超级增强子在C2C12成肌细胞分化中的动态变化研究

Super-enhancers are newly defined transcriptional regulatory elements, with unusually strong enrichment of transcription factors, cofactors, and histone modifications, play a vital role in cell identity-determined process. During myoblast differentiation, identification of super-enhancers and their roles in the molecular regulatory process of cell identity-determined gene are still poorly studied. Here, we first identify genome-wide super-enhancers by performing ChIP-seq for histone modifications H3K4me1 and H3K27ac, using 0, 4, and 8 days cells during C2C12 myoblast differentiation. We then dissect constitutes of super-enhancers by employing single-nucleotide resolution ChIP-exo to profile H3K4me1 and H3K27ac. Furthermore, we test biological effects of varying ChIP-exo signal regions within super-enhancers on activating or enhancing gene expression using both dual-luciferase reporter system and CRISPR-Cas9 genome editing. These results would be helpful for identifying genome-wide super-enhancers and dissecting constitutes of super-enhancers at key time points during myoblast differentiation, as well as elucidating molecular mechanism of super-enhancers in the regulation of cell identity-determined genes during myoblast differentiation.

超级增强子是新近发现的一类重要转录调控元件，富集着大量转录因子、辅因子以及组蛋白修饰，对细胞身份决定有重要影响。在成肌细胞分化过程中，有关超级增强子鉴定及其在细胞身份决定基因的分子调控过程中具体作用的研究还相对缺乏。本研究以小鼠成肌细胞（C2C12）分化过程中三个关键时间节点0、4、8天为研究对象，首先利用组蛋白H3K4me1和H3K27ac ChIP-seq技术鉴定三个关键节点C2C12成肌细胞全基因组内超级增强子分布；再结合单核苷酸分辨率的组蛋白ChIP-exo技术解析超级增强子的组成；最后，利用双荧光素酶报告系统和CRISPR/Cas9基因组编辑技术检测超级增强子中不同ChIP-exo信号区域对激活/增强基因表达过程的生物学效应。本研究结果将有助于揭示超级增强子在成肌细胞分化关键时间节点分布及组成，阐明超级增强子在成肌细胞分化中对细胞身份决定基因的转录调控机理。