TET酶介导的DNA去甲基化在A-T神经元退行病变以及DNA损伤应答中的研究

	TET酶介导的DNA去甲基化在A-T神经元退行病变以及DNA损伤应答中的研究
其他题名	Deciphering the involvement of TET-mediated DNA demethylation in A-T Neurodegeneration and DNA Damage Response
	蒋德伟
学位类型	博士
导师	李家立
	2016-07
学位授予单位	中国科学院研究生院
学位授予地点	北京
关键词	共济失调-毛细血管扩张综合症 5-羟甲基化胞嘧啶 Tet蛋白 Dna损伤应答去甲基化
其他摘要	“共济失调-毛细血管扩张综合症（Ataxia-telangiectasia，A-T）”是一种由单基因Atm（A-T mutated）突变导致的神经退行性疾病。这种常染色体隐性遗传疾病主要影响小脑发育与功能完整，同时累及免疫、生殖和代谢等多个生理系统。目前的研究对A-T患者中枢神经系统病变的脑区特异性病理基础还知之甚少。关于A-T患者小脑神经元退行性病变的一个长期悬而未决的重要问题是：ATM功能缺失后，浦肯野神经元（Purkinje cells，PCs）产生易损性和退行性病变的原因和机理是什么？本研究发现了表观遗传学标记5-hydroxymehylcytocine（5hmC）在A-T患者和Atm-/-小鼠的小脑PCs中选择性地减少。通过全基因组水平的分析和比较，发现了正常人和A-T患者小脑中5hmC和5-methylcytocine（5mC）的分布差异主要集中于神经功能相关基因区域和重复序列等重要调控元件区域。结合GO分析和转录芯片验证，证实了这些表观遗传标记的异常变化导致了神经元发育和功能相关基因的转录或表达失调，特别是PCs功能相关的Pcp2、En1、Hsph1和Cbln1等。干扰Tet1 促进了神经元的细胞周期再入并加剧A-T神经元死亡。同时，通过人为操纵PCs中TET1的活性及表达，证实了提高5hmC水平能改善Atm-/-小鼠PCs的易损性并阻止其退行性病变，并能缓解患病小鼠的运动能力缺陷等病理症状。ATM在DNA损伤应答（DNA damage response，DDR）中的核心调控功能暗示了5hmC 在DDR中的潜在作用。5hmC作为一种独立而稳定的修饰型碱基在基因表达调控等生理过程中有重要作用，但其是否在DNA损伤修复过程中发挥功能还不清楚。本研究发现了5hmC 和TET蛋白能对不同类型DNA损伤产生动态应答，且以ATM-TET1-5hmC和ATR-TET3-5hmC两条通路的形式分别参与了DDR调控。生理生化实验证明了TET1是ATM激酶的特异性底物；ATM对TET1的磷酸化调控了DDR中5hmC 的改变。进一步的实验证实了TET1与TET3在维持细胞基因组稳定性、控制DNA损伤诱导的凋亡和促进存活等过程中具有重要作用。此外，实验还证实了提高5hmC水平和促进去甲基化能够提高细胞的DNA损伤修复能力。总的来说，本研究证实了脑区特异的5hmC选择性丢失影响和决定了A-T小脑PCs的易损性和退行性病变，并且阐明了A-T 中5hmC/5mC 失调的分子机制；同时，本研究发现了TET1/TET3介导的5hmC生成及其偶联的主动去甲基化在DNA损伤修复过程中的重要功能。这些研究结果将为开发新的A-T疾病诊断方法和更有效的药物及治疗打下基础。A-T综合症被认为是研究神经元退行性病变发生与发展机理的最佳模型之一，故本研究也为相关疾病的病理研究和DNA损伤机理的基础研究提供了新的思路。; Ataxia-telangiectasia (A-T) is a hereditary neurodegenerative disease resulting from mutations in the Atm (A-T mutanted) gene. A-T is characterized by multisystemic symptoms involving immunity, reproduction, metabolism, and mainly central nervous system. The earliest and most profound neuropathology involves the Purkinje and granule cells of the cerebellum. However, we know little about the pathological mechanism of specific neuronal cell types in A-T brain. A long-standing mystery surrounding A-T research is why it is mainly cerebellar neurons, Purkinje cells in particular, that appear vulnerable to ATM deficiency.In this study, we found the epigenetic marker 5-hydroxymehylcytocine (5hmC) substantially reduces in human A-T and Atm-/- mouse cerebellar Purkinje cells. Data from genome-wide (h)MeChIP-Seq showed that distribution difference of 5hmC and 5-methylcytocine (5mC) between normal and A-T cerebellum locates mainly in repeatetive regions and important genes for neurons. Combining GO analysis and mRNA chip detection, we further found a series of disregulation genes deriving from 5hmC/5mC changes encompasses neural function and survival pathyways, highlighting genes of particular importance to PC cells such as Pcp2、En1、Hsph1 and Cbln1. While knocking down Tet1 leads to cell cycle reentry and promotes neuron death, over-expressing TET1 enhances DNA repair efficiency of cells. In addition, manipulating in vivo the expression or kinase activity of TET1 in PC cells suggested that 5hmC increase prevents the degeneration of Atm-deficiency PC cells and improves behavioural deficts found in Atm-/- mice.The core function of ATM in DDR implies a potential role of 5hmC in universal DNA repair pathyway. 5hmC is a stable and independent epigenetic marker playing vital roles in viarous biloigical process, however, whether it functions in DDR is unknown. Here, we reported that both double-strand breaks (DSBs) and single-strand breaks (SSBs) lead to dynamic changes in the level of 5hmC and TET protein. ATM-TET1-5hmC and ATR-TET3-5hmC axises mainly participate in DSBs and SSBs related DDR, respectively. We also demonstrated TET1 is a new target of ATM kinase and ATM-mediated TET1 phosphoration is essential for 5hmC responding to DDR. Further experiments showed TET1 and TET3 are important in maintaining genome stability, controlling DNA damage-induced apoptosis and cell survival. Moreover, we found increasing 5hmC and promoting demthylation can improve DNA damage repair of cells.In summary, this study verified selectively brain regions-sepecific 5hmC loss affectes the vulnerability and degeneration of Atm-deficiency PCs and proposed the molecular mechanism of 5hmC/5mC disregulation in A-T cerebellum. Our data further revealed TET1/TET3-miediated 5hmC production and active demethylation function in DNA damage repair process. These findings could aid in differential diagnosis of A-T and discovery of novel therapeutic methods and effective drugs. Given that A-T is generally recognized as one of the most applicable models for reaearch on neurodegenerative diseases, this study also gives new insights into research refering to pathogenesis of other neurodegenerative diseases as well as fundamental mechanism of DNA damage repair.
语种	中文
文献类型	学位论文
条目标识符	http://ir.kiz.ac.cn/handle/152453/11965
专题	科研部门_表观遗传与神经退行性疾病（李家立）
作者单位	中国科学院昆明动物研究所
推荐引用方式 GB/T 7714	蒋德伟. TET酶介导的DNA去甲基化在A-T神经元退行病变以及DNA损伤应答中的研究[D]. 北京. 中国科学院研究生院,2016.

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