Role of TET Proteins in ES Cell Pluripotency and Function

描述(申请人提供)：DNA的甲基化状态影响哺乳动物发育过程中的许多生物学过程，并已知在癌症中高度异常。我们最近发现，Tet蛋白TET1、TET2和TET3组成了一个新的家族，它们依赖于1-酮戊二酸(1 Kg)和Fe(II)，通过催化DNA中5-甲基胞嘧啶(5mC)氧化成5-羟甲基胞嘧啶(5HmC)来改变DNA甲基化状态。Tet1、TET2和5hmC在小鼠胚胎干细胞(ES)和诱导的多能干细胞(IPS)中存在高水平，提示5hmC在多能性中可能发挥作用。此外，5hmC水平和Tet的表达/活性受到严格调控：5hmC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5hmC的水平随着ES细胞的分化而降低。综上所述，这些数据表明，通过Tet蛋白和5hmC的DNA甲基化异常可能在ES细胞的分化和功能中发挥作用。在这里，我们建议分析Tet蛋白在小鼠ES和iPS细胞的基因表达、多能性和细胞命运指定中的生物学作用。在目标1中，我们将利用Tet缺失的ES细胞和小鼠，在体内外研究Tet蛋白在小鼠ES细胞分化和功能中的作用。在目标2中，我们将研究在将小鼠成纤维细胞重新编程为iPS细胞时对Tet蛋白的需求。在目标3中，我们将通过转录图谱确定Tet蛋白在ES细胞中的靶基因，定义5hmC和Tet蛋白的基因组位置，并以单碱基分辨率定位5hmC在基因组中的位置。这些结果将为新碱基5hmC和新发现的Tet酶家族在ES和iPS细胞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：Tet蛋白在ES细胞多能性和功能叙述中的作用除了DNA字母表中的四个主要碱基-A、C、G和T-之外，还有一个非常微小的碱基，即5-甲基胞嘧啶(5mC)，它具有不成比例的关键作用。该碱是由主碱胞嘧啶(C)通过将甲基连接到它的“5”位而产生的。干扰胞嘧啶甲基化可导致许多发育异常、遗传病和癌症。我们最近发现了一类新的蛋白质，称为Tet蛋白，它能将5-甲基胞嘧啶转化为一种名为5-羟甲基胞嘧啶(5HmC)的变体。Tet蛋白和5-HMC在小鼠胚胎干细胞中高表达，当小鼠成纤维细胞被重新编程为诱导的多能干细胞时，Tet蛋白和5-HMC被诱导到高水平。在这项提案中，我们计划研究TET1和TET2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.