 genes were more highly expressed in HL1 cells than were genes that lacked these enhancers (P < 10⁻¹⁶), but expression levels of multiTF and p300 genes were indistinguishable. Gene expression is indicated in log2 scale. (E) MultiTF enhancers were located more proximal to the TSS than p300 enhancers. (F) Gene Ontology (GO) term analysis of MultiTF⁺/p300⁻ and MultiTF⁻/p300⁺ enhancers. Top 10 terms, fraction of positive genes within the set, and Benjamini–Hochberg false discovery rate (FDR) are shown for each class of enhancers.