Cicero Predicts cis-Regulatory DNA Interactions from Single-Cell Chromatin Accessibility Data.

Linking regulatory DNA elements to their target genes, which may be located hundreds of kilobases away, remains challenging. Here, we introduce Cicero, an algorithm that identifies co-accessible pairs of DNA elements using single-cell chromatin accessibility data and so connects regulatory elements to their putative target genes. We apply Cicero to investigate how dynamically accessible elements orchestrate gene regulation in differentiating myoblasts. Groups of Cicero-linked regulatory elements meet criteria of "chromatin hubs"-they are enriched for physical proximity, interact with a common set of transcription factors, and undergo coordinated changes in histone marks that are predictive of changes in gene expression. Pseudotemporal analysis revealed that most DNA elements remain in chromatin hubs throughout differentiation. A subset of elements bound by MYOD1 in myoblasts exhibit early opening in a PBX1- and MEIS1-dependent manner. Our strategy can be applied to dissect the architecture, sequence determinants, and mechanisms of cis-regulation on a genome-wide scale.

Muscle-specific pyruvate kinase isoforms, PKM1 and PKM2, regulate mammalian SWI/SNF proteins and histone 3 phosphorylation during myoblast differentiation.

Pyruvate kinase is a glycolytic enzyme that converts phosphoenolpyruvate and ADP into pyruvate and ATP. There are two genes that encode pyruvate kinase in vertebrates; Pkm and Pkl encode muscle- and liver/erythrocyte-specific forms, respectively. Each gene encodes two isoenzymes due to alternative splicing. Both muscle-specific enzymes, PKM1 and PKM2, function in glycolysis, but PKM2 also has been implicated in gene regulation due to its ability to phosphorylate histone 3 threonine 11 (H3T11) in cancer cells. Here, we examined the roles of PKM1 and PKM2 during myoblast differentiation. RNA-seq analysis revealed that PKM2 promotes the expression of Dpf2/Baf45d and Baf250a/Arid1A. DPF2 and BAF250a are subunits that identify a specific sub-family of the mammalian SWI/SNF (mSWI/SNF) of chromatin remodeling enzymes that is required for the activation of myogenic gene expression during differentiation. PKM2 also mediated the incorporation of DPF2 and BAF250a into the regulatory sequences controlling myogenic gene expression. PKM1 did not affect expression but was required for nuclear localization of DPF2. Additionally, PKM2 was required not only for the incorporation of phosphorylated H3T11 in myogenic promoters but also for the incorporation of phosphorylated H3T6 and H3T45 at myogenic promoters via regulation of AKT and protein kinase C isoforms that phosphorylate those amino acids. Our results identify multiple unique roles for PKM2 and a novel function for PKM1 in gene expression and chromatin regulation during myoblast differentiation.

Impaired myoblast differentiation and muscle IGF-1 receptor signaling pathway activation after N-glycosylation inhibition.

The role of N-glycosylation in the myogenic process remains poorly understood. Here, we evaluated the impact of N-glycosylation inhibition by Tunicamycin (TUN) or by phosphomannomutase 2 (PMM2) gene knockdown, which encodes an enzyme essential for catalyzing an early step of the N-glycosylation pathway, on C2C12 myoblast differentiation. The effect of chronic treatment with TUN on tibialis anterior (TA) and extensor digitorum longus (EDL) muscles of WT and MLC/mIgf-1 transgenic mice, which overexpress muscle Igf-1Ea mRNA isoform, was also investigated. TUN-treated and PMM2 knockdown C2C12 cells showed reduced ConA, PHA-L, and AAL lectin binding and increased ER-stress-related gene expression (Chop and Hspa5 mRNAs and s/uXbp1 ratio) compared to controls. Myogenic markers (MyoD, myogenin, and Mrf4 mRNAs and MF20 protein) and myotube formation were reduced in both TUN-treated and PMM2 knockdown C2C12 cells. Body and TA weight of WT and MLC/mIgf-1 mice were not modified by TUN treatment, while lectin binding slightly decreased in the TA muscle of WT (ConA and AAL) and MLC/mIgf-1 (ConA) mice. The ER-stress-related gene expression did not change in the TA muscle of WT and MLC/mIgf-1 mice after TUN treatment. TUN treatment decreased myogenin mRNA and increased atrogen-1 mRNA, particularly in the TA muscle of WT mice. Finally, the IGF-1 production and IGF1R signaling pathways activation were reduced due to N-glycosylation inhibition in TA and EDL muscles. Decreased IGF1R expression was found in TUN-treated C2C12 myoblasts which was associated with lower IGF-1-induced IGF1R, AKT, and ERK1/2 phosphorylation compared to CTR cells. Chronic TUN-challenge models can help to elucidate the molecular mechanisms through which diseases associated with aberrant N-glycosylation, such as Congenital Disorders of Glycosylation (CDG), affect muscle and other tissue functions.

The transcriptional regulator KLF15 is necessary for myoblast differentiation and muscle regeneration by activating FKBP5.

Successful muscle regeneration following injury is essential for functional homeostasis of skeletal muscles. Krüppel-like factor 15 (KLF15) is a metabolic transcriptional regulator in the muscles. However, little is known regarding its function in muscle regeneration. Here, we examined microarray datasets from the Gene Expression Omnibus database, which indicated downregulated KLF15 in muscles from patients with various muscle diseases. Additionally, we found that Klf15 knockout (Klf15KO) impaired muscle regeneration following injury in mice. Furthermore, KLF15 expression was robustly induced during myoblast differentiation. Myoblasts with KLF15 deficiency showed a marked reduction in their fusion capacity. Unbiased transcriptome analysis of muscles on day 7 postinjury revealed downregulated genes involved in cell differentiation and metabolic processes in Klf15KO muscles. The FK506-binding protein 51 (FKBP5), a positive regulator of myoblast differentiation, was ranked as one of the most strongly downregulated genes in the Klf15KO group. A mechanistic search revealed that KLF15 binds directly to the promoter region of FKBP5 and activates FKBP5 expression. Local delivery of FKBP5 rescued the impaired muscle regeneration in Klf15KO mice. Our findings reveal a positive regulatory role of KLF15 in myoblast differentiation and muscle regeneration by activating FKBP5 expression. KLF15 signaling may be a novel therapeutic target for muscle disorders associated with injuries or diseases.

IGF2 promotes the differentiation of chicken embryonic myoblast by regulating mitochondrial remodeling.

Skeletal muscle is crucial for animal movement and posture maintenance, and it serves as a significant source of meat in the livestock and poultry industry. The number of muscle fibers differentiated from myoblast in the embryonic stage is one of the factors determining the content of skeletal muscle. Insulin-like growth factor 2 (IGF2), a well-known growth-promoting hormone, is crucial for embryonic and skeletal muscle growth and development. However, the specific molecular mechanism underlying its impact on chicken embryonic myoblast differentiation remains unclear. To elucidate the molecular mechanism by which IGF2 regulates chicken myoblast differentiation, we manipulated IGF2 expression in chicken embryonic myoblast. The results demonstrated that IGF2 was upregulated during chicken skeletal muscle development and myoblast differentiation. On the one hand, we found that IGF2 promotes mitochondrial biogenesis through the PGC1/NRF1/TFAM pathway, thereby enhancing mitochondrial membrane potential, oxidative phosphorylation, and ATP synthesis during myoblast differentiation. This process is mediated by the PI3K/AKT pathway. On the other hand, IGF2 regulates BNIP3-mediated mitophagy, clearing dysfunctional mitochondria. Collectively, our findings confirmed that IGF2 cooperatively regulates mitochondrial biogenesis and mitophagy to remodel the mitochondrial network and enhance mitochondrial function, ultimately promoting myoblast differentiation.

Lnc-Malat1 promotes slow myofiber-type transformation through sponging miR-129-5p in C2C12 myotubes.

Long non-coding metastasis-associated lung adenocarcinoma transcript (lnc-Malat1) emerges as a novel regulator in skeletal muscle development, while its function and the related mechanism is not fully revealed yet. In this study, knockdown of lnc-Malat1 by siRNA significantly inhibited the expression of myoblast marker genes (MyHC, MyoD, and MyoG) and slow muscle fiber marker genes (MyHC I), together with repressed expression of mitochondria-related genes COX5A, ACADM, CPTA1, FABP3, and NDUFA1. Overexpression of lnc-Malat1 exerted an opposite effect, promoting myoblast differentiation and slow muscle fiber formation. Dual luciferase reporter assay revealed a direct interaction between lnc-Malat1 and miR-129-5p, and overexpression of lnc-Malat1 significantly inhibited miR-129-5p expression, thereby elevating the expression of Mef2a, miR-129-5p target protein. In addition, enforced expression of lnc-Malat1 restored the inhibitory effect of miR-129-5p on myoblast differentiation and MyHC I expression. Taken together, our results suggest that lnc-Malat1 promotes myoblast differentiation, and maintains the slow muscle fiber phenotype via adsorbing miR-129-5p.

Therapeutic potential of oleic acid supplementation in myotonic dystrophy muscle cell models.

BACKGROUND: We recently reported that upregulation of Musashi 2 (MSI2) protein in the rare neuromuscular disease myotonic dystrophy type 1 contributes to the hyperactivation of the muscle catabolic processes autophagy and UPS through a reduction in miR-7 levels. Because oleic acid (OA) is a known allosteric regulator of MSI2 activity in the biogenesis of miR-7, here we sought to evaluate endogenous levels of this fatty acid and its therapeutic potential in rescuing cell differentiation phenotypes in vitro. In this work, four muscle cell lines derived from DM1 patients were treated with OA for 24 h, and autophagy and muscle differentiation parameters were analyzed. RESULTS: We demonstrate a reduction of OA levels in different cell models of the disease. OA supplementation rescued disease-related phenotypes such as fusion index, myotube diameter, and repressed autophagy. This involved inhibiting MSI2 regulation of direct molecular target miR-7 since OA isoschizomer, elaidic acid (EA) could not cause the same rescues. Reduction of OA levels seems to stem from impaired biogenesis since levels of the enzyme stearoyl-CoA desaturase 1 (SCD1), responsible for converting stearic acid to oleic acid, are decreased in DM1 and correlate with OA amounts. CONCLUSIONS: For the first time in DM1, we describe a fatty acid metabolism impairment that originated, at least in part, from a decrease in SCD1. Because OA allosterically inhibits MSI2 binding to molecular targets, reduced OA levels synergize with the overexpression of MSI2 and contribute to the MSI2 > miR-7 > autophagy axis that we proposed to explain the muscle atrophy phenotype.

UBE2C promotes myoblast differentiation and skeletal muscle regeneration through the Akt signaling pathway.

Ubiquitin-conjugation enzyme E2C (UBE2C) is a crucial component of the ubiquitin-proteasome system that is involved in numerous cancers. In this study, we find that UBE2C expression is significantly increased in mouse embryos, a critical stage during skeletal muscle development. We further investigate the function of UBE2C in myogenesis. Knockdown of UBE2C inhibits C2C12 cell differentiation and decreases the expressions of MyoG and MyHC, while overexpression of UBE2C promotes C2C12 cell differentiation. Additionally, knockdown of UBE2C, specifically in the tibialis anterior muscle (TA), severely impedes muscle regeneration in vivo. Mechanistically, we show that UBE2C knockdown reduces the level of phosphorylated protein kinase B (p-Akt) and promotes the degradation of Akt. These findings suggest that UBE2C plays a critical role in myoblast differentiation and muscle regeneration and that UBE2C regulates myogenesis through the Akt signaling pathway.

Molecular Regulation of Porcine Skeletal Muscle Development: Insights from Research on CDC23 Expression and Function.

Cell division cycle 23 (CDC23) is a component of the tetratricopeptide repeat (TPR) subunit in the anaphase-promoting complex or cyclosome (APC/C) complex, which participates in the regulation of mitosis in eukaryotes. However, the regulatory model and mechanism by which the CDC23 gene regulates muscle production in pigs are largely unknown. In this study, we investigated the expression of CDC23 in pigs, and the results indicated that CDC23 is widely expressed in various tissues and organs. In vitro cell experiments have demonstrated that CDC23 promotes the proliferation of myoblasts, as well as significantly positively regulating the differentiation of skeletal muscle satellite cells. In addition, Gene Set Enrichment Analysis (GSEA) revealed a significant downregulation of the cell cycle pathway during the differentiation process of skeletal muscle satellite cells. The protein-protein interaction (PPI) network showed a high degree of interaction between genes related to the cell cycle pathway and CDC23. Subsequently, in differentiated myocytes induced after overexpression of CDC23, the level of CDC23 exhibited a significant negative correlation with the expression of key factors in the cell cycle pathway, suggesting that CDC23 may be involved in the inhibition of the cell cycle signaling pathway in order to promote the differentiation process. In summary, we preliminarily determined the function of CDC23 with the aim of providing new insights into molecular regulation during porcine skeletal muscle development.

Nonsense-mediated mRNA decay suppresses injury-induced muscle regeneration via inhibiting MyoD transcriptional activity.

Skeletal muscle regeneration is a crucial physiological process that occurs in response to injury or disease. As an important transcriptome surveillance system that regulates tissue development, the role of nonsense-mediated mRNA decay (NMD) in muscle regeneration remains unclear. Here, we found that NMD inhibits myoblast differentiation by targeting the phosphoinositide-3-kinase regulatory subunit 5 gene, which leads to the suppression of the transcriptional activity of myogenic differentiation (MyoD), a key regulator of myoblast differentiation. This disruption of MyoD transcriptional activity subsequently affects the expression levels of myogenin and myosin heavy chain, crucial markers of myoblast differentiation. Additionally, through up-frameshift protein 1 knockdown experiments, we observed that inhibiting NMD can accelerate muscle regeneration in vivo. These findings highlight the potential of NMD as a novel therapeutic target for the treatment of muscle-related injuries and diseases.

Rab44 negatively regulates myoblast differentiation by controlling fusogenic protein transport and mTORC1 signaling.

Skeletal muscle is composed of multinucleated myotubes formed by the fusion of mononucleated myoblasts. Skeletal muscle differentiation, termed as myogenesis, have been investigated using the mouse skeletal myoblast cell line C2C12. It has been reported that several "small" Rab proteins, major membrane-trafficking regulators, possibly regulate membrane protein transport in C2C12 cells; however, the role of Rab proteins in myogenesis remains unexplored. Rab44, a member of "large" Rab GTPases, has recently been identified as a negative regulator of osteoclast differentiation. In this study, using C2C12 cells, we found that Rab44 expression was upregulated during myoblast differentiation into myotubes. Knockdown of Rab44 enhanced myoblast differentiation and myotube formation. Consistent with these results, Rab44 knockdown in myoblasts increased expression levels of several myogenic marker genes. Rab44 knockdown increased the surface accumulation of myomaker and myomixer, two fusogenic proteins required for multinucleation, implying enhanced cell fusion. Conversely, Rab44 overexpression inhibited myoblast differentiation and tube formation, accompanied by decreased expression of some myogenic markers. Furthermore, Rab44 was found to be predominantly localized in lysosomes, and Rab44 overexpression altered the number and size of lysosomes. Considering the underlying molecular mechanism, Rab44 overexpression impaired the signaling pathway of the mechanistic target of rapamycin complex1 (mTORC1) in C2C12 cells. Namely, phosphorylation levels of mTORC1 and downstream mTORC1 substrates, such as S6 and P70-S6K, were notably lower in Rab44 overexpressing cells than those in control cells. These results indicate that Rab44 negatively regulates myoblast differentiation into myotubes by controlling fusogenic protein transport and mTORC1 signaling.

SNAI1 is upregulated during muscle regeneration and represses FGF21 and ATF3 expression by directly binding their promoters.

During skeletal myogenesis, the zinc-finger transcription factors SNAI1 and SNAI2, are expressed in proliferating myoblasts and regulate the transition to terminally differentiated myotubes while repressing pro-differentiation genes. Here, we demonstrate that SNAI1 is upregulated in vivo during the early phase of muscle regeneration induced by bupivacaine injury. Using shRNA-mediated gene silencing in C2C12 myoblasts and whole-transcriptome microarray analysis, we identified a collection of genes belonging to the endoplasmic reticulum (ER) stress pathway whose expression, induced by myogenic differentiation, was upregulated in absence of SNAI1. Among these, key ER stress genes, such as Atf3, Ddit3/Chop, Hspa5/Bip, and Fgf21, a myokine involved in muscle differentiation, were strongly upregulated. Furthermore, by promoter mutant analysis and Chromatin immune precipitation assay, we demonstrated that SNAI1 represses Fgf21 and Atf3 in proliferating myoblasts by directly binding to multiple E boxes in their respective promoter regions. Together, these data describe a new regulatory mechanism of myogenic differentiation involving the direct repressive action of SNAI1 on ER stress and Fgf21 expression, ultimately contributing to maintaining the proliferative and undifferentiated state of myoblasts.

Local GHR roles in regulation of mitochondrial function through mitochondrial biogenesis during myoblast differentiation.

BACKGROUND: Myoblast differentiation requires metabolic reprogramming driven by increased mitochondrial biogenesis and oxidative phosphorylation. The canonical GH-GHR-IGFs axis in liver exhibits a great complexity in response to somatic growth. However, the underlying mechanism of whether local GHR acts as a control valve to regulate mitochondrial function through mitochondrial biogenesis during myoblast differentiation remains unknown. METHODS: We manipulated the GHR expression in chicken primary myoblast to investigate its roles in mitochondrial biogenesis and function during myoblast differentiation. RESULTS: We reported that GHR is induced during myoblast differentiation. Local GHR promoted mitochondrial biogenesis during myoblast differentiation, as determined by the fluorescence intensity of Mito-Tracker Green staining and MitoTimer reporter system, the expression of mitochondrial biogenesis markers (PGC1α, NRF1, TFAM) and mtDNA encoded gene (ND1, CYTB, COX1, ATP6), as well as mtDNA content. Consistently, local GHR enhanced mitochondrial function during myoblast differentiation, as determined by the oxygen consumption rate, mitochondrial membrane potential, ATP level and ROS production. We next revealed that the regulation of mitochondrial biogenesis and function by GHR depends on IGF1. In terms of the underlying mechanism, we demonstrated that IGF1 regulates mitochondrial biogenesis via PI3K/AKT/CREB pathway. Additionally, GHR knockdown repressed myoblast differentiation. CONCLUSIONS: In conclusion, our data corroborate that local GHR acts as a control valve to enhance mitochondrial function by promoting mitochondrial biogenesis via IGF1-PI3K/AKT/CREB pathway during myoblast differentiation. Video Abstract.

CircRNA Profiling of Skeletal Muscle in Two Pig Breeds Reveals CircIGF1R Regulates Myoblast Differentiation via miR-16.

Muscle development is closely related to meat quality and production. CircRNAs, with a closed-ring structure, have been identified as a key regulator of muscle development. However, the roles and mechanisms of circRNAs in myogenesis are largely unknown. Hence, in order to unravel the functions of circRNAs in myogenesis, the present study explored circRNA profiling in skeletal muscle between Mashen and Large White pigs. The results showed that a total of 362 circRNAs, which included circIGF1R, were differentially expressed between the two pig breeds. Functional assays showed that circIGF1R promoted myoblast differentiation of porcine skeletal muscle satellite cells (SMSCs), while it had no effect on cell proliferation. In consideration of circRNA acting as a miRNA sponge, dual-luciferase reporter and RIP assays were performed and the results showed that circIGF1R could bind miR-16. Furthermore, the rescue experiments showed that circIGF1R could counteract the inhibitory effect of miR-16 on cell myoblast differentiation. Thus, circIGF1R may regulate myogenesis by acting as a miR-16 sponge. In conclusion, this study successfully screened candidate circRNAs involved in the regulation of porcine myogenesis and demonstrated that circIGF1R promotes myoblast differentiation via miR-16, which lays a theoretical foundation for understanding the role and mechanism of circRNAs in regulating porcine myoblast differentiation.

Downregulation of Zebrafish Cytosolic Sialidase Neu3.2 Affects Skeletal Muscle Development.

Sialidases remove terminal sialic acids residues from the non-reducing ends of glycoconjugates. They have been recognized as catabolic enzymes that work within different subcellular compartments and can ensure the proper turn-over of glycoconjugates. Four mammalian sialidases (NEU1-4) exist, with different subcellular localization, pH optimum and substrate specificity. In zebrafish, seven different sialidases, with high homology to mammalian counterparts, have been identified. Zebrafish Neu3.2 is similar to the human cytosolic sialidase NEU2, which is involved in skeletal muscle differentiation and exhibits a broad substrate specificity toward gangliosides and glycoproteins. In zebrafish neu3.2, mRNA is expressed during somite development, and its enzymatic activity has been detected in the skeletal muscle and heart of adult animals. In this paper, 1-4-cell-stage embryos injected with neu3.2 splice-blocking morpholino showed severe embryonic defects, mainly in somites, heart and anterior-posterior axis formation. Myog and myod1 expressions were altered in morphants, and impaired musculature formation was associated with a defective locomotor behavior. Finally, the co-injection of Neu2 mouse mRNA in morphants rescued the phenotype. These data are consistent with the involvement of cytosolic sialidase in pathologies related to muscle formation and support the validity of the model to investigate the pathogenesis of the diseases.

Oat ß-glucan alleviates muscle atrophy via promoting myotube formation and suppressing protein degradation.

BACKGROUND: The dangerous inducers of muscle atrophy are inflammatory reaction, oxidative stress, and cachexia, etc. ß-Glucan, an important food derived active ingredient, has been reported to exert anti-inflammatory effects, however, its effects on regulating myoblast differentiation and protein degradation are unclear. This study is aimed to investigate the mechanism of oat ß-glucan on alleviating muscle atrophy. RESULTS: The results showed that oat ß-glucan treatment reversed tumor necrosis factor-α (TNF-α) induced abnormal myoblast differentiation and reduced muscle atrophy related MuRF-1 and Atrogin-1 protein expression. The similar phenomenon was observed after using MCC950 (NLRP3 specific inhibitor) or AS1842856 (FoxO1 specific inhibitor) to suppress NLRP3 and FoxO1 expression, respectively. Exposure to ß-glucan or AS1842856 also inhibited TNF-α induced the activation of TLR4/NF-κB pathway by inactivating FoxO1, and subsequently suppressed the expression of NLRP3. CONCLUSION: Our results indicate that oat ß-glucan exerts essential roles in promoting myoblast differentiation and alleviating muscle atrophy via inactivating FoxO1 and NLRP3 inflammasome signal pathway. © 2023 Society of Chemical Industry.

Down-regulation of pro-necroptotic molecules blunts necroptosis during myogenesis.

Cell death and differentiation are closely related at the molecular level. Differentiation of skeletal muscle cells attenuates susceptibility to apoptosis. Necroptosis has recently been recognized as a form of regulated cell death but its role in myogenesis has not been studied. This study aimed to compare the sensitivity to TNF-induced necroptosis in skeletal muscle at the undifferentiated (myoblasts) and differentiated (myotubes) stages. Surprisingly, our results showed that TNF-induced necroptosis was blunted during myoblast differentiation. Moreover, our data revealed that the key molecules involved in necroptosis, including receptor-interacting serine/threonine protein kinase 1 (RIPK1), RIPK3, and mixed lineage kinase domain-like protein (MLKL), were significantly down-regulated during myogenic differentiation, resulting in suppression of necroptosis signal transduction in differentiated myotubes. In addition, RIPK1, RIPK3, and MLKL expression levels were significantly lower in the skeletal muscle of adult mice than in newborn mice, suggesting that the susceptibility to necroptosis might be attenuated in differentiated muscle tissue. In conclusion, this study revealed that expression of key molecules involved in necroptosis is down-regulated during muscle differentiation, which results in the differentiation of muscles becoming insensitive to necroptotic cell death.

Msx1 cooperates with Runx1 for inhibiting myoblast differentiation.

Myogenesis is an important and complicated biological process, especially during the process of embryonic development. The homeoprotein Msx1 is a crucial transcriptional repressor of myogenesis and maintains myogenic precursor cells in an undifferentiated, proliferative state. However, the molecular mechanism through which Msx1 coordinates myogenesis remains to be elucidated. Here, we determine the interacting partner proteins of Msx1 in myoblast cells by a proteomic screening method. Msx1 is found to interact with 55 proteins, among which our data demonstrate that the cooperation of Runt-related transcription factor 1 (Runx1) with Msx1 is required for myoblast cell differentiation. Our findings provide important insights into the mechanistic roles of Msx1 in myoblast cell differentiation, and lays foundation for the myogenic differentiation process.

Changes in Nup62 content affect contact-induced differentiation of cultured myoblasts.

Differentiation of cultured skeletal myoblasts is induced by extrinsic signals that include reduction in ambient mitogen concentration and increased cell density. Using an established murine myoblast cell line (C2C12), we have found that experimental reduction of the nucleoporin p62 (Nup62) content of myoblasts enhances differentiation in high-mitogen medium, while forced expression of Nup62 inhibits density-induced differentiation. In contrast, differentiation of myoblasts induced by low-mitogen medium was unaffected by ectopic Nup62 expression. Further analyses suggested that Nup62 content affects density-induced myoblast differentiation through a mechanism involving activation of p38 MAP kinase. Nuclear pore complex (NPC) composition, in particular changes in NUP62 content, may be altered during viral infection, differentiation, and in neoplastic growth. The results support a functional role for changes in Nup62 composition in NPCs and density-induced myogenic differentiation, and suggest a link between loss of Nup62 content and induction of an intracellular stress signaling pathways.

Neddylation Regulates Class IIa and III Histone Deacetylases to Mediate Myoblast Differentiation.

As the largest tissue in the body, skeletal muscle has multiple functions in movement and energy metabolism. Skeletal myogenesis is controlled by a transcriptional cascade including a set of muscle regulatory factors (MRFs) that includes Myogenic Differentiation 1 (MYOD1), Myocyte Enhancer Factor 2 (MEF2), and Myogenin (MYOG), which direct the fusion of myogenic myoblasts into multinucleated myotubes. Neddylation is a posttranslational modification that covalently conjugates ubiquitin-like NEDD8 (neural precursor cell expressed, developmentally downregulated 8) to protein targets. Inhibition of neddylation impairs muscle differentiation; however, the underlying molecular mechanisms remain less explored. Here, we report that neddylation is temporally regulated during myoblast differentiation. Inhibition of neddylation through pharmacological blockade using MLN4924 (Pevonedistat) or genetic deletion of NEDD8 Activating Enzyme E1 Subunit 1 (NAE1), a subunit of the E1 neddylation-activating enzyme, blocks terminal myoblast differentiation partially through repressing MYOG expression. Mechanistically, we found that neddylation deficiency enhances the mRNA and protein expressions of class IIa histone deacetylases 4 and 5 (HDAC4 and 5) and prevents the downregulation and nuclear export of class III HDAC (NAD-Dependent Protein Deacetylase Sirtuin-1, SIRT1), all of which have been shown to repress MYOD1-mediated MYOG transcriptional activation. Together, our findings for the first time identify the crucial role of neddylation in mediating class IIa and III HDAC co-repressors to control myogenic program and provide new insights into the mechanisms of muscle disease and regeneration.