An unresolved and critically important question in skeletal muscle biology is how muscle stem cells initiate and regulate the genetic program during muscle development. Epigenetic dynamics are essential for cellular development and organogenesis in early life and it is becoming increasingly clear that epigenetic remodeling may also be responsible for the cellular adaptations that occur in later life. DNA methylation of cytosine bases within CpG dinucleotide pairs is an important epigenetic modification that reduces gene expression when located within a promoter or enhancer region. Recent advances in the field suggest that epigenetic regulation is essential for skeletal muscle stem cell identity and subsequent cell development. This review summarizes what is currently known about how skeletal muscle stem cells regulate the myogenic program through DNA methylation, discusses a novel role for metabolism in this process, and addresses DNA methylation dynamics in adult skeletal muscle in response to physical activity.
The term “epigenetics” literally means “above genetics” and is defined by the NIH Roadmap Epigenomics project as “both heritable changes in gene activity and expression (in the progeny of cells or of individuals) and also stable, long-term alterations in the transcriptional potential of a cell that are not necessarily heritable.” Epigenetics underlies the ability of embryonic stem cells (with an identical DNA code) to commit to the three germ layers (mesoderm, endoderm, and ectoderm) during the early stages of development and eventually commit to specific cell fates to generate all the different cell types in an organism, including skeletal muscle. These biological trait variations are not a result of changes in the DNA code, but rather structural modifications to the DNA and/or histones, or posttranscriptional gene silencing via small RNAs (including miRNA, siRNA, and piRNA) [
Considering the interest surrounding epigenetics and in particular DNA methylation, in the regulation of stem cell identity, this review aims to discuss some of the recent findings regarding methylation, with a particular focus on skeletal muscle stem cells (MuSCs, also referred to as satellite cells). While not discussed in this review, it is worth mentioning that, in addition to direct DNA modifications, structural epigenetic control is conferred at the level of histones. The core histone proteins H2A, H2B, H3, and H4 all contain long N-terminal tails which are highly susceptible to posttranslational modifications including methylation (me), acetylation (ac), phosphorylation (p), SUMOylation (sumo), ubiquitination (ub), ADP-ribosylation (ADP), and citrullination (cit) (reviewed in [
Before discussing the role of DNA methylation in MuSC biology, it is essential to first define the process of methylation. Methylation of DNA is a well-described phenomenon and primarily occurs on the 5′ position of cytosine bases within CpG dinucleotide pairs and leads to the formation of 5-methylcytosine (5mC) and a context specific effect on transcription. DNA methylation within the promoter region of genes is typically linked to transcriptional repression due to recruitment of methyl CpG binding domain (MBD) proteins, which block transcription factor and RNA polymerase access [
The processes of DNA methylation and demethylation are carefully regulated by a family of DNA methyltransferases (DNMTs) and demethylases (the ten-eleven translocation (TET) enzymes) (Figure
Transient DNA methylation and demethylation via specific
DNA methylation was originally thought to occur exclusively during germ cell development and in preimplantation embryos [
Skeletal muscle is derived from a population of mesodermal progenitor cells that undergo proliferation, differentiation, fusion, and maturation to form skeletal muscle fibers, a process known as myogenesis. Importantly, a subpopulation of these cells exit the cell cycle early and enter a state of quiescence (
The paired domain homeobox 3 (Pax3) transcription factor is critical for successful migration of myogenic progenitor cells to the developing limb bud and subsequent muscle formation [
Indeed, in skeletal muscle biology, one of the most intriguing and pressing questions relates to the processes of MuSC activation, specification to the myogenic lineage, and eventual differentiation. Several studies have provided important evidence linking methylation of the promoter and enhancer regions of myogenic regulators to the initiation of the myogenic transcriptional program in the somites [
The differential regulation of DNMT and TET expression and activity during muscle development is critical for understanding the link between environmental cues, intracellular signaling, DNA methylation, and gene expression. Evidence suggests that these methyltransferases and demethylases may be regulated in an isoform-specific manner during myogenesis. Indeed,
Advances in fluorescent activated cell sorting (FACS) techniques, coupled with downstream gene arrays (Affymetrix microarrays) or whole transcriptome sequencing (RNAseq), have allowed for the generation of transcriptome signatures for pure stem cell populations, including MuSCs [
A summary of differential gene expression in DNA methyltransferases and demethylases following MuSC activation (fold change compared to quiescent MuSCs).
Ryall et al. 2015 [ |
Pallafacchina et al. 2010 [ |
Liu et al. 2013 [ |
Pallafacchina et al. 2010 [ |
Pallafacchina et al. 2010 [ | |
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|
↑ 4-fold | ↑ 6-fold | ↑ 6-fold | ↑ 7-fold | ↑ 3-fold |
|
↓ 3-fold | ↓ 3-fold | NA | ↑ 3-fold | |
|
NA | NA | NA | NA | |
|
↓ 10-fold | NA | ↓ 5-fold | NA | NA |
|
↓ 2-fold | ↓ 13-fold | ↓ 3-fold | NA | NA |
|
↓ 2-fold | NA | NA | NA | NA |
DNMT: DNA methyltransferase; Tet: ten-eleven translocase; MuSC: muscle stem cell; NA: not available.
While the methylation status of quiescent MuSCs has not been investigated in detail, several authors have attempted to define a DNA methylation signature in proliferating versus differentiating MuSC cultures. Tsumagari et al. (2013) assessed DNA methylation in proliferating human myoblasts and differentiated myotubes but did not find significant differences between methylation patterns [
The specific enrichment of 5hmC in either gene bodies or enhancer regions is often associated with activation and has been identified in human embryonic stem cells [
Brunk and colleagues were the first to perform studies that linked DNA methylation to muscle cell identity [
Since the seminal work by Brunk and colleagues in 1996, Carrió et al. investigated the methylation status of a 110 kb enhancer region of Myf5/Myf6 (known as a “super-enhancer” because it has a high density of enhancer elements) [
To better characterize the role of DNA demethylation in myogenic development, several studies have utilized 5-azacytidine (5AC), a potent inhibitor of DNA methylation, which acts via the sequestration of DNMT1 and results in global loss of methylation. Mouse fibroblasts (C3H10T1/2) treated with 5AC for 10 days resulted in the emergence of several cell types including those of adipogenic and osteogenic lineages. However, the majority of cells underwent transformation towards the myogenic lineage [
In the immortalized C2C12 myogenic cell line, proliferating myoblasts treated with 5AC exhibited increased expression of muscle-specific genes (including myogenin), enhanced myotube maturation, spontaneous contraction, and Ca2+ transients [
The generation of
In addition to the differential regulation of
Recent work has identified a process of metabolic reprogramming in MuSCs as they move from quiescence to proliferation, with fatty-acid oxidation predominating during quiescence and glycolysis increasing during proliferation [
Several studies have compared CpG methylation patterns in adult skeletal muscle to that in other tissue types in order to define the DNA methylation signature of skeletal muscle. One human study assessed seventeen thousand CpG islands of which 178 were specifically hypermethylated in skeletal muscle compared with other cell types including blood, sperm, brain, and spleen [
It is now accepted that DNA methylation is a dynamic process, and as skeletal muscle is a highly plastic tissue able to rapidly respond to changes in demand, DNA methylation may be a particularly important mediator of these adaptations. Skeletal muscle responds to endurance and resistance training through adaptation of contractile apparatus and metabolic capacity. Barrès and colleagues have previously reported that acute exercise, in humans and mice, is linked to transient DNA demethylation in the promoter region of genes including peroxisome proliferator activated receptor
There is also mounting evidence that during perinatal development skeletal muscle is susceptible to insults or stimuli that may alter the epigenetic program, which has consequences for gene transcription and functional outcomes later in life [
Additional studies are warranted to further characterize how DNA methylation and hydroxymethylation differ between MuSCs in different dynamic states and what specifically regulates these methylation events. In doing so, these studies will reveal novel mechanisms to regulate MuSC identity and growth. Furthermore, the identification of isoform-specific roles for
The authors declare that there is no conflict of interests regarding the publication of this paper.