DNMT1调控人类红细胞发育的阶段特异性作用及分子机制研究

DNA methylation is one of the key epigenetic modifications. However, the roles of DNA methyltransferases (DNMTs), that are the critical enzymes that start and maintain DNA methylation, in the progress of directing differentiation from hematopoietic stem cells (HSC) to erythrocyte remain unclear. In our preliminary study, we found that the expression of DNMT1 significantly up-regulated in the early stage of erythropoiesis, and dramticaly decreased in the late stage; knock-down of DNMT1 in the HSC resulted in markedly impairment in the erythroid proliferation and differentiation. Based on these findings, we propose for the first time that DNMT1 might play important stage-specific regulatory roles in erythropoiesis. In the present project, based on the isolation protocol of human erythroblasts at distinct stages established by our group, we propose to isolate distinct erythroids at successive developmental stages that are diffrentiated from DNMT1 depleted HSC cells. The proliferation, apoptosis and differentiation of the erythroblasts at distinct stage will be investigated using flow cytometry approach to clarify the specific developmental stage that were affected by depletion of DNMT1. Combination of RNA-Seq and DNA methylation-Seq analysis will be performed to check distinct stage of erythroblasts to determine the key molecule that are involved in the modulation effect of DNMT1, and the effect of the key molecule on the progression of erythropoiesis will be further studied using gain and loss of function approaches. The present project will provide novel insight and evidence for further clarifying the regulatory mechanism of erythropoiesis.

DNA甲基化是重要的表观遗传修饰机制，DNA甲基转移酶是启动和维持DNA甲基化的关键酶，目前对于其在人类造血干细胞向红细胞发育中的作用和分子机制尚不明确。前期研究发现，在红细胞发育前期DNA甲基化转移酶1（DNMT1）表达明显上升，而在发育晚期显著下降；在造血干细胞中敲低DNMT1表达后，红系前体细胞数量显著降低，分化明显受阻。由此我们首次提出，DNMT1可能在红系发育进程中起到重要的阶段特异性调控作用。本项目拟利用已构建的红系发育各阶段细胞分离方法，在敲低造血干细胞DNMT1表达后，以流式细胞技术观察各阶段细胞增殖、凋亡和分化情况，以明确DNMT1 影响红系发育进程中的关键阶段及机制；通过DNA甲基化测序和转录组测序联合分析各阶段细胞，以明确DNMT1参与调控的关键分子，并通过基因功能获得和缺失实验证明关键分子对红系发育的影响。本项目将为深入阐明红细胞发育调控机制提供新的思路和依据。

DNA甲基化是重要的表观遗传修饰机制，DNMT1是介导DNA维持甲基化的关键酶，其在人红系发育进程中呈现阶段特异性的表达态势，但对于其在红系发育进程中的作用尚待阐明。本项目采用基于sh-RNA的方法在造血干细胞中敲低DNMT1表达并诱导向红系定向发育，利用分子生物学和细胞生物学的手段观察DNMT1缺失对人红系发育各阶段的影响。结果发现，DNMT1缺失导致红系发育全进程分化延迟、早期阶段细胞周期阻滞、终末阶段凋亡增加以及脱核率下降。为了探究DNMT1敲低后导致红系发育紊乱的机制，对正常和DNMT1敲低的CFU-E及晚幼红细胞进行转录组测序，目的为发现DNMT1缺失影响的关键生物学过程和重要的信号通路。利用生物信息学方法分析比对结合基因功能验证实验，发现在CFU-E阶段DNMT1敲低通过上调CDKN1A表达迟滞了细胞周期途径，而抑制CDKN1A活性可以逆转DNMT1敲低导致的细胞周期异常。此外，在晚幼红细胞中，敲低DNMT1导致RNA剪切相关的基因表达下调。进一步的机制研究证明，DNMT1敲低导致了SF3B1-MKRN1-p53-Caspase3信号轴异常并最终引起终末阶段细胞凋亡上升。以Caspase3抑制剂后可以完全逆转由DNMT1缺失引起的晚幼红细胞凋亡增加。本项目研究结果系统阐明了DNMT1在红系发育进程中的阶段特异性作用和机制，不仅为进一步阐明红系发育调控机制提供新的思路和依据，也能为去甲基化药物的开发和应用提供重要依据。