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The genome exists as an organized, three-dimensional (3D) dynamic architecture, and each cell type has a unique 3D genome organization that determines its cell identity. An unresolved question is how cell type-specific 3D genome structures are established during development. Here, we analyzed 3D genome structures in muscle cells from mice lacking the muscle lineage transcription factor (TF), MyoD, versus wild-type mice. We show that MyoD functions as a “genome organizer” that specifies 3D genome architecture unique to muscle cell development, and that H3K27ac is insufficient for the establishment of MyoD-induced chromatin loops in muscle cells. Moreover, we present evidence that other cell lineage-specific TFs might also exert functional roles in orchestrating lineage-specific 3D genome organization during development. Pioneer transcription factors (TFs) have been proposed to act as protein anchors to orchestrate cell type-specific 3D genome architecture. MyoD is a pioneer TF for myogenic lineage specification. Here the authors provide further support for the role of MyoD in 3D genome architecture in muscle stem cells by comparing MyoD knockout and wild-type mice.

Neat1 is widely expressed in many tissues and cells and exerts pro-proliferation effects on many cancer cells. However, little is known about the function of Neat1 in myogenesis. Here we characterized the roles of Neat1 in muscle cell formation and muscle regeneration. Gain- or loss-of-function studies in C2C12 cells demonstrated that Neat1 accelerates myoblast proliferation but suppresses myoblast differentiation and fusion. Further, knockdown of Neat1 in vivo increased the cross-sectional area of muscle fibers but impaired muscle regeneration. Mechanically, Neat1 physically interacted with Ezh2 mainly through the core binding region (1001–1540 bp) and recruited Ezh2 to target gene promoters. Neat1 promoted myoblast proliferation mainly by decreasing the expression of the cyclin-dependent kinase inhibitor P21 gene but inhibited myoblast differentiation by suppressing the transcription of myogenic marker genes, such as Myog , Myh4 , and Tnni2 . Altogether, we uncover a previously unknown function of Neat1 in muscle development and the molecular mechanism by which Neat1 regulates myogenesis.

The development of ectothermic embryos is strongly affected by incubation temperature, and thermal imprinting of body growth and muscle phenotype has been reported in various teleost fishes. The complex epigenetic regulation of muscle development in vertebrates involves DNA methylation of the myogenin promoter. Body growth is a heritable and highly variable trait among fish populations that allows for local adaptations, but also for selective breeding. Here we studied the epigenetic effects of embryonic temperature and genetic background on body growth, muscle cellularity and myogenin expression in farmed Atlantic salmon (Salmo salar). Eggs from salmon families with either high or low estimated breeding values for body growth, referred to as Fast and Slow genotypes, were incubated at 8°C or 4°C until the embryonic 'eyed-stage' followed by rearing at the production temperature of 8°C. Rearing temperature strongly affected the growth rates, and the 8°C fish were about twice as heavy as the 4°C fish in the order Fast8>Slow8>Fast4>Slow4 prior to seawater transfer. Fast8 was the largest fish also at harvest despite strong growth compensation in the low temperature groups. Larval myogenin expression was approximately 4-6 fold higher in the Fast8 group than in the other groups and was associated with relative low DNA methylation levels, but was positively correlated with the expression levels of the DNA methyltransferase genes dnmt1, dnmt3a and dnmt3b. Juvenile Fast8 fish displayed thicker white muscle fibres than Fast4 fish, while Slow 8 and Slow 4 showed no difference in muscle cellularity. The impact of genetic background on the thermal imprinting of body growth and muscle development in Atlantic salmon suggests that epigenetic variation might play a significant role in the local adaptation to fluctuating temperatures over short evolutionary time.

The fusion of myoblasts is an important step during skeletal muscle differentiation. A recent study in mice found that a transmembrane protein called Myomaker, which is specifically expressed in muscle, is critical for myoblast fusion. However, the cellular mechanism of its roles and the regulatory mechanism of its expression remain unclear. Chicken not only plays an important role in meat production but is also an ideal model organism for muscle development research. Here, we report that Myomaker is also essential for chicken myoblast fusion. Forced expression of Myomaker in chicken primary myoblasts promotes myoblast fusion, whereas knockdown of Myomaker by siRNA inhibits myoblast fusion. MYOD and MYOG, which belong to the family of myogenic regulatory factors, can bind to a conserved E-box located proximal to the Myomaker transcription start site and induce Myomaker transcription. Additionally, miR-140-3p can inhibit Myomaker expression and myoblast fusion, at least in part, by binding to the 3ʹ UTR of Myomaker in vitro. These findings confirm the essential roles of Myomaker in avian myoblast fusion and show that MYOD, MYOG and miR-140-3p can regulate Myomaker expression.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by loss of motor neurons, denervation of target muscles, muscle atrophy, and paralysis. Understanding ALS pathogenesis may require a fuller understanding of the bidirectional signaling between motor neurons and skeletal muscle fibers at neuromuscular synapses. Here, we show that a key regulator of this signaling is miR-206, a skeletal muscle-specific microRNA that is dramatically induced in a mouse model of ALS. Mice that are genetically deficient in miR-206 form normal neuromuscular synapses during development, but deficiency of miR-206 in the ALS mouse model accelerates disease progression. miR-206 is required for efficient regeneration of neuromuscular synapses after acute nerve injury, which probably accounts for its salutary effects in ALS. miR-206 mediates these effects at least in part through histone deacetylase 4 and fibroblast growth factor signaling pathways. Thus, miR-206 slows ALS progression by sensing motor neuron injury and promoting the compensatory regeneration of neuromuscular synapses.

Rhabdomyosarcomas (RMS) are the most frequent soft-tissue sarcoma in children and characteristically show features of developing skeletal muscle. The alveolar subtype is frequently associated with a PAX3-FOXO1 fusion protein that is known to contribute to the undifferentiated myogenic phenotype of RMS cells. Histone methylation of lysine residues controls developmental processes in both normal and malignant cell contexts. Here we show that JARID2 , which encodes a protein known to recruit various complexes with histone-methylating activity to their target genes, is significantly overexpressed in RMS with PAX3-FOXO1 compared with the fusion gene-negative RMS ( t -test; P <0.0001). Multivariate analyses showed that higher JARID2 levels are also associated with metastases at diagnosis, independent of fusion gene status and RMS subtype ( n =120; P =0.039). JARID2 levels were altered by silencing or overexpressing PAX3-FOXO1 in RMS cell lines with and without the fusion gene, respectively. Consistent with this, we demonstrated that JARID2 is a direct transcriptional target of the PAX3-FOXO1 fusion protein. Silencing JARID2 resulted in reduced cell proliferation coupled with myogenic differentiation, including increased expression of Myogenin ( MYOG ) and Myosin Light Chain ( MYL1 ) in RMS cell lines representative of both the alveolar and embryonal subtypes. Induced myogenic differentiation was associated with a decrease in JARID2 levels and this phenotype could be rescued by overexpressing JARID2 . Furthermore, we that showed JARID2 binds to and alters the methylation status of histone H3 lysine 27 in the promoter regions of MYOG and MYL1 and that the interaction of JARID2 at these promoters is dependent on EED, a core component of the polycomb repressive complex 2 (PRC2). Therefore, JARID2 is a downstream effector of PAX3-FOXO1 that maintains an undifferentiated myogenic phenotype that is characteristic of RMS. JARID2 and other components of PRC2 may represent novel therapeutic targets for treating RMS patients.

Embryonal rhabdomyosarcoma (ERMS) is a childhood cancer that expresses myogenic master regulatory factor MYOD but fails to differentiate. Here, we show that the zinc finger transcription factor CASZ1 up-regulates MYOD signature genes and induces skeletal muscle differentiation in normal myoblasts and ERMS. The oncogenic activation of the RAS-MEK pathway suppresses CASZ1 expression in ERMS. ChIP-seq, ATAC-seq and RNA-seq experiments reveal that CASZ1 directly up-regulates skeletal muscle genes and represses non-muscle genes through affecting regional epigenetic modifications, chromatin accessibility and super-enhancer establishment. Next generation sequencing of primary RMS tumors identified a single nucleotide variant in the CASZ1 coding region that potentially contributes to ERMS tumorigenesis. Taken together, loss of CASZ1 activity, due to RAS-MEK signaling or genetic alteration, impairs ERMS differentiation, contributing to RMS tumorigenesis. Embryonal rhabdomyosarcoma (ERMS) is a childhood cancer with impaired myogenic differentiation despite the presence of myogenic transcription factors. Here, the authors show that CASZ1 directly regulates these myogenic factors and the loss of CASZ1 is due to RAS-MEK signaling in ERMS.

The rise in throughput and quality of long-read sequencing should allow unambiguous identification of full-length transcript isoforms. However, its application to single-cell RNA-seq has been limited by throughput and expense. Here we develop and characterize long-read Split-seq (LR-Split-seq), which uses combinatorial barcoding to sequence single cells with long reads. Applied to the C2C12 myogenic system, LR-split-seq associates isoforms to cell types with relative economy and design flexibility. We find widespread evidence of changing isoform expression during differentiation including alternative transcription start sites (TSS) and/or alternative internal exon usage. LR-Split-seq provides an affordable method for identifying cluster-specific isoforms in single cells.

Muscle satellite cell (MSC) isolation, proliferation, and differentiation are the basis of cultured meat (CM) technology, which emerged as a sustainable and moral substitute for conventional animal agriculture. Notwithstanding the encouraging future of CM, there are still a lot of obstacles to overcome, like the high expense of cell culture media and the need for fetal bovine serum (FBS). The goal of this work is to determine whether plant-based nitrogen source soy protein hydrolysate (SPH) can improve myogenic differentiation and functional development in MSCs cultured for CM by acting as a serum substitute. We concentrated on how Angel Yeast Company’s SPH PU041 affected the C2C12 mouse cell line, a useful model for studying muscle biology and the CM sector. Adding PU041 to cell culture media containing different concentrations of FBS was found to promote C2C12 cell proliferation and elongation, with optimal effects observed at 0.5 g/L. Immunofluorescence and flow cytometry analyses revealed that PU041 up-regulated the protein levels of myosin heavy chain (MyHC) and myogenic differentiation factor 1 (MyoD), key biomarkers in myogenesis. Furthermore, quantitative real-time PCR (qPCR) confirmed the up-regulation of MyHC, MyoD, and myogenin (MyoG) mRNA expression, indicating that PU041 induces myogenic differentiation. The findings suggest that SPH PU041 can potentially be used to reduce the costs associated with CM production as a viable serum substitute, thereby facilitating a more sustainable and ethical approach to food production. However, the precise mechanisms underlying PU041’s effects on myogenic differentiation warrant further investigation.

Purpose This study aimed to investigate the additive effects of a combination of metformin and resveratrol on irisin expression in C2C12 cells. Methods The study involved treating C2C12 cells with metformin and resveratrol, either alone or in combination, and analyzing their effects on myogenesis and irisin release. The activation of signaling pathways, including AMPK/SIRT1/PGC1α, as well as the relative mRNA and protein expression levels of MyoD, myogenin, and Myh were also assessed. Results Combination treatment with metformin and resveratrol significantly increased MyoD, myogenin, Myh, and FNDC5 expression compared with the group treated with metformin alone. The increase in irisin production was associated with phosphorylation of AMPK and upregulation of PGC-1α and SIRT1, indicating activation of the AMPK/SIRT1/PGC-1α pathway. The mRNA and protein expression levels of MyoD, myogenin, and Myh were also significantly higher in the combination treatment group compared to the metformin alone group. Conclusion The combination of metformin and resveratrol effectively increased irisin release through the AMPK/Sirt1/PGC-1α pathway, suggesting that this combination treatment could enhance myogenesis.