Functional Crosstalk between Lysine Methyltransferases on Histone Substrates: The Case of G9A/GLP and Polycomb Repressive Complex 2

Significance: Methylation of histone H3 on lysine 9 and 27 (H3K9 and H3K27) are two epigenetic modifications that have been linked to several crucial biological processes, among which are transcriptional silencing and cell differentiation. Recent Advances: Deposition of these marks is catalyzed by H3K9 lysine methyltransferases (KMTs) and polycomb repressive complex 2, respectively. Increasing evidence is emerging in favor of a functional crosstalk between these two major KMT families. Critical Issues: Here, we review the current knowledge on the mechanisms of action and function of these enzymes, with particular emphasis on their interplay in the regulation of chromatin states and biological processes. We outline their crucial roles played in tissue homeostasis, by controlling the fate of embryonic and tissue-specific stem cells, highlighting how their deregulation is often linked to the emergence of a number of malignancies and neurological disorders. Future Directions: Histone methyltransferases are starting to be tested as drug targets. A new generation of highly selective chemical inhibitors is starting to emerge. These hold great promise for a rapid translation of targeting epigenetic drugs into clinical practice for a number of aggressive cancers and neurological disorders

The RNA helicase DDX5 cooperates with EHMT2 to sustain alveolar rhabdomyosarcoma growth

Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma of childhood characterized by the inability to exit the proliferative myoblast-like stage. The alveolar fusion positive subtype (FP-RMS) is the most aggressive and is mainly caused by the expression of PAX3/7-FOXO1 oncoproteins, which are chal-lenging pharmacological targets. Here, we show that the DEAD box RNA helicase 5 (DDX5) is overexpressed in alveolar RMS cells and that its depletion and pharmacological inhibition decrease FP-RMS viability and slow tumor growth in xenograft models. Mechanistically, we provide evidence that DDX5 functions upstream of the EHMT2/AKT survival signaling pathway, by directly interacting with EHMT2 mRNA, modulating its sta-bility and consequent protein expression. We show that EHMT2 in turns regulates PAX3-FOXO1 activity in a methylation-dependent manner, thus sustaining FP-RMS myoblastic state. Together, our findings identify another survival-promoting loop in FP-RMS and highlight DDX5 as a potential therapeutic target to arrest RMS growth

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

Lamin A/C sustains PcG protein architecture, maintaining transcriptional repression at target genes

Beyond its role in providing structure to the nuclear envelope, lamin A/C is involved in transcriptional regulation. However, its cross talk with epigenetic factors--and how this cross talk influences physiological processes--is still unexplored. Key epigenetic regulators of development and differentiation are the Polycomb group (PcG) of proteins, organized in the nucleus as microscopically visible foci. Here, we show that lamin A/C is evolutionarily required for correct PcG protein nuclear compartmentalization. Confocal microscopy supported by new algorithms for image analysis reveals that lamin A/C knock-down leads to PcG protein foci disassembly and PcG protein dispersion. This causes detachment from chromatin and defects in PcG protein-mediated higher-order structures, thereby leading to impaired PcG protein repressive functions. Using myogenic differentiation as a model, we found that reduced levels of lamin A/C at the onset of differentiation led to an anticipation of the myogenic program because of an alteration of PcG protein-mediated transcriptional repression. Collectively, our results indicate that lamin A/C can modulate transcription through the regulation of PcG protein epigenetic factors

Isolation and culture of muscle stem cells

Polycomb group (PcG) proteins are key epigenetic factors responsible for the proper spatiotemporal repression of defi ned transcriptional programs along the process of cell differentiation, including myogenesis. The discovery of the pivotal role played by PcG factors during myogenic differentiation relied on the possibility to culture myogenic cells in vitro. We describe here the methods currently used to isolate muscle stem cells (MuSCs) both from single myofi bers and from bulk muscles by fl uorescence-activated cell sorting (FACS), highlighting experimental details and critical steps. Through these techniques MuSCs can be effi ciently isolated and cultured in vitro to recapitulate the different phases of myogenesis: activation, expansion, differentiation, and self-renewal

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

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

Single Myofiber Isolation and Culture from a Murine Model of Emery-Dreifuss Muscular Dystrophy in Early Post-Natal Development

Autosomal dominant Emery-Dreifuss muscular dystrophy (EDMD) is caused by mutations in the LMNA gene, which encodes the A-type nuclear lamins, intermediate filament proteins that sustain the nuclear envelope and the components of the nucleoplasm. We recently reported that muscle wasting in EDMD can be ascribed to intrinsic epigenetic dysfunctions affecting muscle (satellite) stem cells regenerative capacity. Isolation and culture of single myofibers is one of the most physiological ex-vivo approaches to monitor satellite cells behavior within their niche, as they remain between the basal lamina surrounding the fiber and the sarcolemma. Therefore, it represents an invaluable experimental paradigm to study satellite cells from a variety of murine models. Here, we describe a re-adapted method to isolate intact and viable single myofibers from post-natal hindlimb muscles (Tibialis Anterior, Extensor Digitorum Longus, Gastrocnemius and Soleus). Following this protocol, we were able to study satellite cells from Lamin Δ8-11 -/- mice, a severe EDMD murine model, at only 19 days after birth. We detail the isolation procedure, as well as the culture conditions for obtaining a good amount of myofibers and their associated satellite-cells-derived progeny. When cultured in growth-factors rich medium, satellite cells derived from wild type mice activate, proliferate, and eventually differentiate or undergo self-renewal. In homozygous Lamin Δ8-11 -/- mutant mice these capabilities are severely impaired. This technique, if strictly followed, allows to study all processes linked to the myofiber-associated satellite cell even in early post-natal developmental stages and in fragile muscles