Unknown,Transcriptomics,Genomics,Proteomics

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A KAP1 phosphorylation switch controls MyoD function during skeletal muscle differentiation (ChIP-seq)

ABSTRACT: 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

ORGANISM(S): Mus musculus

SUBMITTER: Evarist Planet

PROVIDER: E-GEOD-62660 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Analysis of Early Myogenesis Reveals an Extensive Set of Transcriptional Regulators Whose Knock-down Can Inhibit Differentiation Myogenesis is a tightly controlled process involving the transcriptional activation and repression of thousands of genes. Although many components of the transcriptional network are known for the later phases of myogenesis, relatively little work has described the transcriptional landscape within the first 24 hours, when myoblasts commit to differentiate. Through dense temporal sampling of differentiating C2C12 myoblasts, we identify 266 transcriptional regulators (TRs) whose expression is altered within the first 12 hours of myogenesis. A high-content shRNA screen of 76 TRs involving 427 stable lines identified 48 genes whose knockdown significantly inhibits differentiation of C2C12 myoblasts. These include known regulators of myogenesis (Myod1, Myog and Myf5), as well as 26 regulators not previously associated with the process. Of the TRs differentially expressed within the first 24 hours, two-thirds inhibited differentiation when knocked down. Surprisingly, a similar proportion (67%) of shRNAs targeting TRs whose expression did not change during differentiation also inhibited myogenesis, suggesting that both stably and differentially expressed TRs are essential for this complex differentiation program. This implies that microarray-based approaches that concentrate functional validation studies on differentially-expressed genes will fail to identify many genes that are critically implicated in complex biological processes. C2C12 myoblasts were differentiated into myotubes and sampled at various time points for gene expression measurement on MOE-430v2 chips. Cells grown in separate plates were harvested at 14 different time points: t_-24h, t_0h, t_0.5h, t_1h, t_1.5h, t_2h, t_3h, t_6h, t_9h, t_12h, t_24h, t_48h, t_96h, t_144h. Cells were also pre-treated with 50uM cycloheximide 1 hour prior to inducing differentiation and harvested at two time points: t_chx_1h, t_chx_3h. All harvests were performed in triplicate using growths from successive passages.

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A KAP1 phosphorylation switch controls MyoD function during skeletal muscle differentiation (ChIP-seq)

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