TET2在正常红系发育及骨髓增生异常综合症红系异常发育中的作用及机制研究

Erythropoiesis is a process by which multipotent hematopoietic stem cells proliferate, differentiate and eventually form mature erythrocytes. It is a complex process that requires tight regulation. Although it is well established that DNA methylation/demethylation influences many biologic processes, its role in erythropoiesis remains largely unexplored. We and others have recently documented that global demethylation occurs during human erythropoiesis. DNA demethylation is mediated by Ten-eleven-translocation (TET) family members. TET2 is expressed in erythroid cells. Mutation of TET2 is the most common mutation in MDS patients. MDS patients have abnormal erythropoiesis. To explore the role of TET2 in DNA demethylation and human erythropoiesis, we knocked down TET2 by shRNA in human CD34+ cells and found that TET2 knockdown resulted in hyper-proliferation of erythroid cells, delayed erythroid differentiation, increased apoptosis of late stage erythroblasts. Interestingly, these changes are characteristics of altered erythropoiesis in myelodysplasticsyndroms (MDS) patients. Based on these findings, we hypothesize that TET2 plays important roles during normal erythropoiesis and that mutation of TET2 in MDS is associated with altered erythropoiesis in MDS. In this application, we will explore the mechanisms by which TET2 regulates erythropoiesis by examining the effects of TET2 knockdown or TET2 mutation on genome wide gene expression and DNA methylation/demethylation. Successful accomplishment of proposed studies will not only provide novel insights into erythroid biology but also help in developing mechanistic understanding of disordered erythropoiesis in MDS.

红系发育是由多潜能造血干细胞增殖、分化成红细胞的过程，需要精细复杂调控。DNA甲基化/去甲基化调控很多生理过程，但它在红系发育过程中的作用却少有研究。本课题组前期发现在人类红系发育过程中出现全基因组去甲基化。DNA去甲基化是由TET家族调控。TET家族成员TET2在红系细胞中表达。骨髓增生异常综合症（MDS）患者红系发育异常，TET2突变是MDS患者中最常见的突变。我们在前期工作中对TET2在红系发育中的作用进行了研究，发现敲低人类CD34+细胞中的TET2导致红系细胞的过度增殖、分化延迟及分化晚期凋亡增加。而这些表型变化与MDS患者红系发育异常类似。由此我们推测，TET2对正常红系发育起着重要的调控作用，TET2突变与MDS中异常红系发育有关。本项目将深入探讨TET2对红系发育的调控机制。该研究不仅为红系研究提供新视野，还有助于阐明MDS患者红系发育异常机制，为 MDS的诊疗提供新策略。

红系发育是指造血干细胞历经多阶段增殖分化最终形成成熟红细胞的复杂调控过程，任何阶段的发育障碍均会导致红细胞异常性疾病的发生。DNA去甲基化酶TET2和TET3在红系发育进程中高表达，它们的主要功能是将5’甲基胞嘧啶（5-mC）转变为5’羟甲基胞嘧啶（5-hmC），进而引起基因组DNA的甲基化水平的下降，但其在红系发育中功能及分子机制尚未可知。.本项目利用体外研究体系，在造血干细胞定向红系发育的过程中分别敲低TET2和TET3的表达，发现TET2和TET3分别特异性地在红系发育的早期和晚期发挥重要调控作用。TET2敲低特异性地引起SCF依赖性的异常Marker-CFU-E的产生，该群细胞c-Kit信号减弱，但ERK和AKT信号增强。进一步研究发现Marker-CFU-E的基因表达模式和总体甲基化水平发生显著改变。Marker-CFU-E中促癌基因AXL的表达显著上调，并且TET2敲低CFU-E中存在约20%高表达AXL具有克隆性增殖能力的细胞。与此同时，TET2突变的MDS患者骨髓单个核细胞中也高表达AXL。综上所述，TET2敲低导致由c-Kit和AXL介导的红系祖细胞克隆性增殖现象。TET3敲低则特异性地引起晚期红系前体细胞发生双核或多核现象及脱核率下降，且与有丝分裂相关基因KLHDC8B表达下调密切相关。Tet3条件性敲除小鼠在应激条件下也出现红系发育障碍。.此外，我们还发现剪切因子SF3B1敲低通过MKRN1-P53-P21信号通路调控红系祖细胞的凋亡；揭示Tmem30a可以通过调节脂筏的形成影响EPOR信号通路的激活调控红系早期阶段发育，而USP7则能通过稳定GATA1在红系发育终末阶段发挥重要作用。不仅如此，我们首次发现EPOR的表达是哺乳动物红系造血岛巨噬细胞（EBI）的共同特征。.本项目的完成为完善复杂的红系分子调控网络提供了大量的数据支撑，并且TET2和SF3B1研究工作的完成为探索MDS的发病机制提供全新的思路和科学依据，而骨髓微环境中EBI分离和鉴定方法的建立为更好地研究EBI和红系发育提供了方法学和数据支撑。