SMCHD1 activates the expression of genes required for the expansion of human myoblasts.

SMCHD1 is an epigenetic regulatory protein known to modulate the targeted repression of large chromatin domains. Diminished SMCHD1 function in muscle fibers causes Facioscapulohumeral Muscular Dystrophy (FSHD2) through derepression of the D4Z4 chromatin domain, an event which permits the aberrant expression of the disease-causing gene DUX4. Given that SMCHD1 plays a broader role in establishing the cellular epigenome, we examined whether loss of SMCHD1 function might affect muscle homeostasis through additional mechanisms. Here we show that acute depletion of SMCHD1 results in a DUX4-independent defect in myoblast proliferation. Genomic and transcriptomic experiments determined that SMCHD1 associates with enhancers of genes controlling cell cycle to activate their expression. Amongst these cell cycle regulatory genes, we identified LAP2 as a key target of SMCHD1 required for the expansion of myoblasts, where the ectopic expression of LAP2 rescues the proliferation defect of SMCHD1-depleted cells. Thus, the epigenetic regulator SMCHD1 can play the role of a transcriptional co-activator for maintaining the expression of genes required for muscle progenitor expansion. This DUX4-independent role for SMCHD1 in myoblasts suggests that the pathology of FSHD2 may be a consequence of defective muscle regeneration in addition to the muscle wasting caused by spurious DUX4 expression.

Long non-coding RNAs and their role in muscle regeneration.

In mammals, most of the genome is transcribed to generate a large and heterogeneous variety of non-protein coding RNAs, that are broadly grouped according to their size. Long noncoding RNAs include a very large and versatile group of molecules. Despite only a minority of them has been functionally characterized, there is emerging evidence indicating long noncoding RNAs as important regulators of expression at multiple levels. Several of them have been shown to be modulated during myogenic differentiation, playing important roles in the regulation of skeletal muscle development, differentiation and homeostasis, and contributing to neuromuscular diseases. In this chapter, we have summarized the current knowledge about long noncoding RNAs in skeletal muscle and discussed specific examples of long noncoding RNAs (lncRNAs and circRNAs) regulating muscle stem cell biology. We have also discussed selected long noncoding RNAs involved in the most common neuromuscular diseases.

Meeting report: The 2023 FSHD International Research Congress.

Facioscapulohumeral muscular dystrophy (FSHD) is one of the most common inherited muscular dystrophies. As part of the FSHD Society's commitment to promote global communication and collaboration among researchers, the Society collaborated with FSHD Europe and convened its 30th annual International Research Congress (IRC) on June 15-16, 2023, in the city of Milan, Italy. Over 240 researchers, clinicians, patients and pharmaceutical company representatives from a wide geographical background participated to hear about the latest developments and breakthroughs in the field. The meeting was structured to provide a mix of basic and clinical research in five sessions: 1. Discovery research & genetics; 2. Outcome assessments; 3. Disease mechanisms & interventional strategies; 4. Clinical studies & trial design; and 5. Pediatric FSHD. The keynote speakers were Professor Baziel van Engelen (on the importance of incorporating the patient's voice to help refine and improve basic laboratory and clinical research) and Dr. Bénédict Chazaud (on the role of the immune system in normal muscle regeneration and in Duchenne muscular dystrophy). The FSHD IRC was preceded by the Industry Collaborative for Therapeutic Development in FSHD meeting and followed by the World FSHD Alliance network of national patient groups and advocacy organizations for FSHD summit. The Congress concluded with the announcement for the 2024 International Research Congress, which will take place on June 13-14, 2024 in Denver, Colorado, USA, and followed by the FSHD Society's flagship educational conference for the FSHD community, the Patient Connect Conference, on June 15-16, 2024.

MATR3 is an endogenous inhibitor of DUX4 in FSHD muscular dystrophy.

Facioscapulohumeral muscular dystrophy (FSHD) is one of the most common neuromuscular disorders and has no cure. Due to an unknown molecular mechanism, FSHD displays overlapping manifestations with the neurodegenerative disease amyotrophic lateral sclerosis (ALS). FSHD is caused by aberrant gain of expression of the transcription factor double homeobox 4 (DUX4), which triggers a pro-apoptotic transcriptional program resulting in inhibition of myogenic differentiation and muscle wasting. Regulation of DUX4 activity is poorly known. We identify Matrin 3 (MATR3), whose mutation causes ALS and dominant distal myopathy, as a cellular factor controlling DUX4 expression and activity. MATR3 binds to the DUX4 DNA-binding domain and blocks DUX4-mediated gene expression, rescuing cell viability and myogenic differentiation of FSHD muscle cells, without affecting healthy muscle cells. Finally, we characterize a shorter MATR3 fragment that is necessary and sufficient to directly block DUX4-induced toxicity to the same extent as the full-length protein. Collectively, our data suggest MATR3 as a candidate for developing a treatment for FSHD.

DUX4-r exerts a neomorphic activity that depends on GTF2I in acute lymphoblastic leukemia.

Translocations producing rearranged versions of the transcription factor double homeobox 4 (DUX4-r) are one of the most frequent causes of B cell acute lymphoblastic leukemia (B-ALL). DUX4-r retains the DNA binding domain of wild-type DUX4 but is truncated on the C-terminal transcription activation domain. The precise mechanism through which DUX4-r causes leukemia is unknown, and no targeted therapy is currently available. We found that the rearrangement leads to both a loss and a gain of function in DUX4-r. Loss of CBP/EP300 transcriptional coactivator interaction leads to an inability to bind and activate repressed chromatin. Concurrently, a gain of interaction with the general transcription factor 2 I (GTF2I) redirects DUX4-r toward leukemogenic targets. This neomorphic activity exposes an Achilles' heel whereby DUX4-r-positive leukemia cells are exquisitely sensitive to GTF2I targeting, which inhibits DUX4-r leukemogenic activity. Our work elucidates the molecular mechanism through which DUX4-r causes leukemia and suggests a possible therapeutic avenue tailored to this B-ALL subtype.

WDR5 is required for DUX4 expression and its pathological effects in FSHD muscular dystrophy.

Facioscapulohumeral muscular dystrophy (FSHD) is one of the most prevalent neuromuscular disorders. The disease is linked to copy number reduction and/or epigenetic alterations of the D4Z4 macrosatellite on chromosome 4q35 and associated with aberrant gain of expression of the transcription factor DUX4, which triggers a pro-apoptotic transcriptional program leading to muscle wasting. As today, no cure or therapeutic option is available to FSHD patients. Given its centrality in FSHD, blocking DUX4 expression with small molecule drugs is an attractive option. We previously showed that the long non protein-coding RNA DBE-T is required for aberrant DUX4 expression in FSHD. Using affinity purification followed by proteomics, here we identified the chromatin remodeling protein WDR5 as a novel DBE-T interactor and a key player required for the biological activity of the lncRNA. We found that WDR5 is required for the expression of DUX4 and its targets in primary FSHD muscle cells. Moreover, targeting WDR5 rescues both cell viability and myogenic differentiation of FSHD patient cells. Notably, comparable results were obtained by pharmacological inhibition of WDR5. Importantly, WDR5 targeting was safe to healthy donor muscle cells. Our results support a pivotal role of WDR5 in the activation of DUX4 expression identifying a druggable target for an innovative therapeutic approach for FSHD.

RNA binding induces an allosteric switch in Cyp33 to repress MLL1-mediated transcription.

Mixed-lineage leukemia 1 (MLL1) is a transcription activator of the HOX family, which binds to specific epigenetic marks on histone H3 through its third plant homeodomain (PHD3) domain. Through an unknown mechanism, MLL1 activity is repressed by cyclophilin 33 (Cyp33), which binds to MLL1 PHD3. We determined solution structures of Cyp33 RNA recognition motif (RRM) free, bound to RNA, to MLL1 PHD3, and to both MLL1 and the histone H3 lysine N6-trimethylated. We found that a conserved α helix, amino-terminal to the RRM domain, adopts three different positions facilitating a cascade of binding events. These conformational changes are triggered by Cyp33 RNA binding and ultimately lead to MLL1 release from the histone mark. Together, our mechanistic findings rationalize how Cyp33 binding to MLL1 can switch chromatin to a transcriptional repressive state triggered by RNA binding as a negative feedback loop.

A regenerative niche for stem cells.

Production of hyaluronic acid allows regenerative signaling in muscle stem cells after injury.

The SUV4-20H Histone Methyltransferases in Health and Disease.

The post-translational modification of histone tails is a dynamic process that provides chromatin with high plasticity. Histone modifications occur through the recruitment of nonhistone proteins to chromatin and have the potential to influence fundamental biological processes. Many recent studies have been directed at understanding the role of methylated lysine 20 of histone H4 (H4K20) in physiological and pathological processes. In this review, we will focus on the function and regulation of the histone methyltransferases SUV4-20H1 and SUV4-20H2, which catalyze the di- and tri-methylation of H4K20 at H4K20me2 and H4K20me3, respectively. We will highlight recent studies that have elucidated the functions of these enzymes in various biological processes, including DNA repair, cell cycle regulation, and DNA replication. We will also provide an overview of the pathological conditions associated with H4K20me2/3 misregulation as a result of mutations or the aberrant expression of SUV4-20H1 or SUV4-20H2. Finally, we will critically analyze the data supporting these functions and outline questions for future research.

Meeting report: the 2021 FSHD International Research Congress.

Facioscapulohumeral muscular dystrophy (FSHD) is the second most common genetic myopathy, characterized by slowly progressing and highly heterogeneous muscle wasting with a typical onset in the late teens/early adulthood [1]. Although the etiology of the disease for both FSHD type 1 and type 2 has been attributed to gain-of-toxic function stemming from aberrant DUX4 expression, the exact pathogenic mechanisms involved in muscle wasting have yet to be elucidated [2-4]. The 2021 FSHD International Research Congress, held virtually on June 24-25, convened over 350 researchers and clinicians to share the most recent advances in the understanding of the disease mechanism, discuss the proliferation of interventional strategies and refinement of clinical outcome measures, including results from the ReDUX4 trial, a phase 2b clinical trial of losmapimod in FSHD [NCT04003974].

DUX4 Role in Normal Physiology and in FSHD Muscular Dystrophy.

In the last decade, the sequence-specific transcription factor double homeobox 4 (DUX4) has gone from being an obscure entity to being a key factor in important physiological and pathological processes. We now know that expression of DUX4 is highly regulated and restricted to the early steps of embryonic development, where DUX4 is involved in transcriptional activation of the zygotic genome. While DUX4 is epigenetically silenced in most somatic tissues of healthy humans, its aberrant reactivation is associated with several diseases, including cancer, viral infection and facioscapulohumeral muscular dystrophy (FSHD). DUX4 is also translocated, giving rise to chimeric oncogenic proteins at the basis of sarcoma and leukemia forms. Hence, understanding how DUX4 is regulated and performs its activity could provide relevant information, not only to further our knowledge of human embryonic development regulation, but also to develop therapeutic approaches for the diseases associated with DUX4. Here, we summarize current knowledge on the cellular and molecular processes regulated by DUX4 with a special emphasis on FSHD muscular dystrophy.

16th Meeting of the Interuniversity Institute of Myology (IIM) - Assisi (Italy), October 17-20, 2019: Foreword, Program and Abstracts.

The 16th Meeting of the Interuniversity Institute of Myology (IIM), October 17-20, 2019, Assisi, Italy brought together scientists, pharma and patient organization representatives discussing new results on muscle research. Internationally renowned Keynote speakers presented advances on muscle development, homeostasis, metabolism, and disease. Speakers selected among submitted abstracts presented their new, unpublished data in seven scientific sessions. The remaining abstracts were showcased in two poster sessions. Young trainees where directly involved in the selection of keynote speakers, the organizing scientific sessions and roundtables discussions tailored to the interests of their peers. A broad Italian, European and North-American audience participated to the different initiatives. The meeting allowed muscle biology researchers to discuss ideas and scientific collaborations aimed at better understanding the mechanisms underlying muscle diseases in order to develop better therapeutic strategies. The active participation of young trainees was facilitated by the friendly and inclusive atmosphere, which fostered lively discussions identifying emerging areas of myology research and stimulated scientific cross-fertilization. The meeting was a success and the IIM community will continue to bring forward significant contributions to the understanding of muscle development and function, the pathogenesis of muscular diseases and the development of novel therapeutic approaches. Here, we report abstracts of the meeting illustrating novel results of basic, translational, and clinical research, which confirms that the Myology field is strong and healthy.

Report and Abstracts of the 17th Meeting of IIM, the Interuniversity Institute of Myology:Virtual meeting, October 16-18, 2020.

In 2020, due to the COVID-19 pandemic, the annual meeting of the Interuniversity Institute of Myology (IIM), took place on a virtual platform. Attendees were scientists and clinicians, as well as pharmaceutical companies and patient organization representatives from Italy, several European countries, Canada and USA. Four internationally renowned Keynote speakers presented recent advances on muscle stem cells regulation, skeletal muscle regeneration, quantitative biology approaches, and metabolic regulation of muscle homeostasis. Novel, unpublished data by young trainees were presented as oral communications or posters, in five scientific sessions and two poster sessions. On October 15, 2020, selected young trainees participated to the High Training Course on "Advanced Myology", organized together with the University of Perugia, Italy. The course, on a virtual platform, showcased lectures on muscle development and regulation of muscle gene expression by international speakers, and roundtables discussions on "Single cell analysis of skeletal muscle" and "Skeletal muscle stem cell in healthy muscle and disease". The Young IIM Committee, composed by young trainee winners of awards in the past IIM Meeting editions, was directly involved in the selection of keynote speakers, the organization of scientific sessions and roundtables discussions tailored to the interests of their peers. A broad audience of Italian, European and North American participants contributed to the different initiatives. The meeting was characterized by a friendly and inclusive atmosphere, facilitating lively and stimulating discussions on emerging areas of muscle research. The meeting stimulated scientific cross-fertilization fostering novel ideas and scientific collaborations aimed at better understanding muscle normal physiology and the mechanisms underlaying muscle diseases, with the ultimate goal of developing better therapeutic strategies. The meeting was a success, and the number of meeting attendees was the highest of all IIM Meeting editions. Despite the current difficulties imposed by the COVID-19 pandemic, we are confident that the IIM community will continue to grow and deliver significant contributions to the understanding of muscle development and function, the pathogenesis of muscular diseases and the development of novel therapeutic approaches. Here, abstracts of the meeting illustrate the new results on basic, translational, and clinical research, confirming that our field is strong and healthy.

The Suv420h histone methyltransferases regulate PPAR-γ and energy expenditure in response to environmental stimuli.

Obesity and its associated metabolic abnormalities have become a global emergency with considerable morbidity and mortality. Epidemiologic and animal model data suggest an epigenetic contribution to obesity. Nevertheless, the cellular and molecular mechanisms through which epigenetics contributes to the development of obesity remain to be elucidated. Suv420h1 and Suv420h2 are histone methyltransferases responsible for chromatin compaction and gene repression. Through in vivo, ex vivo, and in vitro studies, we found that Suv420h1 and Suv420h2 respond to environmental stimuli and regulate metabolism by down-regulating peroxisome proliferator-activated receptor gamma (PPAR-γ), a master transcriptional regulator of lipid storage and glucose metabolism. Accordingly, mice lacking Suv420h proteins activate PPAR-γ target genes in brown adipose tissue to increase mitochondria respiration, improve glucose tolerance, and reduce adipose tissue to fight obesity. We conclude that Suv420h proteins are key epigenetic regulators of PPAR-γ and the pathways controlling metabolism and weight balance in response to environmental stimuli.

A novel L1CAM isoform with angiogenic activity generated by NOVA2-mediated alternative splicing.

The biological players involved in angiogenesis are only partially defined. Here, we report that endothelial cells (ECs) express a novel isoform of the cell-surface adhesion molecule L1CAM, termed L1-ΔTM. The splicing factor NOVA2, which binds directly to L1CAM pre-mRNA, is necessary and sufficient for the skipping of L1CAM transmembrane domain in ECs, leading to the release of soluble L1-ΔTM. The latter exerts high angiogenic function through both autocrine and paracrine activities. Mechanistically, L1-ΔTM-induced angiogenesis requires fibroblast growth factor receptor-1 signaling, implying a crosstalk between the two molecules. NOVA2 and L1-ΔTM are overexpressed in the vasculature of ovarian cancer, where L1-ΔTM levels correlate with tumor vascularization, supporting the involvement of NOVA2-mediated L1-ΔTM production in tumor angiogenesis. Finally, high NOVA2 expression is associated with poor outcome in ovarian cancer patients. Our results point to L1-ΔTM as a novel, EC-derived angiogenic factor which may represent a target for innovative antiangiogenic therapies.

The Trithorax protein Ash1L promotes myoblast fusion by activating Cdon expression.

Myoblast fusion (MF) is required for muscle growth and repair, and its alteration contributes to muscle diseases. The mechanisms governing this process are incompletely understood, and no epigenetic regulator has been previously described. Ash1L is an epigenetic activator belonging to the Trithorax group of proteins and is involved in FSHD muscular dystrophy, autism and cancer. Its physiological role in skeletal muscle is unknown. Here we report that Ash1L expression is positively correlated with MF and reduced in Duchenne muscular dystrophy. In vivo, ex vivo and in vitro experiments support a selective and evolutionary conserved requirement for Ash1L in MF. RNA- and ChIP-sequencing indicate that Ash1L is required to counteract Polycomb repressive activity to allow activation of selected myogenesis genes, in particular the key MF gene Cdon. Our results promote Ash1L as an important epigenetic regulator of MF and suggest that its activity could be targeted to improve cell therapy for muscle diseases.

Amino acid deprivation triggers a novel GCN2-independent response leading to the transcriptional reactivation of non-native DNA sequences.

In a variety of species, reduced food intake, and in particular protein or amino acid (AA) restriction, extends lifespan and healthspan. However, the underlying epigenetic and/or transcriptional mechanisms are largely unknown, and dissection of specific pathways in cultured cells may contribute to filling this gap. We have previously shown that, in mammalian cells, deprivation of essential AAs (methionine/cysteine or tyrosine) leads to the transcriptional reactivation of integrated silenced transgenes, including plasmid and retroviral vectors and latent HIV-1 provirus, by a process involving epigenetic chromatic remodeling and histone acetylation. Here we show that the deprivation of methionine/cysteine also leads to the transcriptional upregulation of endogenous retroviruses, suggesting that essential AA starvation affects the expression not only of exogenous non-native DNA sequences, but also of endogenous anciently-integrated and silenced parasitic elements of the genome. Moreover, we show that the transgene reactivation response is highly conserved in different mammalian cell types, and it is reproducible with deprivation of most essential AAs. The General Control Non-derepressible 2 (GCN2) kinase and the downstream integrated stress response represent the best candidates mediating this process; however, by pharmacological approaches, RNA interference and genomic editing, we demonstrate that they are not implicated. Instead, the response requires MEK/ERK and/or JNK activity and is reproduced by ribosomal inhibitors, suggesting that it is triggered by a novel nutrient-sensing and signaling pathway, initiated by translational block at the ribosome, and independent of mTOR and GCN2. Overall, these findings point to a general transcriptional response to essential AA deprivation, which affects the expression of non-native genomic sequences, with relevant implications for the epigenetic/transcriptional effects of AA restriction in health and disease.

Diversification of the muscle proteome through alternative splicing.

BACKGROUND: Skeletal muscles express a highly specialized proteome that allows the metabolism of energy sources to mediate myofiber contraction. This muscle-specific proteome is partially derived through the muscle-specific transcription of a subset of genes. Surprisingly, RNA sequencing technologies have also revealed a significant role for muscle-specific alternative splicing in generating protein isoforms that give specialized function to the muscle proteome. MAIN BODY: In this review, we discuss the current knowledge with respect to the mechanisms that allow pre-mRNA transcripts to undergo muscle-specific alternative splicing while identifying some of the key trans-acting splicing factors essential to the process. The importance of specific splicing events to specialized muscle function is presented along with examples in which dysregulated splicing contributes to myopathies. Though there is now an appreciation that alternative splicing is a major contributor to proteome diversification, the emergence of improved "targeted" proteomic methodologies for detection of specific protein isoforms will soon allow us to better appreciate the extent to which alternative splicing modifies the activity of proteins (and their ability to interact with other proteins) in the skeletal muscle. In addition, we highlight a continued need to better explore the signaling pathways that contribute to the temporal control of trans-acting splicing factor activity to ensure specific protein isoforms are expressed in the proper cellular context. CONCLUSIONS: An understanding of the signal-dependent and signal-independent events driving muscle-specific alternative splicing has the potential to provide us with novel therapeutic strategies to treat different myopathies.

Report on Abstracts of the 15th Meeting of IIM, the Interuniversity Institute of Myology - Assisi (Italy), October 11-14, 2018.

On October 11-14, 2018, the 15th Meeting of the Interuniversity Institute of Myology (IIM) took place in the city Assisi, Italy. Muscle researchers from Italy, and various European and North-American countries gathered to discuss recent results on the physiology and diseases of skeletal muscle. The program showcased keynote lectures from world-renowned international speakers presenting advances in muscle stem cells, circadian rhythm, organismal development and growth, muscle physiology, and bioengineering. Novel, unpublished results from young trainees were presented as oral communications or posters, based on selection from submitted abstracts. Young trainees where directly involved in several aspects of the meeting by being responsible of organizing a scientific session, arranging three round tables tailored to the interests of their peers and chairing all scientific sessions. The meeting offered a unique opportunity for young researchers to present their work, have feedback from more experienced colleagues and establish collaborations to further understanding of muscular diseases and develop therapeutic strategies. The open, informal and friendly atmosphere of the meeting stimulated lively discussions, instrumental to highlight key areas of muscle research and foster scientific cross-fertilization and new collaborations. The meeting was very successful. A sign that the IIM community will continue to deliver important contributions to the training of young students and fellows, promoting our understanding of muscle formation and activity, the mechanism of muscle diseases and the progress toward therapeutic approaches. The Myology field is strong and articulated in basic, translational and early clinical research, moving toward the development of treatments for several muscle diseases as documented by the abstracts of the IIM meeting.

Polycomb repressive complex 1 provides a molecular explanation for repeat copy number dependency in FSHD muscular dystrophy.

Repression of repetitive elements is crucial to preserve genome integrity and has been traditionally ascribed to constitutive heterochromatin pathways. FacioScapuloHumeral Muscular Dystrophy (FSHD), one of the most common myopathies, is characterized by a complex interplay of genetic and epigenetic events. The main FSHD form is linked to a reduced copy number of the D4Z4 macrosatellite repeat on 4q35, causing loss of silencing and aberrant expression of the D4Z4-embedded DUX4 gene leading to disease. By an unknown mechanism, D4Z4 copy-number correlates with FSHD phenotype. Here we show that the DUX4 proximal promoter (DUX4p) is sufficient to nucleate the enrichment of both constitutive and facultative heterochromatin components and to mediate a copy-number dependent gene silencing. We found that both the CpG/GC dense DNA content and the repetitive nature of DUX4p arrays are important for their repressive ability. We showed that DUX4p mediates a copy number-dependent Polycomb Repressive Complex 1 (PRC1) recruitment, which is responsible for the copy-number dependent gene repression. Overall, we directly link genetic and epigenetic defects in FSHD by proposing a novel molecular explanation for the copy number-dependency in FSHD pathogenesis, and offer insight into the molecular functions of repeats in chromatin regulation.