Abstract 657: Long Noncoding RNA Ppp1r1b Regulates Myogenic Differentiation Through Modulating Histone 3 Methylation

Abstract

Introduction: Emerged as critical epigenetic regulators of the transcriptome, the long noncoding RNAs (lncRNAs) play important roles in cardiac development and might be targeted to treat human cardiomyocyte dysfunction in congenital heart disease. In our work, we identified a novel lncRNA that regulates myogenic differentiation. Methods: Antisense oligonucleotides (GapmeR) were used to suppress Ppp1r1b-lncRNA. Chromatin immunoprecipitation (CHIP) and chromatin isolation by RNA purification (CHIRP) were used to analyze gene specific histone modification level and lncRNA:chromatin interaction, respectively. RNA pull-down and RNA immunoprecipitation (RIP) were used to identify the protein molecules that interact with Ppp1r1b-lncRNA. Results: By silencing Ppp1r1b-lncRNA, C2C12, a skeletal myoblast cell line, and human-induced pluripotent stem cell derived cardiomyocytes failed to develop fully differentiated myotubes. Analysis of GapmeR injected neonatal mouse heart ( in vivo ) and siRNA silenced human myoblasts ( in vitro ) further confirmed the role of Ppp1r1b-lncRNA in normal myogenesis. Members of the MyoD family of muscle-specific transcription factors were not induced during myogenic differentiation in C2C12 cells treated with Ppp1r1b-lncRNA specific GapmeR. Proteins essential for striated muscle development were also suppressed in Ppp1r1b-lncRNA- deficient cells. Histone modification analysis revealed enrichment of histone tri-methylation at Myogenin and MyoD1 promoters in GapmeR treated C2C12 cells. Subsequently, lncRNA- protein complex isolation and lncRNA-DNA interaction assays confirmed Ppp1r1b-lncRNA function. Conclusions: Our findings support important role of Ppp1r1b-lncRNA in promoting myogenic differentiation. Ppp1r1b-lncRNA function is mediated by inhibiting histone methylation on promoters of key myogenic genes. Particularly, the identification of EZH2 in pulled Pp1r1b-lncRNA: protein complex supports that Polycomb repressive complex 2 is involved in Ppp1r1b-lncRNA modulated myoblast differentiation. The recovery of MyoD1 promoter DNA in isolated lncRNA: chromatin DNA interactome implies that Ppp1r1b-lncRNA regulates transcription by recruiting PRC2 complex to its target genes.