Project description:Genome wide gene expression analysis in G9a knockdown myoblasts Illumina WG-6 v2.0 arrays were hybridized with RNA from scrambled control and siG9a myoblasts to determine the gene expression profile murine skeletal myoblasts. All hybridizations were done in biological duplicates
Project description:Lysine methyltransferases G9a and GLP (G9a- Like Protein) form functional heterodimeric complexes that establish mono- and dimethylation on histone H3 lysine 9 (H3K9me1, H3K9me2) in euchromatin. Here we describe unexpected opposite individual roles for G9a and GLP during skeletal muscle terminal differentiation. Indeed, gain- or loss-of-function assays in myoblasts showed that in consistency with previous reports that G9a inhibits terminal differentiation. But GLP plays a more complicated role in muscle differentiation since both knockdown and overexpression inhibits terminal differentiation. Unexpectedly, in contrast to G9a, we show that GLP overexpression promotes abnormal expression of muscle differentiationspecific genes in proliferating myoblasts. Transcriptomic analysis indicates that G9a and GLP regulate different sets of genes. GLP, but not G9a, inhibits at the transcriptional level proteasome subunit-encoding genes resulting in an inhibition of the proteasome activities. Subsequently, GLP, but not G9a, overexpression stabilizes MyoD that is likely to be responsible for muscle markers expression in proliferating myoblasts.
Project description:GATA4 occupancy on the mouse genome of satellite cell-derived primary myoblasts. Proliferating myoblasts cultured in growth medium were immunoprecipitated with anti-GATA4 antibody or control IgG. Precipitated genomic DNAs were subjected to next generation sequencing.
Project description:The transcriptional activator MyoD serves as a master controller of myogenesis. Often in partnership with Mef2, MyoD binds to the promoters of hundreds of muscle genes in proliferating myoblasts, yet activates these targets only upon receiving cues that launch differentiation. What regulates this off/on switch of MyoD function has been incompletely understood, although known to reflect the action of chromatin modifiers. Here, we identify KAP1/TRIM28 as a key regulator of MyoD function. In myoblasts, KAP1 is present with MyoD and Mef2 at many muscle genes, where it acts as a scaffold to recruit not only co-activators such as p300 and LSD1, but also co-repressors such as G9a and HDAC1, with promoter silencing as net outcome. Upon differentiation, MSK1-mediated phosphorylation of KAP1 releases the co-repressors from the scaffold, unleashing transcriptional activation by MyoD/Mef2 and their positive cofactors. Thus, our results reveal KAP1 as a previously unappreciated interpreter of cell signaling, which modulates the ability of MyoD to drive myogenesis. Kap1 and H3K9me3 ChIPseq in proliferating C2C12 cells
Project description:The transcriptional activator MyoD serves as a master controller of myogenesis. Often in partnership with Mef2, MyoD binds to the promoters of hundreds of muscle genes in proliferating myoblasts, yet activates these targets only upon receiving cues that launch differentiation. What regulates this off/on switch of MyoD function has been incompletely understood, although known to reflect the action of chromatin modifiers. Here, we identify KAP1/TRIM28 as a key regulator of MyoD function. In myoblasts, KAP1 is present with MyoD and Mef2 at many muscle genes, where it acts as a scaffold to recruit not only co-activators such as p300 and LSD1, but also co-repressors such as G9a and HDAC1, with promoter silencing as net outcome. Upon differentiation, MSK1-mediated phosphorylation of KAP1 releases the co-repressors from the scaffold, unleashing transcriptional activation by MyoD/Mef2 and their positive cofactors. Thus, our results reveal KAP1 as a previously unappreciated interpreter of cell signaling, which modulates the ability of MyoD to drive myogenesis. Transcriptome profiling of control and Kap1KD in C2C12 during proliferation or at 48hours differentiation stage
Project description:GATA4 occupancy on the mouse genome of satellite cell-derived primary myoblasts. Proliferating myoblasts cultured in growth medium were immunoprecipitated with anti-GATA4 antibody or control IgG. Precipitated genomic DNAs were subjected to next generation sequencing. Paired-end 150 bp sequence reads of GATA4-ChIP and IgG-ChIP using mouse skeletal muscle myoblasts.
Project description:The transcriptional activator MyoD serves as a master controller of myogenesis. Often in partnership with Mef2, MyoD binds to the promoters of hundreds of muscle genes in proliferating myoblasts, yet activates these targets only upon receiving cues that launch differentiation. What regulates this off/on switch of MyoD function has been incompletely understood, although known to reflect the action of chromatin modifiers. Here, we identify KAP1/TRIM28 as a key regulator of MyoD function. In myoblasts, KAP1 is present with MyoD and Mef2 at many muscle genes, where it acts as a scaffold to recruit not only co-activators such as p300 and LSD1, but also co-repressors such as G9a and HDAC1, with promoter silencing as net outcome. Upon differentiation, MSK1-mediated phosphorylation of KAP1 releases the co-repressors from the scaffold, unleashing transcriptional activation by MyoD/Mef2 and their positive cofactors. Thus, our results reveal KAP1 as a previously unappreciated interpreter of cell signaling, which modulates the ability of MyoD to drive myogenesis.
Project description:The transcriptional activator MyoD serves as a master controller of myogenesis. Often in partnership with Mef2, MyoD binds to the promoters of hundreds of muscle genes in proliferating myoblasts, yet activates these targets only upon receiving cues that launch differentiation. What regulates this off/on switch of MyoD function has been incompletely understood, although known to reflect the action of chromatin modifiers. Here, we identify KAP1/TRIM28 as a key regulator of MyoD function. In myoblasts, KAP1 is present with MyoD and Mef2 at many muscle genes, where it acts as a scaffold to recruit not only co-activators such as p300 and LSD1, but also co-repressors such as G9a and HDAC1, with promoter silencing as net outcome. Upon differentiation, MSK1-mediated phosphorylation of KAP1 releases the co-repressors from the scaffold, unleashing transcriptional activation by MyoD/Mef2 and their positive cofactors. Thus, our results reveal KAP1 as a previously unappreciated interpreter of cell signaling, which modulates the ability of MyoD to drive myogenesis.
Project description:Myogenesis is a complex biological process, and understanding the regulatory network of skeletal myogenesis will contribute to the treatment of human muscle related diseases and improvement of agricultural animal meat production. Long noncoding RNAs (lncRNAs) serve as regulators in gene expression networks, and participate in various biological processes. Recent studies have identified functional lncRNAs involved in skeletal muscle development and disease. These lncRNAs regulate the proliferation, differentiation, and fusion of myoblasts through multiple mechanisms, such as chromatin modification, transcription regulation, and microRNA sponge activity. In this review, we presented the latest advances regarding the functions and regulatory activities of lncRNAs involved in muscle development, muscle disease, and meat production. Moreover, challenges and future perspectives related to the identification of functional lncRNAs were also discussed.
Project description:The histone 3 lysine 9 (H3K9)-specific methyltransferase (KMT) Setdb1 is essential for both stem cell pluripotency and terminal differentiation of different cell types. To shed light on Setdb1 role(s) in these mutually exclusive processes, we used mouse skeletal myoblasts as a model of terminal differentiation. Ex vivo studies on isolated single myofibres showed that Setdb1 is required for muscle adult stem cells expansion following activation and in vitro studies on skeletal myoblasts confirmed that Setdb1 suppresses terminal myoblast differentiation. We used genome-wide analyses to identify Setdb1 direct target genes in myoblasts and observed a release of Setdb1 from the promoter of selected target genes upon myoblast terminal differentiation, concomitant to a nuclear export of Setdb1 to the cytoplasm. We demonstrated that both genomic release and cytoplasmic Setdb1 relocalisation during differentiation were dependent on canonical Wnt signalling. Taken together, our findings uncover a functional link between Setdb1 and canonical Wnt signalling in skeletal muscle cells, which affects the expression of a subset of Setdb1 target genes. We revealed Wnt-dependent subcellular relocalisation of Setdb1 as a novel mechanism regulating Setdb1 functions.