Kang X, Zhao Y, Van Arsdell G, Nelson SF, Touma M.
RNA. 2020 Apr;26(4):481-491. doi: 10.1261/rna.073692.119. Epub 2020 Jan 17.
PMID: 31953255 Free PMC Article
Select item 31862220
Long noncoding RNAs (lncRNAs) have emerged as critical epigenetic regulators and play important roles in cardiac development and congenital heart disease. In a previous study, we identified a novel lncRNA, Ppp1r1b, with expression highly correlated with myogenesis. However, the molecular mechanism that underlies Ppp1r1b-lncRNA function in myogenic regulation is unknown. By silencing Ppp1r1b-lncRNA, mouse C2C12 and human skeletal myoblasts failed to develop fully differentiated myotubes. Myogenic differentiation was also impaired in PPP1R1B-lncRNA deficient human-induced pluripotent stem cell-derived cardiomyocytes (hiPSCs-CMs). The expression of myogenic transcription factors, including MyoD, Myogenin, and Tbx5, as well as sarcomere proteins, was significantly suppressed in Ppp1r1b-lncRNA inhibited myoblast cells and neonatal mouse heart. Histone modification analysis revealed increased H3K27 tri-methylation at MyoD1 and Myogenin promoters in GapmeR treated C2C12 cells. Furthermore, Ppp1r1b-lncRNA was found to bind to Ezh2, and chromatin isolation by RNA purification (ChIRP) assay revealed enriched interaction of Ppp1r1b-lncRNA with Myod1 and Tbx5 promoters, suggesting that Ppp1r1b-lncRNA induces transcription of myogenic transcription factors by interacting with the polycomb repressive complex 2 (PRC2) at the chromatin interface. Correspondingly, the silencing of Ppp1r1b-lncRNA increased EZH2 binding at promoter regions of myogenic transcription factors. Therefore, our results suggest that Ppp1r1b-lncRNA promotes myogenic differentiation through competing for PRC2 binding with chromatin of myogenic master regulators during heart and skeletal muscle development.
FIGURE 1. Ppp1r1b-lncRNA is induced during myogenesis and regulates C2C12 myoblast differentiation. (A,C) Quantitative real-time PCR analysis of Ppp1r1b-lncRNA expression after 2 d of differentiation (A) and GapmeR treatment (C). (B) Subcellular localization of Ppp1r1b-lncRNA in myoblasts. U6 RNA was used as a nuclear location control. (D) Light microscope images depict the morphology of C2C12 cells after 2 d of differentiation and Ppp1r1b-GapmeR, or Scramble, treatment. Scale bar, 200 µm. N = 4 biological replicates per condition; error bars, standard error of the mean; (**) P < 0.01.
FIGURE 2. Human PP1R1B-lncRNA promotes differentiation of human skeletal myoblast. (A) Fluorescent microscope images depict inhibition of myogenesis of human skeletal myoblast (myosin positive cells) after PPP1R1B-lncRNA siRNA treatment. Scale bar, 100 µm. (B) Higher magnification images of cells presented in A. (C) Real-time PCR analysis and fusion index analysis of human myoblast before and after differentiation (625 nuclei counted for growth medium; 571 nuclei counted for differentiation medium). (D) RNA fractionation analysis of PPP1R1B-lncRNA down-regulated by siRNA. (E) Real-time PCR analysis and fusion index analysis of human myoblast after PPP1R1B-lncRNA siRNA treatment in differentiation medium (737 nuclei counted for Scramble; 760 nuclei counted for siRNA). N = 4 biological replicates per condition; error bars, standard error of the mean; (*) P < 0.05; (**) P < 0.01.
FIGURE 3. Human PPP1R1B-lncRNA promotes differentiation of human iPSC-derived cardiomyocyte (hiPSC-CM). (A) Microscope images depict the differentiation efficiency of human hiPSC-CM (Myosin positive cells) after PPP1R1B-lncRNA siRNA, or Scramble, treatment. (WGA) Wheat germ agglutinin. Scale bar, 100 µm. (B) Quantitative real-time PCR analysis of PPP1R1B-lncRNA expression after siRNA treatment. (C) Quantitative analysis depicts differentiation efficiency of hiPSC-CMs after siRNA treatment. (EB) Embryonic bodies. N = 4 biological replicates per condition; error bars, standard error of the mean; (*) P < 0.05; (**) P < 0.01.
FIGURE 4. Ppp1r1b-ncRNA regulates expression of myogenic regulatory factors and sarcomere structural genes. Real-time PCR analysis of myogenic regulatory factors and sarcomeric structural genes after Ppp1r1b-lncRNA silencing in Mouse C2C12 myoblasts (A), mouse heart tissue (B), human skeletal myoplasts (C), and human-induced pluripotent stem cell-derived cardiomyocytes (hiPSCs-CM) (D). N = 4 biological replicates per condition. Error bars, standard error of the mean. (*) P < 0.05; (**) P < 0.01.
FIGURE 5. Ppp1r1b-lncRNA inhibits Histone 3 methylation at the promoter regions of myogenic regulatory factors. C2C12 myoblasts were cultured in growth medium (GM) or differentiation medium (DM) and treated by scramble GapmeR or Ppp1r1b-lncRNA GapmeR. After treatment, cells were subjected to ChIP analysis using anti H3K27me3 antibodies. The qPCR data are presented as fold enrichment to the background, indicating the abundance of histone modifications at the promoter regions of the myogenic genes. (A) H3K27me3 level on MyoD1 promoter. (B) H3K27me3 level on Myogenin promoter. (C) Western blot analysis of myogenic regulatory factors and sarcomeric structural proteins from C2C12. Gapdh was used as a loading control. (GM) Growth medium, (DM) differentiation medium. (*) P < 0.05; (**) P < 0.01.
FIGURE 6. Ppp1r1b-lncRNA binds EZH2. (A) RNA pull-down assays were carried out by incubating cell lysate from neonatal mouse heart tissue with biotinylated in vitro transcribed Ppp1r1b-lncRNA. Subsequent western blotting analysis revealed EZH2 in the isolated RNA-protein complex. (B) RNA immunoprecipitation (RIP) demonstrated that Ppp1r1b-lncRNA was immunoprecipitated together with EZH2 in neonatal mouse heart tissue. LncRNA was detected using real-time PCR. (*) P < 0.05.
FIGURE 7. Ppp1r1b-lncRNA binds to MyoD1, Myogenin and Tbx5 promoter region. Chromatin isolation by RNA purification (ChIRP) assays were carried out by incubating cell lysate from mouse neonatal heart tissue and human infantile heart tissue with biotinylated in vitro synthesized Ppp1r1b-lncRNA probes. Promoter DNAs were detected by real-time PCR. (A) Interactions of Ppp1r1b-lncRNA with MyoD1, Myogenin, and Tbx5 promoter DNA in mouse. (B) Interactions of Ppp1r1b-lncRNA with MyoD1, Myogenin, and Tbx5 promoter DNA in human. (*) P < 0.05.