Sex-Based Mhrt Methylation Chromatinizes MeCP2 in the Heart

摘要

class="head no_bottom_margin" id="sec1title">IntroductionLong non-coding RNAs (lncRNAs) have emerged as important regulators of heart development and disease (, ). Several lncRNAs such as Braveheart (), Fendrr (), Upperhand (), and Carmen () are differentially activated at the fetal and adult stages of cardiac development. Although the precise roles of these lncRNAs still remain poorly understood, recent evidence suggests that lncRNAs not only control mRNA expression during the critical transition stages from fetal to adult heart but also function with mRNA stability, decay, and as regulatory sponges (). Not entirely unexpected, in the fetal heart, lncRNAs associate more with genes implicated in processes that involve development and programming, whereas in the adult heart lncRNAs associate more with genes involved in disease. For example, in the adult heart the expression of lncRNAs Chast (), Chaer (), and Wisper () are linked with cardiac hypertrophy and fibrosis. The sarcomeric myosin heavy chain (MHC) isogenes, originally designated α and β (recently renamed Myh6 and Myh7) are predominantly expressed in the heart (, ). During fetal life Myh7 is principally expressed, whereas Myh6 and Myh7 are simultaneously expressed shortly after birth, with Myh6 dominantly expressed in adulthood (, , ). During myocardial hypertrophy Myh7 is selectively expressed under hemodynamic and pressure overload (href="#bib20" rid="bib20" class=" bibr popnode">Izumo et al., 1987, href="#bib29" rid="bib29" class=" bibr popnode">Litten et al., 1982, href="#bib32" rid="bib32" class=" bibr popnode">Lompre et al., 1979).Previously known as antisense β-MHC (href="#bib15" rid="bib15" class=" bibr popnode">Haddad et al., 2003), Myosin heavy-chain-associated RNA transcript (Myheart, or Mhrt) is a myocardium-specific lncRNA regulated by antisense transcription of a specific intergenic promoter that originates from the antisense strand of Myh7 that serves as a switch for Myh6/7 gene expression (href="#bib16" rid="bib16" class=" bibr popnode">Han et al., 2014). Recent studies have shown that Mhrt expression is cardioprotective and behaves as a decoy lncRNA involved in a negative feedback circuit with chromatin remodeling. Interestingly, cardiac hypertrophy induced by pressure overload in a mouse model progressively reduced Mhrt expression. Experiments have shown that restoring Mhrt to prestress levels can attenuate cardiac damage and rescue the heart from pathological hypertrophy (href="#bib16" rid="bib16" class=" bibr popnode">Han et al., 2014). It is hypothesized that under cardiac stress Mhrt restores the Myh6/7 gene shift by virtue of its interaction with the remodeling enzyme, Brg1, an ATP-dependent DNA helicase and a subunit of a much larger SWI/SNF complex. We have recently shown that pri-miR-208b, an lncRNA that originates from Myh7, regulates gene expression during cardiac hypertrophy (href="#bib39" rid="bib39" class=" bibr popnode">Mathiyalagan et al., 2014b). Upon transverse aortic constriction (TAC) in a mouse model of pathological hypertrophy, the left ventricular tissue shows elevated expression of pri-miR-208b that interacts with a Polycomb-group protein, Ezh2, and is part of a Hdac complex that regulates the Myh6/7 gene shift (href="#bib39" rid="bib39" class=" bibr popnode">Mathiyalagan et al., 2014b). The precise mechanisms regulating sex-based expression of lncRNAs such as pri-miR-208b and Mhrt remain poorly characterized.Gender disparity in cardiovascular health and disease is well documented with females generally regarded less vulnerable to pathological cardiac remodeling (href="#bib35" rid="bib35" class=" bibr popnode">Maas and Appelman, 2010). Pathogenic processes such as cardiomyocyte apoptosis and necrosis characteristic of hypertrophy occur predominantly in males (href="#bib14" rid="bib14" class=" bibr popnode">Guerra et al., 1999). Pathological cardiac hypertrophy is characterized by reactivation of fetal gene programming that involves activation of fetal genes and the suppression of genes expressed in adults (href="#bib12" rid="bib12" class=" bibr popnode">Frey and Olson, 2003). This pattern of expression is common to both sexes, whereas molecular remodeling induced by pressure overload hypertrophy occurs predominantly in males (href="#bib70" rid="bib70" class=" bibr popnode">Weinberg et al., 1999, href="#bib74" rid="bib74" class=" bibr popnode">Zhong et al., 2003). Sex differences in the heart are largely determined by steroid hormones; however, several histone modifiers and methyl-binding proteins as well as promoter DNA methylation are closely linked to sex-based gene expression during development and in adult heart (href="#bib26" rid="bib26" class=" bibr popnode">Kurian et al., 2010, href="#bib55" rid="bib55" class=" bibr popnode">Ratnu et al., 2017). The role of lncRNAs in regulating sex-based gene expression by methylation in the heart remain poorly characterized. Because lncRNAs can interact with epigenetic modifiers, our aim was to determine the mechanism of lncRNA methylation in regulating sex-based gene expression in the heart.MeCP2 is a reader of DNA methylation, a component of a co-repressor complex (href="#bib18" rid="bib18" class=" bibr popnode">Harikrishnan et al., 2005) and recently shown to regulate gene expression in chronic heart failure (href="#bib41" rid="bib41" class=" bibr popnode">Mayer et al., 2015). In addition to its high affinity for methylated cytosine sites on DNA, MeCP2 is recognized for its ability to bind RNA and regulate alternative splicing events by interacting with YB-1 (href="#bib72" rid="bib72" class=" bibr popnode">Young et al., 2005, href="#bib33" rid="bib33" class=" bibr popnode">Long et al., 2011, href="#bib21" rid="bib21" class=" bibr popnode">Jeffery and Nakielny, 2004). Although MeCP2 was initially characterized as a reader with high affinity for methylated cytosine in DNA (href="#bib6" rid="bib6" class=" bibr popnode">Bird and Wolffe, 1999), more recent studies have shown that MeCP2 can interact with other RNAs (href="#bib40" rid="bib40" class=" bibr popnode">Maxwell et al., 2013, href="#bib23" rid="bib23" class=" bibr popnode">Khan et al., 2017b, href="#bib24" rid="bib24" class=" bibr popnode">Khan et al., 2017c). For example, MeCP2 has been shown to interact with mRNAs (href="#bib33" rid="bib33" class=" bibr popnode">Long et al., 2011); non-coding RNAs (ncRNAs) such as let-7i, miR-375, and miR-126 (href="#bib24" rid="bib24" class=" bibr popnode">Khan et al., 2017c); as well as Rncr3 and Malat1 (href="#bib40" rid="bib40" class=" bibr popnode">Maxwell et al., 2013). Although these studies suggest that MeCP2 interacts with RNA to suppress gene expression, the mechanism mediated by lncRNA in the myocardium remains poorly understood.One mechanism recently identified in the epigenetic control of lncRNAs is the deposition of 5-methylcytosine (5mC) (href="#bib62" rid="bib62" class=" bibr popnode">Squires et al., 2012, href="#bib3" rid="bib3" class=" bibr popnode">Amort et al., 2013). This epigenetic determinant occurs on coding RNA and ncRNA, and several lncRNAs such as Xist, Hotair, and Malat1 contain 5mC sites and have been shown to regulate ncRNA function (href="#bib3" rid="bib3" class=" bibr popnode">Amort et al., 2013, href="#bib2" rid="bib2" class=" bibr popnode">Amort et al., 2017). The purpose of this study was to explore the sex-based differences in lncRNA expression in the heart. Recent studies have shown that RNA-dependent MeCP2 binding on genes is independent of DNA methylation (href="#bib23" rid="bib23" class=" bibr popnode">Khan et al., 2017b). Therefore, we hypothesized that sex-based interaction of MeCP2 at the pri-miR-208b promoter is associated with Mhrt lncRNA methylation. Close examination of DNA methylation at the pri-miR-208b promoter using bisulfite sequencing identified nine sites of cytosine methylation at the CpG island (CGI). We observed no difference in DNA methylation between the sexes but show dramatic changes in the binding of the methylation reader, MeCP2, on the pri-miR-208b promoter using chromatin immunoprecipitation (ChIP). We identified that sex-specific expression of pri-miR-208b in the female heart is independent of DNA methylation. We provide proof of concept that MeCP2 affinity to chromatin is subject to RNA-methylation-dependent regulation. Sex-specific methylation of Mhrt distinguishes MeCP2 chromatinization of the pri-miR-208b promoter and gene regulation in the female heart.