Epigenetic regulation of Wnt7b expression by the cis-acting long noncoding RNA Lnc-Rewind in muscle stem cells

Skeletal muscle possesses an outstanding capacity to regenerate upon injury due to the adult muscle stem cells (MuSCs) activity. This ability requires the proper balance between MuSCs expansion and differentiation which is critical for muscle homeostasis and contributes, if deregulated, to muscle diseases. Here, we functionally characterize a novel chromatin-associated lncRNA, Lnc-Rewind, which is expressed in murine MuSCs and conserved in human. We find that, in mouse, Lnc-Rewind acts as an epigenetic regulator of MuSCs proliferation and expansion by influencing the expression of skeletal muscle genes and several components of the WNT (Wingless-INT) signalling pathway. Among them, we identified the nearby Wnt7b gene as a direct Lnc-Rewind target. We show that Lnc-Rewind interacts with the G9a histone lysine methyltransferase and mediates the in cis repression of Wnt7b by H3K9me2 deposition. Overall, these findings provide novel insights into the epigenetic regulation of adult muscle stem cells fate by lncRNAs

H3K9 methylation controls Fibro-Adipogenic Progenitors identity and skeletal muscle repair

Fibro-Adipogenic Progenitors (FAPs) are crucial regulators of muscle homeostasis as they possess the intrinsic ability to either support muscle regeneration or to contribute to fibro-adipogenic degeneration of dystrophic muscles. Therefore, the elucidation of the molecular mechanisms controlling their phenotypical plasticity holds therapeutic potential. Here we provide evidence that FAPs are particularly enriched in histone H3 lysine K9 methyltransferases (H3K9 KMTs), G9a, GLP and Prdm16. Our data indicate that H3K9 KMTs safeguard FAPs identity by repressing alternative transcriptional programs through deposition of H3K9 di- methylation (H3K9me2). Specifically, we show that Prdm16 controls G9a/GLP-mediated deposition of H3K9me2 at muscle- specific loci. Of note, we found Prdm16, G9a and GLP particularly enriched at the nuclear lamina (NL) of FAPs, suggesting they organize heterochromatin at the nuclear periphery to maintain the stable repression of genes encoding alternative developmental regulators. Accordingly, pharmacological inhibition or RNAi- mediated knock-down (KD) of H3K9 KMTs de-repress master myogenic genes in FAPs and induce the muscle differentiation program. Together, our findings reveal a FAPs-specific epigenetic axis important to control their identity. These findings are important especially for the possible therapeutic application to conceive strategies aimed to reprogram FAPs fate in vivo to prevent degeneration of diseased muscles

Role of Histone H3 Lysine 9 (H3K9) methyltransferases G9a and GLP in the epigenetic regulation of Fibroadipogenic progenitors (FAPs) differentiation during Duchenne Muscular Dystrophy (DMD) progression

Duchenne muscular dystrophy (DMD) is a severe X-linked neuromuscular degenerative disorder that leads to progressive muscle weakness. This is due to loss of muscle tissue that culminates with its replacement with fat and fibrotic infiltrates, in coincidence with the final stages of disease. Despite recent progresses in genome editing approaches have demonstrated the possibility to correct the genetic defect in vivo, the cure for DMD is still a big challenge. Therefore, pharmacological therapies aimed to counteract the fibro-adipogenic degeneration and to promote the compensatory regeneration that is typical of the early stages of disease hold great promise to slow-down DMD progression. Fibroadipogenic progenitors, FAPs, have been shown to be responsible of fat and fibrotic tissue deposition in degenerating dystrophic muscles, while also contributing to muscle regeneration at early stages of the disease.1,2 As such, understanding the molecular basis of FAP’s differentiation might reveal possible pharmacological targets to manipulate their phenotypical plasticity in vivo, with the ultimate goal to promote muscle regeneration and concomitantly block fibro-adipogenic degeneration. Our results and data from the literature suggest that methylation of Lysine 9 of histone H3 (H3K9) by specific methyltransferases (KMTs), is one of the epigenetic pathway involved in the control of FAPs’ alternative fates. In particular, among the different H3K9 KMTs, the mono- and di- methyltransferases G9a and GLP are of particular relevance in controlling the repression of muscle-specific genes in myogenic precursors,3,4 and likely in FAPs. In fact, our preliminary data show that the in vitro inhibition of H3K9 KMTs in FAPs from mdx mice (the DMD murine model) induces myogenic differentiation at expenses of their adipogenic potential. We show here that FAPs isolated from injured wild type mice treated in vivo with G9a/GLP specific inhibitors display increased expression of myogenic markers and de- regulation of fibroadipogenic genes. Taken together, our results suggest that H3K9 KMTs inhibitors could promote the myogenic potential of muscle progenitor cells and might become a potential new therapeutic approach in the treatment of DMD

Role of Histone H3 lysine 9 methyltransferases during Duchenne Muscular Dystrophy progression

Lysines Methyltransferases (KMTs) have recently raised increased interest as potential targets of therapeutic value thanks to the possibility to revert aberrant epigenetic states associated with human diseases. KMTs catalyzing mono-and di-methylation of lysine 9 on histone 3 (H3K9me1/2) are typically involved in gene repression and heterochromatin formation. In the context of muscle differentiation, the H3K9 KMTs G9a and GLP are emerging as critical epigenetic modulators able to maintain the repression of muscle-specific genes in embryonic precursors and in myoblasts, therefore preventing their premature differentiation.1,2 Our preliminary data suggest that H3K9 KMTs are also involved in the epigenetic control of lineage choice of a population of muscle-resident mesenchymal stem cells, called fibro- adipogenic progenitors (FAPs). FAPs play key roles in Duchenne Muscular Dystrophy (DMD) by both supporting the myogenic differentiation of muscle stem cells in the regenerating phase or by contributing to fibrosis and fat deposition in advanced stages of disease.3,4 However, the molecular regulation governing their lineage determination is largely unknown. We show here that pharmacological inhibition of G9a/GLP, by the use of its specific inhibitor (UNC0642), induce a FAPs’ lineage switch. Indeed, FAPs isolated from young dystrophic (mdx) mice, cultured ex vivo in the presence of UNC0642 unmask a myogenic potential, as suggested by the appearance of MyoD positive cells and increased expression of myogenic genes. This is paralleled by an impaired adipogenic differentiation, as confirmed by a decreased number of FAPs-derived adipocytes, upon UNC0642 treatment. In sum, our preliminary evidence suggest that the H3K9 KMTs G9a/GLP might be involved in maintaining silent the capacity of FAPs to give rise to myogenic cells and indicate these proteins as possible pharmacological targets for therapeutic approaches aimed to promote regeneration, and to prevent fibro-adipogenic degeneration, of dystrophic muscles

Histone 3 lysine 9 methyltransferases G9a and GLP as potential pharmacological targets in skeletal muscle regeneration and Duchenne Muscular Dystrophy

Histone Lysine Methyltransferases (KMTs) are epigenetic modifiers that dynamically control gene expression during stem cell differentiation. Among the different KMTs, EHMT2/G9a and EHMT1/GLP, responsible of mono- and di-methylation of Lysine 9 of histone H3 (H3K9), are of particular relevance in the context of myogenesis since they have been shown to control the repression of muscle-specific genes in myogenic precursors and to prevent their premature differentiation. Modulation of their activity might therefore be exploited to promote the expression of muscle-specific genes and to enhance muscle differentiation in tissues whose myogenic capacity is compromised due to a pathological condition, as in the case of muscles affected by Duchenne Muscular Dystrophy (DMD). DMD is a severe X-linked neuromuscular degenerative disorder that leads to progressive muscle weakness associated with loss of muscle tissue and replacement with adipose and connective infiltrates, in coincidence with the final stages of disease. Despite recent progresses in genome editing approaches, the cure for DMD is still a big challenge and pharmacological therapies aimed to counteract the fibro-adipogenic degeneration and to promote the compensatory regeneration, typical of the early stages of disease, hold great promise to slow-down DMD progression. Here we provide evidence of the pro-regenerative effect of G9a/GLP specific inhibitors in vivo. Our results show that in vivo inhibition of G9a/GLP-mediated H3K9me2 improves skeletal muscle regeneration. This is caused by an accelerated myogenic capacity of muscle stem cells (MuSCs) and by an impaired adipogenic differentiation of fibro-adipogenic progenitors (FAPs), which rather unmasks a previously silent myogenic capacity. Our preliminary results provide proof of concept of the use of H3K9 KMTs specific inhibitors as potential pharmacological strategy to promote the regenerative response of diseased, dystrophic, muscles, while concomitantly blocking their fibro-adipogenic degeneration

Prdm16-mediated H3K9 methylation controls fibro-adipogenic progenitors identity during skeletal muscle repair

H3K9 methylation maintains cell identity orchestrating stable silencing and anchoring of alternate fate genes within the heterochromatic compartment underneath the nuclear lamina (NL). However, how cell type–specific genomic regions are specifically targeted to the NL is still elusive. Using fibro-adipogenic progenitors (FAPs) as a model, we identified Prdm16 as a nuclear envelope protein that anchors H3K9-methylated chromatin in a cell-specific manner. We show that Prdm16 mediates FAP developmental capacities by orchestrating lamina-associated domain organization and heterochromatin sequestration at the nuclear periphery. We found that Prdm16 localizes at the NL where it cooperates with the H3K9 methyltransferases G9a/GLP to mediate tethering and silencing of myogenic genes, thus repressing an alternative myogenic fate in FAPs. Genetic and pharmacological disruption of this repressive pathway confers to FAP myogenic competence, preventing fibro-adipogenic degeneration of dystrophic muscles. In summary, we reveal a druggable mechanism of heterochromatin perinuclear sequestration exploitable to reprogram FAPs in vivo

Statins interfere with the attachment of S. cerevisiae mtDNA to the inner mitochondrial membrane

The 3-hydroxy-3-methylglutaryl-CoA reductase, a key enzyme of the mevalonate pathway for the synthesis of cholesterol in mammals (ergosterol in fungi), is inhibited by statins, a class of cholesterol lowering drugs. Indeed, statins are in a wide medical use, yet statins treatment could induce side effects as hepatotoxicity and myopathy in patients. We used Saccharomyces cerevisiae as a model to investigate the effects of statins on mitochondria. We demonstrate that statins are active in S.cerevisiae by lowering the ergosterol content in cells and interfering with the attachment of mitochondrial DNA to the inner mitochondrial membrane. Experiments on murine myoblasts confirmed these results in mammals. We propose that the instability of mitochondrial DNA is an early indirect target of statins