Figure 8. Rb/p130 is dispensable for Suv39h1-mediated H3K9me3. (A) Rb/p130 deletion did not affect methyltransferase activity of Suv39h1 on H3K9. Comparable reduction of H3K9me3 was detected after depletion of Suv39h1 by siRNA in ACMs isolated from control and IDKO mice hearts 4 wk after TAM treatment. siNS, NS siRNA. (B) Semiquantitative RT-PCR demonstrating increased expression of cdc2 and PLK1 after Suv39h1 is depleted in ACMs with siRNA. CBP, cAMP response element-binding–binding protein. (C) A schematic model depicting Rb/p130-dependent epigenetic modifications associ- ated with cell cycle gene regulation in embryonic cardiac myocytes and ACMs and Rb/p130-dependent heterochromatin formation in ACMs based on our results and published data (Roth et al., 2001; Daniel et al., 2005; Grewal and Jia, 2007; Jin et al., 2011). CM, cardiac myocyte; PCAF, P300/cAMP response element-binding CBP–associated factor. defect appears to be at the level of the myocyte (Fig. S3 D). Of note, heart regeneration in zebrafish, which occurs by car- diac myocyte dedifferentiation and proliferation, is well tolerated, so this contractile defect associated with epigenetic dedifferen- tiation in ACMs may underlie the reason why mammalian car- diac myocytes have evolved to be postmitotic (Jopling et al., 2010; Kikuchi et al., 2010). Heterochromatin is a tightly packed and transcriptionally silent form of DNA, which comes in two varieties: constitutive and facultative. Constitutive heterochromatin is usually repeti- tive and cell type independent and occurs around the chromo- some centromere and near telomeres. In contrast, facultative heterochromatin is an acquired form that is cell type specific and is the result of genes that are silenced through histone methylation (Oberdoerffer and Sinclair, 2007). Although hetero- chromatin formation has been reported in postmitotic skeletal myotubes, the effects of heterochromatin on the expression of proliferation-promoting genes were not determined (Brero et al., 2005). Heterochromatin has a characteristic histone modification profile, which is distinguished by hypoacetylation and enrichment of H3K9me3. Our study demonstrates that the chromatin of proliferating embryonic cardiac myocytes is hyperacetylated (H3K9/14, H3K18, and H3K27), but, after adult differentiation, acetylation decreases, and histone methyl- ation associated with transcriptional repression (H3K9me3 and H3K27me3) predominates. The relationship of H3K9me3 and heterochromatin is well established, and recent studies (Bannister et al., 2001; Dillon, 2004; Daniel et al., 2005; Grewal and Jia, 2007) have demonstrated that Suv39h1-mediated H3K9me3 and the binding of HP1 to H3K9me3 through its chromodomain are necessary for the assembly of facultative heterochromatin. Heterochromatin is a self-assembling framework of tethers (H3K9me3) and adaptors (HP1) where the HP1 molecules bound to different nucleo- somes dimerize through their chromoshadow domains, leading to chromatin condensation (Grewal and Jia, 2007). Although depletion of Rb in p130-null ACMs disrupted heterochromatin formation globally (Fig. 4 C), there was no change in total or E2F-dependent, promoter-associated H3K9me3 or H3K27me3 levels. This implies that these histone modifications may be re- quired but are not sufficient for maintaining heterochromatin in ACMs. Superficially, these results seem to differ from a previ- ous study (Nielsen et al., 2001) in nonmyocytes in which Rb was required for H3K9 methylation of E2F-dependent promoters. 418 JCB • VOLUME 194 • NUMBER 3 • 2011