Silencing of E2F-dependent genes in ACMs is associated with heterochromatin formation and H3K9 and H3K27 trimethylation. (A) To quantify cell cycle genes in ACMs after a growth stimulus, semiquantitative RT-PCR was performed on total RNA isolated from purified primary cardiac myocytes at the indicated developmental time points or 7 d after TAC-induced pressure overload. ANP, atrial natriuretic peptide; AurKB, Aurora kinase B. (B) Western blots on nuclear extracts prepared from purified embryonic cardiac myocytes or ACMs demonstrate that histone H3 becomes hypoacetylated and accumulates H3K9me3 and H3K27me3 modifications with cardiac differentiation. (C) Suv39h1 and Ezh1, along with Rb and p130, are up-regulated in ACMs. (D and E) ACM nuclei demonstrated large, dense heterochromatin foci that have a lack of H3K9/14Ac and are enriched for H3K9me3. Confocal microscopy was performed on myocardial sections from WT C57/BL6 embryonic (E15.5) and adult mice after immunostaining (red, troponin C [TnC]; green, indicated histone H3 modification; blue, DAPI). Bars, 5 µm. (F and G) H3K9me3 (F) and H3K27me3 (G) were increased at promoters of E2F-dependent cell cycle genes after cardiac terminal differentiation. For quantitation of changes, see Fig. S1. ChIP was performed using chromatin extracts from purified myocytes and PCR for E2F-dependent promoters.