In order to understand how Sox6 regulates muscle differentiation at the molecular level, we have performed ChIP-seq analysis to identify Sox6 targets in skeletal myotubes and extended the characterization of the Sox6 null muscle phenotype using muscle specific Sox6 inactivation. Among the 867 Refseq genes found to be associated with Sox6 peaks, the overrepresented GO terms included muscle structure and function, skeletal muscle and heart development, as well as transcriptional regulation. In a concurrently conducted Pol II ChIP-seq analysis, we found that the majority of the Sox6 peak-associated genes exhibited little to no recognizable binding peaks, suggesting that Sox6 mainly functions as a transcription suppressor in developing muscle.
How does Sox6 suppress its target genes? Based on evidence from this and other labs, we can speculate on two possible mechanisms (1 and 2) and, based on evidence accumulated in this report we also demonstrate two other likely mechanisms (3 and 4): (1) Sox6 may fine-tune the transcription of the genes that have been marked by MyoD binding, (2) Sox6 may modulate transcription of its target genes in concert with Tead and Runx factors, (3) Sox6 suppresses transcription by hindering the muscle-specific enhancer activity, and (4) Sox6 also indirectly influences downstream gene expression by regulating the expression of other transcription factors and chromatin modifying enzymes. Below, we will discuss each of these proposed mechanisms in more detail.
MyoD is one of the myogenic regulatory factors and defines the myogenic lineage during development [71
]. In myotubes, MyoD binding events are frequent (~26,000 peaks with a higher cut off, ~60,000 peaks with a lower cut off) and are associated with histone H4 acetylation (H4Ac) [36
], which is a marker of an active chromatin state [73
]. We found that 96% of the Sox6 peaks in fetal myotubes overlapped with, or were in the close vicinity to (within 50 bp), the reported MyoD peaks [34
]. The E-box motifs in the Sox6 peak regions we found were enriched for the CAGCTG E-box sequence (Figure ). Previously, it has been shown that this motif is represented in the peaks more strongly bound in C2C12 myotubes compared to myoblasts, indicating that this E-box motif is mainly associated with the genes regulating muscle differentiation [36
]. Taking this observation together with ours, we speculate that MyoD binding in the myotube would change the chromatin environment in such a way as to allow the approach of additional transcriptional regulators by recruiting the chromatin modifying enzymes [74
], thus allowing the fine-tuning of muscle specific gene expression necessary for the formation of mature skeletal muscle. Sox6 could be one of these additional transcriptional regulators and specify fiber type characteristics during muscle terminal differentiation.
We have previously reported that Sox6 interferes with a MCAT enhancer located in close proximity to the Sox consensus motif in Myh7
, causing suppression of Myh7
]. Tead/MCAT motifs are frequently found in enhancer or promoter regions of muscle specific genes and it has been demonstrated that binding of TEF-1/Tead1 to the MCAT motifs activates transcription of these muscle-specific genes [38
]. In our analysis of the 1,066 Sox6 peaks, we found 203 MCAT motifs. This suggests that the mechanism of Myh7 transcriptional suppression by Sox6 (possibly via physical interference) we reported earlier may be a common mechanism Sox6 uses to suppress genes whose transcription is activated via Tead/MCAT motifs. Our analysis also revealed 559 Runx motifs in the 1,066 Sox6 peaks. Currently, the roles of Runx motif binding factors (Runx-1, -2, and 3) in muscle development are not well known, though there are reports showing that Runx1 plays a role in skeletal muscle differentiation [37
]. In adult skeletal muscle, Runx1 expression is induced by denervation [77
], and muscle-specific Runx1 inactivation leads to accelerated muscle wasting in denervated muscle [76
]. In an earlier stage of muscle differentiation, it has been reported that Runx1 directly interacts with MyoD preferentially in proliferating myoblasts to inhibit terminal differentiation of skeletal muscle [37
]. The authors showed that the Runx1/CBFβ complex recruits suppressive chromatin modifying enzymes (e.g. HDACs), thus inactivating transcription of the MyoD target genes that are necessary for the cell cycle exit and differentiation [37
]. Since the Runx proteins have been shown to function as transcriptional suppressors or activators in different circumstances [78
] (similar to Sox6), the transcriptional outcome of the possible interaction between the Sox6 and Runx proteins needs further investigation.
As demonstrated in the Results section, the Sox6 binding sites in the Tnnc1
first intron and the Tnni
1 5'-upstream region both effectively reduced the activity of the enhancer elements (Figure ). The molecular mechanisms by which Sox6 overrides muscle enhancers is currently under investigation; however, the skeletal muscle MyHC gene clusters may help shed light on this role of Sox6. In the six MyHC isoform genes clustered on the mouse chromosome 11 [Myh3
], only the Myh4
genes were not associated with Sox6 peaks (a Sox6 peak was detected in the 5'-upstream region of Myh13
in one of the two ChIP-seq data sets; data not shown). Therefore, Sox6 may be involved in sequential expression of the MyHC loci, possibly in collaboration with an enhancer element similar to the locus control region (LCR) reported for the globin gene cluster [79
]. This is an appealing hypothesis, because it has been shown that Sox6 (acting as a transcriptional suppressor) regulates sequential expression of the β-globin genes during erythrogenesis [80
] in concert with BCL11A which binds to the globin gene LCR [81
]. There have been reports on transcription factories that unite transcriptionally active genes on separate chromosome regions for coordinated transcription [82
]. It is possible that association of Sox6 with its target sequences inhibits transcriptional initiation by Pol II, thus causing dissociation of Sox6 target genes from transcription factories.
We demonstrated that expression of Tead1
, and Prox1
was upregulated in Sox6 KO skeletal muscle (Figure ), suggesting that Sox6 is a suppressor of these transcriptional regulatory genes. Tead1 (TEF-1) and Tead4 (RTEF-1) are highly expressed in muscle tissues and have been reported to activate muscle specific gene transcription [83
]. Hdac9 is a class IIa HDAC [86
] and functions as a mediator of motor neuron input to skeletal muscle [87
]. Prox1 is expressed in slow muscle in zebrafish [47
]. Since Prox1
is preferentially expressed in slow fiber muscle in control mice (Additional file 1
, Figure S1B) and Sox6 inactivation caused a sizable increase in Prox1
mRNA expression in Sox6 KO muscle, we propose that Prox1 also plays a role in regulation of slow muscle fiber specific gene expression in mice. This observation presents further evidence of evolutionary conservation in the mechanisms regulating muscle fiber type differentiation in vertebrates [19
]. Since there are more transcriptional regulator genes that are closely associated with Sox6 peaks, which we did not have space to discuss in this report, it is likely that Sox6 is part of the transcriptional networks that shape the characteristics of both muscle development and mature muscle functions.
The most striking phenotype of Sox6 null skeletal muscle is the dramatic increase in the expression of multiple slow fiber specific genes. This observation originally led us to hypothesize that Sox6 functions as a transcriptional suppressor of slow fiber specific genes [12
]. In this report, we expanded the gene expression profiling of Sox6 KO skeletal muscle by including cardiac and embryonic muscle isoform genes. Cardiac isoforms Myh6
, as well as embryonic isoforms Myl4
, were upregulated in the Sox6 KO muscle (Figure , Table ). It has been reported that Tnnt2
is upregulated in regenerating dystrophic muscle [89
is expressed in specialized craniofacial muscle, such as jaw and extraocular muscle, but not in limb or other body muscle [90
]. These observations suggest that Sox6 may play a role in not only determining fiber types, but also defining developmental maturity and highly specialized functions of skeletal muscle.
In Sox6 KO muscle, a significant decrease in fast fiber specific gene expression was also observed. This Sox6 KO phenotype could be a secondary effect of the increased slow fiber gene products, or could be regulated indirectly by Sox6. Since we did not find Sox6 peaks associated with fast fiber specific genes, both mechanisms are equally plausible. With regard to indirect regulation, a few possible mechanisms can be hypothesized. For example, expression of the transcription factors Six1 and Six4, activators of fast fiber specific gene expression [29
], could be indirectly suppressed in Sox6 KO muscle during development. Alternatively, downregulation of fast fiber specific genes in Sox6 KO muscle could be caused by changes in microRNA expression. MicroRNAs are known to function as posttranscriptional regulators of gene expression [93
]. A recent report indicates that microRNAs suppress target gene expression predominantly through mRNA degradation [94
], thus, it is plausible to postulate that an increase in microRNAs targeting fast fiber specific genes in Sox6 KO muscle leads to reduced fast fiber specific gene mRNA levels. As described above, we found Sox6 binding peaks associated with Myh6
(Additional file 3
, Figure S2B). In the intron sequences of Myh6
, miR-208a and miR-208b are encoded, respectively [95
]. It has been reported that miR-208 suppresses expression of THRAP1, which promotes fast fiber specific gene expression [96
]. The increased transcription of Myh6
in Sox6 KO muscle, therefore, could lead to upregulation of miR-208, which in turn, suppress fast fiber specific gene expression. However, the actual situation is likely to be more complex. It should be noted that miR-208, along with miR-499, also targets the 3'-UTR region of Sox6
]. MiR-499 is encoded in the intron of Myh7b
], which has a Sox6 binding site in its 5'-upstream region (Additional file 3
, Figure S2C). Since Myh6
, and Myh7b
are all negatively regulated by Sox6 (Figure ), these data suggest that Sox6 and these miRNAs constitute two-way feedback loops.
Figure summarizes both our current results and the reported regulatory mechanisms for Sox6 expression. A recent report on the regulation of Sox6 expression in zebrafish skeletal muscle has demonstrated that Sox6 transcription is positively regulated by MyoD and Myf5, and repression of Sox6 activity in slow fibers is maintained by miR-499 which targets the Sox6 3'-UTR [100
]. We have reported that Sox6 transcription is upregulated when myotube differentiation is induced [13
], therefore, MyoD and Myf5 might also be activating Sox6 transcription during mammalian muscle development. Since MyoD is preferentially expressed in fast fibers in adult mice [59
], it may sustain the higher level of Sox6 expression in adult fast fiber muscles reported here as well as by Quiat et al. [34
]. Although the negative regulation of Sox6 by miR-499 has been already reported in mice [68
], how suppression of Sox6 expression in slow fibers is initiated is not yet understood. Alternatively, it is also possible that Sox6 expression is activated when fast-twitch myotubes emerge during fetal muscle development [101
]. Since fiber type-specific gene expression in mammalian skeletal muscle during development as well as in adult life is very fluid [2
], how Sox6 expression is regulated will be an increasingly important question as we try to understand how muscle fiber type is initially specified, maintained and changed in reseponse to the external signaling.
Figure 9 Summary of the present work concerning the fiber type specification under the control of Sox6. It has been shown that in zebrafish, MyoD and Myf5 are necessary to activate Sox6 gene expression in muscle . This muscle-specific Sox6 activation mechanism (more ...)