转录因子MAX介导三维基因组互作调控猪骨骼肌细胞增殖分化机理研究

Transcription factors play an important role in the growth and development of skeletal muscle, but only a few of them have been well studied. Thus, other important transcription factors have to be studied. Our previous study found that the transcription factors MAX is located in multiple QTLs of porcine, including loin muscle area and lean meat percentage. MAX was highly expressed during the important stages of proliferation and differentiation of porcine embryonic skeletal muscle cells and can promote and repress the proliferation and differentiation of myoblasts. ChIP-Seq binding peaks of MAX in myoblasts were widely distributed in genomic regulatory elements region. We also found that MAX is related to difference of 3D genome structure during skeletal muscle cell proliferation and differentiation, but the specific mechanism remains unclear. Therefore, this project intends to combine the three-dimensional genome research technology LR-ChIA-PET with other experiments to carry out the following research: 1) To identify the 3D genome interaction mediated by MAX in porcine skeletal muscle at different developmental stages, proliferation and differentiation wild-type and MAX knockdown porcine skeletal muscle satellite cells; 2) To analyze the key long range genome interaction and its target genes by which the MAX regulate the proliferation and differentiation of porcine skeletal muscle cells at 3D genome level; 3) Using DNA FISH, CRSPR-Cas9 and other technologies to verify key 3D genome interactions and their regulation role. The results will provide a new basis for the understanding of the growth and development mechanism of porcine skeletal muscle, and provide new candidate genes for the improvement of meat production traits.

转录因子在骨骼肌生长发育过程中发挥重要作用，但目前仅有少数转录因子的调控机制被解析，其他重要转录因子亟待研究和挖掘。申请人发现转录因子MAX位于猪眼肌面积、瘦肉率等多个QTL内，在猪胚胎期骨骼肌细胞增殖分化高峰期高表达，促进成肌细胞增殖抑制分化，成肌细胞ChIP-Seq结合峰广泛分布在基因组调控元件区域，且与骨骼肌细胞增殖分化过程中3D基因组构象变化有关，但具体机理仍不清楚。因此本项目拟运用三维基因组LR-ChIA-PET等技术开展如下研究：1）鉴定不同发育阶段猪骨骼肌、增殖分化期野生型及MAX敲低的猪骨骼肌卫星细胞中MAX介导的3D远程互作；2）筛选MAX在3D基因组水平调控猪骨骼肌细胞增殖分化的关键远程互作位点、靶基因及通路；3）利用DNA FISH、CRSPR-Cas9等技术验证关键3D远程互作及调控作用。研究结果将为解析猪骨骼肌生长发育机理提供新依据，为产肉性状改良提供新候选基因。

本研究的主要任务是：转录因子MAX介导三维基因组互作调控猪骨骼肌细胞增殖分化机理研究。针对项目研究目标，项目组展开了系列研究具体研究结果包括：（1）通过在猪骨骼肌卫星细胞中超表达MAX发现：MAX能显著抑制骨骼肌卫星细胞分化；（2）联合ChIA-PET、Hi-C、ATAC等多组学数据发现MAX在成肌细胞增殖期发生远程互作的调控元件位点的开放染色质区域内显著富集，且在分化过程中以蛋白水平的下调方式减弱染色质的结合，以此调控染色质状态，并以此促进MyoD和MyoG的结合进而促进细胞分化；（3）通过整合ATAC-seq、ChIP-seq及RNA-seq数据发现MAX可协同其他肌源性转录因子调控lncRNA的差异表达，并以此调控骨骼肌细胞的增殖分化。相关研究成功以本项目为第一资助发表了SCI论文一篇（Cell proliferation，2020，IF=6.8）；（4）通过整合2周龄猪骨骼肌组织和脂肪组织ChIP-seq、ATAC-seq及RNA-seq数据分析发现，在大白猪骨骼肌组织中MAX能够通过靶向多个miRNA从而调控骨骼肌组织的基因表达，MAX在骨骼肌组织中的表达明显高于脂肪组织。相关研究结果以学术论文的形式发表在Genomics（2022，IF=4.3）杂志，本项目为论文第一资助。