DNA主动去甲基化过程是近年来DNA甲基化研究的热点领域之一, 该过程的发生依赖于TET-TDG-BER轴紧密有序的协同作用, 但这一过程的调控过程尚未阐明。本研究[1]揭示了UHRF2蛋白在DNA去甲基化过程中发挥的关键调控作用。

之前的研究表明, 虽然UHRF2是UHRF1在哺乳细胞中唯一的同源蛋白, 且它们具有相似的结构域组成, 但UHRF2并不能代偿UHRF1在DNA甲基化维持过程中的作用。晶体结构解析发现它们的SRA结构域对DNA胞嘧啶不同修饰的识别特异性有所差别, UHRF1倾向于结合半甲基化修饰的DNA, 而UHRF2则识别DNA的5-羟甲基化修饰 (5hmC)。

本课题组前期鉴定了UHRF2的相互作用蛋白XRCC1——碱基切除修复复合体 (BER) 的核心组分, 并发现在5hmC存在下, UHRF2能够泛素化XRCC1。在此基础上, 本研究证明UHRF2对XRCC1的泛素E3连接酶活性依赖于其SRA结构域对5hmC的识别。在5hmC激活下, UHRF2催化XRCC1发生K33链类型的多聚泛素化, 这一泛素化修饰并不介导XRCC1的降解。紧接着, 本研究发现在泛素结合蛋白RAD23B的协助下, K33链类型泛素化修饰的XRCC1能够招募TDG, 继而形成TDG-RAD23B-BER复合体。本研究证明了这一由UHRF2介导的TDG-RAD23B-BER复合体的组装对于DNA甲基化的有效去除是必须的。通过整合小鼠胚胎干细胞通量测序的分析结果, 结合基因组整体水平和位点特异的DNA甲基化分析, 我们发现, Uhrf2介导的TDG-RAD23B-BER复合体对DNA去甲基化的调控能够协助完成小鼠胚胎干细胞启动子区域DNA甲基化的去除, 而在神经分化过程中, 这一调控作用则能促进神经谱系相关基因增强子区域DNA甲基化的快速去除, 进而激活谱系相关基因的表达, 决定细胞命运。在神经分化过程中, 缺失Uhrf2会阻碍神经谱系基因增强子区域DNA甲基化的去除, 最终导致神经分化不能正常进行。

概言之, 本研究发现, 在主动去甲基化过程中, UHRF2识别5hmC, 通过其泛素E3连接酶活性调控DNA去甲基化过程的发生。本研究为UHRF2生物学功能的认识提供了新的视角, 为DNA甲基化动态调控提供了新的理论模型, 为DNA去甲基化过程中的表观遗传学动态调控研究提供了新的切入点。

 

[1] 本研究得到国家自然科学基金 (编号：31991164；81730079) 资助。

The transition of oxidized 5-methylcytosine (5mC) intermediates into the base excision repair (BER) pipeline to complete DNA demethylation remains enigmatic. Previous studies have shown that UHRF2, the only paralogue of UHRF1 in mammals who possesses similar domain composition to UHRF1, is neither able to maintain 5mC nor to rescue Uhrf1-/- phenotype. We previously identified the interaction between UHRF2 and XRCC1, the core component of the BER, and found that UHRF2 can ubiquitinate XRCC1 in the presence of 5hmC. Herein, we demonstrate that the E3 ligase activity of UHRF2 depends on the 5hmC recognition by the SRA domain, and that UHRF2 catalyzes the K33-linked polyubiquitination of XRCC1. This nonproteolytic action stimulates the interaction of XRCC1 with the ubiquitin binding domain-bearing RAD23B, leading to the incorporation of TDG into the BER complex. Integrative epigenomic analysis in mouse embryonic stem cells reveals that the Uhrf2-commissioned TDG-RAD23B-BER complex was functionally linked to the DNA demethylation at active promoters, and that during neuronal commitment, Uhrf2-associated and H3K4me1-marked latent enhancers undergo poised-to-active transition, accompanied by complete DNA demethylation. Accordingly, Uhrf2 ablation impedes DNA demethylation and abolishes neuronal differentiation. Together, these observations highlight an essentiality of 5hmC-switched UHRF2 E3 ligase activity in commissioning the accomplishment of active DNA demethylation.

缩略词表 1
第一章 引言 3
第二章 综述 5
2.1 DNA甲基化的去除 5
2.1.1 DNA甲基化修饰概述 5
2.1.2 DNA主动去甲基化 6
2.2 UHRF2蛋白研究概述 9
2.2.1 UHRF蛋白家族 9
2.2.2 UHRF2蛋白研究概述 11
2.3 K33链类型泛素化修饰 13
2.3.1 蛋白质非经典泛素化修饰概述 14
2.3.2 K33链类型泛素化研究进展 15
2.3.3 “泛素密码”研究进展 16
第三章 材料与方法 19
3.1 实验材料 19
3.1.1 质粒 19
3.1.2 菌株 19
3.1.3 细胞系 19
3.1.4 试剂 19
3.1.5 主要仪器 25
3.2 实验方法 26
3.2.1 人HEK-293T细胞培养 26
3.2.2 小鼠胚胎干细胞培养及其神经分化 27
3.2.3 质粒的转化和提取 27
3.2.4 质粒转染 29
3.2.5 RNA干扰 30
3.2.6 细胞总RNA提取及反转录PCR、实时定量PCR 30
3.2.7 Western免疫印迹实验 (Western blot) 32
3.2.8 真核细胞表达的蛋白纯化 33
3.2.9 原核细胞表达的蛋白纯化 33
3.2.10 免疫共沉淀 (Co-IP) 34
3.2.11 免疫亲和纯化联合质谱分析 35
3.2.12 体内泛素化检测 35
3.2.13 体外泛素化检测 36
3.2.14 染色质组分分离实验 36
3.2.15 基因组DNA提取 36
3.2.16 斑点印记试验 (Dot blot) 37
3.2.17 液相色谱质谱联用实验 (LC-MS/MS) 37
3.2.18 逆转录病毒和慢病毒的制备 38
3.2.19 染色质免疫共沉淀 (ChIP) 39
3.2.20 重亚硫酸盐克隆测序 (BSP) 40
3.2.21 甲基化DNA免疫沉淀 (MeDIP) 41
3.2.22 统计学分析 42
第四章 结果 43
4.1 UHRF2泛素化XRCC1依赖其SRA结构域及RING结构域 43
4.2 UHRF2促进XRCC1发生K33链类型多聚泛素化 45
4.3 K33链类型多聚泛素化促进TDG-RAD23B-BER复合体组装 49
4.4 UHRF2介导TDG-RAD23B-BER复合体调控DNA主动去甲基化 54
4.5 Uhrf2的缺失导致小鼠胚胎干细胞DNA甲基化水平升高 56
4.6 Uhrf2促进小鼠胚胎干细胞启动子区域DNA甲基化的去除 58
4.7 Uhrf2促进神经分化过程中静态增强子区域DNA甲基化的去除 61
第五章 讨论 64
5.1 围绕UHRF2及其功能的延伸讨论 65
5.2 围绕BER复合体的延伸讨论 66
第六章 结论 69
附录 70
参考文献 78
学位论文答辩委员会名单 93
个人简历、在学期间发表的学术论文与研究成果 94
致谢 96
北京大学学位论文原创性声明和使用授权说明 98

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