Figure 1. 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 accumu- lates 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. Epigenetic regulation of cardiac proliferation • Sdek et al. 409