In response to muscle injury, muscle stem cells integrate environmental cues in the damaged tissue to mediate regeneration. These environmental cues are tightly regulated to ensure expansion of muscle stem cell population to repair the damaged myofibers while allowing repopulation of the stem cell niche. These changes in muscle stem cell fate result from changes in gene expression that occur in response to cell signaling from the muscle environment. Integration of signals from the muscle environment leads to changes in gene expression through epigenetic mechanisms. Such mechanisms, including post-translational modification of chromatin and nucleosome repositioning, act to make specific gene loci more, or less, accessible to the transcriptional machinery. In youth, the muscle environment is ideally structured to allow for coordinated signaling that mediates efficient regeneration. Both age and disease alter the muscle environment such that the signaling pathways that shape the healthy muscle stem cell epigenome are altered. Altered epigenome reduces the efficiency of cell fate transitions required for muscle repair and contributes to muscle pathology. However, the reversible nature of epigenetic changes holds out potential for restoring cell fate potential to improve muscle repair in myopathies. In this review, we will describe the current knowledge of the mechanisms allowing muscle stem cell fate transitions during regeneration and how it is altered in muscle disease. In addition, we provide some examples of how epigenetics could be harnessed therapeutically to improve regeneration in various muscle pathologies. Keywords: Muscle stem cells, Regeneration, Epigenetics, Cell fate, Duchenne muscular dystrophy Background genome that is used as a blueprint for determining their The development and regeneration of skeletal muscle identity. However, the characteristics of each cell fate are mediated by muscle stem cells (MuSCs), also termed are determined through alternate interpretation of the satellite cells, that act coordinately to ensure the efficient genomic blueprint where epigenetic mechanisms are formation of myofibers while repopulating the niche to used to determine the subset of genes that will be allow for repair after future injuries. To mediate myofi- expressed. These epigenetic mechanisms achieve differ- ber formation, MuSCs must transit through multiple cell ential gene expression by controlling the accessibility of fates before achieving their fully differentiated state. the transcriptional machinery to specific loci. Indeed, Each of these intermediate cell fates share an identical not all genes within the nucleus can be accessible for gene expression as six billion base pairs of genetic infor- mation encoded by human diploid genome must be * Correspondence: firstname.lastname@example.org Sprott Center for Stem Cell Research, Regenerative Medicine Program, highly compacted to reside within the confines of the Ottawa Hospital Research Institute, 501 Smyth Rd, Mailbox 511, Ottawa, ON nuclear membrane. DNA compaction occurs through K1H 8L6, Canada 3 the formation of nucleosomes linking 147 bp and is re- Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, ON K1H 8L6, Canada peated across the genome at 200 bp intervals with the Full list of author information is available at the end of the article © The Author(s). 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Massenet et al. Skeletal Muscle (2021) 11:4 Page 2 of 16 linker histone H1 protein allowing the protection of In contrast to the limited number of characterized inter-nucleosomal DNA. These 10-nm fibers then con- DNA modifications, histones undergo a wide-variety of dense through a disordered self-aggregation to form post-translational modifications (PTMs) including chromatids . While the DNA organization within each acetylation, methylation, phosphorylation, ubiquitina- cell has been suggested to be unique , general rules tion, ADP-ribosylation, and citrullination. Among them, emerge. For instance, genes that are not expressed tend to some histone PTMs are known to allow chromatin com- be associated with the nuclear periphery while transcribed paction while others direct chromatin decompaction. genes tend to aggregate in the nuclear lumen. This distinct For instance, trimethylation of lysine 9 and lysine 27 of nuclear organization between cells is the basis for estab- histone 3 (H3K9me3 and H3K27me3) or trimethylation lishing and maintaining cell-specific gene expression pro- of lysine 20 of histone 4 (H4K20me3) are associated with grams. The chromatin state is fluid to allow expression of local chromatin compaction. This compaction modu- the needed genes in a spacio-temporal manner. One lates TF or RNA polymerase II access to target se- mechanism allowing fluidity of the chromatin structure is quences and leads to the repression of gene expression. the dynamic nature of nucleosome association with DNA In contrast, acetylation of lysine 9 of histone 3 where nucleosome displacement allows incorporation of (H3K9Ac) and lysine 20 of histone 4 (H4K20Ac) and the unmarked histones within newly formed nucleosomes. trimethylation of the lysine 4 of histone 3 (H3K4me3) This histone exchange represents more than a simple lead to relaxation of the chromatin state, improving ac- turnover mechanism, as canonical histones in nucleo- cessibility of the transcription machinery and increasing somes can be replaced by different histone variants. His- local gene expression [4–6]. Presence of relaxed chroma- tone variants alter the chemical properties of nucleosomes tin at gene enhancer caused by monomethylation of ly- and change their stability. sine 20 of histone 3 (H3K20me1) and acetylation of Ability to alter chromatin organization is critical for lysine 27 of histone 3 (H3K27Ac) also modulates gene mediating decisions of cell fate and differentiation. In re- expression [7, 8]. Thus, the relationship between histone sponse to environmental cues, chromatin organization is modifications and transcriptional output reveal how the modified and alters the accessibility of the transcription epigenetic code regulates gene expression. machinery to the gene sequences, which is modulated by In the present review, we will discuss the role for epi- several levels of regulation. The first level lies within the genetic regulators in mediating the regenerative function DNA itself where the presence of specific sequence ele- of MuSCs in adults. The role for diseased and aged ments surrounding the gene can act as promoters or en- muscle environment in modifying the epigenetic land- hancers that serve as binding sites for transcription scape of MuSCs will also be examined. factors (TFs), which can recruit the transcriptional ma- chinery to locus. The ability to recruit the transcriptional MuSCs mediate regeneration of skeletal muscle machinery may be increased by promoter-enhancer in- Residing at the periphery of the muscle fiber between teractions that can occur through chromatin looping to the sarcolemma and the basal lamina , MuSCs lay modulate gene expression. A second level of regulation dormant in a quiescent state, ready to respond to muscle is established through differential accessibility of TFs or injury. The fate of MuSCs is regulated by a series of TFs the transcription machinery to DNA elements. This can that include Pax7 and a family of myogenic basic-Helix- be performed by modulation of chromatin conformation, loop-helix proteins, termed the myogenic regulatory fac- caused by reversible modifications of either DNA or his- tors (MRFs), that include MYOD, MYF5, MRF4, and tones through epigenetic processes. myogenin (MYOG) proteins [10–12]. These muscle TFs DNA methylation is a widely used epigenetic mechan- work with other, more ubiquitous TFs, to establish the ism to regulate chromatin accessibility. The predomin- epigenetic states to regulate muscle regeneration. ant DNA modification in mammals is CpG methylation, MuSC quiescent state is characterized by the expres- where the addition of a methyl group at carbon 5 of the sion of PAX7 and FOXO transcription factors . Myf5 deoxyribonucleotide cytosine alters the affinity of the TF and Myod1 are also transcribed in quiescent MuSCs, but for its DNA element. CpG methylation stabilizes gene si- post-transcriptional regulation prevents their translation + + - - lencing through two potential modes: (I) blocking access (PAX7 FOXO MYF5 MYOD1 ) (Fig. 1)[13, 14]. More of DNA binding proteins required for transcription by specifically, Myf5 mRNA is sequestered in messenger ri- impairing their ability to bind DNA elements or (II) at- bonucleoprotein granules (mRNPs) to avoid its transla- traction of TFs containing methyl-CpG-binding domains tion  while Myod1 mRNA retains an intron that that are able to repress transcription. It has been pro- prevents its translocation out of the nucleus . After posed that DNA methylation does not trigger gene re- muscle injury, MuSC activation leads to symmetric and/ pression but instead stabilizes repression at already or asymmetric division. Asymmetric divisions produce a + + silenced genes . PAX7 MYF5 cell, destined to the myogenic program Massenet et al. Skeletal Muscle (2021) 11:4 Page 3 of 16 Fig. 1 Hierarchy of TF expression during muscle regeneration process. After a muscle injury, muscle stem cells (MuSCs) are activated and exit the quiescence state. Activated MuSC transit to proliferative muscle progenitor cells (myoblasts) which next transit into differentiated myocytes. Myocytes are able to fuse to each other into myotubes, or to newly formed myofibers in order to restore the damaged muscle. In green, expressed genes or proteins; in red, unexpressed genes or proteins + - and a PAX7 MYF5 cell that repopulates the pool of discuss the different epigenetic factors that work in co- quiescent MuSCs. Symmetric division produces two ordination with the myogenic TFs to facilitate transitions + - identical daughter cells: division of Pax7 Myf5 cells ex- in cell fate. We will discuss both in vitro and in vivo ob- pands MuSC pool and division of more committed servations, keeping in mind that studies carried out + + PAX7 MYF5 expands that of MuSCs . Activated in vivo are inherently more difficult to interpret as the + - + + MuSCs exhibit a PAX7 FOXO MYF5 MYOD protein signaling cues can be derived from other cell types expression profile. To achieve this state, Myf5 and within the regenerative muscle environment. Unless Myod1 mRNAs begin to be translated, while PAX7 ex- otherwise stated, studies discussed below were per- pression decreases and FOXO ceases to be expressed. formed in vitro. Activated MuSCs are now poised to rapidly expand their population through continued cell cycle progression in re- Epigenetic regulation of adult myogenesis sponse to environmental signaling cues in the damaged Quiescence and early activation muscle.Asthe MuSCsstart to accumulate,PAX7and Quiescence is a state where cells enter into a reversible MYF5 expression becomes repressed while MYOG becomes cycle arrest in G phase of the cell cycle. Studies investi- -- expressed to drive cell cycle exit and form myocytes (PAX7 gating the role of chromatin and epigenetic regulations - - + + FOXO MYF5 MYOD1 MYOG )(Fig. 1)[13, 18]. Finally, in the maintenance of MuSC quiescence have largely the formation of multinucleated myofibers results in the de- been performed on MuSCs isolated from uninjured creased expression of MYOD1 while MYF6/MRF4 becomes muscle, with the assumption that cells retain the charac- highly expressed in the functional muscle fiber [19–21]. teristics of a quiescent cell during the sorting procedure. The role for MRFs in controlling MuSC fate is well However, recent work has shown the importance of iso- established. However, the myogenic TFs require coord- lation protocols in the study of true quiescence MuSCs ination with a broad range of transcriptional regulators . Indeed, the development of in situ fixation tech- that help modulate the epigenetic landscape that con- niques to lock cells in a quiescent state prior to isolation trols specific gene expression programs. Below, we will has exposed important differences in gene expression as Massenet et al. Skeletal Muscle (2021) 11:4 Page 4 of 16 well as histone post-transcriptional modifications. Exten- (by the addition of H3K4me3 marks at its promoter) sive changes in epigenetic marks on histone H3 are ob- while having no effect on Pax7 expression in quiescent served during the 3-h period needed to isolate MuSCs, MuSCs . though no differences in DNA methylation were observed To maintain quiescence, MuSCs prevent cell cycle within this time frame [16, 22, 23]. Based on these find- entry through the expression of specific cell cycle inhibi- ings, one can assume that most of the epigenetic informa- tors. As mentioned above, the choice of cell cycle inhibi- INK4a tion collected using isolated MuSC analysis do not tors is indispensable, as the expression of p16 cell represent the quiescent state but a transition between qui- cycle inhibitor leads to senescence and permanent cell INK4a escence and activation known as early activation [24, 25]. cycle exit. The repression of p16 in MuSCs is as- Maintenance of the quiescent state requires repression sured by the Polycomb PRC1 complex where the Ring1B of the genes coding for both cell cycle proteins and per- E3 ubiquitin ligase mediates H2A monoubiquitination at manent cell cycle exit. p53 was shown to maintain a re- lysine 119 (H2AUb) of the INK4a locus [26, 32, 33]. In versible cell cycle arrest in quiescent MuSCs whereas addition, the Polycomb PRC2 complex containing the INK4a the activation of tumor suppressor ARF (p16 ) leads EZH2 subunit was shown to bind at the INK4a pro- to a definitive cell cycle arrest and senescence [24, 26]. moter to control its transcription in in vitro culture of To maintain this balance, different pathways contribute mouse embryonic fibroblasts through the depositing of to the quiescent MuSC transcriptional network. In par- the repressive H3K27me3 mark . Regulation of the ticular, the expression of forkhead box (FOXO) tran- INK4a locus by PRC2 is also likely to occur in MuSCs as scription factors, FOXO1, FOXO3A, and FOXO4, were a MuSC-specific KO of EZH2 prevented the expansion reported to be required for the maintenance of the of the stem cell population . MuSC quiescence . FOXO3 maintains the expression MYOD1 has an important function in MuSC commit- of Notch pathway components . Active Notch path- ment, and its expression is needed for MuSC activation way leads to a decreased expression of MDM2 which al- and proliferation. While the gene is expressed at low lows accumulation of p53 to maintain cell cycle arrest levels, a repressive chromatin environment is maintained and quiescence until injury [26–28]. On the other hand, at the Myod1 locus to prevent high-level expression until INK4A p16 needs to be kept in a repressed state to prevent activation. Histone methyl transferase (HMT) Suv4-20 MuSCs from entering into a definitive senescent cell h1 adds H4K20me2 marks at Myod1 promoter and at its cycle arrest . distal regulatory region (DRR), 5 kb upstream of MyoD1 In the quiescent state, transcription levels are relatively transcription starting site. Addition of H4K20me2 at low due to the condensed state of the chromatin . these sites induces heterochromatin formation and de- Nevertheless, many genes are expressed including MRFs creased Myod1 expression in early activated MuSCs which show accumulation of mRNA but not of their . Since this mechanism is necessary to repress encoded proteins. This shows that MuSCs use additional Myod1 expression during quiescence too [29, 30], one mechanisms beyond transcriptional regulation to modulate can assume that these histone PTMs are already present their fate as it is the case for Myf5 and Myod1 mRNA nu- at the quiescent state and maintained during the early clear retention. That being said, Myod1 expression levels activation. The DRR and promoter of Myod1 are also must be minimized. This reduction must be controlled marked by the repressive H3K9me2 modification. While through epigenetic mechanisms. As a matter of fact, ex- the enzyme responsible for H3K9me2 marking is not pression of a H4K20me2 methyltransferase, Suv4-20 h1, is known, some mechanisms maintaining this mark have required for condensation of chromatin and repression of been uncovered. The E3 ubiquitin ligase Deltex2 is es- Myod1 expression in quiescent MuSCs [29, 30]. sential to maintain H3K9me2 mark through the inhib- ition of lysine demethylase jumonji domain containing Histone post-translational regulation of MuSC quiescence 1C (JMJD1c) enzyme . Inhibition of JMJD1c func- and activation tions prevents demethylation to allow maintenance of Though the expression of PAX7 and FOXO transcrip- H3K9me2 marks at the Myod1 promoter and DRR. It is tion factors are key features of quiescent MuSCs, it is noted that the removal of H3K9me2 at these regulatory unclear how their expression is controlled during regions is necessary for the increased expression of quiescence. Myod1 that drives MuSC activation . In activated MuSCs, Pax7 expression is regulated by Finally, the expression of muscle-specific genes is also the antagonism of Polycomb (PcG) and Trithorax repressed in quiescent and activated MuSCs. In this case, (TrxG) group proteins to silence or activate its expres- the PRC2 complex mediates the addition of H3K27me3 sion, respectively. Indeed, the lysine methyltransferase marks to myosin heavy chain 2b (Myh4) and Myogenin MLL1 (a Trithorax group sub-unit) KO mice see a loss (Myog) promoters and to muscle creatine kinase gene of Pax7 expression in activated and proliferating MuSCs (MCK) enhancer, leading to their repression of Massenet et al. Skeletal Muscle (2021) 11:4 Page 5 of 16 expression . While this level of regulation is inferred In proliferating MuSCs, PAX7 is recruited to areas of from early activated MuSCs, it will be important to con- open chromatin and its presence correlates with active firm whether PRC2 is also present at these muscle- histone marks H3K4me1 and H3K27Ac. In particular, specific genes in true quiescent MuSCs. PAX7 facilitates chromatin accessibility at gene loci en- coding MRFs through activation of transcriptional en- hancers . Among these MRFs, MYOD1 plays a key The DNA methylation landscape during MuSC quiescence role in regulating both proliferation and differentiation. and early activation In proliferating cells, the Msh homeobox 1 (MSX1) TF Technical limitations have hindered our understanding was shown to bind a mouse specific isoform of histone of functions of DNA methylation in the maintenance of H1, H1b, at the core enhancer region (CER) of Myod1 in MuSC quiescent state. One could infer that the DNA is order to induce chromatin compaction and to reduce methylated at the MyoD locus to prevent transcription Myod1 expression . Additionally, histone deacety- based on the original lineage conversion studies in fibro- lases (HDACs) are known to be necessary for the main- blast cell lines . However, studies in primary fibro- tenance of proliferation, and class IIA HDACs, HDAC4 blasts have shown that the MyoD locus is not and HDAC5, are recruited by the H3K9me3 methyl- methylated in normal conditions and only becomes transferase SUV39H1 to specifically target Myod1 pro- methylated as part of a genome-wide increase in CpG is- moter. In this way, they modulate Myod1 expression, land methylation in response to crisis . Thus, the which underlines the importance of chromatin shape at role for DNA methylation at MyoD and other genes in Myod1 gene for maintenance of proliferation or entrance regulating the transition between satellite cell quiescence in differentiation (Fig. 2). The accumulation of the and activation is an area that still needs to be explored repressive epigenetic factors at the Myod1 gene in prolif- and will be facilitated as new technologies that allow erating MuSCs is modulated in response to Notch1 sig- analysis of DNA methylation on a small number of cells naling to prevent differentiation where expression of and improvement of techniques to isolate and study qui- Myod1 oscillates due to transient expression of the escent MuSCs become available. Notch-regulated transcriptional inhibitor HES1 [49, 50]. Lahmann et al. showed that decreased HES1 expression Epigenetic histone modifications contributing to the leads to maintenance of MYOD expression and differen- proliferative state of MuSCs tiation . These mechanisms likely work in tandem to MYF5 is a key transcription factor contributing to the orchestrate temporal control of the transition from pro- proliferation of MuSCs. The Myf5 gene is already liferation to differentiation. expressed in quiescent MuSCs but, upon activation, tri- Though expressed at lower levels in proliferating cells, methylation of H3K4 (H3K4me3) at its promoter leads MYOD1 contributes to both proliferation and differenti- to an increase of its expression. Marking of the Myf5 ation. MYOD1 ensures the repression of muscle differ- promoter by H3K4me3 is mediated by the HMT com- entiation genes during proliferation and then the plex, WDR5/ASH2L/MLL1 . This HMT complex is activation of the same genes in response to differenti- recruited by PAX7 through an interaction that requires ation cues . A role for MYOD1 in both proliferation methylation of PAX7 by the CARM1 protein [41, 42]. and differentiation may seem contradictory but is readily HMT recruitment at Myf5 underlies the importance of understood when one considers that this TF can be both PAX7 expression for activation and proliferation of a repressor and an activator at specific genes depending MuSCs. In the same context, MLL1 KO also displays on the context. In this model, MYOD1 and MEF2D diminution of Myf5 gene and protein expression in pro- interact with the scaffold protein KAP1 . In prolifer- liferating primary myoblasts and C2C12 cells [31, 43]. ation conditions, MYOD1, MEF2D, and KAP1 act to- These effects observed in proliferating myoblasts are gether to stabilize the association of both co-repressors consistent with the fact that Pax7 expression needs to be (G9A, HDAC1) [48, 52] and coactivators (P300 and maintained during the transition from quiescence to LSD1) [53, 54] at the muscle differentiation genes. The proliferation and during the proliferation. Maintaining assembly of this enhanceosome-type complex establishes the open state of chromatin at the Pax7 gene is attrib- a poised chromatin state at the promoters where the re- uted to a direct effect of the switch/sucrose nonfermen- pressive enzymes dominate to limit gene expression. table chromatin remodeling complex (SWI/SNF) During differentiation, the increased expression of chromatin remodeling complex. Indeed, the Brg1 SWI/ mitogen-activated protein kinase (MAPK) P38α (P38α) SNF subunit is phosphorylated by casein kinase 2 and leads to activation of MSK1 kinase which phosphorylates contributes to the formation of SWI/SNF complex at KAP1 at serine 473 . The phosphorylated KAP1 pro- Pax7 gene, promoting its expression and leading to tein no longer interacts with the co-repressors, but con- MuSC proliferation [44, 45]. tinues to maintain an interaction with the coactivators, Massenet et al. Skeletal Muscle (2021) 11:4 Page 6 of 16 Fig. 2 Regulation of the myogenin gene control the transition from proliferative myoblasts to differentiated myocytes. To block MyoG expression and prevent early differentiation, a repressive function of MYOD1 is needed. In this repressive action, MYOD1 is recruited on the promoter and is bound by KDMT1A thanks to P38γ phosphorylation at its Ser199 and 200. MYOD1 forms a poised complex with MEF2D, KAP1, G9a, and HDAC. Histone acetyltransferase P300/CAF proteins can also bind to MYOD1/MEF2D complex, but their functions are limited. During induction of differentiation, MYOD1 functions change to allow Myog gene expression. This transition is due to phosphorylation of KAP1, which leads to removal of HDAC1 and G9a proteins from MYOD1/MEF2D complex. In this state, P38α phosphorylates MEF2D at its threonine 308 and 305, which leads to the recruitment of ASH2L and trimethylation of H3K4. At the same time JDP2, JUN and SETD7 are recruited at p300/CAF proteins to allow the establishment of permissive marks H3K4me1 and H3K27/18Ac at the enhancer of Myog and the H3K4me1 mark works as an antagonist to the addition of H3K9me3 by SuV39h1. At the promoter, the MLL/TrxG complex is recruited by MyoD to dimethylate H3R8. In addition, the repressive marks H3K9me2/3 and H3K27me3 are removed by JMJD1c and JMJD2 (KDM6A), respectively, while permissive marks, H3K4me3, H3K36me3, H3R8me2, and H3K27/18Ac, are added at the promoter by MLL2, SETD2, PRMT5, and p300. The presence of these permissive marks allows the recruitment of RNA polymerase II and starting of transcription. At the Myog gene, P38α is responsible of the phosphorylation of MEFD2 as well as of P18 Hamlet (a subunit of SNF2); this phosphorylation leads to the incorporation of unstable H2A.Z variant at the core gene to facilitate transcription leading to the establishment of an open chromatin state coactivators associate with MYOD1, the co-repressors and the expression of muscle target genes (Fig. 2). dominate. Signals from the environment induce phos- In other words, when both co-repressors and phorylation by MSK1 that in turn displaces the co- Massenet et al. Skeletal Muscle (2021) 11:4 Page 7 of 16 repressors from the locus . MYOD1 contribution to Moreover, SNAIl family transcriptional repressor 1, MuSC proliferation directly suggests the possibility of SNAI1, associated with HDAC1/2 can bind E-box at regulation of Myod1 expression level during differentiation-related genes and prevents the binding of proliferation. MYOD1 and target gene expression . This mechan- Additional co-repressors are associated with the ism suggests the importance of the SNAI1/HDAC1/2 MYOD1-MEF2D-KAP1 complex in proliferating cells. complex in promoting proliferation by blocking MYOD1 Heterochromatin proteins HP1α and β interact with from initiating differentiation. MYOD1-MEF2D-KAP1 to repress its activity at target To maintain myoblast proliferation, repression of dif- gene promoters to maintain proliferation . Similarly, ferentiation is not enough. Cells must also maintain the the formation of a HDAC1/MYOD1-KAP1 complex al- expression of genes involved in cell cycle progression. lows deacetylation of MYOD1 target genes. One of their Like in most cell types, E2F protein family plays a key targets, Myog, which is necessary for differentiation, role in regulating cell cycle through the recruitment of shows reduced acetylation and a recruitment of histone acetyltransferase P300/CBP and PCAF/GCN5 SUV39h1 thanks to the presence of HDAC1/MYOD1 histone acetylases to the cyclin genes [61, 62]. In primary complex. In C2C12 cell line, the formation of another mouse myoblasts, the E2F1/PCAF complex mediates complex containing MYOD1/P300/CBP-associated fac- acetylation of histones at E2F1 target genes to allow pas- tor (PCAF) and the HDAC SIRT2 is necessary to main- sage through the G1/S cell cycle checkpoint . In tenance of proliferation and repression of differentiation addition to the recruitment of acetyltransferase, studies . Such as other SIRT enzymes, SIRT2 is dependent in many different cell types have shown that E2F pro- of the level of NAD+, revealing the implication of the teins mediate recruitment of H3K4 histone methyltrans- metabolism for the maintenance of proliferation. The re- ferases from the KMT2 family . In the muscle moval of histone acetylation and the recruitment of system, proliferating C2C12 cells utilize MLL5 to deposit Suv39h1 leads to an enrichment of H3K9me3 marks and H3K4me3 marks at the cyclin A2 gene, a factor neces- maintenance of the chromatin under a closed state, lead- sary for progression through G1/S cell cycle checkpoint ing to repression of Myog expression and impeding the . Finally, H3K36me methyltransferase SET2 KO in start of differentiation (Fig. 2)[57, 58]. Another study re- C2C12 cells compromise G1/S and G2/M phase transi- vealed the function of P38γ MAPK into the phosphoryl- tion by decreasing levels of cyclin D1, CDK4, CDK6, and ation of the Ser199 and 200 of MYOD1 to allow the cyclin E2. This result indicates an essential function of recruitment SUV39h1/KMT1a to the Myog promoter, the H3K36 methyltransferase SET2 for the maintenance reducing its expression during proliferation . of myoblast proliferation . Thus, it is clear that INK4a Fig. 3 Senescence mediated by irreversible cell cycle arrest caused by p16 expression in sarcopenic MuSCs. In young and old MuSCs, the presence of PRC1 complex containing RING1 and BMI1 subunits leads to H2A ubiquitination at the promoter of INK4a locus and the repression of INK4a p16 . This repression allows RB protein phosphorylation and loss of stability of E2F/RB complex. Once free from its complex, E2F induces the INK4a expression of genes promoting the cell cycle. Sarcopenic MuSCs lose their PRC1 repression of p16 , leading to reduction of RB phosphorylation and maintenance of the E2F/RB complex. Without free E2F protein, the expression of genes promoting cell cycle is reduced, causing cell cycle arrest and senescence of MuSCs Massenet et al. Skeletal Muscle (2021) 11:4 Page 8 of 16 epigenetic modifications of histones contributes to main- occur indirectly through MYOD1 functions. Indeed, tain the cell cycle progression though we still lack an in MYOD1 has been shown to control the expression a depth understanding of all the players involved in myo- transcriptional repressor Zinc Finger Protein 238 blast expansion. (ZFP238) in C2C12 cells . This is significant as the The degree to which genes are marked by histone ZFP238 protein is able to recruit DNMT3A and HDAC1 post-translational modifications depends not only on the at the promoter of myogenic genes to repress their ex- presence of epigenetic enzymes, but also on the avail- pression . It is possible that in MuSCs, ZFP238 is ability of key co-factors that contribute to their enzym- also present at Cdkn1c promoter and recruits DNMT3A. atic activity. Indeed, the energy source available to the At the moment, there is no work to support these hy- cell will affect the availability of these co-factors. The pothesis and analysis of the presence of ZFP238 and glycolytic environment of proliferating MuSCs ensures DNMT3A at the Cdkn1c promoter has to be an abundance of several intermediates of the tricarb- investigated. oxylic acid cycle (TCA) that are needed for establish- The continued proliferation of myoblasts is also ment of epigenetic modifications. In particular, oxidative dependent on repression of Myog gene expression to decarboxylation of pyruvate supplies the acetyl-CoA prevent differentiation. This repression was shown to be donor of acetyl utilized by the HAT proteins . Simi- possible thanks to the methylation at the Myog promoter larly, the metabolite α-ketoglutarate is a necessary co- [73, 74]. The Myog promoter has only 1.4% of CpG di- factor for the demethylation of either DNA or histones, nucleotides. However, even if the region is not rich in by ten-eleven translocation (TET) and JMJD proteins, CpG island, bisulphite and methylation-sensitive restric- respectively. The functions of the histone demethylase tion endonuclease analysis revealed a hypermethylation LSD1 and the HDACs also depends on the availability of state during proliferation of C2C12 cells, necessary for FAD+ and NAD+ in the cell . Independently of the Myog repression . Additionally, methylation of the TCA, the intracellular methionine is transformed in S- scattered CpG sites is necessary for the binding of a adenosylmethionine which is necessary for the action of methyl-CpG-binding protein, ZBT38 (CIBZ). The pres- DNA methyltransferase (DNMT) proteins . Under- ence of ZBT38 at CpG islands within the Myog pro- standing these points highlight the importance of the moter acts to inhibit its expression and maintenance of metabolism in the epigenetic regulation of the MuSC. proliferation as the knockout of ZBT38 leads to differen- Indeed, after isolation of mouse MuSCs, during the tran- tiation of C2C12 cells . sition from quiescence to proliferation, a shift between Taken together, these findings show that as MuSCs fatty acid oxidation and glycolysis occurs . This shift start to proliferate, TFs target the epigenetic machinery induces a decrease of NAD+ levels which reduces the to genes necessary to ensure cell cycle progression and activity of SIRT1 family of NAD+-dependent HDAC en- those preventing differentiation. zymes. The activity of SIRT1 is associated with a regula- tion of H4K16Ac marks associated to muscle gene Epigenetic regulation of the differentiation process expression . While characterization of metabolic After the expansion phase, MuSCs undergo cell cycle ar- pathways contributing to the epigenetic regulation of rest and transit towards differentiation . Cell cycle MuSC proliferation remains in its infancy, new tech- arrest is initiated through the expression of the cell cycle nologies in the area of metabolomics make this an excit- inhibitors CDKN1C or CDKN1A (p21) . Moreover, ing area of current research. a progressive increase of MYOD1 protein expression leads to the increased expression of early differentiation The DNA methylation of proliferating MuSCs markers such as Myog and coincides with the abrogation DNA methylation plays a key role in preventing the ex- of MYF5 and PAX7 expression . Coincident with the pression of cell cycle inhibitors during MuSC prolifera- onset of differentiation, MuSCs are marked by hyperace- tion. Among them, repression of cyclin-dependent tylation of histones H3 and H4 while decreased methyla- kinase inhibitor 1C (CDN1C or P57kip2) is required to tion of H3K9 and K27 is observed [79, 80]. The prevent differentiation. Interestingly, deleting DNA epigenetic reprogramming of MuSCs allows the opening methyltransferase DNMT3A in MuSCs leads to a dimin- of chromatin at specific muscle loci needed for ution of proliferation that correlates with increased ex- differentiation. pression of the Cdkn1c gene and decreased DNA methylation at its promoter sequence. This loss of prolif- The control of MuSC differentiation by histone post- eration in the DNMT3A KO myoblasts can be rescued transcriptional with Cdkn1c KD, indicating an important function of de The differentiation of human myoblasts has been shown novo methylation in the maintenance of MuSC prolifera- to result in a large change in the histone PTM landscape tion . The control of Cdkn1c DNA methylation can with a global diminution of H3K9me3 and H4K20me3 Massenet et al. Skeletal Muscle (2021) 11:4 Page 9 of 16 repressive marks. In particular, H3K9me3 is erased from the departure of HDAC enzymes from the promoter. In Myod1 and Myog loci . These results point to the this case, FAK binds the methyl-CpG-binding protein importance of histone modification modulation for myo- MBD2, where it induced phosphorylation of HDAC1 blast differentiation. that breaks up the HDAC1/MBD2 interaction and disso- One of the first steps during the transition to differen- ciates it from the promoter . Once the repressive tiation is the stop of MuSC proliferation by cell cycle ar- marks are cleared, the promoter can then be modified to rest. In this context, the downregulation of Pax7 accumulate transcriptionally permissive marks. One of expression must occur. Indeed, PRC2 interacts with Yin the first marks to appear is the dimethylation at arginine Yang 1 TF (YY1) via the phosphorylation of the threo- 8 in histone 3 (H3R8me2) within the promoter. This is nine 372 of EZH2, a subunit of PRC2 complex, by p38α. achieved through the PRMT5 protein, a type II arginine This interaction allows replacement of H3K4me3 marks methyltransferase. Once the H3R8me2 mark is in place, by H3K27me3 and leads to the formation of a repressive the epigenetic mark acts to allow a stable association of chromatin state at Pax7 promoter and to the repression the chromatin remodeling complex SWI/SNF through of its expression . Among TFs involved in differenti- recognition of modified histone tail by the Brg1 subunit ation, E2F is a family of eight proteins, parts of a com- of the complex. The association of SWI/SNF with the plex including retinoblastoma-associated protein (RB), promoter then allows chromatin decompaction for RNA retinoblastoma-like protein 1 (RBL1) and 2 (RBL2) polymerase II to access the gene . In addition, the pocket proteins. Their function is to control the gene ex- histone methyltransferase SETD7 is targeted to the Myog pression of proteins regulating the cell cycle and pro- promoter by MYOD1 to introduce the H3K4me1 mark. moting differentiation in various tissues [82, 83]. RB, SETD7 is required for differentiation as silencing of which was shown to bind HDAC1 for the maintenance SETD7 leads to a reduced number of myotubes and loss of proliferation, can also regulate cell cycle exit through of expression of Myog [90–92]. The addition of its interaction with E2F4. RB recruits HMT to promote H3K4me1 by SETD7 prevents the reintroduction of re- H3K9me3 and H3K27me3 marks at the gene promoter pressive H3K9me3 repressive marks by blocking of proteins promoting the cell cycle to decrease their ex- Suv39h1 function [90–92]. Without the presence of pression, stop the cell cycle, and start differentiation. HDAC1 at Myog promoter, the histone acetyltransferase This repression is PRC1 and PRC2 dependent where the P300/CAF leads to its enrichment of H3K9 and H3K14 addition of H2AK119Ub1 and H3K27me3 marks estab- acetylation and the expression of Myog . The associ- lish a silent state . This silent state could be main- ation of the histone acetyltransferase P300 with the tained by the recruitment of dimerization partner, RB- Myog promoter is facilitated by chromatin-binding pro- like, E2F, and MuvB (dREAM) chromatin compaction tein, NUPR1 (P8), which also recruits the RNA helicase complex to target genes through interactions between its DDX5 to the locus to promoter high levels of gene ex- L3MBTL1 subunit, E2F4 and HP1γ heterochromatin pression . The introduction of H3K36me3 marks is protein . also essential to high level expression of Myog as silencing Induction of differentiation coincides with an in- of SETD2 blocked its expression and prevented myotube creased expression of MYOD1. The increased Myod1 ex- formation during differentiation . Finally, histone ex- pression is established through JMJD1c-driven changes within the nucleosome can alter the expression of demethylation of H3K9me3 marks in its promoter . the Myog gene. During the differentiation process, the Once MYOD1 is expressed at high levels, a switch be- subunit ZNHI1 (p18Hamlet) of SNF2 complex is phos- tween the SNAI1/HDAC1/2 complex and MYOD1 oc- phorylated by P38α which allows its recruitment to Myog curs at E-box of muscle target genes to promote promoter and allows the replacement of H2A histones by differentiation . its less-stable variant H2A.Z (Fig. 2). Myog expression is also essential for MuSCs to com- For differentiation to proceed, cells must also begin to mit to differentiation (Fig. 2) and is regulated by a super express functional genes that define the muscle lineage. enhancer upstream of the transcription start site . Activation of these MYOD1 target genes requires the re- The ability of TFs such as MYOD1, MEF2D, SIX4, and cruitment of SWI/SNF complex which is facilitated by FOXO3 to create a transcriptional competent state at the presence of histone 4 hyperacetylation that is recog- the myogenin promoter depends upon the combined ac- nized by the bromodomain of the transcription activator tivity of multiple epigenetic enzymes. One of the initial BRG1 (SMCA4), leading to chromatin decompaction events in activating the Myog gene is the removal of the and gene expression . SWI/SNF recruitment is facili- repressive H3K9me2 and H3K9me3 marks from the pro- tated by the incorporation of the MYOD1-associated moter by the action of the lysine demethylase JMJD2/ SMRD3 (BAF60C) subunit into the chromatin remodel- KDM4A . Moreover, focal adhesion kinase (FAK) ing complex . In addition, MyoD recruits the histone helps achieve the open chromatin state by facilitating acetyl transferase P300, the JDP2, the AP1 (JUN) and Massenet et al. Skeletal Muscle (2021) 11:4 Page 10 of 16 RUNX1 TFs, and SETD7 HMT, leading to active histone reveals the refined roles of distinct epigenetic enzymes in modification marks H3K27Ac, H3K18ac, and H3K4me1 ensuring the temporal expression of genes during muscle that target RNA polymerase II to the promoter region of differentiation. muscle differentiation genes (Fig. 2). Interestingly, Finally, when fully differentiated, myocytes fuse thanks SETD7 methylates non-histone proteins such as the TF to Myomaker and Myomerger proteins to form multinu- SRF. SRF acetylation promotes its binding to its serum cleated myofibers. While the epigenetic regulation of the response element at the muscle-specific gene Acta1 to Myomaker and Myomerger proteins has yet to be eluci- promote its expression. This regulation is necessary for dated, the membrane protein CDON which positively differentiation and is revered by KDM2B . These regulates fusion was shown to be regulated by the data suggest an indirect function of SETD7 in the regu- Trithorax HMT ASH1L which deposits dimethylation of lation of differentiation. However, apart from Acta1,no lysine 36 of histone 3 (H3K36me2) at the transcription other SRF targets have been identified in MuSCs so far. start site to prevent Polycomb-mediated repression While many repressive enzymes are removed from the . Interestingly, the absence of ASH1L provokes a MYOD1 target genes during differentiation, some com- diminution of fusion capacities of mouse and human plexes remain to permit repression of gene expression in myocytes in vitro without impairment of myosin heavy response to a changing environment. An example of this chain (MHC) protein expression. This suggests a direct is the Myh4, Myog, and Ckm genes which are marked by control of fusion by a Trithorax complex and one can the Polycomb PRC2 mediated H3K27me3 modifications imagine that understanding of the regulation of fusion in proliferating MuSCs to repress their expression and by epigenetic will emerge soon. to prevent differentiation. As differentiation initiates, a switch occurs between subunits in the PRC2 complex DNA methylation modifications during differentiation where the EZH2 subunit in proliferation (PRC2-EZH2) Several studies have revealed the important role of DNA is replaced by a functionally inactive EZH1-containing methylation in regulating muscle differentiation. Indeed, PRC2 complex that lacks the EED subunit (PRC2-EZH1) during differentiation, the whole DNA methylation land- [37, 99]. To permit gene expression during differenti- scape was reported to decrease . Several years ago, ation, removal of the H3K27me3 modifications is medi- sodium arsenic treatment of C2C12 was reported to re- ated by the H3K27 demethylase KDM6A (also known as duce differentiation capacities of the cells. This was cor- UTX), a H3K27 demethylase that opens the chromatin related with increased DNA methylation of the CpG site to allow the expression of Myog and the entrance in dif- of Myog promoter and a diminution of Myog expression ferentiation [100, 101]. The stable association of PRC2- . Hypermethylation of Myog promoter in C2C12 EZH1 complex that lacks methyltransferase activity cells decreases quickly after the induction of differenti- helps to maintain the transcriptional permissive state ation . DNA methylation is known to repress TF [37, 99]. In response to cellular stresses such as muscle binding. However, using a model of 293 T cells express- atrophy, the PRC2-EZH1 complex incorporates an EED ing luciferase reporter construct and different TFs, the subunit to form a functional HMT complex that can re- binding of Sine oculis homeobox homolog 1 (SIX1) and introduce H3K27me3 marks at these muscle genes to MEF2A at Myog promoter was confirmed. Silencing prevent their expression [37, 99]. SIX1 in C2C12 leads to increased methylation of Myog As MYOG begins to push the differentiation program promoter, suggesting that SIX1 could have a role in the forward, additional epigenetic events will lead to the ex- repression of the methylation [74, 107]. In the last few pression of terminal differentiation-related genes and fu- years, TET proteins have been shown to catalyze the sion of differentiated myocytes into myotubes. TrxG conversion of DNA 5-methylcytosine into different oxi- complexes, containing a H3K4me3 methyl transferase dized forms, 5-hydroxymethylcytosine, 5-formylcytosine, ASH2L, are recruited by MEF2D to muscle-specific and finally in 5-carboxylcytosine, demonstrating active genes such as a muscle cytoplasmic enzyme, the muscle demethylation capacities [108, 109]. Supporting the idea creatine kinase, where they mediate the addition of of a decrease in DNA methylation during differentiation, H3K4me3 marks that promote gene expression . TET1 and TET2 expression are significatively increased This recruitment is modulated through phosphorylation in myoblasts after induction of differentiation. Interest- of MEF2D at threonine 308 and 315 by P38α MAPK. ingly, inhibition of TET2 but not TET1 by siRNA in Interestingly, the function of a histone arginine methyl- C2C12 results in increased DNA methylation of Myog, transferase Prmt5 is critical for early differentiation but is Myf6/Mrf4, and Mymk (Myomaker) gene promoters. dispensable for late differentiation while the type I argin- The increased DNA methylation at the promoter of ine methyltransferase, CARM1/PRMT4 is necessary for these genes correlates with a decreased expression and late differentiation as it deposits dimethylation of arginine an abrogation of C2C12 differentiation . The pres- 17 at histone 3 to permit gene activation [89, 103]. This ence of CpG methylation at Myog promoter is necessary Massenet et al. Skeletal Muscle (2021) 11:4 Page 11 of 16 for the binding of ZBT38 protein and the decreased ex- damages are associated with an important inflammatory pression of Myog. The diminution of methylation at environment causing elevated levels of TNF, leading to Myog promoter may lead to ZBT38 removal and abroga- diminution of the Notch1 expression, an important medi- tion of its repression . In addition to Myog promoter ator of activation and proliferation of MuSCs [115, 116]. demethylation, a general diminution of DNA methyla- The repression of Notch1 protein is due to an increase of tion at the CpG sites of Myod1 promoter occurs after 3 DNA methylation of Notch1 led by EZH2-dependent re- days of differentiation in C2C12 cells . DNA methy- cruitment of DNMT3b . Diminution of Notch signal- lation changes during differentiation are not only attrib- ing could impair MuSC maintenance in the quiescence uted to demethylation of the promoter of genes required state by modification of DNA methylation landscape. The for myogenic differentiation. The addition of DNA absence of a functional dystrophin complex also conducts methylation at CpG sites on the promoter of specific to an alteration of its related nitric oxide (NO) pathway. genes is also necessary to allow differentiation of myo- The histone acetyl transferase CBP/P300 was shown to be blasts. In C2C12 cells, after 3 days of differentiation, an downregulated in the zebrafish model of DMD while its increase in DNA methylation of CpG sites of Pax7 and overexpression in embryonic development rescues the Myf5 promoters was shown. Interestingly, after 5 days of phenotype . This suggests the importance of epigen- differentiation, the DNA methylation is still higher as etic factors in the ability of the muscle to resist damages compared with the proliferative state but slightly re- in dystrophy. Moreover, an abnormal pattern of histone duced as compared to day 3 of differentiation . modifications is present in proliferative myoblasts of hu- Genetic deletion of DNMT3A in mice resulted in fibro- man DMD and the mdx mouse model of DMD. This glo- sis and reduction of cross-section area of the muscle bal change in histone PTMs is characterized by an after regeneration from an acute injury. The diminution increased level of H3K14 and H3K9 acetylation, augmen- of DNMT3A is correlated with a diminution of pro- tation of H3K79me2 marks, and an increase of phosphor- moter DNA methylation and the expression of Gdf5,an ylation of H3 serine 10 . The decreased expression of important muscle gene. Interestingly, Gdf5 increased ex- BMI1, a subunit of PRC1 complex, was revealed in human pression does not change proliferation or differentiation DMD myoblasts as compared with healthy myoblasts. capacities. However, a diminution of myofiber size, Interestingly, BMI1 overexpression reduces oxidative length, and nuclei number and a decreased expression of stress and DNA damages and increases ATP production differentiation-related genes was reported, suggesting in DMD myoblasts . It is interesting to note that the that DNA methylation of Gdf5 promoter is necessary to absence of BMI1 expression causes dysregulation of INK4a avoid undesired muscle atrophy . p16 and early senescence of MuSCs . Thus, epi- Modification of DNA methylation during differenti- genetic changes in MuSCs from DMD patients lead to ation is also correlated with histone modifications. In functional exhaustion of the stem cell pool. particular, heterochromatin protein HP1γ recognizes Another example of a myopathy in which epigenome and binds H3K9me3 over the genome, interacts with is altered is Emery-Dreifuss muscular dystrophy. In this DNMT1, and recruits HMTs [55, 113]. During this disease, mutations in Lamin A/C gene, encoding for the phenomenon, the protein level of HP1γ does not change, nuclear envelop protein Lamin A/C, alter chromatin but its spatial localization does and is correlated with the condensation in MuSCs. Furthermore, the altered inter- presence of a methyl-CpG-binding protein, MECP2. actions between chromatin and nuclear lamina cause de- These functions allow the maintenance of specific gene regulation of the H3K27me3-mediating Polycomb silencing by the addition of DNA methylation during complex in MuSCs . This change in Polycomb differentiation and suggest interactions between DNA complex position leads to a diminution of self-renewal methylation and changes in histone PTMs . and exhaustion of MuSCs . Age-related inflammation also creates an altered muscle Changes of the muscle epigenome in muscle environment that changes MuSC epigenome. Among diseases these changes, DNA methylation marks are well known to Aged and diseased muscles present an altered cell envir- change with age and environmental exposure, suggesting onment as compared with healthy adult muscles. There- a potential modification of MuSC epigenome over time fore, different cues trigger epigenome changes which . In human, a comparison of DNA methylation re- will in turn alter the ability of MuSCs to maintain their vealed hypomethylation near the 5′ region and hyperme- functions. thylation at the middle and 3′ gene regions of genes in old Epigenome changes are well studied in the context of (68–89 years old) versus young (18–27 years old) human Duchenne muscular dystrophy (DMD). In this disease, skeletal muscle . Moreover, hypermethylation of in- loss of the dystrophin gene induces important muscle- tragenic regions of genes involved in motor neuron junc- degenerative phenotype in vivo. Constant myofiber tions and myofibers formation was shown . A Massenet et al. Skeletal Muscle (2021) 11:4 Page 12 of 16 correlation was found between gene underexpression and altered muscle environment in myopathies and aging hypermethylation of intragenic, 5′ and at transcription might give rise to alternate MuSC fates. start regions. Opposingly, upregulation of genes is corre- In recent years, evidence started to accumulate that lated with hypomethylation of intragenic, 5′ and tran- MuSCs can give rise to fibroblasts in DMD and aging. scription start regions . Such gene deregulation can An extracellular component of the aged muscle was be correlated to the loss of motor units and denervation shown to induce a lineage conversion of MuSCs towards observed in muscle in sarcopenia. Gene deregulation re- the fibroblast lineage . Lineage conversion in re- lated to the modification of the DNA methylation during sponse to environmental cues was confirmed in lineage CreER YFP aging is observed in muscles of older subjects [123, 124]. tracing experiment using PAX7 ;R26R bred with In humans, the increased DNA methylation was observed mdx mouse, in which 7 to 20% of MuSCs acquire a at the promoter of genes coding for components of mito- fibroblast phenotype . Additionally, mdx mouse chondrial respiratory chain, COX7A1 and NDUFB6, muscle shows elevated TGFβ and Wnt signaling, which which is related to decrease expression of the genes in the induces the myogenic to fibrogenic conversion. Indeed, elderly. These alterations exhibit a direct effect of DNA Wnt signaling controls TGFβ2 expression to induce methylation alteration with age [125, 126]. A study per- fibrogenic conversion [137, 138]. Accordingly, inhibition formed in MuSCs isolated from young and old mice sug- of WNT-TGFβ2 signaling prevents the lineage conversion gested that a change of DNA methylation during aging is and reduces the expression of fibrogenic genes [137–139]. a stochastic event. These events occur at gene promoters Mechanisms sustaining such changes of MuSC fate due to and drive inter-variability gene expression between myo- altered environment are still unknown and need to be in- blasts . An important decline of muscle regenerative vestigated, as well as their evidence in human since fate functions is also observed. Studies performed on young conversion was demonstrated in the mouse. (2–6 months), old (18–24 months), and geriatric (26–36 Finally, similar to the problem discussed in for Emery- months) mice exposed loss of MuSC functions with aging, Dreifuss muscular dystrophy, a loss of functional MuSCs INK4a even when the cells were transplanted in young muscle, due to p16 expression has also been shown in sar- suggesting intrinsic alterations of MuSCs. These changes copenia. In this case, downregulation of BMI1 leads to INK4a arecausedbyderegulationof p16 by PRC1 subunit the displacement of PRC1 complex from the promoter INK4a BMI1, which leads geriatric MuSCs to a switch in a pre- of p16 in geriatric MuSCs. In the absence of PRC1, senescence state (Fig. 3)[26, 128]. This conversion of inappropriate removal of PRC1 complex decreases the MuSCs to a senescent state has the unwanted conse- presence of its repressive marks H2Aub, increases INK4a quence of reducing the number of functional stem cells p16 expression, leading MuSCs to senescence . available for repair of muscle wasting. Thus, changes in While similar approaches of dead Cas9 (dCas9)-Cbx4 to ink4a the muscle environment in both aging and disease lead to repress the p16 gene would also be effective, the use functional exhaustion of MuSCs. of gene therapy to prevent aging pushes the ethical boundaries. Instead, small molecule treatments to main- Altered cell fate in muscle disease and aging tain Bmi1 expression in aged MuSCs would seem to be As discussed above, the identity of a cell is defined by a more appropriate means to ensure PRC1 mediated re- INK4a tissue-specific gene expression programs that are deter- pression of the p16 promoter to reduce senescence mined through epigenetic mechanisms. As such, an al- in aged MuSCs. tered cell environment may alter MuSC fate through epigenetic mechanisms. Cell plasticity due to changes in Conclusion the epigenome was established in experiments showing The study of mechanisms allowing total regeneration of that treatment of fibroblast cell line with the DNA muscles by MuSCs had started a few decades ago. After methylation inhibitor 5-azacytidine triggers the activa- the discovery of MRFs, the understanding of epigenetic tion of MYOD1 and the formation of multinucleated mechanisms added new insights in the transition of each myotubes [38, 129, 130]. Cell plasticity in response to step of myogenesis. Separately, DNA methylation and specific stimuli has since been more extensively studied histone post-transcriptional regulation have been well in terms of pluripotency where fibroblasts were repro- studied in muscle, although many unanswered aspects grammed to the embryonic state by the formation of in- still remain. Recent studies highlighted the importance duced pluripotent stem cells [131–133]. While the of considering that these regulations are changing in a induced pluripotent stem cells were first derived using a spacio/temporal manner. Because of its complexity, tight combination of pluripotency TFs, later studies showed dysregulation of any of these mechanisms can lead to an that the lineage conversion mechanisms can be similarly abnormal myogenic program and incapacity to correctly driven by exposure to a variety of small molecule inhibi- regenerate the muscle. Alterations of the MuSC epige- tors [134, 135]. As such, it should not be surprising that nome encountered during aging or muscle pathology Massenet et al. Skeletal Muscle (2021) 11:4 Page 13 of 16 conduct gene dysregulations and to the diminution of Received: 16 October 2020 Accepted: 20 December 2020 the capacity of MuSCs to regenerate. Recently, modifica- tions of these regulations were reported to alter the maintenance of a healthy MuSC fate in mouse models References 1. Ou HD, Phan S, Deerinck TJ, Thor A, Ellisman MH, O’Shea CC. ChromEMT: [137, 139]. 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Skeletal Muscle – Springer Journals
Published: Jan 11, 2021