猪TCF12互作转录因子鉴定及其在骨骼肌能量代谢中的作用机理研究

Skeletal muscle energy metabolism (SMEM) is one of the key factors of feed efficiency in pigs. We have found that TCF12 could promote the energy metabolism level and differentiation in myoblast. TCF12 gene has also been considered as the candidate gene of feed intake and some growth traits based on QTL and GWAS studies in pigs. Also, our selective sweep study showed that TCF12 contained strong selection signaling in western pig breeds. Therefore, TCF12 should be an important candidate gene for SMEM and feed efficiency in pigs. At present, the mechanism of the SMEM regulated by TCF12 is largely unknown in pigs. We found that TCF12 could interact with YY1 based on the results of bioinformatic analysis of the ChIP data of human TCF12 gene. It has been confirmed that YY1 could regulated the energy metabolism through interaction with PGC1α directly. Based on these results, we hypothesis that TCF12 firstly binds to its DNA motif, and then TCF12 recruits and promotes the DNA binding of YY1 through forming complex, and then the complex promotes SMEM of pigs through recruiting the PGC1α, and finally affects the feed efficiency of pigs. In this study, 1) We will validate the interaction of TCF12 and YY1 in pigs. 2) Identification of mechanism of energy metabolism regulated by TCF12/YY1 complex in pigs. 3) Identify the functional mutation located at the selective signaling regions of TCF12 in pigs. 4) Identification of the correlation between functional mutation and the feed efficiency traits in pigs. This study will reveal the mechanism of skeletal energy metabolism regulated by the TCF12/YY1 complex in pigs. Also, we will identify some important novel genetic markers for feed efficiency improvement in pigs.

骨骼肌能量代谢（SMEM）是影响猪饲料效率的重要因素。申请人发现转录因子TCF12正调控成肌细胞能量代谢及分化。QTL及GWAS研究表明TCF12与猪采食量、生长性状相关，且在西方猪中受到强烈选择，很可能是调控猪SMEM及饲料效率的关键基因。目前TCF12调控猪SMEM分子机理尚不清楚。申请人分析人ChIP数据发现TCF12能与YY1互作。YY1已被证实能够招募PGC1α调控能量代谢。据此我们假设：猪TCF12结合DNA后招募并促进YY1与DNA结合，二者复合物进一步招募PGC1α调控猪SMEM，影响猪饲料效率。本项目拟开展：1）验证猪TCF12与YY1互作；2）阐明TCF12/YY1互作调控猪SMEM的分子机理；3）鉴定猪TCF12选择信号区功能突变；4）验证功能突变对猪饲料效率的作用。结果将系统揭示TCF12/YY1互作调控猪SMEM的分子机理，为猪饲料效率性状改良提供新的分子标记。

现代养猪生产中，饲料成本占总生产成本的60%以上，提高猪饲料效率（Feed efficiency, FE），降低饲料成本，是提高养猪生产效益的重要手段之一。目前，猪饲料效率性状的研究还相对较少，其分子遗传机理也不甚清楚。TCF12基因是项目主持人鉴定的一个与猪骨骼肌能量代谢密切相关的重要功能基因，前期研究表明骨骼肌能量代谢是影响猪饲料效率的重要因素之一，为了揭示TCF12基因对骨骼肌发育及能量代谢调控作用分子机理，揭示其对猪饲料效率性状的调控作用，开展了本项目研究。本项目的主要研究内及结果如下：1）细胞及个体水平揭示了TCF12基因生物学功能。研究表明TCF12基因促进成肌分化及骨骼肌能量代谢，骨骼肌组织特异敲除TCF12基因抑制肌肉生长，降低骨骼肌卫星细胞增殖和分化能力，骨骼肌损伤修复能力减弱；2）系统揭示TCF12互作蛋白及其调控骨骼肌发育分子机理。本研究通过Co-IP实验发现TCF12能够与生肌因子MyoD、MyoG互作调控骨骼肌发育及能量代谢；3）阐明TCF12转录调控分子机理。ChIP-seq数据分析发现TCF12基因转录调控区存在MyoD、MyoG转录因子结合位点，这些结果表明TCF12与MyoD、MyoG形成复合体调控其自身转录；4）揭示了TCF12基因组调控元件及其互作关系。通过ChIP-seq、HI-C等技术对TCF12基因的三维结构进行了研究，鉴定出了猪TCF12基因的增强子和启动子区域，并揭示了这些区域的互作关系，研究还发现TCF12转录因子对基因组三维结构存在调控作用；5）鉴定TCF12基因关键突变，筛选具有育种价值的分子标记；通过整合了猪ENCODE、转录因子结合Motif、选择信号、QTL等信息，对TCF12基因重要突变位点进行了筛选，鉴定到了954个重要的候选SNP位点，其中有3个改变了转录因子Motif结合位点， GWAS分析发现与TCF12基因连锁的SNP位点与猪饲料效率性状存在关联。本研究系统揭示了TCF12调控骨骼肌发育的分子机理，研究结果可为哺乳动物骨骼肌发育、再生研究提供重要参考，鉴定的SNP位点也可为猪育种提供重要分子标记；另外，本研究从基因组、表观组、转录组、个体等多个层次解析基因功能策略，也可为其他基因功能研究提供参考。