ANKRD9 negatively regulates chicken myogenesis through ubiquitin-mediated regulation of IMPDH2

Skeletal muscle development is regulated by transcriptional and post-translational mechanisms. While ankyrin repeat proteins participate in post-translational modifications, their role in myogenesis remains unclear. This study identifies ankyrin repeat domain-containing protein 9 (ANKRD9) as a novel negative regulator of chicken skeletal muscle development. ANKRD9 showed dynamic expression during postnatal muscle growth and downregulated cell cycle and DNA replication-related genes. Functionally, ANKRD9 overexpression inhibited myoblast proliferation and differentiation, while its knockdown enhanced these processes. In vivo, siRNA-mediated knockdown of ANKRD9 markedly increased muscle mass and myofiber diameter in chicks. Mechanistically, ANKRD9 bound directly to inosine monophosphate dehydrogenase 2 (IMPDH2), a rate-limiting enzyme in purine synthesis, and promoted its ubiquitin-mediated degradation without affecting mRNA levels. Crucially, rescue experiments confirmed that restoring IMPDH2 expression effectively reversed the inhibitory effects of ANKRD9 on myoblast proliferation and differentiation. Thus, this study unveils a novel regulatory axis in which ANKRD9 negatively regulates skeletal myogenesis by mediating the ubiquitination of IMPDH2. This discovery not only provides new insights into the post-translational regulatory network governing muscle development but also offers a potential target for genetic improvement of meat yield in poultry.