CBP介导RNA-染色质互作调控猪骨骼肌能量代谢的分子机理研究

Skeletal muscle energy metabolism is the key factor determines the feed efficiency and meat quality in pig. We discovered CBP (CREB binding protein) gene was located in the QTL responsible for feed efficiency and up-regulated in high feed-efficiency populations. Our experimental data suggest that CBP negatively regulated the energy metabolism in porcine muscle satellite cells. Therefore , CBP is closely related to feed efficiency in pigs. Previous studies have shown that CBP is an RNA-binding protein and the CBP-binding RNAs could interact with chromatin to regulate target gene expression. Based on these studies, we hypothesis that CBP could control the energy metabolism regulatory regions through RNA-mediated CBP-RNA-chromatin interaction, thereby affecting pig feed efficiency. In this study, we will first identify the CBP-bound RNAs in porcine skeletal muscle satellite cells by GOLD-CLIP technique. Then uncover the CBP induced RNA-chromatin interactions by GRID-seq method. Finally, we will identify the CBP-RNA-chromatin interactions controlling the energy metabolism through integration analysis to unravel the regulatory mechanism of CBP on porcine skeletal muscle energy metabolism in vivo. This work will not only help us to understand the regulation of CBP on porcine skeletal muscle metabolism, but also provide potential genetic markers and candidate gene for feed efficiency improvement in pigs.

研究证实骨骼肌能量代谢是影响猪饲料效率性状的重要因素。申请人发现CBP基因位于饲料效率QTL位点，在饲料效率好的猪中上调表达，且负调控猪骨骼肌卫星细胞能量代谢。这些研究表明CBP基因与饲料效率密切相关。功能研究表明CBP是一个重要的RNA互作蛋白，与CBP结合的RNA能够与染色质互作。据此我们推测：CBP很可能是通过RNA介导识别能量代谢基因调控区域，通过形成CBP-RNA-染色质复合物，调控猪骨骼肌能量代谢基因表达及其能量代谢，进而影响猪饲料效率。为此，本项目拟在猪骨骼肌卫星细胞中，通过GOLD-CLIP技术鉴定CBP结合的RNA；再通过GRID-seq技术鉴定RNA-DNA互作；最后通过整合分析鉴定CBP-RNA-染色质互作关系及其调控的能量代谢基因。研究结果将揭示CBP调控骨骼肌能量代谢的分子机理，为深入理解猪骨骼肌能量代谢提供重要理论依据，也为猪遗传改良提供新的候选基因及分子靶点。

猪骨骼肌能量代谢是影响猪饲料效率的关键因素，CBP调控猪骨骼肌能量代谢及成肌分化。功能研究表明CBP是一个重要的RNA互作蛋白，与CBP结合的RNA能够与染色质互作调控基因转录。但是，CBP蛋白结合哪些RNA参与调控猪骨骼肌卫星能量代谢及成肌分化及其分子机理不甚清楚。鉴于此，本项目对抑制CBP蛋白表达的增殖期和分化期猪骨骼肌卫星细胞进行ATAC-seq和RNA-seq测序，通过多组学整合分析鉴定到CBP通过调控线粒体生成信号通路，影响猪骨骼肌卫星能量代谢。接下来，我们对于CBP介导哪些RNA调控能量代谢及成肌分化进行深入研究。我们利用GRID-seq技术鉴定到在成肌分化过程中，caRNA的相互作用明显增强，并发现了CBP可能介导非编码RNA Malat1改变染色质三维结构，从而调控成肌分化。此外，对猪骨骼肌的GRID-seq和BL-HiChIP等多维组学数据整合分析，发现CBP可能结合MNBL1 RNA，靶向ATP合成信号通路，进而影响骨骼肌能量代谢。以上成果较系统的阐明了CBP调控猪骨骼肌能量代谢及成肌分化的分子机理，确定了CBP是影响猪影响猪饲料效率的关键候选基因。在本项目的资助下发表SCI(IF>5)论文1篇，申请专利3项。