DNA甲基化和去甲基化在神经干细胞向星形胶质细胞分化过程中的作用机制

Neural stem/progenitor cells(NPCs) can differentiate into neurons and glia. The principle of NPC differentiation follows "neuron fist, glia second"role. Previous studies from our lab has revealed that a de nove DNA methyltransferase, Dnmt3a, is involved in epigenetic regulation of the sequential lineage differentiation of neurons and glia. It remains a challenge, however, to reveal detailed underlying molecular mechanisms by which DNA methylation and demethylation regulate astrogliogenesis in a coordinated manner. In this research project, we propose to study the function of Dnmt3a and Tet family members in regulating astroglial differentiation, via gain and lose function analyses, both in vitro and in vivo. In addition, using whole genome oppupancy analyses through chromatin immunoprecipitation coupled with next-generation seguencing (ChIP-seq) as well as MeDIP-seq and 5OHMeDIP-seq to reveal the genome-wide DNA methylation/demethylation network that ochestrates the gliogenesis process. Lastly, we will study a potential upstream regulator, olig2, for tet family proteins, which are tightly regulated during NPC differentiation along the astroglial lineage. This proposed study will reveal the coordination between the two opposing DNA　methylation/demethylation epigenetic forces in regulating cell fate specification of NPCs along the astroglial lineage.

神经干细胞具有分化为神经元和神经胶质细胞的潜能，其分化遵循神经元分化在先，神经胶质分化在后的原则。本课题组前期研究发现DNA从头（de novo）甲基化酶Dnmt3a参与调控这一分化过程。为了深入研究DNA甲基化和去甲基化对星形胶质细胞分化的影响，本项目拟采运用功能获得和缺失实验，在Dnmt3a基因敲除小鼠和ShRNA-tet转染的神经干细胞模型中，探究甲基化酶Dnmt3a与去甲基化酶Tet在神经干细胞向星形胶质细胞分化过程中的功能及分子机制；采用全基因组Dnmt3a和Tet占位分析以及MedIP-seq,50H-MedIP-seq和组蛋白修饰的ChIP-chip技术，揭示其在星形胶质细胞分化关键基因上的作用方式。最后，我们将研究上游潜在调控元件Olig2在神经干细胞向星形胶质细胞分化中对Tet的作用。本项目的实施将揭示神经干细胞分化过程中DNA甲基化和去甲基化酶协同调控的表观遗传机制。

神经干细胞具有分化为神经元和神经胶质细胞的潜能，在神经发育过程中其分化遵循神经元分化在先，神经胶质分化在后的原则。本课题组前期研究发现DNA de novo甲基化酶 Dnmt3a参与调控这一分化过程。为了深入研究 DNA 甲基化和去甲基化对星形胶质细胞分化的影响，本项目运用功能获得和缺失实验，在Dnmt3a基因敲除小鼠和ShRNA-tet 转染的神经干细胞模型中，研究了甲基化酶Dnmt3a与去甲基化酶 Tet 在神经干细胞向星形胶质细胞分化过程中的功能及分子机制；采用全基因组Dnmt3a 和Tet占位分析以及MedIP-seq,50H-MedIP-seq和组蛋白修饰的ChIP-chip技术,发现Dnmt3a与组蛋白甲基转移酶G9a互作，可抑制NPC向星形胶质细胞分化;并发现Olig2直接靶向Tet基因的启动子区调控其表达，Tet 2可降低星形胶质发生相关基因TSS区的甲基化水平，促进NPC向星形胶质细胞分化。本项目的完成解析了Dnmt3a甲基化抑制NPC向星形胶质细胞分化和Tet去甲基化促进NPC向星形胶质细胞分化的分子机制；并构建出NPC向星形胶质细胞分化过程中的DNA甲基化和去甲基化的表观遗传调控网络。