JCB: Article Y G O L O I B L L E C F O L A N R U O J E H T Rb and p130 control cell cycle gene silencing to maintain the postmitotic phenotype in cardiac myocytes Patima Sdek,1 Peng Zhao,1 Yaping Wang,1 Chang-jiang Huang,2 Christopher Y. Ko,1 Peter C. Butler,2 James N. Weiss,1 and W. Robb MacLellan1 1Cardiovascular Research Laboratory, Department of Medicine and Department of Physiology, and 2Larry Hillblom Islet Research Center, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095 T soon after birth. Adult cardiac myocytes (ACMs) he mammalian heart loses its regenerative potential permanently exit the cell cycle, and E2F-dependent genes are stably silenced, although the underlying mech- anism is unclear. Heterochromatin, which silences genes in many biological contexts, accumulates with cardiac differentiation. H3K9me3, a histone methylation charac- teristic of heterochromatin, also increases in ACMs and at E2F-dependent promoters. We hypothesize that genes relevant for cardiac proliferation are targeted to hetero- chromatin by retinoblastoma (Rb) family members inter- acting with E2F transcription factors and recruiting heterochromatin protein 1 (HP1) proteins. To test this hypothesis, we created cardiac-specific Rb and p130 inducible double knockout (IDKO) mice. IDKO ACMs showed a decrease in total heterochromatin, and cell cycle genes were derepressed, leading to proliferation of ACMs. Although Rb/p130 deficiency had no effect on total H3K9me3 levels, recruitment of HP1- to promoters was lost. Depleting HP1- up-regulated proliferation-promoting genes in ACMs. Thus, Rb and p130 have overlapping roles in maintaining the postmitotic state of ACMs through their interaction with HP1- to direct heterochromatin formation and silencing of proliferation-promoting genes. Introduction Some amphibians, as well as teleost fish, have a robust capacity to regenerate their hearts after injury throughout their life (Poss et al., 2002). Adult zebrafish, for example, can fully regenerate their heart without scar formation after up to 20% of the ventri- cle has been resected. This regenerative response is thought to occur primarily through dedifferentiation and proliferation of existing myocytes (Jopling et al., 2010; Kikuchi et al., 2010). Recent studies have demonstrated that the embryonic mamma- lian heart is also capable of mounting a hyperplastic response to injury but that this is lost rapidly after birth (Drenckhahn et al., 2008; Porrello et al., 2011). Although some studies have sug- gested that the adult mammalian heart possesses a very limited capacity for new cardiomyocyte formation, the source has been Correspondence to W. Robb MacLellan: rmaclellan@mednet.ucla.edu Abbreviations used in this paper: ACM, adult cardiac myocyte; ANF, atrial natriuretic factor; ChIP, chromatin IP; HP1, heterochromatin protein 1; IDKO, inducible double knockout; IP, immunoprecipitation; ITS, insulin-transferrin- selenium; MCM, MerCreMer; MHC, myosin heavy chain; NI, nonionic; NRVM, neonatal rat ventricular myocyte; NS, nonspecific; PSG, penicillin-streptomycin- glutamine; Rb, retinoblastoma; TAC, trans-aortic constriction; TAM, tamoxifen; TLVM, time-lapse video microscopy; WT, wild type. The Rockefeller University Press $30.00 J. Cell Biol. Vol. 194 No. 3 407–423 www.jcb.org/cgi/doi/10.1083/jcb.201012049 postulated to be endogenous cardiac stem cells, not existing myocytes (Pasumarthi and Field, 2002; Hsieh et al., 2007; Bergmann et al., 2009). Thus, the vast majority of cardiac myo- cytes permanently exits the cell cycle soon after birth in mam- mals, and, when adult cardiac myocytes (ACMs) are subjected to a growth stimulus, they undergo an increase in cell size (hypertrophy) but do not proliferate. The molecular basis for the postmitotic state of ACMs is unknown, but the subset of E2F-dependent genes specifically involved in regulating G2/M and cytokinesis is not up-regulated after growth stimuli in ACMs. The mechanisms that irreversibly silence these genes are un- known; however, stable transcriptional silencing in eukaryotic cells is often caused by specific histone modifications that result in heterochromatin formation around a gene, making it inacces- sible to transcriptional machinery. © 2011 Sdek et al. This article is distributed under the terms of an Attribution– Noncommercial–Share Alike–No Mirror Sites license for the first six months after the pub- lication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/). JCB 407