Enhancer Enh483 regulates myoblast proliferation and differentiation of buffalo myoblasts by targeting FAXC.

A detailed understanding of the precise regulatory mechanisms governing buffalo skeletal muscle is crucial for improving meat quality and yield. Proper skeletal muscle fate decisions necessitate the accurate regulation of key enhancers. This study screened nine potential enhancers linked to muscle development by analysing ATAC-seq data from buffalo myoblasts during the proliferative and differentiative phases. The enhancer activity of these candidates was confirmed in buffalo myoblasts, C2C12, and human skeletal muscle myoblasts using a dual-luciferase reporter system. The CRISPRi system and RT-qPCR were used to test the effects of 9 candidate enhancers on buffalo myoblasts. The active enhancer, Enh483, was selected based on its significant impact. Upon successful inhibition of Enh483 using CRISPRi, decreases in the expression of buffalo myogenic proliferation marker genes (PCNA, CyclinD1, and CDK2) were observed via RT-qPCR and Western blot. Subsequent proliferation assays using CCK-8 and EdU confirmed the promotive effect of Enh483 on buffalo myogenic cell proliferation. Following a 5-day differentiation induction period, changes in the expression of differentiation marker genes (MyoD1, MyoG, and MyHC) were analysed using RT-qPCR and Western blot. Additionally, fused myotube numbers were quantified, and the impact of Enh483 on buffalo myogenic cell differentiation was assessed through immunofluorescence. Our findings indicate that Enh483 facilitates buffalo myogenic cell differentiation. Further interaction analysis utilising 3C-PCR revealed a direct association between Enh483 and the FAXC promoter. In summary, the results from this study lay a foundational framework for deciphering the intricate regulatory mechanisms underpinning buffalo muscle development.