Non-Coding SNPs Regulate Bovine Muscle Satellite Cell Proliferation and Differentiation by Modulating PENK Expression

The functions of noncoding variants associated with complex traits in livestock remain poorly understood. In this study, we investigated two candidate noncoding variants within the XKR4-CHCHD7 locus identified from our previous analysis. Dual-luciferase reporter assays demonstrated allele-specific regulatory activity of these two regions in bovine muscle satellite cells (BMSCs), 293T cells, and C2C12 cells. Endogenous deletion of the candidate regions using a clustered regularly interspaced short palindromic repeats (CRISPR)-based high-fidelity Cas12Max (hfCas12Max) system revealed that the region containing chr14:22840845 (SNP-0845) exerted broader effects on BMSC function including reduced proliferation and migration, altered cell-cycle progression, and enhanced myogenic differentiation. Expression screening of candidate effector genes further identified PENK and TMEM68 as downstream candidate genes for SNP-0845. Rescue experiments further showed that PENK exerted stronger recovery effects than TMEM68 on the proliferation and migration defects caused by deletion of this region, supporting PENK as a major candidate effector downstream of the SNP-0845. Functional assays showed that PENK knockdown impaired BMSC proliferation and migration while promoting myogenic differentiation, whereas PENK overexpression partially reversed these effects. In vivo Penk knockdown reduced quadriceps femoris weight and altered muscle fiber composition in mice. Collectively, our findings suggest that a noncoding regulatory region modulates BMSC fate and muscle growth-related processes through PENK.