Role of TET proteins in myeloid malignancies

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。在哺乳动物细胞中，DNA甲基化主要发生在二核苷酸CpG背景下胞嘧啶的对称甲基化，启动子处高水平的5-甲基胞嘧啶（5mC）的存在通常与基因表达的减少相关。我们最近发现TET蛋白TET1， TET2和TET3构成了一个新的双加氧酶家族，利用分子氧和辅助因子Fe（II）和2-氧葡萄糖酸盐将DNA中的5mC氧化为5-羟甲基胞嘧啶（5hmC）。因此，TET蛋白以一种前所未有的新方式改变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（如慢性髓单核细胞白血病（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和多梳组蛋白ASXL1的基因，ASXL1是去泛素化H2A复合物的组成部分。在本研究中，我们将使用合适的小鼠模型，在分子水平上探索Tet2和Tet3在造血和骨髓功能中的作用（目的1）。在Aims 2和Aims 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方法并生成了许多与这些拟议研究相关的试剂，包括用于测量MDS/ MPN/ CMML/继发性AML患者骨髓样本中5hmC总体基因组水平的定量方法；以及在单碱基分辨率下绘制5hmC基因组位置和分析5hmC的创新策略。我们培育了Tet2和Tet3基因有条件破坏的小鼠；并发现了5hmC和多梳复合体之间的新关系。我们已经证明，在胚胎干细胞中，5hmC主要存在于基因的启动子上，这些基因(i)不活跃，但“准备”在胚胎干细胞分化时表达；（ii）具有双重（“二价”）H3K4me3和H3K27me3标记；（iii）由多梳复合物PRC1和PRC2的组分结合（H3K27me3标记由PRC2沉积）。通过定义DNA甲基化、DNA羟甲基化和组蛋白修饰的全基因组变化，这些变化是由于Tet2和Tet3功能丧失以及Dnmt3a和Asxl1中选择的白血病相关突变所导致的，我们提出的研究将为这些蛋白质如何控制染色质结构和靶基因的表观遗传景观提供基本见解。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如何导致人类骨髓癌的易感性。

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.