DNA羟甲基化修饰改变：唐氏综合征神经系统发育异常的新机制？

Down's syndrome (DS), results from the presence of an extra copy of chromosome 21, affects one in every 1000 to 1100 newborns in different populations. Each year approximately twenty to thirty thousands children with DS are born in China. Mental retardation caused by the abnormal nervous development is the most notable feature in DS patients. However, the molecular mechanisms of this abnormality is still not well understood. DNA methylation at the 5-position of cytosine (5mC) in mammals is essential in a variety of biological processes. 5-hydroxymethylcytosine (5hmC) was discovered as another cytosine modification, which coverted from 5mC by the ten-eleven translocation (Tet) family proteins, in mouse neurons and embryonic stem cells in 2009. A number of recent studies have shown that 5hmC levels vary substantially between different cell types and tissues and are highest in the brain, in particular in neurons. In addtition, formation of 5hmC promotes neuronal differentiation by modulating the expression of genes. Therefore, the hypothesis is reasonable that change of hydroxylmethylcytosine profile might play a major role in the abnormality of nervous system in DS. In this study, peripheral blood samples are collected from DS and normal children. Genome-wide profiles of 5hmC and 5mC are assessed by MeDIP-chip, and the 5hmC levels of differentiated modified genes which are involved in the neuronal development and differentiation are detected with ox-BS pyrosequencing. Meanwhile, the expression pattern is also identified by microarray, and the relationship between above epigenetic modification and expression level is investigated. The regulation of 5hmC levels conducted by TET enzymes is analyzed by in vitro transfected experiments on human monocytes. To investigate the contribution of hydroxymethylation on the neuronal development and differentiation of DS, Tc1 mouse model which contains a freely-segregating copy of 92% of human chromosome 21 is used. The brains are obtained from the fetus, newborn and adult Tc1 mice, normal mice are also sacrificed for control. Pathology is measured for the analysis of the abnormality of nervous system. Then DNA and RNA samples are extracted from these mice. Profiles of 5hmC modification and gene expression are identified by microarray, pyrosequencing and qRT-PCR, the signal pathway focus on neuronal development and differentiation is analyzed with bioinformatics softwares. Finally, iPS cells induced from fibroblasts of Tc1 mice are cultured and neurons are produced from induced differentiation. The effect of differatiated hydroxymethylcytosine modification on the abnormality of neuronal development and differentiation is assessed. Characteristic 5-hydroxymethylcytosine profiles and its regulatory mechansim on development of nervous system of DS would be shown in this study.

唐氏综合征（DS）是新生儿最常见的染色体病，神经系统发育异常引起的智力低下是最主要的临床表现，然而其分子机制仍不清楚。近年研究显示，DNA羟甲基化修饰是神经系统发育重要的调控因子，异常修饰可能导致神经系统疾病。本研究以Tc1模型小鼠（具有DS绝大部分表型）为主要对象，采用体内和体外实验结合的途径，综合应用芯片、oxBS-焦磷酸测序、体外转染、qRT-PCR、CHIP等方法分析不同发育时期的Tc1小鼠大脑中羟甲基化修饰、基因表达与正常对照的差异；研究其与催化羟甲基化修饰的TET蛋白及其他转录因子的相互作用及调节网络；并在DS患儿中验证相关结果。进而分析Tc1小鼠iPS细胞（已制备成功）定向分化为神经细胞过程中，羟甲基化修饰模式的改变及其对神经分化的调控。本研究将首次揭示DS个体特征性的羟甲基化修饰模式，动态地阐明其对DS神经系统发育的影响及调控机制，为DS患儿智力低下的早期干预提供理论依据。

唐氏综合征（DS）是新生儿最常见的染色体病，21号染色体三体导致的基因组甲基化/羟甲基化修饰改变可能是其神经系统发育异常发生的重要因素。已有的研究工作表明，唐氏综合征患儿胚胎发育早期即出现神经系统发育的异常，因此本项目将DS新生儿来源的iPS细胞和具有DS绝大部分表型的Tc1模型小鼠iPS细胞为主要对象，获取其特征性的甲基化和羟甲基化修饰谱，并分析相关作用网络。同时，深入DS患儿外周血样本中神经系统发育相关基因PRDM8的甲基化/羟甲基化修饰模式及其对表达的影响。.经过4年的研究，本项目取得以下成果：.1. 与正常对照相比，DS-iPS细胞中存在全基因组高甲基化和高羟甲基化修饰，差异修饰基因涉及多个神经系统发育相关的通路。其中RunX1基因的高表达会显著影响iPS细胞的线粒体功能；.2. DS-iPS细胞中存在全基因组表达谱改变，其中lncRNA的异常表达会显著影响线粒体功能；.3. DS患儿外周血样本中神经系统发育相关基因PRDM8在内部启动子区存在高甲基化和高羟甲基化修饰，修饰改变与其表达的异常密切相关；.4. 成功地制备了来源于Tc1小鼠的iPS细胞，细胞具有发育成个体的全能性。Tc1-iPS细胞的分析结果表明，额外一条人21号染色体的存在会导致Tc1-iPS细胞中呈现全基因组高甲基化修饰模式，高甲基化导致的神经元分化相关基因（Myt1l，Ascl1，Pou3f2）的表达下调，可能通过调控Wnt和Notch信号通路的相关基因表达来影响神经系统发育。.上述结果表明，唐氏综合征患儿样本，尤其是早期发育的胚胎样本中存在显著的DNA甲基化和羟甲基化修饰改变，所引发的相关基因表达变化与神经系统发育和分化异常密切相关，其中线粒体功能损伤是重要的介导途径。本项目的研究结果为唐氏综合征神经系统异常发生的分子机制研究提供了科学的数据。