Kbtbd5 is regulated by MyoD and restricted to the myogenic lineage

• Identification of a novel muscle-specific gene during embryogenesis, Kbtbd5.
• Characterization of the Kbtbd5 promoter.
• Kbtbd5 is a direct downstream target gene of MRFs.
• Kbtbd5 interacts with the ubiquitination ligase Cullin 3.
• Knockdown of Kbtbd5 results in the perturbed myogenic differentiation.

BTB–BACK–Kelch (BBK) proteins play broad roles in cellular and molecular regulation. The role of BBK proteins in the skeletal muscle lineage and myogenesis remains an active area of research. Herein, we report a novel BBK gene, Kbtbd5, which we discovered and found to be restricted to the myogenic lineage. We observed that Kbtbd5 was absent in proliferating myoblasts and upregulated upon myogenic differentiation. In situ hybridization analysis revealed that Kbtbd5 was restricted to the skeletal muscle lineage during embryogenesis. We identified a conserved 1.2 kb upstream region, which directs reporter expression to the developing skeletal muscle lineage. Transcriptional and mutagenesis assays demonstrated that the E-box motifs contribute to the Kbtbd5 promoter activity. We have also demonstrated the in vivo and in vitro binding between MRFs and the E-box motif in the 1.2 kb promoter of the Kbtbd5 gene. Our studies have revealed that the Myod family can transactivate the 1.2 kb-luc reporter through the E-box motifs. In addition, we have shown that Kbtbd5 can recruit the Cullin 3 complex in vivo. Using shRNA knockdown, our study has revealed that Kbtbd5 plays an important role in the myogenic differentiation. In summary, we have demonstrated that Kbtbd5 is the direct downstream target gene of the Myod family and regulates myogenic differentiation. Our results further support the notion that Kbtbd5 may serve as an adapter of Cul3 during myogenic differentiation.