Genome-wide mapping of Sox6 binding sites in skeletal muscle reveals both direct and indirect regulation of muscle terminal differentiation by Sox6

Division of Cardiovascular Medicine, Department of Internal Medicine, University of California, Davis, One Shields Avenue, Davis, California 95616, USA.
BMC Developmental Biology (Impact Factor: 2.67). 10/2011; 11(1):59. DOI: 10.1186/1471-213X-11-59
Source: PubMed


Sox6 is a multi-faceted transcription factor involved in the terminal differentiation of many different cell types in vertebrates. It has been suggested that in mice as well as in zebrafish Sox6 plays a role in the terminal differentiation of skeletal muscle by suppressing transcription of slow fiber specific genes. In order to understand how Sox6 coordinately regulates the transcription of multiple fiber type specific genes during muscle development, we have performed ChIP-seq analyses to identify Sox6 target genes in mouse fetal myotubes and generated muscle-specific Sox6 knockout (KO) mice to determine the Sox6 null muscle phenotype in adult mice.
We have identified 1,066 Sox6 binding sites using mouse fetal myotubes. The Sox6 binding sites were found to be associated with slow fiber-specific, cardiac, and embryonic isoform genes that are expressed in the sarcomere as well as transcription factor genes known to play roles in muscle development. The concurrently performed RNA polymerase II (Pol II) ChIP-seq analysis revealed that 84% of the Sox6 peak-associated genes exhibited little to no binding of Pol II, suggesting that the majority of the Sox6 target genes are transcriptionally inactive. These results indicate that Sox6 directly regulates terminal differentiation of muscle by affecting the expression of sarcomere protein genes as well as indirectly through influencing the expression of transcription factors relevant to muscle development. Gene expression profiling of Sox6 KO skeletal and cardiac muscle revealed a significant increase in the expression of the genes associated with Sox6 binding. In the absence of the Sox6 gene, there was dramatic upregulation of slow fiber-specific, cardiac, and embryonic isoform gene expression in Sox6 KO skeletal muscle and fetal isoform gene expression in Sox6 KO cardiac muscle, thus confirming the role Sox6 plays as a transcriptional suppressor in muscle development.
Our present data indicate that during development, Sox6 functions as a transcriptional suppressor of fiber type-specific and developmental isoform genes to promote functional specification of muscle which is critical for optimum muscle performance and health.

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    • "The slow-fiber type program appears to be partially promoted by PGC-1 (Rasbach, Gupta et al. 2010, Summermatter, Thurnheer et al. 2012), PPARγ (Luquet, Lopez-Soriano et al. 2003) and MEF2 (Wu, Naya et al. 2000). At the transcriptional level the slow program can be enhanced by Pdrm1a which represses the transcriptional factor Sox 6 (von Hofsten, Elworthy et al. 2008, Wang, Ono et al. 2011), a transcriptional repressor of the slow-fiber type program (Hagiwara, Ma et al. 2005, Quiat, Voelker et al. 2011). The fast-fiber type program is dependent upon the six transcriptional complex (STC), where elimination of Six1, Six4 and the cofactor Eya1 can prevent fast-twitch muscle fiber formation (Grifone, Laclef et al. 2004, Niro, Demignon et al. 2010, Richard, Demignon et al. 2011). "
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    ABSTRACT: Satellite cells derived from fast and slow muscles have been shown to adopt contractile and metabolic properties of their parent muscle. Mouse muscle shows less distinctive fiber-type profiles than rat or rabbit muscle. Therefore, in this study we sought to determine whether three-dimensional muscle constructs engineered from slow soleus (SOL) and fast tibialis anterior (TA) from mice would adopt the contractile and metabolic properties of their parent muscle. Time-to-peak tension (TPT) and half-relaxation time (1/2RT) was significantly slower in SOL constructs. In agreement with TPT, TA constructs contained significantly higher levels of fast myosin heavy chain (MHC) and fast troponin C, I, and T isoforms. Fast SERCA protein, both slow and fast calsequestrin isoforms and parvalbumin were found at higher levels in TA constructs. SOL constructs were more fatigue resistant and contained higher levels of the mitochondrial proteins SDH and ATP synthase and the fatty acid transporter CPT-1. SOL constructs contained lower levels of the glycolytic enzyme phosphofructokinase but higher levels of the β-oxidation enzymes LCAD and VLCAD suggesting greater fat oxidation. Despite no changes in PGC-1α protein, SOL constructs contained higher levels of SIRT1 and PRC. TA constructs contained higher levels of the slow-fiber program repressor SOX6 and the six transcriptional complex (STC) proteins Eya1and Six4 which may underlie the higher in fast-fiber and lower slow-fiber program proteins. Overall, we have found that muscles engineered from predominantly slow and fast mouse muscle retain contractile and metabolic properties of their native muscle. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
    Journal of Cellular Physiology 10/2014; 230(8). DOI:10.1002/jcp.24848 · 3.84 Impact Factor
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    • "Different studies have demonstrated that Sox6-null muscle has increased levels of I/β slowMyHC and a general switch towards a slower phenotype [79–81]. It has been shown that Sox6 exerts its function by direct binding to the I/β slowMyHC promoter [79, 80, 82]. Moreover, the group of Olson identified a microRNA (miR)-mediated transcriptional regulatory network. "
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    ABSTRACT: Skeletal myogenesis has been and is currently under extensive study in both mammals and teleosts, with the latter providing a good model for skeletal myogenesis because of their flexible and conserved genome. Parallel investigations of muscle studies using both these models have strongly accelerated the advances in the field. However, when transferring the knowledge from one model to the other, it is important to take into account both their similarities and differences. The main difficulties in comparing mammals and teleosts arise from their different temporal development. Conserved aspects can be seen for muscle developmental origin and segmentation, and for the presence of multiple myogenic waves. Among the divergences, many fish have an indeterminate growth capacity throughout their entire life span, which is absent in mammals, thus implying different post-natal growth mechanisms. This review covers the current state of the art on myogenesis, with a focus on the most conserved and divergent aspects between mammals and teleosts.
    Cellular and Molecular Life Sciences CMLS 03/2014; 71(16). DOI:10.1007/s00018-014-1604-5 · 5.81 Impact Factor
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    • "Because both Trip12 knockdown and 26S proteasome inhibition in myotube cultures resulted in increased Sox6 protein levels (Figures 4A, 4B, 5D and 5E), and decreased mRNA levels of the Sox6 target Myh7 (Figures 4C and 5F), we hypothesized that knockdown of Trip12 would result in fiber type-specific gene expression changes opposite to those observed in the Sox6 KO mouse, i.e., an increase in fast fiber-specific gene expression and a decrease in slow fiber-specific gene expression [9,12]. "
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    ABSTRACT: Background A sophisticated level of coordinated gene expression is necessary for skeletal muscle fibers to obtain their unique functional identities. We have previously shown that the transcription factor Sox6 plays an essential role in coordinating muscle fiber type differentiation by acting as a transcriptional suppressor of slow fiber-specific genes. Currently, mechanisms regulating the activity of Sox6 in skeletal muscle and how these mechanisms affect the fiber phenotype remain unknown. Methods Yeast two-hybrid screening was used to identify binding partners of Sox6 in muscle. Small interfering RNA (siRNA)-mediated knockdown of one of the Sox6 binding proteins, Trip12, was used to determine its effect on Sox6 activity in C2C12 myotubes using quantitative analysis of fiber type-specific gene expression. Results We found that the E3 ligase Trip12, a HECT domain E3 ubiquitin ligase, recognizes and polyubiquitinates Sox6. Inhibiting Trip12 or the 26S proteasome activity resulted in an increase in Sox6 protein levels in C2C12 myotubes. This control of Sox6 activity in muscle cells via Trip12 ubiquitination has significant phenotypic outcomes. Knockdown of Trip12 in C2C12 myotubes led to upregulation of Sox6 protein levels and concurrently to a decrease in slow fiber-specific Myh7 expression coupled with an increased expression in fast fiber-specific Myh4. Therefore, regulation of Sox6 cellular levels by the ubiquitin-proteasome system can induce identity-changing alterations in the expression of fiber type-specific genes in muscle cells. Conclusions Based on our data, we propose that in skeletal muscle, E3 ligases have a significant role in regulating fiber type-specific gene expression, expanding their importance in muscle beyond their well-established role in atrophy.
    Skeletal Muscle 05/2013; 3(1):11. DOI:10.1186/2044-5040-3-11
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