Genome-wide DNA-methylation and transcriptome change due to lncRNA H19 knockdown in myotubes
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ABSTRACT:
ORGANISM(S): Mus musculus
SUBMITTER: Na Zhang
PROVIDER: E-GEOD-73014 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:We used Methyl-MiniSeq platform from Zymo Research company to identify genome-wide methylation changes affected by lncRNA H19 knockdown in myotubes. Following H19 knockdown, we observed extensive genome-wide mthylation pattern changes relative to siCon cells, with some genes showing incresed methylation, others showing decreased methylation, and a third group with no significant change. Myotubes differentiated from mouse C3H myoblasts were transfected with either control siRNA or siH19, 48h later, cellular genomic DNA was extracted and subjected to genome-scale DNA methylation mapping using the platform of an improved version of Reduced Representation Bisulfite Sequencing (RRBS).
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Project description:We used high throughput sequencing to analyze the transcriptional profiling of myotubes. By comparing the transcriptional profiling of myotubes with or without H19 knockdown,1131 genes or transcripts were found at least 1.5-fold up-regulated in siH19 relative to siCon cells, meanwhile 508 genes or transcripts were found at least 1.5-fold down-regulated in siH19 over siCon cells. 40h following initiation of differentiation, myotubes were transfected with either control siRNA or siH19. 48h later after transfection, total RNA was extracted for library preparation and RNA-seq analysis to compare trancript profiles between siCon and siH19 cells.
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Project description:Ubiquitination is a post-translational protein modification that has been shown to have a range of effects, including regulation of protein function, interaction, localization, and degradation. We have previously shown that the muscle-specific ubiquitin E3 ligase, ASB2β, is down-regulated in models of muscle growth and that overexpression ASB2 is sufficient to induce muscle atrophy. To gain insight into the effects of increased ASB2 expression on skeletal muscle mass and function, we used liquid chromatography coupled to tandem mass spectrometry to investigate ASB2-mediated changes to the skeletal muscle proteome and ubiquitinome, via a parallel analysis of remnant di-Gly-modified peptides. The results show that viral vector-mediated ASB2β overexpression in murine muscles causes progressive muscle atrophy and impairment of force-producing capacity, while ASB2β knockdown induces mild muscle hypertrophy. ASB2β-induced muscle atrophy and dysfunction were associated with the early downregulation of mitochondrial and contractile protein abundance, and the upregulation of proteins involved in proteasome-mediated protein degradation (including other E3 ligases), protein synthesis and the cytoskeleton/sarcomere. The overexpression ASB2β also resulted in marked changes in protein ubiquitination, however, there was no simple relationship between changes in ubiquitination status and protein abundance. To investigate proteins that interact with ASB2 and, therefore, potential ASB2 targets, Flag-tagged wild type ASB2, and a mutant ASB2 lacking the C-terminal SOCS box domain (dSOCS) were immunoprecipitated from C2C12 myotubes and subjected to label-free proteomic analysis to determine the ASB2 interactome. ASB2β was found to interact with a range of cytoskeletal and nuclear proteins. When combined with the in vivo ubiquitinomic data, our studies have identified novel putative ASB2β target substrates that warrant further investigation. These findings provide novel insight into the complexity of proteome and ubiquitinome changes that occur during E3 ligase-mediated skeletal muscle atrophy and dysfunction.
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Project description:By applying ChIP-seq, we generated genome-wide maps of YY1 in skeletal myoblasts and myotubes. We found that YY1 binds to 1820 confident target with a large portion residing in the intergenic regions. In addition, YY1 was found to activate many loci, and there is no significant overlap between YY1 and Ezh2 targets, suggensting a Ezh2-independent manner. Further detailed study revealed that YY1 can regulate some lincRNAs which are fucntional in skeletal myogenesis. In this study, we identified a YY1-Yam-1-miR-715 (TF-lincRNA-miRNA) regulatory curcuit in myogensis. Examination of YY1 targets in myoblast versus myotubes
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Project description:Ubiquitylation of H2B on lysine 120 (H2Bub) is associated with active transcriptional elongation. H2Bub has been implicated in histone cross-talk and is generally regarded to be a prerequisite for H3K4 and H3K79 tri-methylation in both yeast and mammalian cells. We performed a genome-wide analysis of epigenetic marks during muscle differentiation, and, strikingly, we observed a near-complete loss of H2Bub in the differentiated state. We examined the basis for global loss of this mark and found that the H2B ubiquitin E3 ligase, RNF20, was depleted from chromatin in differentiated myotubes, indicating that recruitment of this protein to genes substantially decreases upon differentiation. Remarkably, during the course of myogenic differentiation, we observed retention and acquisition of H3K4 tri-methylation on a large number of genes in the absence of detectable H2Bub. The Set1 H3K4 trimethylase complex was efficiently recruited to a subset of genes in myotubes in the absence of detectable H2Bub, accounting in part for H3K4 tri-methylation in myotubes. Our studies suggest that H3K4me3 deposition in the absence of detectable H2Bub in myotubes is mediated via Set1 and, perhaps, MLL complexes, whose recruitment does not require H2Bub. Thus, muscle cells represent a novel setting in which to explore mechanisms that regulate histone cross-talk. Mapping of H2Bub in growing myoblasts (MB) and fully differentiated myotubes (MT).
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Project description:Rhabdomyosarcoma is a pediatric tumor of skeletal muscle that expresses the myogenic basic helix-loop-helix protein MyoD but fails to undergo terminal differentiation. Prior work has determined that DNA binding by MyoD occurs in the tumor cells, but myogenic targets fail to activate. Using MyoD chromatin immunoprecipitation coupled to high-throughput sequencing and gene expression analysis in both primary human muscle cells and RD rhabdomyosarcoma cells, we demonstrate that MyoD binds in a similar genome-wide pattern in both tumor and normal cells but binds poorly at a subset of myogenic genes that fail to activate in the tumor cells. Binding differences are found both across genomic regions and locally at specific sites that are associated with binding motifs for RUNX1, MEF2C, JDP2, and NFIC. These factors are expressed at lower levels in RD cells than muscle cells and rescue myogenesis when expressed in RD cells. MEF2C is located in a genomic region that exhibits poor MyoD binding in RD cells, whereas JDP2 exhibits local DNA hypermethylation in its promoter in both RD cells and primary tumor samples. These results demonstrate that regional and local silencing of differentiation factors contributes to the differentiation defect in rhabdomyosarcomas. ChIP-Seq profiling of MyoD in human myotube, myoblast and rhabdomyosarcoma cells
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