However, they are consistent with the conclusion that the re- cruitment of HP1 to H3K9 methylation is Rb/p130 dependent. We have explored this issue in ACMs and demonstrated that deleting Rb reduced H3K9me2 but had no effect on H3K9me3 levels (Fig. 4 B). Thus, whether the previous study was related to differences in the precise H3K9 methylation or was related to cell type–specific utilization or interaction of these factors will need to be determined. Regardless, our data suggest that H3K9me3 alone is not sufficient to recruit HP1 in ACMs, and additional factors such as Rb family members are required for efficient binding. The ability to reverse the postmitotic state was also shown in a recent study in which concomitant inactivation of p14ARF and Rb led to skeletal myotube cell cycle reentry, loss of differentiation properties, and up-regulation of cytokinetic machinery (Pajcini et al., 2010). Although these authors did not examine the epigenetic impact of these gene manipulations, our data would suggest that Rb may be the key to recruiting a mul- timeric chromatin-remodeling complex in postmitotic cells and explain why certain genes are specifically targeted for incorpo- ration into heterochromatin. Irreversible growth arrest in myo- genic differentiation requires the LXCXE-binding motif in Rb (Chen and Wang, 2000), indicating that at least one additional factor is necessary for Rb to induce terminal differentiation, as the LXCXE pocket is not required for Rb–E2F interactions. HP1 proteins contain an LXCXE (or LXCXD) motif (Williams and Grafi, 2000) and interact with both Rb and p130 (Panteleeva et al., 2007), and their recruitment to methylated H3K9 is Rb dependent (Nielsen et al., 2001). HP1 belongs to a highly conserved family of chromatin proteins, with homologues that are found from fission yeast to mammals (Huisinga et al., 2006). The role of HP1 family mem- bers during differentiation has received limited investigation but appears to be complex and developmentally dependent (Zhang et al., 2002; Cammas et al., 2004; Feldman et al., 2006; Agarwal et al., 2007; Panteleeva et al., 2007; Yahi et al., 2008; Takanashi et al., 2009). HP1- can negatively regulate differentiation by inhibiting tissue-specific factors at an early developmental stage (Zhang et al., 2002; Yahi et al., 2008) while promoting differen- tiation at later stages by mediating repression of cell cycle genes and cell cycle exit (Panteleeva et al., 2007). We have detected the expression of all three family members of HP1 in post- mitotic cardiac myocytes; however, depletion of HP1-, but not HP1- or HP1-, resulted in reexpression of E2F-dependent genes in ACMs. HP1- is also present at the promoters of G2/M and cytokinesis genes in ACMs, and this binding is Rb and p130 dependent. Loss of HP1- recruitment appeared to be the key factor in the disruption of heterochromatin and the ability to reenter the cell cycle. Our results demonstrate that HP1- is specifically essential for stably repressing proliferation-promoting genes in ACMs and, although HP1 family members share simi- lar structure (HP1- and HP1-), could not compensate for the loss of function of HP1- in cardiac myocytes. This result dem- onstrates that HP1 proteins, in contrast to Rb family members, are not functionally redundant. Why HP1- was specifically re- cruited to G2/M and cytokinesis genes in ACMs versus other E2F-dependent promoters is unclear. However, E2F binding specificity to target promoters is effected by numerous factors including nonconsensus genomic sequences and interactions of E2F with other factors that bind to the target promoters (Rabinovich et al., 2008). Binding of specific E2F family mem- bers and their interactions with other factors can differentially regulate subsets of E2F-dependent cell cycle genes (Caretti et al., 2003). Determining how particular HP1 family members are recruited to specific subsets of genes and their differential effects on transcription is a critical question. ACMs also displayed higher levels of H3K27me3, the other repressive histone mark associated with heterochromatin, compared with embryonic cardiac myocytes. We chose to focus on the regulation of H3K9me3, Rb, and HP1-, given their well-established roles in permanent gene silencing and localiza- tion to heterochromatin. However, H3K27me3 can also be important for stable gene repression, including suppression of E2F-dependent genes, in certain contexts (Blais et al., 2007). Given that H3K27me3 is relatively enriched in ACM euchroma- tin, not heterochromatin, it will need to be determined what role, if any, it has in heterochromatin formation in ACMs and whether it contributes to the irreversible cell cycle exit seen in ACMs. In summary, we have demonstrated that epigenetic modi- fications play a critical role in cardiac growth and differentia- tion. Actively proliferating embryonic cardiac myocytes enriched for H3K9/14Ac express high levels of proliferation-promoting genes. Terminally differentiated ACMs express high levels of Rb and p130, which serve as a bridge to link H3K9me3 and heterochromatin formation through their interaction with HP1-. Heterochromatin stably represses the expression of proliferation- promoting genes and maintains the postmitotic phenotype of ACMs (Fig. 8 C). The ability to restore ACMs’ proliferative potential in vivo could lead to regenerative approaches similar to what occurs naturally in zebrafish hearts (Jopling et al., 2010; Kikuchi et al., 2010). These findings have important implica- tions for promoting regeneration of the injured mammalian hearts and suggest that epigenetic manipulation of cardiac myocytes may be a viable therapeutic approach. Materials and methods Animal studies CRbL/L;p130/ mice have been previously described (MacLellan et al., 2005). To create inducible, cardiac-specific Rb- and p130-null mice, RbL/L;p130/ mice were bred to -MHC–MCM mice, resulting in MCM;RbL/L;p130+/ mice. Rb was deleted in ACMs after the Cre activation by TAM (Zhong et al., 2006). 8-wk-old mice carrying MCM were fed with TAM- containing chow (400 mg TAM citrate/kg supplemented standard diet Global 2016 pellets; Harlan Teklad Europe) for 14 d. Experiments were performed 4 wk after stopping TAM treatment unless specifically indicated. For TAC, a fixed pressure overload was obtained by surgically constricting the transverse aorta as previously described (Xiao et al., 2001). In brief, adult mice weighing 20–25 g were anesthetized by an i.p. injection of 60 mg/kg sodium pentobarbital. Microsurgical procedures were performed under a dissecting microscope (PZM III; World Precision Instruments, Inc.). Endotracheal intubation was performed using a blunt 20-gauge needle that was then connected to a volume-cycled rodent ventilator (SAR-830/P; IITC Life Science) with a tidal volume of 0.2 ml and a respiratory rate of 120/min. The chest was entered in the second intercostal space at the top left aortic arch, the transverse aorta was isolated, and aortic constriction was performed by tying a 7–0 nylon suture ligature against a 20-guage needle. The needle was then removed to yield a constriction of 0.4 mm in diameter. All animals were han- dled and maintained in accordance with institutional guidelines and the Na- tional Institutes of Health Guide for the Care and Use of Laboratory Animals. Epigenetic regulation of cardiac proliferation • Sdek et al. 419