TET enzymes as guardians of genome stability

项目摘要/摘要 2009年，我的实验室发现了三种哺乳动物Tet(Ten-Eleven-Translo-Tet)的酶活性。 阳离子)蛋白、TET1、TET2和TET3。从那时起，我们自己和其他组织的工作就牵涉到了Tet 调节基因表达、细胞谱系特征、胚胎发育、神经元功能的蛋白质 和癌症。Tet蛋白是一种双加氧酶，可将5-甲基胞嘧啶(5mC)氧化为5-羟甲基胞嘧啶 (5hmC)和进一步氧化的甲基胞嘧啶(oxi-MC)。它们有两个生化功能：产生氧- MC和促进DNA去甲基化。然而，Tet蛋白发挥其多样性的机制 对生物效应的了解要少得多。 TET2突变在髓系恶性肿瘤中经常被观察到，但许多其他癌症被记录为 具有较低的5hmC水平，这意味着即使在没有Tet编码区突变的情况下，Tet功能也会严重丧失。 特兹。由于Tet功能丧失与DNA甲基化增加有关，因此Tet的肿瘤抑制作用 Tet蛋白的活性被认为与它们维持DNA去甲基化状态的能力有关。然而， 在我们实验室开发的强大的、可诱导的小鼠模型中，我们已经证明了两者的急性缺失 TET2和TET3在4周内迅速诱导侵袭性、可传播性髓系白血病，且100% 洞察力。使用这个系统，我们发现虽然DNA甲基化的平均水平增加 在早期造血干细胞/前体细胞中表达的基因中，如预期的那样，很少或没有相关性。 DNA甲基化的增加或减少与基因表达的上调或下调或与癌基因的表达 创世纪。相反，我们观察到致癌转化与磷酸化H_2AX的增加有很强的相关性。 以及受损的DNA损伤修复。在这里，我们建议扩展这些研究，以解决这些机制 牵涉其中。 在这个项目中，我们将使用小鼠模型以及体外系统来分析肿瘤发生的机制。 由Tet功能丧失所致。我们将考察Tet催化活性的作用，并比较它们的结论。 Tet蛋白功能丧失与DNA甲基转移酶(DNMT)的关系我们将研究 OXi-MC在扩张细胞中丢失与复制应激、基因组发展的动力学关系 不稳定和染色体异常。如果可行，我们将进行RNAi/CRISPR筛查以确定 调节由Tet功能丧失引起的细胞扩张的重要角色。我们将把我们的发现扩展到 高、低5hmC的人类肿瘤。我们的研究有可能改变目前的范式和 通过定义Tet功能与基因组联系的机制，提出新的治疗方法 稳定性。

Project summary/ abstract In 2009, my laboratory discovered the enzymatic activities of the three mammalian TET (Ten-Eleven-Translo- cation) proteins, TET1, TET2 and TET3. Since then, work from our own and other groups has implicated TET proteins in regulating gene expression, cell lineage specification, embryonic development, neuronal function and cancer. TET proteins are dioxygenases that oxidize 5-methylcytosine (5mc) to 5-hydroxymethylcytosine (5hmC) and further oxidized methylcytosines (oxi-mC). They have two biochemical functions: to generate oxi- mC and to facilitate DNA demethylation. However, the mechanisms by which TET proteins exert their diverse biological effects are much less understood. TET2 mutations are frequently observed in myeloid malignancies, but many other cancers are documented to have low 5hmC levels, implying profound loss of TET function even in the absence of TET coding region muta- tions. Because TET loss-of-function is associated with increased DNA methylation, the tumor suppressive role of TET proteins has been assumed to involve their ability to maintain DNA in a demethylated state. However, in powerful, inducible mouse models developed in our laboratory, we have shown that acute deletion of both Tet2 and Tet3 rapidly induces an aggressive, transmissible myeloid leukemia within 4 weeks and with 100% penetrance. Using this system, we have found that while the average level of DNA methylation increases across expressed genes in early hematopoietic stem/precursor cells as expected, there is little or no correla- tion of increased or decreased DNA methylation with up- or down-regulation of gene expression or with onco- genesis. Instead, we observe a strong correlation of oncogenic transformation with increased phospho-H2AX and impaired DNA damage repair. Here we propose to extend these studies to address the mechanisms involved. In this project we will use mouse models as well as in vitro systems to analyze the mechanisms of oncogenesis induced by TET loss-of-function. We will examine the role of TET catalytic activity, and compare the conse- quences of loss-of-function of TET proteins versus DNA methyltransferases (DNMTs). We will examine the kinetic relation between loss of oxi-mC in expanding cells and the development of replication stress, genome instability and chromosomal aberrations. As feasible, we will perform RNAi/CRISPR screens to identify important players that regulate cell expansion induced by TET loss-of-function. We will extend our findings to human cancers with high and low 5hmC. Our studies have the potential to change current paradigms and suggest new therapeutic approaches, by defining the mechanisms by which TET function is linked to genome stability.