Project description:Identifying tissue and condition-specific gene regulatory elements and the mechanisms by which they associate with their target genes in human cells remains a significant challenge, largely due to the vast amount of non-protein-coding sequence in the human genome. Despite increasing evidence of physical interactions between distant regulatory regions and gene promoters in a variety of mammalian cell types, many searches for regulatory regions still consider only genomic regions proximal to the gene promoter. We identify a set of putative cis-regulatory modules (CRMs) of human skeletal muscle differentiation by combining new ChIP-chip data measuring the binding of myogenic TFs MyoG, MyoD, and SRF over the timecourse of differentiation with previously generated histone modification data. A substantial proportion of these putative CRMs are located distant (> 20 kb) from muscle gene promoters, and these distantly located CRMs are more likely than proximal promoter regions to show differentiation-specific changes in myogenic TF binding. Examination of two distant CRMs, which were previously shown to activate transcription in differentiating muscle cells, revealed that they interact physically with their upstream or downstream gene promoters (PDLIM3 and ACTA1) specifically upon myogenic differentiation. Our results highlight the importance of considering CRMs located distant from their target genes in investigations of mammalian gene regulation and further support the hypothesis that enhancer-promoter looping contacts are a general mechanism of regulation by distant CRMs. ChIP-chip experiments were performed using a custom Agilent 1x244K oligonucleotide array format (AMADID # 015037) to achieve 25-bp resolution tiling of 100 kb surrounding each of 104 selected human genes as well as positive and negative control regions. Biological and technical replicates of ChIP-chip experiments were performed using antibodies for MyoG and SRF in primary human skeletal muscle myoblasts crosslinked at 0 and 48 h relative to the removal of serum to induce differentiation. Additional technical replicate ChIP-chip experiments were performed for MyoD at 0 and 48 hours post-differentiation and p300 at 48 h post-differentiation. Total Input DNA for each experiment was labeled with Cy3 and used to create log-ratios with the Cy5 labeled IP data. Negative control no antibody mock-IP ChIP experiments were performed at 0 and 48 h post-differentiation.

Project description:STAC3 was identified as a nutritionally regulated gene from an Atlantic salmon subtractive hybridization library with highest expression in skeletal muscle. Salmon STAC3 mRNA was highly correlated with myogenin and myoD1a expression during differentiation of a salmon primary myogenic culture and was regulated by amino acid availability. In zebrafish embryos, STAC3 was initially expressed in myotomal adaxial cells and in fast muscle fibres post-segmentation. Morpholino knockdown resulted in defects in myofibrillar protein assembly, particularly in slow muscle fibres, and decreased levels of the hedgehog receptor patched. The function of STAC3 was further characterized in vitro using the mammalian C2C12 myogenic cell line. STAC3 mRNA expression increased during the differentiation of the C2C12 myogenic cell line. Knockdown of STAC3 by RNAi inhibited myotube formation, and microarray analysis revealed that transcripts involved in cell cycle, focal adhesion, cytoskeleton and the pro-myogenic factors IGFBP-5 and IGF2 were down regulated. RNAi-treated cells had suppressed AKT signalling and exogenous IGF2 was unable to rescue the phenotype, however, IGF/AKT signalling was not blocked. Overexpression of STAC3, which results in increased levels of IGFBP-5 mRNA, did not lead to increased differentiation. In synchronized cells, STAC3 mRNA was most abundant during the G1 phase of the cell cycle. RNAi-treated cells were smaller, had higher proliferation rates and decreased proportion of cells in G1 phase when compared to controls, suggesting a role in the G1 phase checkpoint. These results identify STAC3 as a new gene required for myogenic differentiation and myofibrillar protein assembly in vertebrates. We identified STAC3 as being essential for myogenic differentiation. The goal of the microarray analysis was to identify genes that were up- or down-regulated in C2C12 cells when STAC3 expression was inhibited by interfering RNA (RNAi). The microarray compares the C2C12 mouse myogenic cell line between control samples to samples treated with an interfering RNA directed at STAC3. The mouse C2C12 cell line was used at two time points: day 0, which was 24 hours after adding the RNAi, with cells growing in growth media (non-differentiating), and day 1, which was 48 hours after the interfering RNAi was added, and 24 hours after switching to differentiation media. RNAi-treated cells were transfected with an RNAi directed against STAC3, and control cells were transfected with a high-GC content control RNAi. Total RNA was extracted from control and RNAi-treated samples at two time points, at day 0 (24 h after treating with RNAi, while samples were still in growth media), and at day 1 (cells grown in differentiation media for 24h, 48 h after RNAi treatment). At each time point, STAC3 RNAi-treated cells were compared to controls. For the array comparison, at day 0, triplicate biological samples + one technical replicate were used for the controls, and four biological samples were used for the RNAi-treated cells. At day 1, four biological samples and one technical replicate were used for controls and RNAi-treated cells. Technical replicate identifiers are appended by _r.

Project description:The adult skeletal muscle is a plastic tissue with a remarkable ability to adapt to different levels of activity by altering its excitability, its contractile and metabolic phenotype and its mass. Knowledge on the mechanisms responsible for muscle mass comes primarily from models of muscle inactivity or denervation or from genetic models of muscle diseases. Given that the underlying exercise-induced transcriptional mechanisms regulating muscle mass are not fully understood, here we investigated the cellular and molecular adaptive mechanisms taking place in fast skeletal muscle of adult zebrafish in response to swimming. Fish were trained at low swimming speed (0.1 m/s; non-exercised) or at their optimal swimming speed (0.4 m/s; exercised). A significant increase in fibre cross-sectional area (1,290 M-BM-1 88 vs. 1,665 M-BM-1 106 M-NM-<m2) and vascularization (298 M-BM-1 23 vs. 458 M-BM-1 38 capillaries/mm2) was found in exercised over non-exercised fish. Gene expression profiling evidenced the transcriptional activation of a series of complex networks of extracellular and intracellular signaling molecules and pathways involved in the regulation of muscle mass, myogenesis and angiogenesis, many (e.g. BMP, TGFM-oM-^AM-", FGF, Notch, Wnt, MEF2, Shh, EphrinB2) not previously associated with exercise-induced contractile activity, and that recapitulate in part the transcriptional events occurring during skeletal muscle regeneration. These results demonstrate that fibre hypertrophy is responsible for the growth-promoting effects of exercise accompanied by a switch to a more oxidative capacity of white muscle fibres to fuel the increased energy demands. Importantly, our study identified novel molecular mechanisms regulating muscle mass and function in vertebrates. Adult zebrafish were subjected or not to a swim training regime consisting of swimming at the optimal swimming speed for this species for 6 h/day, 5 days/week for a total of 4 weeks (20 experimental days). Total RNA of fast muscle from individual non-exercised (n = 8) and exercised (n = 8) zebrafish was analyzed.

Project description:STAC3 was identified as a nutritionally regulated gene from an Atlantic salmon subtractive hybridization library with highest expression in skeletal muscle. Salmon STAC3 mRNA was highly correlated with myogenin and myoD1a expression during differentiation of a salmon primary myogenic culture and was regulated by amino acid availability. In zebrafish embryos, STAC3 was initially expressed in myotomal adaxial cells and in fast muscle fibres post-segmentation. Morpholino knockdown resulted in defects in myofibrillar protein assembly, particularly in slow muscle fibres, and decreased levels of the hedgehog receptor patched. The function of STAC3 was further characterized in vitro using the mammalian C2C12 myogenic cell line. STAC3 mRNA expression increased during the differentiation of the C2C12 myogenic cell line. Knockdown of STAC3 by RNAi inhibited myotube formation, and microarray analysis revealed that transcripts involved in cell cycle, focal adhesion, cytoskeleton and the pro-myogenic factors IGFBP-5 and IGF2 were down regulated. RNAi-treated cells had suppressed AKT signalling and exogenous IGF2 was unable to rescue the phenotype, however, IGF/AKT signalling was not blocked. Overexpression of STAC3, which results in increased levels of IGFBP-5 mRNA, did not lead to increased differentiation. In synchronized cells, STAC3 mRNA was most abundant during the G1 phase of the cell cycle. RNAi-treated cells were smaller, had higher proliferation rates and decreased proportion of cells in G1 phase when compared to controls, suggesting a role in the G1 phase checkpoint. These results identify STAC3 as a new gene required for myogenic differentiation and myofibrillar protein assembly in vertebrates. We identified STAC3 as being essential for myogenic differentiation. The goal of the microarray analysis was to identify genes that were up- or down-regulated in C2C12 cells when STAC3 expression was inhibited by interfering RNA (RNAi). The microarray compares the C2C12 mouse myogenic cell line between control samples to samples treated with an interfering RNA directed at STAC3. The mouse C2C12 cell line was used at two time points: day 0, which was 24 hours after adding the RNAi, with cells growing in growth media (non-differentiating), and day 1, which was 48 hours after the interfering RNAi was added, and 24 hours after switching to differentiation media.

Dataset Information

Transcriptome analysis of zebrafish mononuclear myogenic cells (zMNCs) during myogenic differentiation

Publications

Isolation and transcriptome analysis of adult zebrafish cells enriched for skeletal muscle progenitors.

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