Role of TET Proteins in ES Cell Pluripotency and Function

描述（由申请人提供）：DNA的甲基化状态影响哺乳动物发育期间的许多生物学过程，并且已知在癌症中高度异常。我们最近发现，泰特蛋白TET 1、TET 2和TET 3构成1-酮戊二酸（1 KG）-和Fe（II）-依赖性双加氧酶的新家族，其通过催化DNA中5-甲基胞嘧啶（5 mC）氧化为5-羟基-甲基胞嘧啶（5 hmC）来改变DNA甲基化状态。Tet 1、Tet 2和5 hmC在小鼠胚胎干（ES）细胞和诱导多能干（iPS）细胞中以高水平存在，表明5 hmC在多能性中的潜在作用。此外，5 hmC水平和泰特表达/活性受到严格调控：5 hmC存在于未分化的ES细胞的基因组DNA中，但不存在于几种分化的细胞类型中，并且5 hmC水平在ES细胞分化后降低。总之，这些数据表明，通过泰特蛋白和5 hmC的DNA甲基化失调可能在ES细胞分化和功能中起作用。在这里，我们建议分析泰特蛋白在小鼠ES和iPS细胞的基因表达，多能性和细胞命运规范的生物学作用。目的1：利用Tet缺失的小鼠ES细胞和小鼠，研究泰特蛋白在小鼠ES细胞分化和功能中的作用。在目标2中，我们将研究在将鼠成纤维细胞重编程为iPS细胞中对泰特蛋白的需求。在目标3中，我们将通过转录谱确定ES细胞中泰特蛋白的靶基因，确定5 hmC和泰特蛋白的基因组位置，并以单碱基分辨率定位5 hmC在基因组中的位置。这些结果将为新型碱基5 hmC和新发现的泰特家族酶在ES和iPS细胞中的多能性和干细胞功能中的作用提供新的见解。 公共卫生相关性：泰特蛋白在ES细胞多能性和功能中的作用除了DNA字母表中的四个主要碱基- A、C、G和T -之外，还有一个非常小的碱基，称为5-甲基胞嘧啶（5 mC），其具有不成比例的关键作用。该碱基由主要碱基胞嘧啶（C）通过将甲基连接到其“5”位而产生。胞嘧啶甲基化的干扰可导致许多发育异常、遗传疾病和癌症。我们最近鉴定了一类新的蛋白质，称为泰特蛋白质，其将5-甲基胞嘧啶转化为称为5-羟甲基胞嘧啶（5 hmC）的变体。泰特蛋白和5-hmC在小鼠胚胎干（ES）细胞中强烈表达，并且当小鼠成纤维细胞重编程为诱导多能干（iPS）细胞时被诱导至高水平。在这个提议中，我们计划研究TET 1和TET 2蛋白在ES和iPS细胞中的作用。

DESCRIPTION (provided by applicant): The methylation status of DNA influences many biological processes during mammalian development, and is known to be highly aberrant in cancer. We recently discovered that the TET proteins TET1, TET2 and TET3 constitute a new family of 1-ketoglutarate (1KG)- and Fe(II)-dependent dioxygenases that alter DNA methylation status by catalysing the oxidation of 5-methylcytosine (5mC) to 5-hydroxy- methylcytosine (5hmC) in DNA. Tet1, Tet2 and 5hmC are present at high levels in mouse embryonic stem (ES) cells and induced pluripotent stem (iPS) cells, suggesting a potential role for 5hmC in pluripotency. Moreover, 5hmC levels and Tet expression/ activity are tightly regulated: 5hmC is present in genomic DNA of undifferentiated ES cells but not several differentiated cell types, and 5hmC levels diminish upon ES cell differentiation. Together these data suggest that dysregulation of DNA methylation via TET proteins and 5hmC may have a role in ES cell differentiation and function. Here we propose to analyze the biological roles of Tet proteins in gene expression, pluripotency and cell fate specification in mouse ES and iPS cells. In Aim 1, we will study the roles of Tet proteins in mouse ES cell differentiation and function in vitro and in vivo using Tet-deleted ES cells and mice. In Aim 2, we will examine the requirement for Tet proteins in reprogramming murine fibroblasts to iPS cells. In Aim 3, we will identify target genes for Tet proteins in ES cells by transcriptional profiling, defining the genomic locations of 5hmC and Tet proteins, and mapping the location of 5hmC at single- base resolution in the genome. The results should provide new insights into the role of the novel base, 5hmC, and the newly-discovered TET family of enzymes, in pluripotency and stem cell function in ES and iPS cells. PUBLIC HEALTH RELEVANCE: Role of TET proteins in ES cell pluripotency and function Narrative In addition to the four major bases in the DNA alphabet - A, C, G and T - there is also a very minor base known as 5-methylcytosine (5mC) that has a disproportionately crucial role. This base is produced from the major base cytosine (C) by attaching a methyl group to its "5" position. Interference with cytosine methylation can lead to a number of developmental abnormalities, genetic diseases and cancer. We recently identified a new class of proteins known as TET proteins that convert 5-methyl- cytosine to a variant known as 5-hydroxymethylcytosine (5hmC). TET proteins and 5-hmC are strongly expressed in mouse embryonic stem (ES) cells, and are induced to high levels when mouse fibroblasts are reprogrammed into induced pluripotent stem (iPS) cells. In this proposal we plan to investigate the role of TET1 and TET2 proteins in ES and iPS cells.