5-methylcytosine oxidation in development and disease

项目总结：发育和疾病中的5-甲基胞嘧啶氧化 基因启动子和增强子的DNA甲基化通常表明基因沉默，而 通过TET介导的5-甲基胞嘧啶（5 mC）氧化途径的甲基化丧失可导致基因 activation.泰特蛋白和5 mC氧化途径是已知的，在胚胎发育中起关键作用 然而，TET介导的5 mC氧化的调节机制是完全未知的。 我们最近发现蛋白SMCHD 1是泰特活性的潜在负调节因子。在 在人类中，SMCHD 1缺失激活DUX 4基因，导致细胞毒性、肌肉变性和 面肩肱型肌营养不良症（FSHD）。我们假设SMCHD 1的一个关键功能是控制 5 mC氧化酶活性和SMCHD 1的缺乏导致TET介导的表观遗传功能的过度激活， 和DUX 4的激活。本项目的目的是测试这一假设，并获得一个基本的 了解SMCHD 1如何与正常人中TET介导的表观遗传功能相互作用和控制 胚胎发育和疾病。在目标1中，我们将使用小鼠胚胎干细胞和人类诱导的 多能干细胞，以确定SMCHD 1调节正常人泰特功能的基本机制 发展我们将使用SMCHD 1敲除细胞和小鼠系，并确定泰特蛋白如何与SMCHD 1基因结合。 基因组中的分布改变;哪些位点受到影响;如果氧化的5-甲基胞嘧啶碱基的分布 （5 hmC）对SMCHD 1结合位点具有特异性;（合子）基因组的哪些区域在SMCHD 1后被激活 如果小鼠和人类多能性细胞之间存在共享的SMCHD 1介导的调控途径， 细胞这些实验的结果将确立SMCHD 1在正常人中的负调节功能。 开发并为目标2中的疾病特异性研究奠定基础。在目标2中，我们将在 人肌肉细胞，并确定SMCHD 1在细胞分化和疾病中的表观遗传作用， 确定基因表达变化、染色质结构和全基因组DNA甲基化状态。我们 预期SMCHD 1介导的泰特抑制和基因表达调节在人类中是相似的， 小鼠细胞，并且在SMCHD 1敲除细胞中耗尽泰特活性将至少部分逆转该效应 SMCHD 1功能丧失表型。在目标3中，我们将进行SMCHD 1-泰特的生化研究 蛋白质-蛋白质相互作用，以鉴定关键结合结构域，并确定泰特抑制剂是否可以沉默DUX 4 在缺乏功能性SMCHD 1的细胞系中以及在源自FSHD患者的肌细胞中表达。如果 如果成功，这些数据将为泰特抑制剂可能对FSHD具有治疗益处提供原理证明 患者总之，该项目的结果将产生第一个泰特的机械调节模型， 在小鼠和人中介导的5 mC氧化途径，以及新的TET-SMCHD 1轴在正常人中的作用 发展和疾病。

PROJECT SUMMARY: 5-methylcytosine oxidation in development and disease DNA methylation at gene promoters and enhancers is usually an indication that genes are silenced, whereas loss of methylation through the TET-mediated 5-methylcytosine (5mC) oxidation pathway can lead to gene activation. TET proteins and the 5mC oxidation pathway are known, pivotal actors in embryonic development and disease, yet the mechanisms by which TET-mediated 5mC oxidation is regulated are completely unknown. We recently discovered that the protein SMCHD1 is a potential negative regulator of TET activity. In humans, SMCHD1 depletion activates the DUX4 gene(s) that cause(s) cell toxicity, muscle degeneration, and facioscapulohumeral muscular dystrophy (FSHD). We hypothesize that one key function of SMCHD1 is to control 5mC oxidase activities and that lack of SMCHD1 leads to an over-activation of TET-mediated epigenetic function, and the activation of DUX4. The objective of this project is to test this hypothesis and obtain a fundamental understanding of how SMCHD1 interacts with and controls TET-mediated epigenetic function in normal embryonic development and disease. In Aim 1, we will use mouse embryonic stem cells and human induced pluripotent stem cells to determine the basic mechanisms of SMCHD1-regulated TET function in normal development. We will employ SMCHD1-knockout cell and mouse lines and determine how TET protein distribution in the genome is altered; which loci are affected; if the distribution of oxidized 5-methylcytosine bases (5hmC) is specific to SMCHD1 binding sites; which regions of the (zygotic) genome are activated upon SMCHD1 loss; and if there are shared SMCHD1-mediated regulatory pathways between mouse and human pluripotent cells. The results of these experiments will establish the negative regulatory function of SMCHD1 in normal development and set the stage for disease-specific studies in Aim 2. In Aim 2, we will inactivate SMCHD1 in human muscle cells and determine the epigenetic role of SMCHD1 in cell differentiation and disease by determining gene expression changes, chromatin structure, and genome-wide DNA methylation status. We expect that SMCHD1-mediated TET inhibition and regulation of gene expression will be similar in human and mouse cells, and that depleting TET activity in SMCHD1 knockout cells will at least partially reverse the effects of SMCHD1 loss-of-function phenotypes. In Aim 3, we will perform biochemical studies of SMCHD1-TET protein-protein interactions to identify key binding domains, and determine if TET inhibitors can silence DUX4 expression in cell lines lacking functional SMCHD1, and in muscle cells derived from FSHD patients. If successful, these data will provide proof of principle that TET inhibitors may have therapeutic benefit for FSHD patients. Together, the results of this project will generate the first mechanistic, regulatory model of the TET- mediated 5mC oxidation pathway in mouse and man, and the role of the novel TET-SMCHD1 axis in normal development and disease.