Role of TET proteins in myeloid malignancies

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。在哺乳动物细胞中，DNA甲基化主要是在二核苷酸CpG的背景下发生的胞嘧啶的对称甲基化，启动子上存在高水平的5-甲基胞嘧啶(5mC)通常与基因表达减弱相关。我们最近发现，Tet蛋白TET1、TET2和TET3是一个新的双加氧酶家族，它利用分子氧和辅因子Fe(II)和2-氧化戊二酸将DNA中的5mC氧化为5-羟甲基胞嘧啶(5HmC)。因此，Tet蛋白以一种新颖的、迄今为止前所未有的方式改变了DNA甲基化状态。同时，一些实验室报道，TET2突变经常与骨髓增生异常综合征(MDS)、骨髓增生性肿瘤(MPN)和髓系恶性肿瘤(如慢性粒单核细胞白血病(CMML)和急性髓系白血病(AML))相关。在这些患者中经常发生突变的另外两个基因包括编码DNA甲基转移酶DNMT3A的基因和多梳蛋白ASXL1，ASXL1是一种去泛素化过氧化氢的复合体的组成部分。在这项提案中，我们将使用适当的小鼠模型在分子水平上探索TET2和TET3在造血和髓系功能中的作用(目标1)。在目标2和目标3中，我们将分别研究TET2/Tet3与DNMT3A和ASXL1的关系。我们已经开发了许多与这些研究相关的方法和试剂，包括定量方法来测量MDS/MPN/CMML/继发性AML患者骨髓样本中5hmC的总体基因组水平；以及以单碱基分辨率绘制5hmC基因组位置和分析5hmC的创新策略。我们已经产生了TET2和Tet3基因有条件中断的小鼠；并发现了5hmC和多梳复合体之间的一种新的关系。我们已经在ES细胞中证明了5hmC主要存在于基因的启动子上，这些基因(I)不活跃，但在ES细胞分化时“准备”表达；(Ii)具有双重(“双价”)H3K4me3和H3K27me3标记；以及(Iii)被多梳复合体PRC1和PRC2的成分结合(H3K27me3标记由PRC2沉积)。通过定义由于TET2和Tet3的功能丧失以及DNMT3A和ASXL1的特定白血病相关突变而发生的全基因组DNA甲基化、DNA羟甲基化和组蛋白修饰的变化，我们拟议的研究将为这些蛋白质如何控制染色质结构和其目标基因的表观遗传格局提供基本的见解。这些信息将帮助我们理解干细胞中这些基因产物的体细胞突变所导致的变化可能会如何诱发人类的髓系癌症。

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. In mammalian cells, DNA methylation occurs primarily as symmetrical methylation of cytosine in the context of the dinucleotide CpG, and the presence of high levels of 5-methyl- cytosine (5mC) at promoters is generally correlated with diminished gene expression. We recently discovered that the TET proteins TET1, TET2 and TET3 constitute a new family of dioxygenases that utilize molecular oxygen and the cofactors Fe(II) and 2-oxoglutarate to oxidize 5mC to 5-hydroxymethylcytosine (5hmC) in DNA. As a result, TET proteins alter DNA methylation status in a novel and hitherto unprecedented way. Simultaneously, several labs reported that TET2 mutations are frequently associated with myelodysplastic syndromes (MDS), myeloproliferative neoplasms (MPN) and myeloid malignancies such as chronic myelomonocytic leukemia (CMML) and acute myeloid leukemia (AML). Two other genes frequently mutated in these patients include those encoding the DNA methyltransferase DNMT3A and the polycomb group protein ASXL1, a component of a complex that deubiquitinates H2A. In this proposal we will explore, at a molecular level using appropriate mouse models, the roles of Tet2 and Tet3 in hematopoiesis and myeloid function (Aim 1). In Aims 2 and 3, we will investigate the relation of Tet2/ Tet3 to Dnmt3a and Asxl1 respectively. We have developed many methods and generated many reagents relevant to these proposed studies, including quantitative methods to measure overall genomic levels of 5hmC in bone marrow samples from patients with MDS/ MPN/ CMML/ secondary AML; and innovative strategies for mapping the genomic location of 5hmC and profiling 5hmC at single-base resolution. We have generated mice with conditional disruption of the Tet2 and Tet3 genes; and have uncovered a novel relation between 5hmC and the polycomb complex. WE have shown in ES cells that 5hmC is present predominantly at the promoters of genes that are (i) inactive but "poised" to be expressed upon ES cell differentiation; (ii) bear dual ("bivalent") H3K4me3 and H3K27me3 marks; and (iii) are bound by components of the polycomb complexes PRC1 and PRC2 (the H3K27me3 mark is deposited by PRC2). By defining the genome-wide changes in DNA methylation, DNA hydroxymethylation and histone modifications that occur as a result of loss of function of Tet2 and Tet3 and selected leukemia-associated mutations in Dnmt3a and Asxl1, our proposed studies will provide fundamental insights into how these proteins control chromatin structure and the epigenetic landscape at their target genes. This information will help us understand how changes that occur as a result of somatic mutations in these gene products in stem cells might predispose to myeloid cancers in humans.