MyoD介导SKIP的染色质重塑激活控制猪骨骼肌卫星细胞成肌分化的机制研究

In domestic animals, a change in the number of fibers that form during myogenesis could have a profound effect on the total muscle mass of the adult animal and long-term growth potential and meat quality of the animal. According to research, the MyoD-dependent chromatin remodeling activation of muscle-specific gene controlled by PI3K/Akt and p38 signaling pathway is an important way to control myogenesis. Based on our previous studies, SKIP was a candidate gene for muscle fiber number, and its up-regulation in myogenesis is dependent on MyoD binding site in the 5′-flanking region of its own. As we known, SKIP is a negative regulator of PI3K/Akt signaling pathway. Consequently, we speculate that MyoD mediates the chromatin remodeling of SKIP gene to increase its latter expression, which in turn negative regulates the myogenesis process. Taken together, CHIP and MeDIP technologies will be employed to identify the chromatin conformation at SKIP 5’-flanking region and the underling mechanism of MyoD-dependent SKIP transcriptional activation at the onset of the differentiation program. Additionally, RNAi and overexpression methods will be used to illuminate the regulatory mechanism by SKIP in myogenesis.This study will have important implications for elucidating the regulatory mechanism and function of SKIP expression, constructing the regulation network of MyoD, and revealing the molecular mechanism of muscle fiber number.

骨骼肌纤维的数目由出生前肌源细胞的增殖程度和成肌分化进度所决定，直接影响家畜的生长潜能和肉质。研究发现，由PI3K/Akt和p38信号介导、MyoD依赖的肌肉特异性基因的染色质重塑激活是控制成肌分化的重要方式。申请者前期工作表明，影响肌纤维数目的候选基因SKIP在成肌分化时的上调表达依赖于5′调控区MyoD的结合元件。而SKIP同时又负调控PI3K/Akt信号。我们推测，MyoD通过介导SKIP的染色质重塑上调其表达，从而对自身的促分化机制进行负控制。本研究利用CHIP和MeDIP技术鉴定SKIP基因在成肌分化前后的染色质开放状态，并通过候选法解析MyoD转录激活SKIP的模式，再运用RNA干扰和过表达技术探讨SKIP对细胞增殖和成肌分化的控制机制。本项目对于阐明SKIP基因在成肌分化时的表达调控机制和功能，构建MyoD控制成肌分化的自调控网络，揭示猪肌纤维数目的控制机理均具有重要意义。

骨骼肌纤维的数目由出生前肌源细胞的增殖程度和成肌分化进度所决定，直接影响家畜的生长潜能和肉质。研究发现，由PI3K/Akt和p38信号介导、MyoD依赖的肌肉特异性基因的染色质重塑激活是控制成肌分化的重要方式。申请者前期工作表明，影响肌纤维数目的候选基因SKIP在成肌分化时的上调表达依赖于5′调控区MyoD的结合元件。而SKIP同时又负调控PI3K/Akt信号。我们推测，MyoD通过介导SKIP的染色质重塑上调其表达，从而对自身的促分化机制进行负控制。本研究通过亚硫酸氢盐法以及CHIP、ATAC-seq技术确定了成肌分化前后SKIP的染色质开放状态、甲基化状态及转录因子结合情况：发现SKIP染色质在成肌分化过程中由关闭和抑制状态转变为开放状态，甲基化分布和状态发生改变，H3K27me3在SKIP启动子上的结合减少，MyoD在SKIP启动子上的结合增加。通过候选法和CHIP技术解析了成肌分化过程中转录因子MyoD转录激活SKIP的模式：即SKIP的转录激活是由MyoD通过与MEF2、p300、E47、BAF60等形成蛋白复合体共同介导。通过信号通路抑制剂的运用明确了SKIP的转录调控通路：发现SKIP的转录表达受PI3K-AKT和p38信号共同调控，且PI3K/Akt信号是通过调控转录因子MyoD与SKIP启动子的结合影响成肌分化过程中SKIP基因的转录。最后通过超表达SKIP阐明了其在成肌分化进度中的作用以及其对成肌分化的控制机制：免疫荧光分析发现SKIP 控制着肌动蛋白应激纤维的重排；细胞周期检测和分化标志蛋白MHC的间接免疫荧光分析表明，超表达SKIP不能重建p38信号缺失导致的分化受阻；SKIP的过表达使分化72 h的 MHC和分化48 h 的Myogenin表达量显著降低，使分化48 h及72 h的AKT磷酸化程度显著降低，并抑制了分化定向时MyoD与p300的给合，说明SKIP的过表达抑制了成肌分化以及MyoD介导的染色质重塑复合体的形成，并且有可能通过PI3K/Akt信号通路来影响这一过程。通过本研究，我们构建了MyoD控制成肌分化的自调控网络，明确了MyoD在分化晚期通过上调表达SKIP负反馈PI3K/Akt信号，从而负调控成肌分化。本项目对于阐明SKIP基因在成肌分化时的表达调控机制和功能，构建MyoD控制成肌分化的自调控网络具有重要意义。