Dissection of the cellular function of the ZBED6 transcription factor in mouse myoblast cells using gene editing, RNAseq and proteomics
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SUMMARYThe transcription factor ZBED6 acts as a repressor ofIgf2and affects directly or indirectly the transcriptional regulation of thousands of genes. Here, we use gene editing in mouse C2C12 myoblasts and show that ZBED6 regulatesIgf2exclusively through its binding site 5-GGCTCG-3 in intron 1 ofIgf2. Deletion of this motif (Igf2GGCT) or complete ablation ofZbed6leads to ~20-fold up-regulation of IGF2 protein. Quantitative proteomics revealed an activation of Ras signaling pathway in bothZbed6-/-andIgf2GGCTmyoblasts, and a significant enrichment of mitochondrial membrane proteins among proteins showing altered expression inZbed6-/-myoblasts. BothZbed6-/-andIgf2GGCTmyoblasts showed a faster growth rate and developed myotube hypertrophy. These cells exhibited an increased O2consumption rate, due to IGF2 up-regulation. Transcriptome analysis revealed ~30% overlap between differentially expressed genes inZbed6-/-andIgf2GGCTmyotubes, with an enrichment of up-regulated genes involved in muscle development. In contrast, ZBED6-overexpression in myoblasts led to cell apoptosis, cell cycle arrest, reduced mitochondrial activities and ceased myoblast differentiation. The similarities in growth and differentiation phenotypes observed inZbed6-/-andIgf2GGCTmyoblasts demonstrates that ZBED6 affects mitochondrial activity and myogenesis largely through its regulation of IGF2 expression. This study suggests that the interaction between ZBED6-Igf2may be a therapeutic target for human diseases where anabolism is impaired.
