Linlin Zhang

Objective: This study investigates the molecular mechanisms by which the deubiquitinase MYSM1 regulates the proliferation and differentiation of bovine skeletal muscle satellite cells (BSMSCs), thereby providing new theoretical insights into the regulation of muscle growth and development in beef cattle.Methods: An in vitro model of BSMSCs was established to investigate the role of MYSM1. The expression pattern of MYSM1 during cell proliferation and differentiation was analyzed using quantitative real-time polymerase chain reaction (qRT-PCR). MYSM1 knockdown models were generated, and the expression of proliferation markers PAX7 and Ki67, as well as differentiation markers MYHC and MYOG, were examined by qRT-PCR and Western blotting. Analysis of protein–protein interactions identified BRG1 as a potential MYSM1 interactor, and its function was subsequently evaluated. Downstream signaling activity was evaluated by examining phosphorylation changes in key components of the AKT/mTOR pathway. In addition, global histone ubiquitination H2AK119ub1 and the histone methylation markers H3K4me3 and H3K27me3 were analyzed following MYSM1 knockdown.Results: MYSM1 expression was dynamically regulated, exhibiting significant upregulation during differentiation, reaching its highest at days 2–3 (p<0.05). Silencing of MYSM1 significantly (p<0.05) decreased the expression of PAX7, Ki67, MYHC, and MYOG. Histone modification analyses demonstrated elevated levels of H2AK119ub1 and H3K27me3, along with reduced H3K4me3 (p<0.05). Mechanistic studies showed that MYSM1 knockdown significantly reduced BRG1 expression (p<0.05), and BRG1 silencing similarly decreased proliferation and differentiation markers. Moreover, interference with either MYSM1 or BRG1 significantly attenuated the activation of the AKT/mTOR/NF-κB signaling pathway, as evidenced by decreased phosphorylation of AKT, mTOR, and p65 (p<0.05).Conclusion: MYSM1 promotes the proliferation and differentiation of BSMSCs through BRG1-mediated epigenetic regulation and activation of the AKT/mTOR/NF-κB signaling cascade. These findings establish a dual-target framework that advances the understanding of muscle development in beef cattle and offers potential strategies for regenerative therapies.

The developmental efficiency of swine somatic cell nuclear transfer (SCNT) embryos remains limited, primarily due to the lack of physiologically relevant in vitro culture conditions that can fully support reprogramming and early embryogenesis. In this study, we established a co-culture system using swine oviduct epithelial cells (OECs), including untreated and hormone-pretreated OECs with estradiol and progesterone (EP-OECs), to better mimic the oviductal environment. Compared with the control group, EP-OECs exhibited elevated expression of the oviduct-specific marker OVGP1. Moreover, SCNT embryos co-cultured with EP-OECs displayed a significantly higher blastocyst formation rate (control: 18.6% ± 0.01; OEC: 24.5% ± 0.01; EP-OEC: 30.5% ± 0.03). Although the total blastocyst cell number did not increase, co-culture significantly elevated intracellular glutathione (GSH) levels and reduced oxidative stress at key developmental stages. Single-cell transcriptomics (SMART-seq2) analysis revealed that the co-culture activated multiple metabolic pathways, including the pentose phosphate pathway and lipid metabolism, thereby improving redox regulation and energy utilization. Moreover, embryonic development was enhanced through the modulation pluripotency-associated factors, including SOX2, and activation of the PI3K–AKT signaling cascade. Notably, OEC co-culture induced PI3K upregulation at the 8-cell stage and further affected PDK expression. OEC/EP-OEC co-culture treatment suggests PI3K-AKT signaling pathway activation in embryos, which may be a key mechanism promoting embryonic development. These findings indicate that hormone-primed OEC co-culture provides a microenvironment that closely resembles in vivo conditions, offering an effective strategy for improving the efficiency of swine cloning and a new insight into the molecular mechanisms underlying embryonic development in vitro.

MSTN has been used as a candidate gene in the genetics, breeding, and improvement of animal breeds. However, the possible mechanism by which the MSTN gene regulates muscle development through PSMA6 is not well understood. Previous methylome and transcriptome sequencing analyses of gluteal muscle tissues from MSTN+/−Luxi cattle and wild-type Luxi cattle identified that the PSMA6 gene exhibited a negative correlation between methylation levels and transcriptional activity. To investigate whether MSTN expression regulates PSMA6 gene expression, we examined the effects of MSTN on DNA methyltransferases (DNMT1, DNMT2, DNMT3A, and DNMT3B) and DNA demethylases (TET1, TET2, and TET3). Additionally, chromatin immunoprecipitation (ChIP) assays were performed to detect the binding interaction between PSMA6 and TET2. In this paper, we first established an MSTN knockdown cellular model to preliminarily validate its regulatory effect on PSMA6 expression. Subsequently, the developmental impact of PSMA6 on bovine skeletal muscle satellite cells was further investigated through both knockdown and overexpression of the PSMA6 gene. Furthermore, we examined changes in the expression of key components of the AKT/mTOR signaling pathway to elucidate the mechanisms underlying the PSMA6-mediated regulation of satellite cell development. The results demonstrate that myostatin (MSTN) inhibition significantly decreased proteasome 20S subunit alpha-6 (PSMA6) gene expression, while increasing demethylase expression, particularly ten-eleven translocation-2 (TET2), which exhibited the most pronounced changes. During the cell proliferation stage, the markers Paired Box 7 (PAX7) and Ki-67 exhibited no significant changes, whereas the PSMA6 gene was either overexpressed or disrupted. Conversely, PSMA6 overexpression altered the myogenic differentiation markers, causing the differential regulation of myosin heavy chain (MyHC) and myogenin (MyoG) expression, with MyHC upregulation and concurrent MyoG downregulation. PSMA6 gene overexpression led to the downregulation of AKT1 and Rac1, as well as the activation of the AKT/mTOR pathway, including key factors such as mTOR, p-mTOR, RPS6, p-RPS6, and RhoA. PSMA6 interference resulted in the downregulation of p-mTOR and the upregulation of p-RPS6. Gene expression profiling in our study revealed that the myostatin (MSTN) knockout model significantly reduced the transcriptional levels of the proteasome α6 subunit (PSMA6) (p < 0.05), with the regulatory intensity showing a significant negative correlation with MSTN expression. This molecular evidence substantiates a negative regulatory axis between MSTN and PSMA6. Functional experiments demonstrated that PSMA6 overexpression specifically enhanced myotube formation rates in bovine skeletal muscle satellite cells, whereas siRNA-mediated PSMA6 knockdown exhibited no significant effects on cellular proliferation, indicating the functional specificity of this gene in myogenic differentiation. Mechanistic investigations further revealed that PSMA6 activates the canonical AKT/mTOR signaling transduction cascade through the phosphorylation of AKT and its downstream effector mTOR, thereby mediating the expression of myogenic regulatory factors MyoD and myogenin. Collectively, these findings demonstrate that MSTN deficiency alleviates the transcriptional repression of PSMA6, remodels skeletal muscle differentiation-associated signaling networks, and ultimately drives the directional differentiation of satellite cells toward myofiber specification.

The CRISPR-Cas system functions as an adaptive immune mechanism in archaea and bacteria, providing defense against the invasion of foreign nucleic acids. Most CRISPR-Cas systems are classified into class 1 or class 2, with further subdivision into several subtypes. The primary distinction between class 1 and class 2 systems lies in the assembly of their effector modules. In class 1 systems, the effector complex consists of multiple proteins with distinct functions, whereas in class 2 systems, the effector is associated with a single protein. Class 1 systems account for approximately 90% of the CRISPR-Cas repertoire and are categorized into three types (type I, type IV, and type III) and 12 subtypes. To date, various CRISPR-Cas systems have been widely employed in the field of genetic engineering as essential tools and techniques for genome editing. Type I CRISPR-Cas systems remain a valuable resource for developing sophisticated application tools. This review provides a comprehensive review of the characteristics, mechanisms of action, and applications of class 1 type I CRISPR-Cas systems, as well as transposon-associated systems, offering effective approaches and insights for future research on the mechanisms of action, as well as the subsequent development and application of type I CRISPR-Cas systems.

Heterogeneous nuclear ribonucleoproteins (hnRNPs) are a superfamily of RNA-binding proteins consisting of more than 20 members. These proteins play a crucial role in various biological processes by regulating RNA splicing, transcription, and translation through their binding to RNA. In the context of muscle development and regeneration, hnRNPs are involved in a wide range of regulatory mechanisms, including alternative splicing, transcription regulation, miRNA regulation, and mRNA stability regulation. Recent studies have also suggested a potential association between hnRNPs and muscle-related diseases. In this report, we provide an overview of our current understanding of how hnRNPs regulate RNA metabolism and emphasize the significance of the key members of the hnRNP family in muscle development. Furthermore, we explore the relationship between the hnRNP family and muscle-related diseases.

Skeletal muscle satellite cells (MuSCs) can proliferate, differentiate, and self-renew, and can also participate in muscle formation and muscle injury repair. Long noncoding RNAs (lncRNAs) can play an important role with the RNA binding protein and microRNAs (miRNAs) to regulate the myogenesis of bovine MuSCs, however, its molecular mechanism is still being explored. In this study, differentially expressed 301 lncRNAs were identified during the myogenic differentiation of cells based on an in vitro model of induced differentiation of bovine MuSCs using RNA sequencing (RNA-seq). Based on the ability of miR-206 to regulate myogenic cell differentiation, a new kind of lncRNA-lncA2B1 without protein-coding ability was found, which is expressed in the nucleus and cytoplasm. Subsequently, lncA2B1 inhibited cell proliferation by downregulating the expression of the proliferation marker Pax7 and promoted myogenic differentiation by upregulating the expression of the differentiation marker MyHC, whose regulatory function is closely related to miR-206. By RNA pulldown/LC-MS experiments, heterogeneous ribonucleoprotein A2/B1 (HNRNPA2B1), and DExH-Box Helicase 9 (DHX9) were identified as common binding proteins of lncA2B1 and miR-206. Overexpression of lncA2B1 and miR-206 significantly upregulated the expression level of HNRNPA2B1. Downregulation of HNRNPA2B1 expression significantly decreased the expression level of the differentiation marker MyHC, which indicates that miR-206 and lncA2B1 regulate myogenic differentiation of bovine MuSCs by acting on HNRNPA2B1. This study screened and identified a novel lncRNA-lncA2B1, which functions with miR-206 to regulate myogenesis via the common binding proteins HNRNPA2B1. The results of this study provide a new way to explore the molecular mechanisms by which lncRNAs and miRNAs regulate muscle growth and development.

Myostatin (MSTN) is a negative regulator of skeletal muscle development and plays an important role in muscle development. Fluctuations in gene expression influenced by DNA methylation are critical for homeostatic responses in muscle. However, little is known about the mechanisms underlying this fluctuation regulation and myogenic differentiation of skeletal muscle. Here we report a genome-wide analysis of DNA methylation dynamics in bovine skeletal muscle myogenesis after myostatin editing. We show that, after myostatin editing, an increase in TETs (DNA demethylases) and a concomitant increase in the receptor for activated C kinase 1 (RACK1) control the myogenic development of skeletal muscle. Interestingly, enhancement of PI3K/AKT/mTOR signaling by RACK1 appears to be an essential driver of myogenic differentiation, as it was associated with an increase in myogenic differentiation marker factors (MyHC and MyoG) during muscle differentiation. Overall, our results suggest that loss of myostatin promotes the myogenic differentiation response in skeletal muscle by decreasing DNA methylation of RACK1.

Myoblast differentiation is essential for the formation of skeletal muscle myofibers. Profilin1 (Pfn1) has been identified as an actin-associated protein, and has been shown to be critically important to cellular function. Our previous study found that PFN1 may inhibit the differentiation of bovine skeletal muscle satellite cells, but the underlying mechanism is not known. Here, we confirmed that PFN1 negatively regulated the myogenic differentiation of bovine skeletal muscle satellite cells. Immunoprecipitation assay combined with mass spectrometry showed that Cdc42 was a binding protein of PFN1. Cdc42 could be activated by PFN1 and could inhibit the myogenic differentiation like PFN1. Mechanistically, activated Cdc42 increased the phosphorylation level of p2l-activated kinase (PAK), which further activated the phosphorylation activity of c-Jun N-terminal kinase (JNK), whereas PAK and JNK are inhibitors of myogenic differentiation. Taken together, our results reveal that PFN1 is a repressor of bovine myogenic differentiation, and provide the regulatory mechanism.