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: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:Histone chaperones affect chromatin structure and gene expression through interaction with histones and RNA polymerase II (PolII). Here, we report that the histone chaperone Spt6 counteracts H3K27me3, an epigenetic mark deposited by the Polycomb Repressive Complex 2 (PRC2) and associated with transcriptional repression. We found that Spt6 is required for proper engagement and function of the H3K27 demethylase KDM6A (UTX) on muscle genes and regulates muscle gene expression and cell differentiation. ChIP-Seq experiments revealed an extensive genome-wide overlap of Spt6, PolII and KDM6A at transcribed regions that are devoid of H3K27me3. Mammalian cells and zebrafish embryos with reduced Spt6 display increased H3K27me3 and diminished expression of the master regulator MyoD, resulting in myogenic differentiation defects. As a confirmation for an antagonistic relationship between Spt6 and H3K27me3, inhibition of PRC2 permits MyoD re-expression in myogenic cells with reduced Spt6. Our data indicate that, through cooperation with PolII and KDM6A, Spt6 orchestrates removal of H3K27me3, thus effectively controlling developmental gene expression and cell differentiation. Examination of Spt6 and KDM6A levels in a skeletal muscle cells at various developmental stages
Project description:Transcription factors and DNA regulatory binding motifs are fundamental components of the gene regulatory network (GRN). Here, by using genome-wide occupancy profiling of master regulators of MyoGenesis (MyoD and MyoGenin), we show their extensive occupancy in the extragenic enhancer regions coinciding with RNA synthesis (i.e. eRNA). In particular, multiple regions coding for eRNAs were observed within regulatory region of MYOD1, including previously characterized Distal Regulatory Regions (DRR) and Core Enhancer (CE). While CERNA enhanced RNA polymerase II (PolII) occupancy and transcription at MYOD1, DRRRNA acted in trans to activate the downstream MyoGenic GRN. The deployment of transcriptional machinery to appropriate loci is contingent on chromatin accessibility, a rate-limiting step preceding PolII assembly. By nuclease sensitivity assay, we show that eRNAs increase genomic access to the transcriptional complex at defined regulatory regions. In conclusion, our data suggest eRNAs establish a cell-type-specific transcriptional circuitry by directing chromatin-remodeling events. Examination of MyoD and MyoG binding events and production of RNA at the enhancer sites during myogenic differentiation. We performed RNAi against enhancer-derived RNA (DRRi) which resulted in reduction of chromatin accessiblity and RNA polymerase II occupancy at defined regulatory elements. In complementary experiments, overexpression of DRR-RNA (pHAN_DRR1.2) resulted in early activation of MyoG. GFPi and GFP overexpression (pHAN_GFP) were used as control in these experiments, respectively.
Project description:Polycomb group (PcG) proteins initiate the formation of repressed chromatin domains and regulate developmental gene expression. A mammalian PcG protein, Enhancer of Zeste homolog 2 (Ezh2), triggers transcriptional repression by catalyzing the addition of methyl groups onto lysine-27 of histone H3 (H3K27me2/3)1. This action facilitates the binding of other PcG proteins to histone H3 and compaction of chromatin. Interestingly, there exists a paralog of Ezh2, termed Ezh1, whose primary function remains unclear. Here, we provide evidence for genome-wide association of Ezh1 with active epigenetic marks, RNA polymerase II (PolII) and mRNA production. Ezh1 depletion reduced global PolII occupancy within gene bodies and resulted in delayed transcriptional activation during differentiation of skeletal muscle cells. Conversely, ectopic expression of wild-type Ezh1 led to premature gene activation and rescued PolII-elongation defects in Ezh1-depleted cells. Collectively, these findings reveal an unanticipated role of a PcG protein in promoting mRNA transcription. Examination of 3 different histone modifications, 3 modified forms of RNA polymerase II, Ezh1, Ezh2 and mRNA levels in a skeletal muscle cells at various developmental stages.
Project description:Nrf2 (NF-E2-related-factor-2) contributes to the maintenance of glucose homeostasis in vivo. Nrf2 suppresses blood glucose levels by protecting pancreatic β-cells from oxidative stress and improving peripheral tissue glucose utilization. To elucidate the molecular mechanisms by which Nrf2 contributes to the maintenance of glucose homeostasis, we generated skeletal muscle (SkM)-specific Keap1-knockout (Keap1MuKO) mice that express abundant Nrf2 in SkM and then examined Nrf2-target gene expression in this tissue. In Keap1MuKO mice, blood glucose levels were significantly downregulated, and the levels of glycogen branching enzyme (Gbe1) mRNA, along with those of glycogen branching enzyme (GBE) protein, were significantly upregulated in mouse SkM. Consistent with this result, chemical Nrf2-inducers promoted Gbe1 mRNA expression in both mouse SkM and C2C12 myotubes. Chromatin-immunoprecipitation analysis demonstrated that Nrf2 binds the Gbe1 upstream promoter regions. In Keap1MuKO mice, muscle glycogen content was strongly reduced, and forced GBE expression in C2C12 myotubes promoted glucose uptake. Therefore, our results demonstrate that Nrf2-induction in SkM increases GBE expression and reduces muscle glycogen content, resulting in improved glucose tolerance. Chromatin occupancy of Nrf2 under CDDO-Im-treated condition were generated by deep sequencing, in dupliplicate
Project description:We use tag-based next generation sequencing analysis to quantify gene expression levels during myogenic differentiation and focused on the identifcation of novel 3' ends with DeepSAGE and novel 5' ends with DeepCAGE. Illumina DeepCAGE and DeepSAGE with C2C12 cells at time points T0 (proliferating) and T9 (differentiated) days of differentiation.
Project description:High reproducibility with TaqMan microRNA array (qPCR-array) was demonstrated by comparing replicate results from the same RNA sample. Pre-amplification of the miRNA cDNA improved sensitivity of the qPCR-array and increased the number of detectable miRNAs. Furthermore, the relative expression levels of miRNAs were maintained after pre-amplification. When the performance of qPCR-array and microarrays were compared using different aliquots of the same RNA, a low correlation between the two methods (r = -0.443) indicated considerable variability between the two assay platforms. Higher variation between replicates was observed in miRNAs with low expression in both assays. Finally, a higher false positive rate of differential miRNA expression was observed using the microarray compared to the qPCR-array. Replicate preparations (n =4) using different aliquots of a single C2C12 RNA sample (500 ng) were used to determine the reproducibility of the reverse transcription process as well as the results of the same reverse transcription products performed on different days. TaqMan microRNA Array A was used for this purpose. To assess the reliability of pre-amplification, miRNA expression profiles obtained with and without pre-amplification using MiRNA TaqMan Array B were performed. Independent miRNA expression profiling studies using uParaflo microfluidic biochips were performed by an independent company to determine the relationship between results obtained with qPCR-array and microarrays. Aliquots of the same C2C12 RNA were used in both the qPCR-array (500 ng) and microarrays (8 µg).