Figure 7. Rb/p130-dependent recruitment of HP1- to E2F-responsive promoters is essential for irreversible silencing of proliferation-associated genes in ACMs. (A) HP1- bound to G2/M and cytokinesis genes in an Rb/p130-dependent manner as shown by ChIP using chromatin extracts from purified ACMs. (B) Quantitation of HP1- enrichment at the indicated promoters by real-time PCR. Error bars represent the SEM from three replicates. *, P = 0.018; **, P = 0.003. (C) A Western blot showing HP1-, -, and - expression in ventricles from mice with the indicated genotypes. (D) HP1- colocalized with heterochromatin in control ACM. IDKO ACM lost heterochromatin, and HP1- staining is diffused. TnC, troponin C. Bar, 5 µm. resulted in a comparable reduction of H3K9me3 in both genotypes. Knockdown of Suv39h1 had a similar effect on cell cycle gene expression as was seen in HP1-–deficient cells (Fig. 8 B). Cdc2 and PLK1 mRNA levels increased 1.5–2-fold (P < 0.05). The binding of HP1 to H3K9 is Suv39h1 dependent (Bannister et al., 2001; Lachner et al., 2001; Peters et al., 2001). Prolifera- tion-associated genes were up-regulated either by depleting Suv39h1 or Rb/p130, suggesting that both are required for per- manent gene silencing by HP1- (Fig. 8 C). Discussion It has been recognized for some time that many lower verte- brates have a robust capacity to regenerate organs, including the heart. This was felt to be lost in mammals, but a recent study has suggested that it is retained through embryonic and peri- natal life (Porrello et al., 2011). However, it is clear that the adult mammalian heart has lost this regenerative capacity. If there is limited new cardiac myocyte formation, it arises from a non- myocyte pool of progenitor cells (Davis et al., 2006; Bergmann et al., 2009). In the present study, we have investigated the molecular basis of this postmitotic state of ACM and demon- strated that heterochromatin accumulates in ACMs as they lose proliferative potential. We show that Rb and p130 are critical for targeting and maintaining a subset of E2F-dependent genes in heterochromatin in ACMs. Rb and p130 permanently silence these G2/M and cytokinesis genes via the recruitment of HP1-, an essential component of heterochromatin to the promoters of these genes. Reversing these epigenetic changes was also asso- ciated with reexpression of fetal cardiac genes, which we be- lieve is analogous to the reexpression of Gata4 that was seen in dedifferentiated cardiac myocytes in zebrafish (Kikuchi et al., 2010). Left ventricular dysfunction was also seen, but whether this is a direct consequence of the epigenetic manipulations we made or is a secondary effect related to the dedifferentiation of IDKO ACMs will need to be determined; however, the contractile Epigenetic regulation of cardiac proliferation • Sdek et al. 417