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

Abstract

BackgroundSox6 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.ResultsWe 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.ConclusionsOur 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.

Loss ofProx1in striated muscle causes slow to fast skeletal muscle fiber conversion and dilated cardiomyopathy

“…4A), which is ordinarily enriched in slow-twitch muscle (5,29). In contrast, levels of the fast-twitch MyHC genes Myh2 and Myh1 were significantly up-regulated, although Myh4-the fastest MyHC gene-expression remained equivalent between all genotypes (Fig.…”

“…Recently, Prospero-related homeobox factor 1 (Prox1) expression was shown to be enriched in murine slow-twitch skeletal muscle (5), and this is conserved in the zebrafish, where Prox1 is required for slow-twitch myofibril assembly (6). Previously we demonstrated an essential role for Prox1 in normal heart development in the mouse, with cardiac-specific Prox1 mutant embryos characterized by impaired cardiac growth, reduced fetal cardiomyocyte hypertrophy, persistent ventricular septal defects (VSDs), and myofibrillar disarray (7).…”

“…These include hormones, such as thyroid hormone (9), as well as epigenetic modifications, micro RNAs (miRs), and a cohort of transcription factors (10,11). Several loss-of-function studies, including targeting of Histone deacetylases 1 and 2 (HDAC 1/2) and miR-208a deletion in the mouse heart and skeletal-muscle-specific knockout of Sox6 and miR-208b/miR-499, have reported aberrant expression of fasttwitch skeletal MyHC, MyLC, and troponin complex gene isoforms (5,12,13). In the slow-twitch skeletal muscle of the zebrafish, these fast-twitch genes are negatively regulated by the transcription factor Prdm1 (Blimp-1), which also inhibits the expression of Sox6, a transcriptional repressor of slow-twitch contractile genes (6,14).…”

Positive feedback loop between Sox2 and Sox6 inhibits neuronal differentiation in the developing central nervous system

“…How Sox6 exactly performs its function also is an issue to be resolved. A recent genome-wide mapping of Sox6 targets by ChIP-seq analysis in skeletal muscles revealed that Sox6 binds and represses a group of genes to be specifically expressed in slow-fiber muscle cells, indicating that Sox6 regulates the balance between slow-twitch and fast-twitch muscle fiber groups (18). It would be interesting to see if a parallel mechanism is in place for the regulation of CNS development, and genomewide mapping studies for Sox6 binding and Sox6-dependent expression profiling assays in NPC should add to our understanding of the self-renewal of NPC in the developing CNS.…”

Prokaryotic expression and purification of porcine Sox6

“…In Sox6 null-p100H mutant mice, all fetal muscle fibers have slow-twitch fiber characteristics, and the slow-twitch myosin heavy chain is increasingly expressed (Hagiwara et al, 2007). The knockdown of Sox6 in skeletal muscle causes a dramatic increase in slow-twitch fibers and a significant decrease in fast-twitch fibers in fetal as well as in adult mice (Hagiwara et al, 2007;An et al, 2011;Quiat et al, 2011). These findings indicate that Sox6 plays a critical role in embryonic muscle development and adult fast myofiber maintenance.…”