TET enzymes as guardians of genome stability

TET 酶作为基因组稳定性的守护者

• 批准号: 9761480
• 负责人: Anjana Rao
• 金额: $ 104.76万
• 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/9761480
• 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TET（Ten-Eleven-Translo- 阳离子）蛋白质、TET1、TET2 和 TET3。从那时起，我们自己和其他小组的工作就涉及到 TET 调节基因表达、细胞谱系规范、胚胎发育、神经元功能的蛋白质 和癌症。 TET 蛋白是将 5-甲基胞嘧啶 (5mc) 氧化为 5-羟甲基胞嘧啶的双加氧酶 (5hmC) 和进一步氧化的甲基胞嘧啶 (oxi-mC)。它们有两个生化功能：产生氧化 mC 并促进 DNA 去甲基化。然而，TET 蛋白发挥其多样性的机制 生物效应的了解要少得多。 TET2 突变常见于骨髓恶性肿瘤，但许多其他癌症也有记录 5hmC 水平较低，这意味着即使没有 TET 编码区突变，TET 功能也会严重丧失 系统蒸发散。由于 TET 功能丧失与 DNA 甲基化增加有关，因此肿瘤抑制作用 TET 蛋白的功能被认为涉及其维持 DNA 去甲基化状态的能力。然而， 在我们实验室开发的强大的诱导小鼠模型中，我们已经证明，两种基因的急性缺失 Tet2 和 Tet3 在 4 周内迅速诱发侵袭性、传染性骨髓性白血病，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，几乎没有或没有相关性 DNA 甲基化的增加或减少与基因表达的上调或下调或肿瘤相关 起源。相反，我们观察到致癌转化与磷酸化 H2AX 增加有很强的相关性 以及 DNA 损伤修复受损。在这里，我们建议扩展这些研究以解决机制 涉及。 在这个项目中，我们将使用小鼠模型和体外系统来分析肿瘤发生的机制 由 TET 功能丧失引起。我们将研究 TET 催化活性的作用，并比较结果 TET 蛋白与 DNA 甲基转移酶 (DNMT) 功能丧失的序列。我们将检查 扩增细胞中 oxi-mC 丢失与复制应激、基因组发展之间的动力学关系 不稳定和染色体畸变。如果可行，我们将进行 RNAi/CRISPR 筛选来识别 调节 TET 功能丧失引起的细胞扩张的重要参与者。我们将把我们的发现扩展到 5hmC 高和低的人类癌症。我们的研究有可能改变当前的范式 通过定义 TET 功能与基因组关联的机制，提出新的治疗方法 稳定。