Dissection of the cellular function of the ZBED6 transcription factor in mouse myoblast cells IGF2–GGCT, Medium

The transcription factor ZBED6 acts as a repressor of Igf2 and affects directly or indirectly the transcriptional regulation of thousands of genes. Here, we use gene editing in mouse C2C12 myoblasts and show that ZBED6 regulates Igf2 exclusively through its binding site 5′-GGCTCG-3′ in intron 1 of Igf2. Deletion of this motif (Igf2ΔGGCT) or complete ablation of Zbed6 leads to ~20-fold up-regulation of IGF2 protein. Quantitative proteomics revealed an activation of Ras signaling pathway in both Zbed6-/- and Igf2ΔGGCT myoblasts, and a significant enrichment of mitochondrial membrane proteins among proteins showing altered expression in Zbed6-/- myoblasts. Both Zbed6-/- and Igf2ΔGGCT myoblasts showed a faster growth rate and developed myotube hypertrophy. These cells exhibited an increased O2 consumption rate, due to IGF2 up-regulation. Transcriptome analysis revealed ~30% overlap between differentially expressed genes in Zbed6-/- and Igf2ΔGGCT myotubes, 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 in Zbed6-/- and Igf2ΔGGCT myoblasts demonstrates that ZBED6 affects mitochondrial activity and myogenesis largely through its regulation of IGF2 expression. This study suggests that the interaction between ZBED6-Igf2 may be a therapeutic target for human diseases where anabolism is impaired.

转录因子ZBED6可作为胰岛素样生长因子2（Igf2）的转录阻遏因子，直接或间接调控数千个基因的转录。本研究通过对小鼠C2C12成肌细胞进行基因编辑，证实ZBED6仅通过Igf2第一内含子中5′-GGCTCG-3′的结合位点调控Igf2的表达。删除该结合基序（Igf2ΔGGCT）或完全敲除Zbed6基因，均可使IGF2蛋白的表达量上调约20倍。定量蛋白质组学分析显示，在Zbed6敲除（Zbed6-/-）与Igf2ΔGGCT成肌细胞中，Ras信号通路均被激活；且在Zbed6-/-成肌细胞中，表达量发生改变的蛋白显著富集于线粒体膜蛋白类群。Zbed6-/-与Igf2ΔGGCT两种成肌细胞均表现出更快的生长速率，并可形成肌管肥大表型。由于IGF2的表达上调，这两类细胞的氧气消耗速率均有所提升。转录组分析结果表明，Zbed6-/-与Igf2ΔGGCT成肌管间的差异表达基因重叠率约为30%，其中上调基因显著富集于肌肉发育相关通路。与之相反，在成肌细胞中过表达ZBED6会引发细胞凋亡、细胞周期阻滞，降低线粒体活性，并抑制成肌细胞分化。Zbed6-/-与Igf2ΔGGCT成肌细胞在生长与分化表型上的高度相似性，证实ZBED6主要通过调控IGF2的表达来影响线粒体活性与肌发生过程。本研究提示，ZBED6与Igf2的相互作用或许可作为合成代谢受损相关人类疾病的治疗靶点。