Chemical modulators of Tet-family proteins

Tet 家族蛋白的化学调节剂

• 批准号: 7820122
• 负责人: Anjana Rao
• 金额: $ 50万
• 依托单位: IMMUNE DISEASE INSTITUTE, INC.
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7820122
• 关键词: Acute; Acute Lymphocytic Leukemia; Adult; Affect; Antibodies; Appearance; Area; Behavior; Behavioral; Binding; Binding Proteins; Biological; Biological Process; Biology; Brain; Catalytic Domain; Cell physiology; Cells; Cerebellum; Chemicals; Chromatin; Clinic; CpG dinucleotide; Cytosine; DNA; DNA Methylation; DNA Modification Process; DNA Repair Pathway; Data; Development; Dioxygenases; Disease; Dysmyelopoietic Syndromes; Enzymes; Epigenetic Process; Family; Female; Figs - dietary; Gene Expression; Generations; Genes; Genomics; Goals; Hematopoietic; Hereditary Disease; Hippocampus (Brain); Human; Hydroxylation; Image; In Vitro; Insecta; Investigation; Laboratories; Language; Learning; Link; Lymphoblastic Leukemia; Malignant Neoplasms; Mammalian Cell; Memory; Messenger RNA; Methods; Methyl-CpG-Binding Protein 2; Methylation; Minor; Mole the mammal; Monitor; Motor; Mus; Mutation; Myelodysplastic/Myeloproliferative Disease; Myelogenous; Myeloproliferative disease; Nervous System Physiology; Nervous system structure; Neuraxis; Neurobiology; Neurons; Nuclear; Oligonucleotides; Oncogenic; Organism; Outcome; Pathologic Processes; Pathway interactions; Phenotype; Physiological; Principal Investigator; Production; Protein Family; Proteins; Public Health; Purkinje Cells; Pyramidal Cells; Reagent; Recombinants; Repetitive Sequence; Research; Retrotransposon; Rett Syndrome; Role; Staining method; Stains; Stem cells; Synaptic plasticity; System; Tetanus Helper Peptide; Text; Tumor Suppressor Genes; Undifferentiated; X Chromosome; X Inactivation; alpha ketoglutarate; autism spectrum disorder; base; cancer cell; cancer stem cell; cell transformation; cell type; clinical application; demethylation; ds-DNA; embryonic stem cell; high throughput screening; imprint; induced pluripotent stem cell; inhibitor/antagonist; interest; loss of function; motor control; novel; overexpression; pluripotency; programs; public health relevance; research study; self-renewal; small molecule; stem cell differentiation; telomere; tumorigenesis

DESCRIPTION (provided by applicant): The methylation status of DNA influences many biological processes during mammalian development, including retrotransposon silencing, X-inactivation and the asymmetric expression of parentally imprinted genes. In mammalian cells, DNA methylation occurs almost exclusively as symmetrical methylation of cytosine in the context of the dinucleotide CpG. 5-methylcytosine (5mC) is found at high levels at repetitive elements, telomeres and the inactive X-chromosome in females, and its presence correlates with diminished gene expression. In cancer, DNA methylation has been linked to aberrant silencing of tumor suppressor genes; in the central nervous system (CNS), it has been implicated, among other things, in learning, memory and synaptic plasticity. We have recently discovered that the TET proteins TET1, TET2 and TET3 constitute a new family of 2-oxoglutarate (2OG)- and Fe(II)-dependent dioxygenases that catalyse hydroxylation of 5mC to 5-hydroxymethylcytosine (hmC) in DNA. hmC levels and TET expression/ activity appear to be tightly regulated. (i) hmC is present in genomic DNA of undifferentiated ES cells but not differentiated cell types, and hmC levels diminish upon ES cell differentiation. (ii) TET1 is an MLL partner in acute myeloid and lymphoid leukemia (AML, ALL), and TET2 is implicated in myelodysplastic syndromes and AML. (iii) In the CNS, hmC is present at high levels in cerebellar Purkinje neurons; moreover, TET1 and TET2 mRNA are expressed in the brain, with TET1 mRNA being particularly high in Purkinje cells and hippocampal pyramidal cells. Together these data suggest potential functions of TET proteins and hmC in (a) pluripotency and stem cell function; (b) oncogenic transformation, especially of haematopoietic cells; and (c) motor control, learning and memory. Here we propose to perform two separate high-throughput screens to identify small-molecule activators and inhibitors of TET-family proteins. The first is a completely in vitro screen in which we will use recombinant catalytic domains of TET1, TET2 or TET3 expressed in insect cells to convert 5mC to hmC in methylated double-stranded DNA oligonucleotides. The appearance of hmC will be monitored by using an antibody to hmC. The second is a cell-based screen which relies on the fact that overexpression of TET1, TET2 or TET3 catalytic domains in HEK293 cells yields cellular phenotypes that are readily assayed by high-throughput imaging: loss of 5mC staining, acquisition of hmC staining, and increased nuclear size. Together these studies should identify small molecules that modulate the functions of this new and interesting class of enzymes, and may be used as reagents to investigate their biological functions in cells and in organisms. PUBLIC HEALTH RELEVANCE: In addition to the four major bases in the DNA alphabet - A, C, G and T - there is also a minor fifth base, 5-methylcytosine (5mC) that has a disproportionately important role. Abnormal production, distribution and recognition of 5mC have been linked to developmental abnormalities and genetic diseases, including Rett syndrome, an autism spectrum disorder. We recently identified a new family of proteins, the TET proteins that convert 5mC to a sixth base, 5-hydroxymethylcytosine (hmC). TET proteins have been linked to cancer, and hmC is found at high levels in embryonic stem cells and certain classes of neuronal cells. In this proposal we will devise methods to find chemical activators and inhibitors of TET proteins. These molecules will be useful for further investigations into the biological functions of TET proteins, and may eventually be used in the clinic to treat Rett syndrome and other diseases of DNA methylation.

描述（由申请人提供）：DNA的甲基化状态会影响哺乳动物发育过程中许多生物过程，包括返回跨跨跨座孔沉默，X灭活和父育印度基因的不对称表达。在哺乳动物细胞中，DNA甲基化几乎完全作为在二核苷酸CpG的背景下作为胞嘧啶的对称甲基化。 5-甲基环胞嘧啶（5MC）在雌性中的重复元素，端粒和非活性X染色体下发现，其存在与基因表达降低相关。在癌症中，DNA甲基化与肿瘤抑制基因的异常沉默有关。在中枢神经系统（CNS）中，它与学习，记忆和突触可塑性有关。我们最近发现，TET蛋白TET1，TET2和TET3构成了一个新的2-氧化甲酸酯（2OG） - 和Fe（II）依赖性二氧酶，可在DNA中催化5MC将5MC催化为5-羟基甲基胞质（HMC）。 HMC水平和TET表达/活性似乎受到严格调节。 （i）HMC存在于未分化的ES细胞的基因组DNA中，但没有分化的细胞类型，而HMC水平在ES细胞分化后降低。 （ii）TET1是急性髓样和淋巴白血病（AML，ALL）的MLL伴侣，而TET2与骨髓增生性综合征和AML有关。 （iii）在中枢神经系统中，HMC存在于小脑Purkinje神经元中的高水平；此外，TET1和TET2 mRNA在大脑中表达，TET1 mRNA在Purkinje细胞和海马锥体细胞中特别高。这些数据一起表明TET蛋白和HMC在（a）多能性和干细胞功能中的潜在功能。 （b）致癌细胞的致癌转化； （c）电动机控制，学习和记忆。在这里，我们建议执行两个单独的高通量筛选，以鉴定TET-tem蛋白质的小分子激活剂和抑制剂。第一个是一个完全的体外屏幕，在其中，我们将使用在昆虫细胞中表达的TET1，TET2或TET3的重组催化结构域在甲基化的双链DNA寡核苷酸中转化为HMC。 HMC的出现将通过使用HMC抗体来监测。第二个是基于细胞的屏幕，它依赖于HEK293细胞中TET1，TET2或TET3催化域的过表达产生的细胞表型，这些表型很容易通过高通量成像进行测定：5MC染色的丢失，HMC染色的获得，HMC染色以及HMC染色的获取以及获得。核大小增加。这些研究共同确定了调节这种新的有趣类酶功能的小分子，并且可以用作试剂，以研究其在细胞和生物体中的生物学功能。 公共卫生相关性：除了DNA字母中的四个主要基础外，A，C，G和T还包括一个次要的第五碱基，5 -Methylcytosine（5MC），其作用不成比例。 5MC的异常产生，分布和识别与发育异常和遗传疾病有关，包括自闭症谱系疾病，包括RETT综合征。我们最近确定了一个新的蛋白质家族，即将5MC转换为第六碱基5-羟基甲基胞嘧啶（HMC）的TET蛋白。 TET蛋白与癌症有关，并且在胚胎干细胞和某些类型的神经元细胞中发现HMC。在此提案中，我们将设计方法来找到TET蛋白的化学激活剂和抑制剂。这些分子将有助于进一步研究TET蛋白的生物学功能，并最终可以在临床中用于治疗RETT综合征和其他DNA甲基化疾病。