CBX7 is a polycomb group protein, and despite being implicated in many diseases, its role in cell proliferation has been controversial: some groups described its pro-proliferative properties, but others illustrated its inhibitory effects on cell growth. To date, the reason for the divergent observations remains unknown. While several isoforms for CBX7 were reported, no studies investigated whether the divergent roles of CBX7 could be due to distinct functions of CBX7 isoforms. In this study, we newly identified mouse CBX7 transcript variant 1 (mCbx7v1), which is homologous to the human CBX7 gene (hCBX7v1) and is expressed in various mouse organs. We revealed that mCbx7v1 and hCBX7v1 encode a 36 kDa protein (p36 CBX7) whereas mCbx7 and hCBX7v3 encode a 22 kDa protein (p22 CBX7). This study further demonstrated that p36 CBX7 was localized to the nucleus and endogenously expressed in proliferating cells whereas p22 CBX7 was localized to the cytoplasm, induced by serum starvation in both human and mouse cells, and inhibited cell proliferation. Together, these data indicate that CBX7 isoforms are localized in different locations in a cell and play differing roles in cell proliferation. This varying function of CBX7 isoforms may help us understand the distinct function of CBX7 in various studies. Cell proliferation is a finely tuned process and its dysregulation is associated with many diseases including cancers, cardiovascular diseases, neurodegenerative diseases, immune diseases, and congenital disorders 1,2. While genes directly control cell growth and division as evidenced by genetic alterations in proto-oncogenes or tumor suppressor genes in various cancers 3 , epigenetics control cell proliferation by modulating accessibility of transcription machinery to DNA 4 and the resulting gene expression patterns 5. Thus, genome-wide epigenetic modifications are one of the major hallmarks of cells with dysregulated proliferation 6,7 and epigenetic changes are causative factors for various diseases where cell proliferation is aberrant 8. One class of epigenetic regulators is polycomb group (PcG) proteins. First discovered in fruit flies, they are known to govern cell proliferation 9,10. They epigenetically control transcription of cell cycle-regulatory genes such as CDKN2A 11 , PTEN 12 , and c-MYC 13. The canonical mechanism of PcG-mediated epigenetic silencing involves coordinated actions of two major types of polycomb repressive complex (PRC), PRC1 and PRC2 14. PRC2 initiates the repressing process by tri-methylation of Histone 3 tail (H3K27me3) 15. PRC1 is then recruited and stabilizes this silencing process via mono-ubiquitination of H2A tail (H2Aub) 16. Finally, H2Aub serves as a binding site for PRC2 which further propagates the H3K27me3 repressive histone mark on H2Aub nucleosomes, generating a positive feedback loop 17. CBX family proteins are subunits of PRC1 and recognize trimethylated histone tail, thereby determining target specificity 18,19. There are five mammalian orthologues in the CBX family: CBX2, 4, 6, 7 ...

BACKGROUND: Shortly after birth, cardiomyocytes exit the cell cycle and cease proliferation. At present, the regulatory mechanisms for this loss of proliferative capacity are poorly understood. CBX7 (chromobox 7), a polycomb group (PcG) protein, regulates the cell cycle, but its role in cardiomyocyte proliferation is unknown. METHODS: We profiled CBX7 expression in the mouse hearts through quantitative real-time polymerase chain reaction, Western blotting, and immunohistochemistry. We overexpressed CBX7 in neonatal mouse cardiomyocytes through adenoviral transduction. We knocked down CBX7 by using constitutive and inducible conditional knockout mice ( Tnnt2-Cre;Cbx7 fl/+ and Myh6-MCM;Cbx7 fl/fl , respectively). We measured cardiomyocyte proliferation by immunostaining of proliferation markers such as Ki67, phospho-histone 3, and cyclin B1. To examine the role of CBX7 in cardiac regeneration, we used neonatal cardiac apical resection and adult myocardial infarction models. We examined the mechanism of CBX7-mediated repression of cardiomyocyte proliferation through coimmunoprecipitation, mass spectrometry, and other molecular techniques. RESULT: We explored Cbx7 expression in the heart and found that mRNA expression abruptly increased after birth and was sustained throughout adulthood. Overexpression of CBX7 through adenoviral transduction reduced proliferation of neonatal cardiomyocytes and promoted their multinucleation. On the other hand, genetic inactivation of Cbx7 increased proliferation of cardiomyocytes and impeded cardiac maturation during postnatal heart growth. Genetic ablation of Cbx7 promoted regeneration of neonatal and adult injured hearts. Mechanistically, CBX7 interacted with TARDBP (TAR DNA-binding protein 43) and positively regulated its downstream target, RBM38 (RNA Binding Motif Protein 38), in a TARDBP-dependent manner. Overexpression of RBM38 inhibited the proliferation of CBX7-depleted neonatal cardiomyocytes. CONCLUSIONS: Our results demonstrate that CBX7 directs the cell cycle exit of cardiomyocytes during the postnatal period by regulating its downstream targets TARDBP and RBM38. This is the first study to demonstrate the role of CBX7 in regulation of cardiomyocyte proliferation, and CBX7 could be an important target for cardiac regeneration.

Introduction: Cardiomyocyte (CM) proliferation notably decreases during the perinatal period. Regulatory mechanisms for this loss of proliferative capacity are poorly understood. CBX7, a polycomb group (PcG) protein, regulates the cell cycle but its role in CM proliferation is unknown. Methods: We profiled CBX7 expression in the mouse hearts via qRT-PCR, western blotting, and immunohistochemistry. We overexpressed CBX7 in neonatal mouse CMs via adenoviral transduction. We knocked down CBX7 by using constitutive and inducible conditional knockout mice (Myh6-cre;Cbx7 fl/+ and Myh6-MCM;Cbx7 fl/fl , respectively). We measured CM proliferation by immunostaining of proliferation markers such as Ki67, phospho-histone 3, and cyclin B1. We examined the mechanism of CBX7-mediated repression of CM proliferation via co-immunoprecipitation, mass spectrometry, and other molecular techniques. Results: The mRNA expression of Cbx7 was increased at the perinatal stage and sustained in the postnatal heart (fold increase of adult vs. prenatal: 32.1 ± 3.3, P < 0.01). Overexpression of CBX7 in neonatal mouse CMs reduced CM proliferation (% of Ki67 + CMs: Ad-Mock, 22.3 ± 1.7 vs. Ad-CBX7, 7.7 ± 0.7, P < 0.001). The haplodeficiency of CBX7 (Tnnt2-Cre;Cbx7 fl/+ ) enhanced proliferation of neonatal CMs in vivo (% of Ki67 + CMs: wild-type, 7.9 ± 0.9% vs. mutants, 24.7 ± 1.2%, P < 0.01), leading to increased myocardial wall thickness, cardiomegaly and neonatal lethality. Genetic deletion of CBX7 in CMs (Myh6-MCM;Cbx7 fl/fl ) at P1 resulted in increased CM proliferation (% of Ki67 + CMs: vehicle, 9.5 ± 1.4% vs. tamoxifen, 18.3 ± 1.8%, P < 0.01), leading to cardiomegaly at 3 months. Mechanistically, CBX7 interacted with TAR DNA-binding protein 43 (TARDBP) and positively regulated its downstream target, RNA Binding Motif Protein 38 (RBM38). Rbm38 was perinatally upregulated in the mouse hearts and overexpression of RBM38 reduced proliferation of neonatal CMs. Conclusions: CBX7 expression is perinatally increased in the mouse hearts and inhibits proliferation of CMs by controlling TARDBP/Rbm38 pathway. This is the first study to demonstrate the role of CBX7 in regulation of CM proliferation and CBX7 could be an important target for cardiac regeneration.

Cardiomyocyte (CM) proliferation notably decreases during the perinatal period. At present, regulatory mechanisms for this loss of proliferative capacity is poorly understood. CBX7, a polycomb group (PcG) protein, regulates the cell cycle but its role in CM proliferation is unknown. Here, we report that CBX7 inhibits proliferation of perinatal CMs by controlling TARDBP/Rbm38 pathway. Gene expression profiling demonstrated that CBX7 expression in the heart was low during the prenatal period, abruptly increased during the perinatal period, and sustained constantly throughout the adulthood. CBX7, when overexpressed via adenoviral transduction in neonatal CMs, reduced proliferation and promoted multinucleation of the CMs. Mutant mice carrying targeted inhibition of CBX7 in CMs exhibited cardiomegaly with increased proliferation of CMs at postnatal stages. Mechanistically, CBX7 interacted with TAR DNA-binding protein 43 (TARDBP) and positively regulated its downstream target, RNA Binding Motif Protein 38 (RBM38). Rbm38 was upregulated in the postnatal hearts and overexpression of RBM38 reduced proliferation of neonatal CMs. Together, this study provides a novel insight into the role of CBX7 in regulation of CM proliferation during the perinatal period.