LIM domain binding 3 (LDB3) serves as a striated muscle-specific Z-band alternatively spliced protein that plays an important role in mammalian skeletal muscle development, but its regulatory role and molecular mechanism in avian muscle development are still unclear. In this study, we reanalyzed RNA sequencing data sets of 1415 samples from 21 chicken tissues published in the NCBI GEO database. First, three variants (LDB3-X, LDB3-XN1, and LDB3-XN2) generated by alternative splicing of the LDB3 gene were identified in chicken skeletal muscle, among which LDB3-XN1 and LDB3-XN2 are novel variants. LDB3-X and LDB3-XN1 are derived from exon skipping in chicken skeletal muscle at the E18-D7 stage and share three LIM domains, but LDB3-XN2 lacks a LIM domain. Our results preliminarily suggest that the formation of three variants of LDB3 is regulated by RBM20. The three splice isomers have divergent functions in skeletal muscle according to in vitro and in vivo assays. Finally, we identified the mechanism by which different variants play different roles through interactions with IGF2BP1 and MYHC, which promote the proliferation and differentiation of chicken myoblasts, in turn regulating chicken myogenesis. In conclusion, this study revealed the divergent roles of three LDB3 variants in chicken myogenesis and muscle remodeling and demonstrated their regulatory mechanism through protein-protein interactions.

Deposition of the exon junction complex (EJC) upstream of exon-exon junctions helps maintain transcriptome integrity by preventing spurious re-splicing events in already spliced mRNAs. Here we investigate the importance of EJC for the correct splicing of the 2.2-megabase-long human DMD pre-mRNA, which encodes dystrophin, an essential protein involved in cytoskeletal organization and cell signaling. Using targeted RNA-seq, we show that knock-down of the eIF4A3 and Y14 core components of EJC in a human muscle cell line causes an accumulation of mis-splicing events clustered towards the 3\' end of the DMD transcript (Dp427m). This deregulation is conserved in the short Dp71 isoform expressed ubiquitously except in adult skeletal muscle and is rescued with wild-type eIF4A3 and Y14 proteins but not with an EJC assembly-defective mutant eIF4A3. MLN51 protein and EJC-associated ASAP/PSAP complexes independently modulate the inclusion of the regulated exons 71 and 78. Our data confirm the protective role of EJC in maintaining splicing fidelity, which in the DMD gene is necessary to preserve the function of the critical C-terminal protein-protein interaction domain of dystrophin present in all tissue-specific isoforms. Given the role of the EJC in maintaining the integrity of dystrophin, we asked whether the EJC could also be involved in the regulation of a mechanism as complex as skeletal muscle differentiation. We found that eIF4A3 knockdown impairs myogenic differentiation by blocking myotube formation. Collectively, our data provide new insights into the functional roles of EJC in human skeletal muscle.

Myogenesis is a crucial process governing skeletal muscle development and homeostasis. Lead (Pb) exposure impaired the development and the health of bones, which slows the growth of children. However, it is far from clear what exactly the effects of Pb on skeletal muscle development are. In this study, C2C12 cells are commonly used as an in vitro model of muscle regeneration due to their ability to transition from a proliferative phase into differentiated myofibers. The dose of 1, 5, and 10 μM Pb were adopted to study the toxicity of Pb on C2C12 proliferation and differentiation. First, the effects of Pb on cell viability were detected and the results demonstrated that 5 μM and 10 μM Pb exposure decreased cell viability, while 1 μM Pb exposure has no obvious effects on cell viability. Then, 1-10 μM Pb exposure seriously reduced the C2C12 myoblasts differentiation, with the decrease of myogenic differentiation marker genes expression, including Muscle creatine kinase (MCK), Myosin Heavy Chain 4 (MYH4), Myogenin (MYOG), Myogenic Differentiation (MYOD). What\'s more, it was found that the epigenetic modifier histone deacetylase-2 (HDAC2) was upregulated after Pb exposure on C2C12 myoblasts. Further studies conclusively showed knockdown of HDAC2 ameliorated Pb-damaged C2C12 myoblasts differentiation, indicating HDAC2 plays a vital role in the Pb-induced C2C12 myoblasts differentiation deficits. In summary, these results demonstrated that Pb exposure inhibited C2C12 myoblasts differentiation by regulating HDAC2.

Meat quality and meat composition are not separated from the influences of animal genetic improvement systems; the growth and development of skeletal muscle are the primary factors in agricultural meat production and meat quality. Though the muscle-type cofilin (CFL2) gene has a crucial influence on skeletal muscle fibers and other related functions, the epigenetic modification mechanism of the CFL2 gene regulating meat quality remains elusive. After exploring the spatiotemporal expression data of CFL2 gene in a group of samples from fetal bovine, calf, and adult cattle, we found that the level of CFL2 gene in muscle tissues increased obviously with cattle age, whereas DNA methylation levels of CFL2 gene in muscle tissues decreased significantly along with cattle age by BSP and COBRA, although DNA methylation levels and mRNA expression levels basically showed an opposite trend. In cell experiments, we found that bta-miR-183 could suppress primary bovine myoblast differentiation by negatively regulated CFL2. In addition, we packaged recombinant adenovirus vectors for CFL2 gene knockout and overexpression and found that the CFL2 gene could promote the differentiation of primary bovine myoblasts by regulating marker genes MYOD, MYOG and MYH3. Therefore, CFL2 is an essential mediator for promoting myogenic differentiation by regulating myogenic marker genes in cattle myoblasts.

p53, with its family members p63 and p73, have been shown to promote myoblast differentiation by regulation of the function of the retinoblastoma protein and by direct activation of p21Cip/Waf1 and p57Kip2, promoting cell cycle exit. In previous studies, we have demonstrated that the TAp63γ isoform is the only member of the p53 family that accumulates during in vitro myoblasts differentiation, and that its silencing led to delay in myotube fusion. To better dissect the role of TAp63γ in myoblast physiology, we have generated both sh-p63 and Tet-On inducible TAp63γ clones. Gene array analysis of sh-p63 C2C7 clones showed a significant modulation of genes involved in proliferation and cellular metabolism. Indeed, we found that sh-p63 C2C7 myoblasts present a higher proliferation rate and that, conversely, TAp63γ ectopic expression decreases myoblasts proliferation, indicating that TAp63γ specifically contributes to myoblasts proliferation, independently of p53 and p73. In addition, sh-p63 cells have a defect in mitochondria respiration highlighted by a reduction in spare respiratory capacity and a decrease in complex I, IV protein levels. These results demonstrated that, beside contributing to cell cycle exit, TAp63γ participates to myoblasts metabolism control.

The myogenic regulatory factors (MRFs) and myocyte enhancer factor 2 (MEF2) transcription factors have been extensively studied as key transcription factors that regulate myogenic gene expression. However, few reports on the molecular mechanism that modulates chromatin remodeling during skeletal muscle differentiation are available. We reported here that the expression of the H3-K9 methyltransferase Suv39h1 was decreased during myoblast differentiation. Ectopic expression of Suv39h1 could inhibit myoblast differentiation, increasing H3-K9 methylation levels, whereas knockdown of Suv39h1 stimulated myoblast differentiation. Furthermore, Suv39h1 interacted with MEF2C directly and inhibited MEF2 transcription activity in a dose-dependent manner. Together, our studies revealed a molecular mechanism wherein Suv39h1 modulated myogenic gene expression and activation during skeletal muscle differentiation.