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PAX7 is a paired-homeobox transcription factor that specifies the myogenic identity of muscle stem cells and acts as a nodal factor by stimulating proliferation while inhibiting differentiation. We previously found that PAX7 recruits the H3K4 methyltransferases MLL1/2 to epigenetically activate target genes. Here we report that in the absence of Mll1, myoblasts exhibit reduced H3K4me3 at both Pax7 and Myf5 promoters and reduced Pax7 and Myf5 expression. Mll1-deficient myoblasts fail to proliferate but retain their differentiation potential, while deletion of Mll2 had no discernable effect. Re-expression of PAX7 in committed Mll1 cKO myoblasts restored H3K4me3 enrichment at the Myf5 promoter and Myf5 expression. Deletion of Mll1 in satellite cells reduced satellite cell proliferation and self-renewal, and significantly impaired skeletal muscle regeneration. Pax7 expression was unaffected in quiescent satellite cells but was markedly downregulated following satellite cell activation. Therefore, MLL1 is required for PAX7 expression and satellite cell function in vivo. Furthermore, PAX7, but not MLL1, is required for Myf5 transcriptional activation in committed myoblasts., PAX7 transcription factor specifies the myogenic identity of muscle stem cells and acts as a nodal factor by stimulating proliferation while inhibiting differentiation. Here authors find that Mll1 deletion in myoblasts in mice results in reduced H3K4me3 at both Pax7 and Myf5 promoters, reduced Pax7 and Myf5 expression, and proliferation defects.

Muscle stem cell function has been suggested to be regulated by Acetyl-CoA and NAD+ availability, but the mechanisms remain unclear. Here we report the identification of two acetylation sites on PAX7 that positively regulate its transcriptional activity. Lack of PAX7 acetylation reduces DNA binding, specifically to the homeobox motif. The acetyltransferase MYST1 stimulated by Acetyl-CoA, and the deacetylase SIRT2 stimulated by NAD +, are identified as direct regulators of PAX7 acetylation and asymmetric division in muscle stem cells. Abolishing PAX7 acetylation in mice using CRISPR/Cas9 mutagenesis leads to an expansion of the satellite stem cell pool, reduced numbers of asymmetric stem cell divisions, and increased numbers of oxidative IIA myofibers. Gene expression analysis confirms that lack of PAX7 acetylation preferentially affects the expression of target genes regulated by homeodomain binding motifs. Therefore, PAX7 acetylation status regulates muscle stem cell function and differentiation potential to facilitate metabolic adaptation of muscle tissue., The acetyltransferase MYST1 stimulated by acetyl-CoA, and the deacetylase SIRT2 stimulated by NAD+, regulate PAX7 acetylation in muscle stem cells, which in turn, regulates stem cell self-renewal and regeneration following injury in mouse skeletal muscle.

Satellite cells are required for the growth, maintenance, and regeneration of skeletal muscle. Quiescent satellite cells possess a primary cilium, a structure that regulates the processing of the GLI family of transcription factors. Here we find that GLI3, specifically, plays a critical role in satellite cell activation. Primary cilia-mediated processing of GLI3 is required to maintain satellite cells in a G0 dormant state. Strikingly, satellite cells lacking GLI3 enter GAlert in the absence of injury. Furthermore, GLI3 depletion or inhibition of its processing stimulates symmetrical division in satellite cells and expansion of the stem cell pool. As a result, satellite cells lacking GLI3 display rapid cell-cycle entry, increased proliferation and augmented self-renewal, and markedly enhanced long-term regenerative capacity. Therefore, our results reveal an essential role for primary cilia processing of GLI3 in regulating muscle stem cell activation and fate.

Satellite cells are required for the growth, maintenance, and regeneration of skeletal muscle. Quiescent satellite cells possess a primary cilium, a structure that regulates the processing of the GLI family of transcription factors. Here we find that GLI3 processing by the primary cilium plays a critical role for satellite cell function. GLI3 is required to maintain satellite cells in a G

Exercise training improves skeletal muscle function, notably through tissue regeneration by muscle stem cells. Here, we hypothesized that exercise training reprograms the epigenome of muscle cell, which could account for better muscle function. Genome-wide DNA methylation of myotube cultures established from middle-aged obese men before and after endurance exercise training identified a differentially methylated region (DMR) located downstream ofGremlin 1(GREM1), which was associated with increasedGREM1expression. GREM1 expression was lower in muscle satellite cells from obese, compared to lean mice, and exercise training restored GREM1 levels to those of control animals. We show that GREM1 regulates muscle differentiation through the negative control of satellite cell self-renewal, and that GREM1 controls muscle lineage commitment and lipid oxidation through the AMPK pathway. Our study identifies novel functions of GREM1 and reveals an epigenetic mechanism by which exercise training reprograms muscle stem cells to improve skeletal muscle function.

The Myogenic Regulatory Factors (MRFs) Myf5, MyoD, myogenin and MRF4 are members of the basic helix-loop-helix family of transcription factors that control the determination and differentiation of skeletal muscle cells during embryogenesis and postnatal myogenesis. The dynamics of their temporal and spatial expression as well as their biochemical properties have allowed the identification of a precise and hierarchical relationship between the four MRFs. This relationship establishes the myogenic lineage as well as the maintenance of the terminal myogenic phenotype. The application of genome-wide technologies has provided important new information as to how the MRFs function to activate muscle gene expression. Application of combined functional genomics technologies along with single cell lineage tracing strategies will allow a deeper understanding of the mechanisms mediating myogenic determination, cell differentiation and muscle regeneration.

The transcription factor PAX7 is a critical regulator of satellite cell survival, self-renewal and proliferation. PAX7 protein is regulated by different post-translational modifications that affect its transcriptional activity, and consequently, influence muscle regeneration. Here, we report two novel acetylation sites on the PAX7 protein, which regulate its transcriptional activity and chromatin binding. Abolishing PAX7 acetylation using CRISPR/Cas9 mutant mice impairs muscle regeneration and leads to progressive satellite cell exhaustion. We identified molecular regulators of PAX7 acetylation, the acetyltransferase MYST1 as well as with the deacetylase SIRT2, both of which control PAX7 acetylation levels and PAX7 target gene expression in myoblasts. MYST1 and SIRT2 are also important players in regulating the balance between satellite stem cell symmetric versus asymmetric division, therefore controlling satellite cell expansion, self-renewal and commitment. Our data demonstrate that acetylation levels regulate PAX7 transcriptional activity and function in satellite cells.

Adult satellite cells are quiescent, but are poised for activation in response to exercise, injury, or disease allowing adult muscle growth or repair. Once activated, satellite cells proliferate extensively to produce enough myogenic progenitors in order to regenerate the muscles. In order to self-renew, a subset of activated satellite cells can resist the myogenic differentiation and return to quiescence to replenish the satellite cell pool. These cellular processes that normally occur during skeletal muscle regeneration can be recapitulated ex vivo using isolated and cultured myofibers. Here, we describe a protocol to isolate single myofibers from the extensor digitorum longus muscle. Moreover, we detail experimental conditions for analyzing satellite cells in quiescence and progression through the myogenic lineage.

Adult satellite cells are quiescent, but are poised for activation in response to exercise, injury, or disease allowing adult muscle growth or repair. Once activated, satellite cells proliferate extensively to produce enough myogenic progenitors in order to regenerate the muscles. In order to self-renew, a subset of activated satellite cells can resist the myogenic differentiation and return to quiescence to replenish the satellite cell pool. These cellular processes that normally occur during skeletal muscle regeneration can be recapitulated ex vivo using isolated and cultured myofibers. Here, we describe a protocol to isolate single myofibers from the extensor digitorum longus muscle. Moreover, we detail experimental conditions for analyzing satellite cells in quiescence and progression through the myogenic lineage.

The regenerative capacity of skeletal muscle is due to a population of satellite cells known as satellite stem cells. Owing to their ability to generate both stem cells and committed myogenic progenitors, satellite stem cells allow self-renewal of the satellite cell reservoir and provide myogenic progenitor cells to repair the muscle tissue. Increasing numbers of studies have highlighted the indispensable role of the niche in the regulation of the stem cell functions. The niche maintains the muscle stem cell in a quiescent state but in response to muscle injury, the niche actively generates signals for satellite cell activation, proliferation, and differentiation. The cross talk between the muscle stem cell and its niche is critical and alterations of the niche components result in defective regeneration and muscle pathologies. Here, we describe how the satellite cell niche regulates satellite cell functions, namely commitment and self-renewal, in resting, injured, and pathologic muscle.

Summary Loss of dystrophin expression in Duchenne muscular dystrophy (DMD) causes progressive degeneration of skeletal muscle, which is exacerbated by reduced self-renewing asymmetric divisions of muscle satellite cells. This, in turn, affects the production of myogenic precursors and impairs regeneration and suggests that increasing such divisions may be beneficial. Here, through a small-molecule screen, we identified epidermal growth factor receptor (EGFR) and Aurora kinase A (Aurka) as regulators of asymmetric satellite cell divisions. Inhibiting EGFR causes a substantial shift from asymmetric to symmetric division modes, whereas EGF treatment increases asymmetric divisions. EGFR activation acts through Aurka to orient mitotic centrosomes, and inhibiting Aurka blocks EGF stimulation-induced asymmetric division. In vivo EGF treatment markedly activates asymmetric divisions of dystrophin-deficient satellite cells in mdx mice, increasing progenitor numbers, enhancing regeneration, and restoring muscle strength. Therefore, activating an EGFR-dependent polarity pathway promotes functional rescue of dystrophin-deficient satellite cells and enhances muscle force generation.

Age-related frailty may be due to decreased skeletal muscle regeneration. The role of TGF-β molecules myostatin and GDF11 in regeneration is unclear. Recent studies showed an age-related decrease in GDF11 and that GDF11 treatment improves muscle regeneration, which were contrary to prior studies. We now show that these recent claims are not reproducible and the reagents previously used to detect GDF11 are not GDF11 specific. We develop a GDF11-specific immunoassay and show a trend toward increased GDF11 levels in sera of aged rats and humans. GDF11 mRNA increases in rat muscle with age. Mechanistically, GDF11 and myostatin both induce SMAD2/3 phosphorylation, inhibit myoblast differentiation, and regulate identical downstream signaling. GDF11 significantly inhibited muscle regeneration and decreased satellite cell expansion in mice. Given early data in humans showing a trend for an age-related increase, GDF11 could be a target for pharmacologic blockade to treat age-related sarcopenia.

This review describes the recent findings on epigenetic regulation in satellite stem cells and committed myoblasts. It also addresses the potential of epigenetic drugs, such as histone deacetylase inhibitors, and their molecular mechanism of action in muscle cells., Skeletal muscle regeneration is initiated by satellite cells, a population of adult stem cells that reside in the muscle tissue. The ability of satellite cells to self-renew and to differentiate into the muscle lineage is under transcriptional and epigenetic control. Satellite cells are characterized by an open and permissive chromatin state. The transcription factor Pax7 is necessary for satellite cell function. Pax7 is a nodal factor regulating the expression of genes associated with satellite cell growth and proliferation, while preventing differentiation. Pax7 recruits chromatin modifiers to DNA to induce expression of specific target genes involved in myogenic commitment following asymmetric division of muscle stem cells. Emerging evidence suggests that replacement of canonical histones with histone variants is an important regulatory mechanism controlling the ability of satellite cells and myoblasts to differentiate. Differentiation into the muscle lineage is associated with a global gene repression characterized by a decrease in histone acetylation with an increase in repressive histone marks. However, genes important for differentiation are upregulated by the specific action of histone acetyltransferases and other chromatin modifiers, in combination with several transcription factors, including MyoD and Mef2. Treatment with histone deacetylase (HDAC) inhibitors enhances muscle regeneration and is considered as a therapeutic approach in the treatment of muscular dystrophy. This review describes the recent findings on epigenetic regulation in satellite stem cells and committed myoblasts. The potential of epigenetic drugs, such as HDAC inhibitors, as well as their molecular mechanism of action in muscle cells, will be addressed. Significance This review summarizes recent findings concerning the epigenetic regulation of satellite cells in skeletal muscle.

Dystrophin is expressed in differentiated myofibers, in which it is required for sarcolemmal integrity, and loss-of-function mutations in the gene that encodes it result in Duchenne muscular dystrophy (DMD), a disease characterized by progressive and severe skeletal muscle degeneration. Here we found that dystrophin is also highly expressed in activated muscle stem cells (also known as satellite cells), in which it associates with the serine-threonine kinase Mark2 (also known as Par1b), an important regulator of cell polarity. In the absence of dystrophin, expression of Mark2 protein is downregulated, resulting in the inability to localize the cell polarity regulator Pard3 to the opposite side of the cell. Consequently, the number of asymmetric divisions is strikingly reduced in dystrophin-deficient satellite cells, which also display a loss of polarity, abnormal division patterns (including centrosome amplification), impaired mitotic spindle orientation and prolonged cell divisions. Altogether, these intrinsic defects strongly reduce the generation of myogenic progenitors that are needed for proper muscle regeneration. Therefore, we conclude that dystrophin has an essential role in the regulation of satellite cell polarity and asymmetric division. Our findings indicate that muscle wasting in DMD not only is caused by myofiber fragility, but also is exacerbated by impaired regeneration owing to intrinsic satellite cell dysfunction.