超级增强子及其关联主转录因子影响胚胎干细胞分化倾向性的研究

The development of robust and stable in vitro differentiation protocols has been hindered by the functional variability of pluripotent stem cells, which further has an impact on stem cells application in translational medicine research. Several studies have recently reported that master transcription factors (TFs) play pivotal roles in cells fate decision, and the concept of super-enhancers (SEs), which can drive expression of genes that control and define cell identity, has been defined by the master TFs binding signal density. Although master TFs remain unknown for a large variety of cell types, H3K27ac modification of chromatin histones could be used as a surrogate mark to identify SEs. In this study, we will utilize both spontaneous and direction differentiation methods, into ectoderm, mesoderm and endoderm lineages, to assess early differentiation capacity differences among 6 human embryonic stem cell lines. To explore the relationship between SE-associated TF genes and embryonic stem cell propensity, cells at day1, 3, 5 differentiation stages will be taken for both chromatin immunoprecipitation sequencing(ChIP-seq) and RNA-seq analysis to investigate H3K27ac modification and gene expression changes in a time-wise and cell-wise manner. SEs for each hESC line and three germ layers will be identified through H3K27ac-enriched region analysis and SEs-associated genes will be selected as mater TF candidates through gene function bioinformatics annotation. Then we will verify master TF candidate function via CRISPR/Cas9 system which has been demonstrated as an easy-handle, highly specific and efficient approach for eukaryotic genome engineering. Finally we aim to build a stable and efficient platform that could facilitate master TF identification in various cell types.

多能干细胞分化倾向性的差异阻碍高效稳定体外分化系统的建立，影响干细胞在转化医学研究中的应用。近期研究表明主转录因子在细胞命运决定中起重要作用，依据其在染色质上的结合位点密度定义了超级增强子概念，超级增强子驱动能定义细胞身份相关基因的表达。目前多数细胞类型的主转录因子未知，可用H3K27ac表观修饰作为定义超级增强子的替代物。本项目我们将结合自主和定向分化确定不同胚胎干细胞系向外、中和内三胚层分化效率的差异，利用染色质免疫共沉淀测序技术和RNA-seq研究分化过程中H3K27ac表观修饰和基因表达的变化，分析各胚胎及胚层细胞系特异的超级增强子及关联基因，阐明备选主转录因子和胚胎干细胞定向分化差异的相关性；同时对备选主转录因子利用CRISPR-Cas9体系进行功能分析，逆向寻找决定三胚层状态的主转录因子，建立高效稳定的主转录因子功能分析平台，为其他细胞类型主转录因子发现和功能分析奠定好的基础。

多能干细胞分化倾向性的差异阻碍高效稳定体外分化系统的建立，影响干细胞在转化医学研究中的应用。近期研究表明主转录因子在细胞命运决定中起重要作用，依据其在染色质上的结合位点密度定义了超级增强子概念，超级增强子驱动能定义细胞身份相关基因的表达。目前多数细胞类型的主转录因子未知，可用H3K27ac表观修饰作为定义超级增强子的替代物。本项目：（1）我们分析了早期外胚层、中胚层和内胚层细胞H3K27ac表观修饰（ChIP-seq），分别获得了41, 49 和 55个自调控转录因子，这些自调控转录因子是各胚层主转录因子重要的候选基因；（2）我们分析了不同胚胎干细胞转录组水平（RNA-seq）上的差异，结果表明基因表达差异和分化倾向性相关联,并且三胚层超级增强子关联的转录因子，尤其是自调控转录因子在不同胚胎干细胞间显示了更为明显的差异表达;（3）进一步利用神经祖细胞和心肌细胞体外定向分化技术探讨了分化倾向性的差异对定向分化的影响，结果表明分化倾向性差异会显著影响体外定向分化的效率或靶标细胞的产率。因而，本项目的研究从表达水平上解释了引起不同多能干细胞分化倾向性的分子机理，同时针对特定靶标细胞在体外诱导备选多能干细胞系的筛选进而进行体外定向分化体系的建立具有很好的指导意义。