Cellular Polycomb Repressive Complex 2 Regulates Normal Development of the Mouse Heart

Rationale: Epigenetic marks are crucial for organogenesis, but their role in heart development is poorly understood. Polycomb repressive complex 2 (PRC2) trimethylates histone H3 at lysine 27, which establishes H3K27me3 repressive epigenetic marks that promote tissue-specific differentiation by silencing ectopic gene programs. Objective: We studied the function of PRC2 in murine heart development using a tissue-restricted conditional inactivation strategy. Methods and Results: Inactivation of the PRC2 subunit Ezh2 by Nkx2–5 Cre (Ezh2 NK ) caused lethal congenital heart malformations, namely, compact myocardial hypoplasia, hypertrabeculation, and ventricular septal defect. Candidate and genome-wide RNA expression profiling and chromatin immunoprecipitation analyses of Ezh2 NK heart identified genes directly repressed by EZH2. Among these were the potent cell cycle inhibitors Ink4a/b (inhibitors of cyclin-dependent kinase 4 A and B), the upregulation of which was associated with decreased cardiomyocyte proliferation in Ezh2 NK . EZH2-repressed genes were enriched for transcriptional regulators of noncardiomyocyte expression programs such as Pax6 , Isl1 , and Six1 . EZH2 was also required for proper spatiotemporal regulation of cardiac gene expression, because Hcn4 , Mlc2a , and Bmp10 were inappropriately upregulated in ventricular RNA. PRC2 was also required later in heart development, as indicated by cardiomyocyte-restricted TNT-Cre inactivation of the PRC2 subunit Eed . However, Ezh2 inactivation by TNT-Cre did not cause an overt phenotype, likely because of functional redundancy with Ezh1 . Thus, early Ezh2 inactivation by Nk2–5 Cre caused later disruption of cardiomyocyte gene expression and heart development. Conclusions: Our study reveals a previously undescribed role of EZH2 in regulating heart formation and shows that perturbation of the epigenetic landscape early in cardiogenesis has sustained disruptive effects at later developmental stages. ( Circ Res . 2012;110:406-415.) Libraries sequenced on an Illumina HiSeq 2000. ChIP-seq peaks were identified by Sole-Search 17 with default parameters. Genomic intervals were annotated, combined, and analyzed for gene ontology term enrich- ment by use of the completeMOTIFs pipeline. deposited Gene Expression Omnibus, accession number GSE29997 Cardiovascular Development Consortium ChIP-qPCR Thus, our results demonstrate an essential role for PRC2 and epigenetic transcriptional regulation in heart development. Our data suggest that perturbation of the epigenetic landscape early in cardiogenesis has sustained, disruptive effects at later stages. These results provide new insights into the transcriptional regulation of heart development and the pathogenesis of congenital heart disease. ,

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