TET2通过介导染色质状态转变调控红系发育的作用及机制研究

Dynamic DNA methylation/demethylation play critical roles in the regulation of erythropoiesis by maintaining the precise spatial and temporal control of gene expression. TET2 has been well established to act as the key enzyme that mediate DNA demethylation, mutation of TET2 is the most common mutation in MDS patients. Abnormal erythropoiesis is one of the characters of MDS patients. However, the roles and mechanism of TET2 in the erythropoiesis remains largely unexplored. In our preliminary study, we found that knock down of TET2 resulted in markedly impairment in the cell function of distinct stage erythroid, which mimics the phenotype of MDS related abnormal erythropoiesis. However, the most striking finding is TET2 deficiency show no significant effect of global DNA demethylation. By conducting further exploration, we found that TET2 deficiency leads to the abnormal expression of various histone proteins at distinct stages, and resulted in significant change of chromatin opening state as well. Based on these findings, we propose for the first time that TET2 might play important stage-specific regulatory roles in erythropoiesis by modulating the dynamic chromatin states. In the present project, the in vitro culture system, samples from MDS patients with TET2 mutation and the TET2 knock off rat will be comprehensively utilized, by conducting multi-omics analysis, we propose to firstly construct an atlas of transcriptional, chromatin accessibility, and dynamic chromatin states in erythropoiesis. Based on this, we will further elucidate the regulatory roles and mechanism of TET2 in the dynamic chromatin state during erythropoiesis. The present project will provide novel insight for further clarifying the multi-mechanism cooperative regulation of erythropoiesis, and provide help for the precise diagnosis and therapy of MDS.

DNA甲基化/去甲基化的动态平衡是调控红系发育进程的关键机制之一。TET2是介导DNA去甲基化的关键酶，TET2突变是骨髓增生异常综合症（MDS）中最常见突变，红系发育障碍是MDS发病重要特征，但目前对TET2在MDS红系发育障碍中的作用机制了解极少。本课题前期研究发现，TET2表达缺失引起红系发育进程多个阶段细胞功能显著异常，但并未导致整体甲基化水平改变。进一步研究发现，TET2敲低后红系发育不同阶段组蛋白表达发生改变，并且导致染色质可及性态势异常。由此，我们首次提出TET2可能通过影响染色质状态转变在红系发育调控中起到阶段特异性的重要作用。本项目拟从在体和离体水平，通过多组学联合分析，在建立正常和TET2缺失条件下染色质状态动态图谱的基础上，阐明红系发育进程中TET2调控染色质状态转变的作用和机制。本项目将为多途径协同调控红系发育的机制研究提供新视野，并为MDS的精准诊疗提供帮助。

TET2作为DNA去甲基化酶中的重要成员，在调控红系祖细胞的发育分化中发挥重要作用，但其在红系终末分化中的作用尚不清楚。本项目利用体外诱导分化体系，利用RNA干扰技术敲低TET2的表达，发现TET2表达缺失延迟红系终末分化。此外，我们利用RNA-Seq、ATAC-seq联合分析发现TET2表达缺失导致红系终末分化阶段组蛋白修饰相关通路异常，且引起染色质可接近性改变。为进一步明确DNA甲基化和组蛋白修饰协同调控的作用和机制，我们利用敲低调控甲基供给关键酶AHCY的表达或抑制其活性的策略，观察全局甲基化受阻以及DNA甲基化或组蛋白甲基化分别受阻对红系分化发育的影响。研究AHCY功能受阻能够导致红系祖细胞分化延迟，且能导致红系前体细胞去分化。进一步的机制研究发现，以地西他滨处理抑制DNA甲基化虽能抑制红系祖细胞的分化发育，但并不影响红系终末分化和细胞去分化。随后我们对终末阶段的研究发现，AHCY失能能够导致关键组蛋白修饰分子，如H3K4me3、H3K9me3、H3K27Me3、H3K36Me2、H3K79Me2的丰度显著下调。而与此相对应，TET2敲低亦会引起红系终末分化阶段H3K4Me3水平显著下调。总之，本项目研究发现DNA去甲基化酶TET2调控红系终末分化是通过影响H3K4me3的表达，而不依赖于其调控DNA甲基化的功能；并且关键组蛋白修饰分子的异常可能影响红系发育终末阶段的细胞命运决定。本项目研究发现不仅有助于进一步揭示TET2缺失导致红系发育障碍的分子机制，还能够为阐释红系细胞的命运决定机制提供新的线索。