转录因子Mesp1结合组蛋白甲基转移酶Ezh2调控人类多能干细胞心血管定向分化的分子机制

The transcription factor Mesp1 is a master regulator for cardiovascular cell-fate determination of pluripotent stem cells. Mesp1 not only directly activates many genes related to the core cardiac transcriptional machinery, but also directly represses expression of other genes promoting early mesoderm and endoderm cell-fate specification. Currently the precise mechanism of how Mesp1 regulates gene expression remains unclear. To further clarify the mechanism, we searched for the interacting proteins of Mesp1 by yeast two-hybrid assays and identified for the first time that Mesp1 interacted directly with the histone methyltransferase Ezh2. Ezh2 is the catalytic unit of PRC2 (polycomb repressive complex 2) which catalyzes the epigenetic "silencing" signal of the trimethylation of histone H3 at lysine 27 (H3K27me3). In this study, we propose that Mesp1 interacts with Ezh2 and modulates its chromosomal distribution and/or enzyme activities to regulate key genes expression, and eventually stimulate cardiovascular lineage commitment of pluripotent stem cells. We will test our hypothesis and further dissect the molecular roles of Ezh2 in cardiovascular specification, as well as how Mesp1 regulates chromosomal distribution and enzyme activities of Ezh2. Understanding the molecular mechanisms by which Mesp1 and Ezh2 act in cardiovascular specification will help us improve the production of cardiovascular cells from human pluripotent stem cells for regenerative therapy.

转录因子Mesp1是调控多能干细胞向心血管细胞定向分化的核心分子，它既能直接激活多个心血管发育核心转录因子的表达，也能直接抑制多个促进早期中胚层、内胚层发育的基因表达。为了明确Mesp1调控基因表达的具体机制，申请人通过酵母双杂交实验筛选与Mesp1相互作用的蛋白，首次发现Mesp1与组蛋白甲基转移酶Ezh2直接结合。Ezh2是PRC2复合体的催化亚基，能够催化组蛋白H3第27位赖氨酸的三甲基化修饰，从而抑制基因表达。本项目中申请人提出假说：Mesp1结合Ezh2，通过影响Ezh2的染色质分布和（或）酶活性来调控相关基因表达，从而促进多能干细胞心血管定向分化。我们将验证以上假设，深入探讨Ezh2在多能干细胞心血管定向分化中的作用以及Mesp1对Ezh2分布和酶活性的调控，从而阐明Mesp1结合Ezh2调控人类多能干细胞心血管定向分化的分子机制，促进人类多能干细胞心血管定向分化效率的提高。

转录因子Mesp1是调控多能干细胞向心肌等心血管细胞定向分化的核心分子，它既能直接激活多个心肌发育核心转录因子的表达，也能直接抑制多个促进早期中胚层、内胚层发育的基因表达。为了明确Mesp1调控基因表达的具体机制，申请人同时敲除Mesp1及其同源基因Mesp2，发现Mesp1/2的同时敲除会导致分化过程中，中胚层非心肌谱系基因表达的升高，促进非心肌细胞的分化。申请人通过酵母双杂交实验筛选与Mesp1相互作用的蛋白，首次发现Mesp1与组蛋白甲基转移酶Ezh2直接结合。Ezh2是PRC2复合体的催化亚基，能够催化组蛋白H3第27位赖氨酸的三甲基化修饰，从而抑制基因表达。染色质免疫沉淀-测序研究发现，在心血管定向分化的各个时期，Ezh2结合在多个中胚层基因处，并在非心肌谱系基因处结合增多从而抑制基因表达；而Ezh2敲除细胞系与Mesp1/2的同时敲除表型相似，会导致中胚层非心肌谱系基因表达的去抑制，影响心肌的正常分化。以上表明Mesp1与Ezh2通过抑制同一组非心肌基因的表达在心肌分化中发挥重要作用。目前，我们还在通过染色质免疫沉淀检测Mesp1是否与Ezh2共定位以及Mesp1/2的同时敲除对Ezh2染色质分布的影响，从而阐明Mesp1结合Ezh2调控人类多能干细胞心血管定向分化的分子机制，增进对于Ezh2和Mesp1在心脏发育和心脏疾病中作用的认识，并促进人类多能干细胞心血管定向分化效率的提高。