Tributyrin, a Butyrate Pro-Drug, Primes Satellite Cells for Differentiation by Altering the Epigenetic Landscape

Satellite cells (SC) are a population of muscle resident stem cells that are responsible for postnatal muscle growth and repair. With investigation into the genomic regulation of SC fate, the role of the epigenome in governing SC myogenesis is becoming clearer. Histone deacetylase (HDAC) inhibitors have been demonstrated to be effective at enhancing the myogenic program of SC, but their role in altering the epigenetic landscape of SC remains undetermined. Our objective was to determine how an HDAC inhibitor, butyrate, promotes myogenic differentiation. SC from tributyrin treated neonatal piglets showed a decrease relative to SC from control animals in the expression of enhance of zeste homologue-2 (EZH2), a chromatin modifier, ex vivo. Chromatin Immunoprecipitation-Sequencing (ChIP-Seq) analysis of SC isolated from tributyrin treated pigs showed a global reduction of the tri-methylation of lysine 27 of histone H3 (H3K27me3) repressive chromatin mark. To determine if reductions in EZH2 was the primary mechanism through which butyrate affects SC behavior, SC were transfected with siRNA targeting EZH2, treated with 0.5 mM butyrate, or both. Treatment with butyrate reduced paired-box-7 (Pax7) and myogenic differentiation-1 (MyoD) gene expression, while siRNA caused reductions in EZH2 had no effect on their expression. EZH2 depletion did result in an increase in differentiating SC, but not in myotube hypertrophy. These results indicate that while EZH2 reduction may force myogenic differentiation, butyrate may operate through a parallel mechanism to enhance the myogenic program.

BAF complexes drive proliferation and block myogenic differentiation in fusion-positive rhabdomyosarcoma

Rhabdomyosarcoma (RMS) is a pediatric malignancy of skeletal muscle lineage. The aggressive alveolar subtype is characterized by t(2;13) or t(1;13) translocations encoding for PAX3- or PAX7-FOXO1 chimeric transcription factors, respectively, and are referred to as fusion positive RMS (FP-RMS). The fusion gene alters the myogenic program and maintains the proliferative state while blocking terminal differentiation. Here, we investigated the contributions of chromatin regulatory complexes to FP-RMS tumor maintenance. We define the mSWI/SNF functional repertoire in FP-RMS. We find that SMARCA4 (encoding BRG1) is overexpressed in this malignancy compared to skeletal muscle and is essential for cell proliferation. Proteomic studies suggest proximity between PAX3-FOXO1 and BAF complexes, which is further supported by genome-wide binding profiles revealing enhancer colocalization of BAF with core regulatory transcription factors. Further, mSWI/SNF complexes localize to sites of de novo histone acetylation. Phenotypically, interference with mSWI/SNF complex function induces transcriptional activation of the skeletal muscle differentiation program associated with MYCN enhancer invasion at myogenic target genes, which is recapitulated by BRG1 targeting compounds. We conclude that inhibition of BRG1 overcomes the differentiation blockade of FP-RMS cells and may provide a therapeutic strategy for this lethal childhood tumor.

RNA-Binding Proteins in the Post-transcriptional Control of Skeletal Muscle Development, Regeneration and Disease

Embryonic myogenesis is a temporally and spatially regulated process that generates skeletal muscle of the trunk and limbs. During this process, mononucleated myoblasts derived from myogenic progenitor cells within the somites undergo proliferation, migration and differentiation to elongate and fuse into multinucleated functional myofibers. Skeletal muscle is the most abundant tissue of the body and has the remarkable ability to self-repair by re-activating the myogenic program in muscle stem cells, known as satellite cells. Post-transcriptional regulation of gene expression mediated by RNA-binding proteins is critically required for muscle development during embryogenesis and for muscle homeostasis in the adult. Differential subcellular localization and activity of RNA-binding proteins orchestrates target gene expression at multiple levels to regulate different steps of myogenesis. Dysfunctions of these post-transcriptional regulators impair muscle development and homeostasis, but also cause defects in motor neurons or the neuromuscular junction, resulting in muscle degeneration and neuromuscular disease. Many RNA-binding proteins, such as members of the muscle blind-like (MBNL) and CUG-BP and ETR-3-like factors (CELF) families, display both overlapping and distinct targets in muscle cells. Thus they function either cooperatively or antagonistically to coordinate myoblast proliferation and differentiation. Evidence is accumulating that the dynamic interplay of their regulatory activity may control the progression of myogenic program as well as stem cell quiescence and activation. Moreover, the role of RNA-binding proteins that regulate post-transcriptional modification in the myogenic program is far less understood as compared with transcription factors involved in myogenic specification and differentiation. Here we review past achievements and recent advances in understanding the functions of RNA-binding proteins during skeletal muscle development, regeneration and disease, with the aim to identify the fundamental questions that are still open for further investigations.

Feedback regulation of Notch signaling and myogenesis connected by MyoD–Dll1 axis

Muscle precursor cells known as myoblasts are essential for muscle development and regeneration. Notch signaling is an ancient intercellular communication mechanism that plays prominent roles in controlling the myogenic program of myoblasts. Currently whether and how the myogenic cues feedback to refine Notch activities in these cells are largely unknown. Here, by mouse and human gene gain/loss-of-function studies, we report that MyoD directly turns on the expression of Notch-ligand gene Dll1 which activates Notch pathway to prevent precautious differentiation in neighboring myoblasts, while autonomously inhibits Notch to facilitate a myogenic program in Dll1 expressing cells. Mechanistically, we studied cis-regulatory DNA motifs underlying the MyoD–Dll1–Notch axis in vivo by characterizing myogenesis of a novel E-box deficient mouse model, as well as in human cells through CRISPR-mediated interference. These results uncovered the crucial transcriptional mechanism that mediates the reciprocal controls of Notch and myogenesis.

Fibroblast fusion to the muscle fiber regulates myotendinous junction formation

Vertebrate muscles and tendons are derived from distinct embryonic origins yet they must interact in order to facilitate muscle contraction and body movements. How robust muscle tendon junctions (MTJs) form to be able to withstand contraction forces is still not understood. Using techniques at a single cell resolution we reexamine the classical view of distinct identities for the tissues composing the musculoskeletal system. We identify fibroblasts that have switched on a myogenic program and demonstrate these dual identity cells fuse into the developing muscle fibers along the MTJs facilitating the introduction of fibroblast-specific transcripts into the elongating myofibers. We suggest this mechanism resulting in a hybrid muscle fiber, primarily along the fiber tips, enables a smooth transition from muscle fiber characteristics towards tendon features essential for forming robust MTJs. We propose that dual characteristics of junctional cells could be a common mechanism for generating stable interactions between tissues throughout the musculoskeletal system.

Muscle Enriched Lamin Interacting Protein (Mlip) Binds Chromatin and Is Required for Myoblast Differentiation

Muscle-enriched A-type lamin-interacting protein (Mlip) is a recently discovered Amniota gene that encodes proteins of unknown biological function. Here we report Mlip’s direct interaction with chromatin, and it may function as a transcriptional co-factor. Chromatin immunoprecipitations with microarray analysis demonstrated a propensity for Mlip to associate with genomic regions in close proximity to genes that control tissue-specific differentiation. Gel mobility shift assays confirmed that Mlip protein complexes with genomic DNA. Blocking Mlip expression in C2C12 myoblasts down-regulates myogenic regulatory factors (MyoD and MyoG) and subsequently significantly inhibits myogenic differentiation and the formation of myotubes. Collectively our data demonstrate that Mlip is required for C2C12 myoblast differentiation into myotubes. Mlip may exert this role as a transcriptional regulator of a myogenic program that is unique to amniotes.

Vitamin D Restores Skeletal Muscle Cell Remodeling and Myogenic Program: Potential Impact on Human Health

Skeletal muscle cells, albeit classified as vitamin D receptor (VDR)-poor cells, are finely controlled by vitamin D through genomic and non-genomic mechanisms. Skeletal muscle constantly undergoes cell remodeling, a complex system under multilevel regulation, mainly orchestrated by the satellite niche in response to a variety of stimuli. Cell remodeling is not limited to satisfy reparative and hypertrophic needs, but, through myocyte transcriptome/proteome renewal, it warrants the adaptations necessary to maintain tissue integrity. While vitamin D insufficiency promotes cell maladaptation, restoring vitamin D levels can correct/enhance the myogenic program. Hence, vitamin D fortified foods or supplementation potentially represents the desired approach to limit or avoid muscle wasting and ameliorate health. Nevertheless, consensus on protocols for vitamin D measurement and supplementation is still lacking, due to the high variability of lab tests and of the levels required in different contexts (i.e., age, sex, heath status, lifestyle). This review aims to describe how vitamin D can orchestrate skeletal muscle cell remodeling and myogenic programming, after reviewing the main processes and cell populations involved in this important process, whose correct progress highly impacts on human health. Topics on vitamin D optimal levels, supplementation and blood determination, which are still under debate, will be addressed.

BAF complexes drive proliferation and block myogenic differentiation in fusion-positive rhabdomyosarcoma

Rhabdomyosarcoma (RMS) is a pediatric malignancy of skeletal muscle lineage. The aggressive alveolar subtype is characterized by t(2;13) or t(1;13) translocations encoding for PAX3- or PAX7-FOXO1 chimeric transcription factors, respectively, and are referred to as fusion positive RMS (FP-RMS). The fusion gene alters the myogenic program and maintains the proliferative state wile blocking terminal differentiation. Here we investigated the contributions of chromatin regulatory complexes to FP-RMS tumor maintenance. We define, for the first time, the mSWI/SNF repertoire in FP-RMS. We find that SMARCA4 (encoding BRG1) is overexpressed in this malignancy compared to skeletal muscle and is essential for cell proliferation. Proteomic studies suggest proximity between PAX3-FOXO1 and BAF complexes, which is further supported by genome-wide binding profiles revealing enhancer colocalization of BAF with core regulatory transcription factors. Further, mSWI/SNF complexes act as sensors of chromatin state and are recruited to sites of de novo histone acetylation. Phenotypically, interference with mSWI/SNF complex function induces transcriptional activation of the skeletal muscle differentiation program associated with MYCN enhancer invasion at myogenic target genes which is reproduced by BRG1 targeting compounds. We conclude that inhibition of BRG1 overcomes the differentiation blockade of FP-RMS cells and may provide a therapeutic strategy for this lethal childhood tumor.

miRNAs and Muscle Stem Cells

Skeletal muscle represents between 30 and 38% of the human body mass. Both the maintenance and repair of adult muscle tissue are directed by satellite cells (SCs). SCs are located beneath the basal lamina of the skeletal muscle myofiber. They are quiescent for most of their life but, in response to physiological stimuli or muscle trauma, they activate, proliferate, and enter the myogenic program via generating myogenic progenitors (myoblasts) that fuse to existing myofibers or de novo myofibers. MicroRNAs (miRNAs or miRs) play a critical role in regulating muscle regeneration and stem cell behavior. In this chapter, we review the pivotal role in the regulation of SC quiescence, activation, and differentiation in the context of muscular dystrophies.

Integrated glycoproteomics identifies a role of N-glycosylation and galectin-1 on myogenesis and muscle development

Many cell surface and secreted proteins are modified by the covalent addition of glycans that play an important role in the development of multicellular organisms. These glycan modifications enable communication between cells and the extracellular matrix via interactions with specific glycan-binding lectins and the regulation of receptor-mediated signaling. Aberrant protein glycosylation has been associated with the development of several muscular diseases suggesting essential glycan- and lectin-mediated functions in myogenesis and muscle development but our molecular understanding of the precise glycans, catalytic enzymes and lectins involved remain only partially understood. Here, we quantified dynamic remodeling of the membrane-associated proteome during a time-course of myogenesis in cell culture. We observed wide-spread changes in the abundance of several important lectins and enzymes facilitating glycan biosynthesis. Glycomics-based quantification of released N-linked glycans confirmed remodeling of the glycome consistent with the regulation of glycosyltransferases and glycosidases responsible for their formation including a previously unknown di-galactose-to-sialic acid switch supporting a functional role of these glycoepitopes in myogenesis. Furthermore, dynamic quantitative glycoproteomic analysis with multiplexed stable isotope labelling and analysis of enriched glycopeptides with multiple fragmentation approaches identified glycoproteins modified by these regulated glycans including several integrins and growth factor receptors. Myogenesis was also associated with the regulation of several lectins most notably the up-regulation of galectin-1 (LGALS1). CRISPR/Cas9-mediated deletion of Lgals1 inhibited differentiation and myotube formation suggesting an early functional role of galectin-1 in the myogenic program. Importantly, similar changes in N-glycosylation and the up-regulation of galectin-1 during postnatal skeletal muscle development were observed in mice. Treatment of new-born mice with recombinant adeno-associated viruses to overexpress galectin-1 in the musculature resulted in enhanced muscle mass. Our data form a valuable resource to further understand the glycobiology of myogenesis and will aid the development of intervention strategies to promote healthy muscle development or regeneration.