Epigenetic regulation of satellite cell activation during muscle regeneration

Satellite cells are a population of adult muscle stem cells that play a key role in mediating muscle regeneration. Activation of these quiescent stem cells in response to muscle injury involves modulating expression of multiple developmentally regulated genes, including mediators of the muscle-specific transcription program: Pax7, Myf5, MyoD and myogenin. Here we present evidence suggesting an essential role for the antagonistic Polycomb group and Trithorax group proteins in the epigenetic marking of muscle-specific genes to ensure proper temporal and spatial expression during muscle regeneration. The importance of Polycomb group and Trithorax group proteins in establishing chromatin structure at muscle-specific genes suggests that therapeutic modulation of their activity in satellite cells could represent a viable approach for repairing damaged muscle in muscular dystrophy.


Introduction
Skeletal muscle regeneration is mediated by myogenic cell populations that reside in the muscle and behave as adult stem cells [1][2][3]. In the present article we will focus on satellite cells, which represent the best characterized population of adult muscle stem cells. Satellite cells are a population of mononuclear cells that reside between the muscle fi ber and the basal lamina [1,4].
While satellite cells spend most of their lifetime in a quiescent state, upon muscle damage they can re-enter the cell cycle and either: undergo a symmetric cell division to self-renew and expand the satellite cell popu lation; or undergo an asymmetric cell division that results in the cell on the basal lamina side maintaining the satellite cell identity, while the cell adjacent to the muscle fi ber enters the myogenic diff erentiation program [5,6]. Cell fate deci sions undertaken by the satellite cells upon muscle damage are thought to be regulated through epigenetic mechanisms that modify the structure of chromatin without changing the DNA sequence. Th ese epigenetic changes lead to altered gene expression profi les that contribute to defi ning cellular identity. Understanding the nature, origin and raison d'être of these epigenetic modifi cations in the regenerating muscle will be critical to determining how satellite cells can be maintained ex vivo such that this adult stem cell population can be amplifi ed for therapeutic use to treat muscle-wasting diseases.

Polycomb group and Trithorax group proteins in muscle regeneration
Genetic screens for mutations that caused patterning defects in Drosophila led to the identifi cation of Polycomb group (PcG) proteins, which act to repress developmentally regulated gene expression [7,8]. Further screening to identify genes that rescued the Polycomb phenotype resulted in the identifi cation of an antagonistic group of proteins, termed Trithorax group (TrxG) proteins, which act to establish high levels of trans cription from these same developmentally regulated loci. Over the past 5 years, studies in human and mouse embry onic stem cells have suggested that PcG and TrxG families of epigenetic regulators modulate pluripotency and lineage restriction of a number of cell types [9].
While not all PcG and TrxG proteins have been extensively studied, the role of the PcG and TrxG histone methyl transferases in regulating gene expression is well characterized. Th ese histone methyltransferases include the lysine methyltransferase family 6 (KMT6) enzymes Ezh1 and Ezh2 that act as the active subunit of the polycomb repressor complex 2 (PRC2), and the TrxG lysine methyltransferase family 2 (KMT2) members (that is, MLL1, MLL2, MLL3, MLL4, hSET1A, and hSET1B) that act as the active subunit of Ash2L-containing methyltransferase complexes. Th e KMT6 family of Abstract Satellite cells are a population of adult muscle stem cells that play a key role in mediating muscle regeneration. Activation of these quiescent stem cells in response to muscle injury involves modulating expression of multiple developmentally regulated genes, including mediators of the muscle-specifi c transcription program: Pax7, Myf5, MyoD and myogenin. Here we present evidence suggesting an essential role for the antagonistic Polycomb group and Trithorax group proteins in the epigenetic marking of muscle-specifi c genes to ensure proper temporal and spatial expression during muscle regeneration. The importance of Polycomb group and Trithorax group proteins in establishing chromatin structure at musclespecifi c genes suggests that therapeutic modulation of their activity in satellite cells could represent a viable approach for repairing damaged muscle in muscular dystrophy.
methyl transferases is involved in depositing the transcriptionally repressive mark trimethyl histone H3 at lysine 27 (H3K27me3) on developmentally regulated genes, where as the transcrip tionally permissive mark trimethy lation of H3 at lysine 4 (H3K4me3) is mediated by the KMT2 family of methyltransferases. As the repressive H3K27me3 mark is heritably transmitted to daughter cells [10], and is domi nant over H3K4me3 [11], the activation of transcription at developmentally regulated genes requires the activity of a third family of enzymatic proteins, which act as H3K27me3 de methylases -namely, lysine demethylase family 6 (KDM6) members UTX and JMJD3 [12][13][14]. Th e KMT6 family of enzymes thus establishes gene silencing at developmentally regu lated loci, while the KDM6 and KMT2 families of enzymes work together to antagonize this repressive activity and permit gene expression in specifi c cell types. Reciprocally, KMT6-mediated methylation of histones is used to silence developmentally regulated genes as lineage restriction takes place [15].
Several developmentally regulated, lineage-specifi c regu lators have been defi ned in muscle regeneration. Th ese include Pax7 in the quiescent and activated satellite cells, MyoD and Myf5 in the proliferating myoblasts, and myogenin (Myog) in the fusion-competent myocytes that repair the damaged fi ber (see Figure 1). While the complete pathway of epigenetics that modulate the temporal and spatial expression of these lineage-specifi c regulators remains to be elucidated, strong evidence exists showing a role for PcG/TrxG antagonism in modulating the expression of these muscle-specifi c transcriptional regulators at the diff erent stages of muscle regeneration.
In quiescent satellite cells, the Pax7 gene is expressed while modulators of cell-cycle progression and musclespecifi c transcriptional regulators remain silenced. To date, epigenetic analysis of quiescent satellite cells has been limited by technical challenges. Firstly, the current techniques for explanting muscle tissue and growing progenitors ex vivo are by themselves suffi cient to trigger satellite cell diff erentiation, altering the epigenomic profi le. Secondly, the limited number of quiescent satellite cells present on a muscle fi ber [16] has to date precluded chromatin immunoprecipitation analysis to determine the role of PcG and TrxG proteins in establishing the epigenetic state of these cells. Th e existence of histone modifi cations at developmentally regulated genes during the later stages of myogenesis, however, implies a regulation through the antagonistic functions of PcG and TrxG proteins. For instance, a transition from a transcriptionally permissive H3K4me3 mark to a repressive H3K27me3 mark induced by Ezh2 was observed on the Pax7 gene as proliferating myoblasts turn off this important marker of satellite cell identity, and prepare for diff erentiation [17]. Similarly, genes involved in cell-cycle and Trithorax group (TrxG) methyltransferase complexes at developmentally regulated loci is depicted. Histone modifi cations or the presence of PcG/ TrxG complexes on the gene highlighted in grey are predicted and have not been formally shown (please see text for rationale on the predictions). (a) Cells undergoing symmetrical cell division will express Pax7 and the genes involved in cell-cycle progression. These genes are predicted to be marked by TrxG-mediated H3K4me3, while the repressed MyoD/Myf5 and Myog genes would be marked by the repressive H3K27me3 mark. In the case of Myf5/MyoD, it will be interesting to determine whether these are bivalently marked genes poised for transcription. (b) During asymmetrical cell division, one of the two cells will go on to become a proliferating myoblast. The proliferating myoblast will express genes involved in cell-cycle progression, as well as Pax7, and Myf5/MyoD. These genes are known to be marked by H3K4me3 in proliferating myoblasts, and in the case of Myf5 it has been shown that this mark is established through the recruitment of TrxG proteins by Pax7. (c) In terminally diff erentiating cells that will fuse to the damaged fi ber, Pax7 is silenced along with genes involved in cell-cycle progression. This repression involves PcGmediated incorporation of H3K27me3 into the chromatin at these genes. At this time, the Myog gene becomes expressed as MyoD collaborates with Mef2d and Six4 to establish the transcriptionally permissive state of H3K4me3. MRF, muscle regulatory factor.  progression are enriched for the permissive H3K4me3 mark in proliferation conditions [18], and then become enriched for the repressive H3K27me3 mark [19] through a process involving the E2F family of transcription factors and the retinoblastoma protein as the cells exit the cell cycle to terminally diff erentiate [20]. A role for PRC2mediated repression at the Myf5 locus in quiescent satellite cells can also be inferred from the observation that this gene becomes marked by the antagonizing TrxG-mediated mark of H3K4me3 in proliferating myoblasts [21]. While these fi ndings are strongly sugges tive of a role for TrxG and PcG in maintaining the quiescent state, confi rmation of this mechanism will require the use of more sensitive detection techniques such as chromatin immunoprecipitation sequencing for H3K4me3 and H3K27 on satellite cells obtained by laser-capture microdissection of fi xed muscle tissue.
Upon muscle injury, satellite cells become activated and re-enter the cell cycle. Th ese cells begin to express cell-cycle regulatory genes, which become marked by H3K4me3 [18]. Satellite cells that divide in the planar orientation along the fi ber will undergo a symmetrical cell division and give rise to two satellite cells that can reenter the quiescent state [5]. In contrast, cells that divide in the apical-basal plane will undergo asymmetric cell division to give rise to one satellite cell (which returns to the quiescent state), and one proliferating myoblast [5]. Th e proliferating myoblast is characterized by the expression of Myf5/MyoD [5] as well as genes that regulate cell-cycle progression while the satellite cell marker Pax7 is progressively silenced. As described above, the activation of transcription at Myf5 and genes involved in cell-cycle progression coincides with enrichment of the transcriptionally permissive H3K4me3 mark within their chromatin [21]. In contrast, the Pax7 gene transitions from a transcriptionally permissive state of H3K4me3 to a repressive state of H3K27me3 as the cell proceeds through diff erentiation [17].
Th e formation of multinucleated myotubes requires the downregulation of Pax7, Myf5, and cell-cycle regulatory genes, and the activation of Myog. Expression of the Myog gene coincides with the removal of the repressive H3K27me3 mark [22,23] and the appearance of the transcriptionally permissive H3K4me3 mark within the 5' end of the gene [23,24]. Coincident with the terminal diff erentiation, myoblasts exit the cell cycle as regulators of this process are silenced through incorporation of the H3K27me3 modifi cation into chromatin marking their respective genes [19]. While our knowledge of epigenetic marking of chromatin in proliferating and diff erentiating myoblasts is currently restricted to a limited number of genes, advances in high-throughput sequencing should soon provide the epigenetic status for the entire muscle transcriptome at diff erent stages of muscle regeneration.

Targeting Polycomb group and Trithorax group proteins to muscle-specifi c genes
Th e H3K27me3 mark is established by proteins of the KMT6 (Ezh1 and Ezh2) family of PcG proteins. In 2004, Caretti and colleagues were the fi rst to demonstrate the involvement of PcG proteins in myogenic gene silencing [25]. Th ey showed that the expression of two terminal muscle diff erentiation genes, Myh10 (myosin, heavy polypeptide 10, nonmuscle) and Ckm (muscle creatine kinase), are silenced via PcG repression in proliferating myoblasts, and that this silencing is lifted upon diff erentiation. An interesting aspect of these fi ndings is that the recruitment of Ezh2 (KMT6B), the catalytic subunit of PRC2, to its target genes in precursor cells is mediated by the sequence-specifi c transcription factor YY1. Th e mechanism that allows YY1-mediated targeting of Ezh2 to these muscle-specifi c genes is intriguing, as both proteins are ubiquitously expressed. Furthermore, as hundreds of genes are coordinately induced upon myogenic diff erentiation [26][27][28][29], it will be important to identify those genes that are silenced by PRC2 in precursor cells, and to determine whether YY1 mediates KMT6 recruitment in all such instances.
Studies of the Ezh2-mediated repression of Notch1 expression in TNFα-treated satellite cells did not examine the mechanism of PRC2 recruitment [30]. Studies of the Pax7 gene, however, showed that YY1 also mediates the recruitment of Ezh2 to the transcriptional regulatory region of this marker of satellite cell identity to silence its expression in proliferating myoblasts [17]. Th is recruitment of Ezh2 to the Pax7 gene is modulated through mitogen-activated protein kinase (MAPK) signaling, where Ezh2 is phosphorylated by p38 MAPK to permit its interaction with the enhancer bound YY1. In contrast, recruitment of Ezh2 by YY1 to the Myh10 gene is not modulated by p38 MAPK signaling [17]. Furthermore, Ezh2 departs the Myh10 and CKm genes when p38 MAPK signaling is activated upon terminal diff erentiation [25]. An impor tant question raised by these studies, therefore, is how p38 MAPK can stimulate YY1 and Ezh2 recruitment to a given locus (Pax7, silenced in diff erentiated cells) but not to another (Myh10, silenced in proliferating cells). Possibly the com position of multiprotein complexes at the regulatory region of these genes is not entirely defi ned, and p38-mediated stimulation depends on additional unidentifi ed factors that might be diff erentially recruited to these loci.
Evidence suggests a role for additional factors in targeting Ezh2 to specifi c loci since high-throughput studies in embryonic stem cells show that the genomic binding profi les of PRC2 and YY1 do not overlap [31]. In these pluripotent cells, the histone demethylase Jarid2 has been shown to mediate recruitment of PRC2 (Ezh2) to specifi c genes [32][33][34]. Jarid2 could also be involved in targeting Ezh2 to muscle-specifi c genes as it is expressed in satellite cells before being downregulated twofold during diff erentiation (FJD and AB, unpublished observa tions based on published datasets [35,36]) Determining the relative role of these two pathways to the establishment of PRC2-mediated transcription repression during muscle regeneration will be of future interest, and will require satellite cell-specifi c knockout/knockdown of YY1 and/or Jarid2.
An important implication of the fi ndings on gene repression mediated by PRC2 is that this silencing of muscle development genes must be lifted for diff erentiation to occur. Removal of the H3K27me3 mark is mediated by KDM6 family members UTX (KDM6A) and JMJD3 (KDM6B) [12][13][14]. Interestingly, the demethylase UTX can associate with TrxG proteins, which antagonize PcG function by marking genes for activation [37]. To date, the recruitment of KDM6 family members to muscle-specifi c genes has only been examined in terminally diff erentiating myoblasts. In these cells, UTX is fi rst recruited to the promoter region of the Myog gene, where it then associates with the elongating RNA polymerase II to demethylate a region extending over the entire length of the gene [22,23]. Interestingly, recruitment of the UTX demethylase to the Myog locus is mediated by the homeodomain transcription factor Six4 [22,23]. Importantly, Six1 and Six4 factors are involved in upregulating the expression of Pax3, MyoD, Myf5 and Myog [38][39][40] and of fast-twitch muscle-function genes [41] during muscle development. Furthermore, Six1 and Six4 are essential for terminal diff erentiation in adult myoblasts [38,42] and they function in part by cooperating with the muscle regulatory factors (MRFs) MyoD and Myog in activating target gene transcription [42]. Genome-wide profi ling of Six1 binding in myoblasts revealed a strong correlation between Six binding and target gene activation during diff erentiation [42]. Th is observation suggests that Six factors may have a global function in recruiting UTX complexes to developmentally regulated genes during myoblast diff er entiation.
Following removal of the repressive H3K27me3 modifica tion, developmentally regulated genes become enriched for the transcriptionally permissive H3K4me3 mark to establish high levels of gene expression. Indeed, studies in proliferating myoblasts have shown that Pax7 is responsible for recruitment of the TrxG complex Ash2L into the Myf5 locus to mark the gene by H3K4me3 [21]. In terminally diff erentiating myoblasts, targeting of the Ash2L complex to the Myog promoter is mediated by the MADS-domain transcription factor Mef2d [23,24]. Several diff erent transactivators can thus clearly recruit Ash2L complexes to developmentally regulated genes to mediate the marking of chromatin by H3K4me3 during muscle regeneration. Importantly, the recruitment of Ash2L to the Myog gene has been shown to be modulated by p38 MAPK signaling through a direct phosphorylation of Mef2d [23,24]. Th is ability to modulate the recruitment of Ash2L to the Myog promoter through inhibition of p38 MAPK signaling suggests a possible mechanism to regulate gene expression therapeutically.

DNA methylation in muscle regeneration
In addition to the repressive H3K27me3 mark mediated by PRC2/Ezh2 [10], methylation of CpG dinucleotides (5-methylcytosine) within a gene regulatory region can be heritably transmitted to daughter cells to block transcription [43,44]. Th e importance of this methylation of DNA in myogenesis has been established from early studies showing that treatment of fi broblast with the inhi bitor of DNA methylation (5-azacytidine) caused cells to diff erentiate towards the muscle lineage [45]. Subse quently, the Weintraub group used a genomic library obtained from 5-azacytidine-treated fi broblasts to clone the master regulator of muscle gene expression, MyoD [46].
Reciprocally, more recent studies have shown that treatment of C2C12 cells with an inhibitor of DNA methylation (zebularine) caused the cells to diff erentiate into a smooth muscle lineage [47]. Th is observation provides strong evidence that DNA methylation plays an important role in repressing factors involved in estab lishing alternate cell fates. Interestingly, the two repres sive marks of CpG methylation and H3K27me3 have been shown to co-exist at specifi c genes in cells of restricted/limited potency [9,48]. Moreover, co-existence of methylated H3K27 and CpG dinucleotides within transcriptional regulatory regions is not coincidental because Ezh2 has been shown to target the de novo DNA methyltransferase enzymes DNMT3a and DNMT3b to specifi c genes [49]. Th is combination of epigenetic marks is proposed to provide a more stable repression of trans cription at genes coding for either mediators of pluri potency or determination factors that are specifi c to alternate cell lineages [9,48]. Not all Ezh2 target genes, however, are marked by methylated CpG dinucleotides. Instead, genes with nonmethylated CpG dinucleotides are repressed through a bivalent chromatin state of nucleosomes doubly marked by methylated H3K4 and H3K27 that remain poised for activation [50]. Th e presence of methy lated H3K4 within the nucleosome is proposed to block the recruitment of DNTM3a/DNTM3b to chromatin [51,52] and to maintain the ability of these PcG marked genes to be activated later in lineage commitment. As a general rule, therefore, genes no longer required for lineage progres sion would be targeted for stable repression by a combi nation of H3K27me3 and CpG methylation, while genes required for further lineage progression would be bivalently marked by H3K4me3 and H3K27me3.
Th e importance of bivalent chromatin domains in regulating expression of muscle-specifi c genes remains to be established. Th e fi nding that methyl-CpG binding proteins mediate reorganization of chromatin during terminal myogenesis, however, confi rms an essential role for this epigenetic mark in muscle regeneration [53]. Recent studies have shown the involvement of Ezh2 and DNMT3b in establishing repression at the Notch-1 promoter during satellite cell activation [30]. Indeed, downregulation of Notch-1 occurs in an Ezh2-dependent manner and results in accumulation of the repressive H3K27me3 mark as well as recruitment of DNMT3b to mediate DNA methylation within the promoter region of this gene [30]. It remains to be determined whether Ezh2 or DNMT3b plays a role in downregulating other mediators of satellite cell function such as Pax7 in proliferating myoblasts. Over laying genome-wide DNA methylation (obtained using either bisulfi te sequencing or MeDIP) and H3K27me3 patterns (obtained using chromatin immuno preci pitation) in satellite cells will permit a full appreciation of the extent to which these complementary epigenetic marks modulate the myogenic gene expression program.

The function of Pax7 in satellite cells
Mice defi cient in Pax7 expression are characterized by low-weight, skeletal muscle of small caliber and by null or very low numbers of satellite cells [54]. Surprisingly, it was recently reported in adult mice that myogenic regeneration occurs in the absence of Pax7 (and/or Pax3), suggesting that the homeodomain transcription factor would only be essential for growth and regeneration during the juvenile period [55]. However, considering the role of Pax7 in establishing the H3K4me3 marks at muscle regulatory genes such as Myf5 [21], an important role for Pax7 in the epigenetic modifi cation of histones in adult satellite cells is likely to exist.
In light of the fact that satellite cells can regenerate damaged muscle in the absence of Pax7, we propose that this transcriptional regulator could act prior to the onset of adulthood to establish stable epigenetic modifi cation of chromatin whose infl uence on gene expression continues after its expression has been ablated. Th is idea of epigenetic marking of chromatin to maintain cellular memory is supported by studies in Myf5-Cre/ROSA26-YFP mice, where it was shown that YFP + satellite cells (which had previously expressed Myf5 and represent 90% of the satellite cell population) turn on expression of the endogenous Myf5 gene with faster kinetics than YFPsatellite cells [5]. Consistent with this, we propose a model in which Pax7-dependent epigenetic marks set up during the juvenile growth phase would establish satellite cell identity permanently. As these epigenetic marks could persist over successive cycles of proliferation/ quiescence in satellite cells, such a scenario would make Pax7 expression dispensable in adult cells. However, the identifi cation of Pax7-dependent marks in juvenile satellite cells, and of Pax7-bound genomic loci, will be required to verify this hypothesis formally.
How might Pax7 act to epigenetically mark genes of the muscle transcriptome? Pax7 could participate in the establishment of a bivalent state at muscle genes (such as Myf5) in quiescent satellite cells where the H3K4me3 mark co-exists with the repressive H3K27me3 mark to poise them for activation [50]. In such a case, activation of the muscle genes would no longer require Pax7 in the adult satellite cells as the chromatin would already be marked by H3K4me3 in juvenile satellite cells. Th is mark would persist through rounds of proliferation/quiescence, but would be counteracted at specifi c genes (depending on cellular context) by the regulated removal of the H3K27me3 mark. Recruitment of a KDM6 family histone demethylase specifi c to the gene by an additional transcription factor such as Six4 would thus be suffi cient to establish expression of muscle development genes.
Alternatively, Pax7 could epigenetically mark genes of the muscle transcriptome through the introduction of variant histones within its target genes. Previous studies have shown that Pax7 can interact with HIRA, a chaperone specifi c for the variant histone H3.3 [56]. Because nucleosomes enriched in histone H3.3 are generally found at the start sites of transcribed genes [57] and are involved in epigenetic memory [58], the Pax7-HIRA interaction could prevent the permanent silencing of its target genes by marking them with H3.3. Indeed, the MyoD gene is marked by H3.3 in proliferating myoblasts [59]. Interestingly, this mark is stable enough to permit expression of MyoD in Xenopus oocytes that have undergone nuclear transfer using a nucleus from a muscle donor cell [58]. Th ese two scenarios, which are not mutually exclusive, could explain how Pax7 would establish the inheritance of an active chromatin state at important loci in juvenile satellite cells, prior to their transcriptional activation.

Modulating epigenetics as a therapeutic approach to muscular dystrophy
Th e importance of the epigenetic pathways in modulation of tissue-specifi c gene expression makes them excellent candidate targets for disease interventions. Several drugs that attempt to modify epigenetic mechanisms are currently undergoing clinical trial [60,61]. Th ese include histone deacetylase inhibitors [61], histone methyltransferase inhibitors [62], as well as the inhibitor of DNA methylation 5-azacytidine [63].
In the case of muscular dystrophy, histone deacetylase inhibitors are currently being examined using the mdx mouse model for their ability to improve the dystrophic phenotype [64]. Here it is believed that deacetylase inhibi tors prevent the eff ects of disrupted nitric oxide signaling on acetylation at chromatin within the diseased muscle [65]. Th e eff ects of prolonged treatment with drugs that inhibit these ubiquitously required chromatinmodifying enzymes, however, are of potential concern. As an alternative or complement to this strategy, the identifi cation of small molecules that promote or disrupt the specifi c protein-protein interactions required for targeting the epigenetic enzymes to determined loci within the genome could have a similar benefi t without the side eff ect of modifying gene expression in other cell types. Along this line of thought, a cell-permeable small molecule that inhibits the protein-protein interaction between bromo domain-containing protein BRD4 and histones H3-acetylated at lysine 14 has recently been reported [66]. Th e broad-reaching eff ects of blocking this inter action, however, maintain the same caveats described above for blocking the enzymatic activity of ubiquitously expressed epigenetic proteins.
Future screens should be directed at disrupting the interactions between the PcG and TrxG proteins and the transcriptional regulators that target these enzymes to muscle-specifi c genes. As many of the PcG and TrxG activities are present in multiprotein complexes, the screening of molecules to disrupt this targeted recruitment to muscle-specifi c genes will fi rst require the delineation of specifi c subunits that mediate direct interactions with the transcriptional regulator of interest. Th e use of small molecules to disrupt interactions between transcriptional regulators and PcG and Trx proteins will thus require extensive research before they can be developed to treat muscular dystrophy.
An alternative approach to targeting PcG and TrxG activities to specifi c genes is the use of artifi cial zincfi nger transcription factors [67]. Th is technique has recently been used to target the VP16 transactivation domain to a 9-base-pair sequence within the utrophin promoter, allowing for an upregulation of expression from the endogenous gene in the mdx mouse [68]. In this case, a three-zinc-fi nger array fused to VP16 was expressed in transgenic animals using the muscle-specifi c myosin light-chain promoter. While a 9-base-pair target sequence is not suffi cient to ensure a single genomic targeting event, artifi cial activators have been generated containing six zinc fi ngers that permit the targeting of a transactivation domain to an 18-base-pair sequence of the γ-globin gene that is unique in the genome [69]. As an alternative to the VP16 fusion with the gene-specifi c zinc fi nger array, an enzyme such as Ezh2, UTX, or MLL1 could be fused to these artifi cial DNA binding domains. In this way, TrxG or PcG fusion proteins could be targeted to individual loci within the genome to mediate silencing or activation of specifi c genes.
While utrophin is a therapeutically important gene for treatment of muscular dystrophy, an alternative target has been suggested by the recent fi nding that the discrepancy between the mild dystrophic phenotype observed in mdx mice and the severe phenotype observed in humans can be explained through the inactivation of the telomerase in the latter [70]. An artifi cial transcriptional zinc-fi nger-mediated upregulation of telomerase activity through epigenetic mechanisms specifi cally in satellite cells could perhaps lead to increased self-renewal such that the stem cells do not become depleted as the need for repair continues over the lifetime of the patient. A similar approach has recently been explored to repress expression of telomerase in transformed cells using artifi cial zinc fi ngers fused to the transcriptional repressor domain of KRAB [71]. Epigenetic enzymes could thus represent a viable target for future gene therapies to permit muscle repair in muscular dystrophy patients. However, current limitations associated with gene therapy remain -we must ensure that these zinc fi nger proteins are targeted to muscle cells effi ciently while also ensuring that they do not activate muscle genes in other cell types.

Conclusions
Th ere is little doubt that the incredible ability of certain structural features of chromatin to be perpetuated over several cell divisions is at play in controlling the fate of adult muscle stem cells. Th e elucidation of epigenetic mechanisms regulating the function of satellite cell function is still just beginning, but signifi cant progress is being made at an exponential pace, thanks in part to our increasing knowledge of how these molecular pathways are laid out in embryonic stem cells. Moreover, technical advances are constantly emerging, speeding up our study of the inner workings of the epigenetic control machinery and helping with the design of new therapeutic approaches based on this knowledge. While most muscular illnesses are not epigenetic diseases per se, we can envision a near future where epigenetic therapies will be part of a successful treatment regimen for dystrophic patients.