Promoter methylation modification and transcriptional regulatory mechanism analysis of the MSTN gene in Guanling cattle

【Objective】 This study aimed to clarify the differential expression of the myostatin （MSTN） gene in diffe-rent tissues of young and adult Guanling cattle， the methylation of their promoters， and their associations with the Smad3 signaling pathway， so as to provide a theoretical basis for revealing the multi-level regulatory network of the MSTN gene in cattle muscle development， and new insights for improving beef yield and quality traits. 【Method】 Tissue samples （heart， liver， spleen， kidney， longissimus dorsi muscle， and subcutaneous fat） were collected from young （3-day-old） and adult （3-year-old） Guanling cattle. The expression of the MSTN gene and its related regulatory genes （FOXO1， MYOD1， and TET1） were detected by real-time fluorescence quantitative PCR. The methylation of the CpG island in the MSTN gene promoter was analyzed using bisulfite sequencing. The phosphorylation of Smad3-Ser423/425 in different tissues of Guanling cattle was measured by enzyme-linked immunosorbent assay （ELISA） to assess Smad3 signaling pathway activity. 【Result】 The relative expression of the MSTN gene in the longissimus dorsi muscle of Guanling cattle was significantly higher than that in other tissues （P<0.01， the same below）， and its relative expression in the longissimus dorsi muscle of young cattle was significantly higher than that in adult cattle （P<0.05）， indicating a stronger regulatory role of this gene during the rapid muscle growth period. Furthermore， the relative expressions of MYOD1 and TET1 genes in the longissimus dorsi muscle of young cattle were significantly higher than those in adult cattle， while the relative expression of the FOXO1 gene showed no significant difference between young and adult cattle （P>0.05）. The methylation rate of the CpG island in the MSTN gene promoter in the longissimus dorsi muscle was significantly lower than that in other tissues in both young and adult cattle， and the methylation rate in calves was significantly lower than that in adult cattle. Similarly， the phosphorylation of Smad3-Ser423/425 in the longissimus dorsi muscle was significantly greater than that in other tissues， and the phosphorylation of Smad3-Ser423/425 in young cattle was significantly higher than that in adult cattle， indicating a positive correlation between Smad3-Ser423/425 phosphorylation and MSTN gene expression. 【Conclusion】 The MSTN gene in Guanling cattle shows tissue-specific high expression in the longissimus dorsi muscle； its regulation is related to the synergistic effect of promoter methylation modification and the Smad3 signaling pathway， and is influenced by age. Low hypomethylation may maintain the basal transcriptional activity of the MSTN gene in muscle， while Smad3 phosphorylation enhances its downstream inhibitory effects， thereby promoting normal muscle development.