TET enzymes as guardians of genome stability

TET 酶作为基因组稳定性的守护者

• 批准号: 9978730
• 负责人: Anjana Rao
• 金额: $ 108万
• 依托单位: LA JOLLA INSTITUTE FOR IMMUNOLOGY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-01 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9978730
• 关键词: Acute; Address; Biochemical; Biological; Biological Models; CRISPR screen; Cell Lineage; Cells; Chromosome abnormality; Code; Cytosine; DNA; DNA Methylation; DNA Modification Methylases; DNA Repair; Development; Dioxygenases; Embryonic Development; Enzymes; Gene Expression; Genes; Genetic Diseases; Genome Stability; Genomic Instability; Hematopoietic; Human; Impairment; In Vitro; Kinetics; Laboratories; Link; Malignant Neoplasms; Methylation; Mutation; Myeloid Leukemia; Myeloproliferative disease; Neurons; Oncogenic; Oxides; Penetrance; Positioning Attribute; Protein translocation; Proteins; RNA Interference; Role; System; Tetanus Helper Peptide; Up-Regulation; Work; base; demethylation; gene repression; loss of function; methyl group; mouse model; novel therapeutic intervention; precursor cell; protein function; replication stress; stem; tumor; tumorigenesis

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.

项目概要/摘要 2009年，我的实验室发现了三种哺乳动物泰特（Ten-Eleven-Translo-）的酶活性 阳离子）蛋白、TET 1、TET 2和TET 3。从那时起，我们自己和其他团体的工作就涉及到了泰特 调节基因表达的蛋白质，细胞谱系特化，胚胎发育，神经功能 和癌症泰特蛋白是将5-甲基胞嘧啶（5 mc）氧化成5-羟甲基胞嘧啶的双加氧酶 （5 hmC）和进一步氧化的甲基胞嘧啶（oxi-mC）。它们有两种生物化学功能：产生氧- mC和促进DNA去甲基化。然而，泰特蛋白发挥其多样性的机制， 对生物学效应的了解要少得多。 TET 2突变经常在骨髓恶性肿瘤中观察到，但许多其他癌症被证明是 5 hmC水平较低，意味着即使在不存在穆塔泰特编码区的情况下，泰特功能也严重丧失。 选择。由于泰特功能丧失与DNA甲基化增加有关，因此肿瘤抑制作用 泰特蛋白质的功能被认为与其维持DNA去甲基化状态的能力有关。然而，在这方面， 在我们实验室开发的强大的可诱导小鼠模型中，我们已经表明， Tet 2和Tet 3在4周内迅速诱导侵袭性、可传播的髓系白血病，并且100% 外显率使用这个系统，我们发现，虽然DNA甲基化的平均水平增加， 正如预期的那样，在早期造血干细胞/前体细胞中表达的基因中， DNA甲基化的增加或减少与基因表达的上调或下调或与肿瘤的发生有关。 创世纪相反，我们观察到致癌转化与磷酸化-H2 AX增加的强相关性。 和受损的DNA损伤修复在这里，我们建议扩展这些研究，以解决机制 涉案 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 由泰特功能丧失引起。我们将检查泰特催化活性的作用，并比较结果 泰特蛋白相对于DNA甲基转移酶（DNMT）的功能丧失序列。我们会研究 扩增细胞中oxi-mC的丢失与复制应激的发展之间的动力学关系，基因组 不稳定性和染色体畸变。在可行的情况下，我们将进行RNAi/CRISPR筛选， 调节由泰特功能丧失诱导的细胞扩增的重要参与者。我们将把我们的发现扩展到 高和低5 hmC的人类癌症。我们的研究有可能改变当前的范式， 提出了新的治疗方法，通过定义泰特功能与基因组相关的机制， 稳定