少突胶质细胞早期发育的表观遗传调控—TET1非氧合酶活性依赖的作用机制研究

Demyelination in the central nervous system (CNS) is associated with many demyelinating disorders and nervous system injury. Oligodendrocyte (OL) is the only myelinating cell in the CNS and the failure of OL remyelination impairs rapid propagation of action potentials and leads to nerve degeneration. To promote remyelination, it is important to understand the mechanism for OL differentiation. Epigenetic modification has been identified as an important player in the regulation of OL differentiation. Recently, we have identified the expression of TET dioxygenase family members during OL differentiation, in which three members show different pattern and TET1 is highly expressed in oligodendrocyte precursor cells (OPCs) and early differentiated immature OL. In TET1 conditional knockout mice (TET1 loxP/loxP; Olig1-cre, TET1 CKO), which loss TET1 function in OL lineage, the specification and early differentiation of OL are repressed. But, as the product of TET dioxygenase, 5-hydroxymethylcytosine (5-hmc) level starts to rise from immature OLs and therefore is thought to be involved in later maturation of OL. These observations suggest that the stage-specific appearance of TET1 might regulate certain target genes expression through 5-hmc independent ways. With the facility of TET1CKO mice, we plan to map the target genes of TET1 in OLs and study the chromatin regulator complexes that TET1 might involve in, especially TET1/Ezh2, TET1/Sin3A/HDAC and TET1/OGT/HCF1/SET/COMPASS. After confirming the regulatory role of these complexes in the expression of TET1 target genes, we will overexpress or knockdown target genes in TET1CKO embryo or OL cultures to test the rescue effect in OL lineage formation and early differentiation. This study reveals a new epigenetic category in understanding OL differentiation and might be applied in further study on promoting remyelination in CNS related diseases.

中枢神经系统(CNS)脱髓鞘是多种神经系统疾病的重要病理特征，研究CNS髓鞘形成细胞—少突胶质细胞(OL)的分化成熟机制对促进再髓鞘化非常重要。近年来表观遗传调控在OL发育中的作用逐渐受到重视。我们前期观察到，一种氧合酶家族分子—TET1在少突前体和未成熟细胞中高表达，且在OL中条件性敲除该分子(TET1CKO)将影响细胞谱系形成和早期分化。但作为TET的催化产物—DNA羟甲基胞嘧啶(5-hmc)，在少突前体细胞中修饰水平较低。因此我们推测TET1可能通过非5-hmc修饰依赖的方式调控相关靶基因的表达并促进OL谱系形成。我们计划利用TET1CKO小鼠，从TET1在OL基因组中结合的靶基因入手，探讨三种TET1可能参与的转录调控复合物对相应靶基因的调控作用，并验证靶基因对OL分化的影响。本研究将为全面认识少突分化的调控机制提供崭新的思路，并为促进髓鞘缺失疾病的成功再髓鞘化提供重要的实验依据。

中枢神经系统(CNS)髓鞘损伤是多种神经系统疾病的重要病理变化之一，脱髓鞘通常伴有运动，认知，焦虑抑郁等多种脑功能异常。研究CNS髓鞘形成细胞的分化成熟以及再髓鞘化机制对于相关疾病治疗有着重要意义。.本项目深入研究了DNA羟甲基化（5hmC）修饰酶TET1蛋白在少突胶质细胞髓鞘化过程中的作用和机制。首先利用不同分化阶段少突胶质细胞纯化培养物，我们对TET1结合的靶蛋白和靶基因进行了分析，免疫沉淀、蛋白质谱结合免疫印迹发现了HDAC1、Olig2和OGT等调控少突胶质细胞分化的转录因子均可以与少突TET1结合；染色质沉淀结合高通量测序发现TET1随少突胶质细胞分化可以与不同靶基因结合。并且同基因表达谱的组学串联分析后，GSEA分析（GO聚类分析）发现与髓鞘发育、细胞周期调控、钙离子转运等相关的基因均是TET1的靶分子。我们利用钙成像技术对少突胶质细胞的钙活性进行分析发现，Tet1cKO小鼠来源的少突胶质细胞中钙活动降低。进一步研究TET1的靶分子Itpr2，一种定位于内质网的IP3依赖钙通道，发现，体外敲减Itpr2的表达将抑制少突胶质细胞分化的水平，Itpr2的条件性基因敲除小鼠髓鞘发育受阻。以上结果提示，TET1可分别通过影响少突前体细胞的细胞周期和钙活动变化调控细胞分化进程。.更有意思的是，Tet1条件性敲除小鼠出现一系列异常行为表型，包括焦虑水平降低、空间记忆力变差、感觉门控功能降低等精神分裂症样的行为学表型。.以上结果不但揭示了TET1调控少突胶质细胞分化的详细机制，更为为我们进一步深入探究TET1介导的表观遗传机制在少突胶质细胞和神经元互作中如何发挥的功能，以及少突胶质细胞如何参与神经系统疾病中神经环路的正常功能发挥奠定了重要的基础。