Role of TET Proteins in ES Cell Pluripotency and Function

TET 蛋白在 ES 细胞多能性和功能中的作用

• 批准号: 8107190
• 负责人: Anjana Rao
• 金额: $ 14.14万
• 依托单位: IMMUNE DISEASE INSTITUTE, INC.
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-04-01 至 2011-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8107190
• 关键词: Acute; Affect; Alleles; Animals; Atlases; Biological; Biological Process; Brain; Cell physiology; Cerebellum; Cytosine; DNA; DNA Methylation; Data; Development; Dioxygenases; Dysmyelopoietic Syndromes; Embryo; Embryonic Development; Enzymes; Family; Fibroblasts; Gene Expression; Gene Targeting; Generations; Genome; Genomics; Hematopoietic stem cells; Hereditary Disease; Hippocampus (Brain); Human; Hydroxylation; In Vitro; Laboratories; Lead; Learning; Location; Lymphoblastic Leukemia; Malignant Neoplasms; Mammals; Maps; Memory; Methylation; Minor; Molecular Profiling; Mus; Myelogenous; Myeloproliferative disease; Neurons; Oncogenic; Pattern; Plants; Positioning Attribute; Process; Property; Proteins; Purkinje Cells; Pyramidal Cells; Resolution; Role; Stem cells; Tetanus Helper Peptide; Undifferentiated; Variant; alpha ketoglutarate; base; cell fate specification; cell type; chromatin immunoprecipitation; embryonic stem cell; genome-wide; in vivo; induced pluripotent stem cell; insight; interest; loss of function; methyl group; motor control; novel; overexpression; oxidation; pluripotency; promoter; protein function; stem; stem cell differentiation

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.

描述（由申请人提供）：DNA 的甲基化状态影响哺乳动物发育过程中的许多生物过程，并且已知在癌症中高度异常。我们最近发现 TET 蛋白 TET1、TET2 和 TET3 构成了一个新的 1-酮戊二酸 (1KG) 和 Fe(II) 依赖性双加氧酶家族，它们通过催化 DNA 中 5-甲基胞嘧啶 (5mC) 氧化为 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 (ES) 细胞和诱导多能干 (iPS) 细胞中高水平存在，表明 5hmC 在多能性中的潜在作用。此外，5hmC 水平和 Tet 表达/活性受到严格调控：5hmC 存在于未分化 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 水平在 ES 细胞分化时减少。这些数据共同表明，通过 TET 蛋白和 5hmC 引起的 DNA 甲基化失调可能在 ES 细胞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、多能性和细胞命运规范中的生物学作用。在目标 1 中，我们将使用 Tet 缺失的 ES 细胞和小鼠在体外和体内研究 Tet 蛋白在小鼠 ES 细胞分化和功能中的作用。在目标 2 中，我们将研究将小鼠成纤维细胞重编程为 iPS 细胞时对 Tet 蛋白的需求。在目标 3 中，我们将通过转录分析鉴定 ES 细胞中 Tet 蛋白的靶基因，定义 5hmC 和 Tet 蛋白的基因组位置，并以单碱基分辨率绘制基因组中 5hmC 的位置。这些结果应该为新碱基 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。 公共健康相关性：TET 蛋白在 ES 细胞多能性和功能中的作用 叙述 除了 DNA 字母表中的四个主要碱基 - A、C、G 和 T - 之外，还有一个非常次要的碱基，称为 5-甲基胞嘧啶 (5mC)，它具有极其重要的作用。该碱基是通过将甲基连接到其“5”位置而由主要碱基胞嘧啶 (C) 产生的。干扰胞嘧啶甲基化可导致许多发育异常、遗传疾病和癌症。我们最近发现了一类新的蛋白质，称为 TET 蛋白，可将 5-甲基胞嘧啶转化为称为 5-羟甲基胞嘧啶 (5hmC) 的变体。 TET 蛋白和 5-hmC 在小鼠胚胎干 (ES) 细胞中强烈表达，当小鼠成纤维细胞重编程为诱导多能干 (iPS) 细胞时，TET 蛋白和 5-hmC 被诱导至高水平。在本提案中，我们计划研究 TET1 和 TET2 蛋白在 ES 和 iPS 细胞中的作用。