Chromatin is remodeled into active (euchromatin) and si- lent (heterochromatin) regions through covalent modification of DNA and histones. These modifications regulate chromatin structure and gene expression. Histone acetylation, established by histone acetyltransferases, generally correlates with gene ac- tivation and is essential for animal development. Two distinct families of histone acetyltransferases, GCN5 and PCAF (P300/ CBP-associated factor; acetyltransferases of H3K9) and CBP and p300 (acetyltransferases of H3K18/27), have been demon- strated to be involved in E2F-dependent gene expression, and the deletion of either of them in mice leads to early embryonic lethality (Lang et al., 2001; Roth et al., 2001; Wang et al., 2007; Jin et al., 2011). The role of histone acetylation in cardiac myo- cytes has received much attention as a means of promoting eu- chromatin (Shogren-Knaak et al., 2006) and activating gene expression in the heart (Haberland et al., 2009). In contrast, the role of histone methylation in cardiac growth has been largely ignored despite its critical role in regulating gene expression in many cell types. Both the position and the degree of methyla- tion may either activate or silence gene expression (Martin and Zhang, 2005; Shi, 2007). In animals, both H3K9me2 and H3K9me3 are related to gene silencing (Krauss, 2008); how- ever, H3K9me3 is found predominantly within heterochroma- tin, whereas H3K9me2 is mostly associated with silent domains within the euchromatic regions (Peters et al., 2001; Lehnertz et al., 2003). H3K9me1 and H3K9me2 are catalyzed by the his- tone methyltransferase G9a, whereas H3K9me3 is established by the histone methyltransferase Suv39h1 (Brenner and Fuks, 2007; Chin et al., 2007), which is often expressed in senescence and postmitotic cells (Rice et al., 2003). Trimethylation of lysine 27 on histone H3 (H3K27me3) has also been associated with heterochromatin formation and transcriptional repression (Dillon, 2004). The establishment of H3K27me3 is a result of the methyltransferase activity of polycomb group multiprotein complexes PRC1 or PRC2. Ezh2, the catalytic subunit of PRC2, had been thought to be the sole H3K27 methyltransferase (Schuettengruber et al., 2007). Recently, however, Ezh1 was also shown to have H3K27 methyltransferase activity (Shen et al., 2008). Ezh1 and Ezh2 form similar PRC2 complexes but exhibit contrasting repressive roles (Margueron et al., 2008). Ezh2 expression is associated with proliferation, whereas Ezh1 is typically more abundant in nonproliferative adult organs (Laible et al., 1997). One of the primary mechanisms by which histone modifi- cations transmit their biological signals is through binding of effector proteins that recognize distinct modifications (Fischle et al., 2003a; Daniel et al., 2005). H3K9me3 and H3K27me3 are preferentially recognized by the chromodomain-containing proteins heterochromatin protein 1 (HP1; Lachner et al., 2001) or Pc2 (Fischle et al., 2003b; Min et al., 2003), respectively. HP1 is a family of proteins (, , and ) that plays an important role in gene silencing in many organisms (James and Elgin, 1986; Kellum, 2003) by establishing and maintaining hetero- chromatin (Daniel et al., 2005), but their role in the heart is un- known. HP1 family members typically differ in their subcellular localization and interaction partners and thus likely have dis- tinct cellular functions (Minc et al., 1999, 2001; Auth et al., 2006). 408 JCB • VOLUME 194 • NUMBER 3 • 2011 The structure of Pc2 bound to H3K27me3 is very similar to that of HP1-binding H3K9me3. Functionally, the Pc2-containing PRC1 complex stabilizes the repression of chromatin structure by antagonizing the switch–sucrose nonfermentable complex (Shao et al., 1999). The mechanisms whereby distinct histone modifications or chromatin is localized to specific genes are an area of in- tense research. Retinoblastoma (Rb) family members can direct H3K9me3 and H3K27me3 to specific promoter regions, pro- viding a means to target cell cycle genes to heterochromatin through their ability to interact with E2F family members and recruit repressive chromatin remodeling factors to E2F-dependent promoters (Nielsen et al., 2001; Blais et al., 2007). Rb can po- tentially associate with multiple chromatin remodeling factors, including histone deacetylases (Luo et al., 1998; Magnaghi- Jaulin et al., 1998), Suv39h1 (Nielsen et al., 2001), HP1 (Nielsen et al., 2001), and Pc2 (Dahiya et al., 2001). In senescent cells, Rb promotes heterochromatin formation at E2F-responsive genes by recruiting HP1 to H3K9 trimethylated promoters, thereby producing a permanent insensitivity to mitogenic sig- nals (Nielsen et al., 2001; Narita et al., 2003). Pc2 has also been implicated in the repression of the E2F-dependent genes and can cooperate with Rb to inhibit the expression of cyclin A and cdc2 (Dahiya et al., 2001). Despite numerous descriptive studies (Soonpaa and Field, 1994; Li et al., 1996; Bergmann et al., 2009; Walsh et al., 2010) characterizing the limited ability of ACMs to proliferate, almost no mechanistic data exist to explain why ACMs do not reenter the cell cycle in response to mitogenic stimuli. We have impli- cated Rb family members in mediating cardiac cell cycle exit (MacLellan et al., 2005), but what role they play in maintaining the postmitotic state of ACMs and the mechanisms underlying their importance are unknown. Indeed, recent studies have questioned whether Rb family members actually play any role in the maintenance of terminal differentiation (Camarda et al., 2004; Huh et al., 2004; Mantela et al., 2005; Blais et al., 2007). Here, we demonstrate that heterochromatin accumulation and targeting of proliferation-promoting genes to these transcrip- tionally silent regions are the molecular basis of the postmitotic phenotype of ACMs. Rb and p130 target E2F-dependent genes to heterochromatin in ACMs, silencing these proliferation- promoting genes through the recruitment of HP1- to H3K9 tri- methylated promoters. Results Silencing of E2F-dependent genes in ACMs is associated with H3K9me3 and heterochromatin formation To clarify the expression pattern of cell cycle genes in cardiac myocytes, we compared expression levels of cell cycle genes in purified embryonic (embryonic day 15.5 [E15.5]) cardiac myo- cytes, ACMs, and growth-stimulated ACMs. To provide a growth stimulus for ACMs in vivo, trans-aortic constriction (TAC) was performed, which leads to pressure overload and primarily hyper- trophic growth. Embryonic cardiac myocytes expressed high levels of all cell cycle genes consistent with their proliferative