Regulation of T-cell DNA Methylation

T 细胞 DNA 甲基化的调节

• 批准号: 6886309
• 负责人: JOHN T ATTWOOD
• 金额: $ 12.85万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-05-01 至 2007-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6886309
• 关键词: DNA methylation; RNA binding protein; T lymphocyte; biological signal transduction; enzyme biosynthesis; genetic regulation; laboratory mouse; lymphocyte proliferation; methyltransferase; polymerase chain reaction

DESCRIPTION (provided by applicant): The long-term objectives of this grant proposal are to allow the applicant to further the understanding of the role of DNA methyltransferases in T cells. This grant will also allow the applicant to pursue a career as a research scientist in the field of T cell biology and the specific aims and experimental design of the proposal will support the development of his career as an independent investigator. The outcome of this research will promote better understanding of dysregulated DNA methylation that occurs in autoimmune disease. Specific Aim 1: Examine the hypothesis that DNA methyltransferases regulate gene expression in both resting and activated T cells and that proteins involved in DNA methylation are differentially expressed in T cell subsets, as follows: (i) the location of DNA methyltransferases in resting and activated T cells determined by immunofluorescence; (ii) the role of Dnmt1 and Dnmt3a in regulating gene function in resting T cellsdetermined by treatment with morpholino antisense oligonucleotides and measurement of T cell proliferation and gene array analysis; (iii) proteins that complex with Dnmt1 and Dnmt3a in vivo identified by co-immunoprecipitation; and (iv) differential expression of DNA methyltransferases, a putative demethylase and methylcytosine binding proteins determined in CD4+ and CD8+ T cell subsets by real time RT-PCR and western blotting. Specific Aim 2: Examine the hypothesis that the regulation of expression of DNA methyltransferase 1 occurs at the level of transcript stability involving RNA-binding proteins as follows: (i) the kinetics of Dnmt1 upregulation in T cells determined at the protein level by western blotting; (ii) changes in Dnmt1 mRNA stability in resting and activated T cells confirmed by treatment with actinomycin D and real time RT-PCR; (iii) transcriptional upregulation of Dnmt1 mRNA during T cell activation determined by measurement of primary transcripts in resting and activated T cells using real time RT-PCR; (iv) determine whether RNA-binding proteins mediate changes in Dnmt1 transcript stability in activated T cells by UV cross-linking analysis; (v) identify RNA-proteins by affinity chromatography, polyacrylamide gel electrophoresis and protein microsequencing; and (vi) determine intracellular signaling pathways involved in the upregulation of Dnmt1 mRNA by treatment with specific inhibitors of signaling pathways.

描述（由申请人提供）：本资助提案的长期目标是让申请人进一步了解DNA甲基转移酶在T细胞中的作用。该补助金还将允许申请人在T细胞生物学领域从事研究科学家的职业，该提案的具体目标和实验设计将支持他作为独立研究者的职业发展。这项研究的结果将促进更好地了解发生在自身免疫性疾病中的DNA甲基化失调。具体目标1：检查DNA甲基转移酶调节静息和活化T细胞中的基因表达以及涉及DNA甲基化的蛋白质在T细胞亚群中差异表达的假设，如下：（i）通过免疫荧光测定静息和活化T细胞中DNA甲基转移酶的位置;（ii）Dnmt 1和Dnmt 3a在静息T细胞中调节基因功能的作用，通过用吗啉代反义寡核苷酸处理和测量T细胞增殖和基因阵列分析来确定;（iv）通过真实的时间RT-PCR和蛋白质印迹法测定的在CD 4+和CD 8 + T细胞亚群中DNA甲基转移酶、推定的脱甲基酶和甲基胞嘧啶结合蛋白的差异表达。具体目标二：检验DNA甲基转移酶1表达的调节发生在涉及RNA结合蛋白的转录物稳定性水平的假设，如下：（i）通过蛋白质印迹在蛋白质水平测定的T细胞中Dnmt 1上调的动力学;（ii）通过用放线菌素D处理和真实的时间RT-PCR证实的静息和活化T细胞中Dnmt 1 mRNA稳定性的变化;（iii）通过使用真实的时间RT-PCR测量静息和活化的T细胞中的初级转录物来确定在T细胞活化期间Dnmt 1 mRNA的转录上调;（v）通过亲和层析、聚丙烯酰胺凝胶电泳和蛋白质微测序鉴定RNA-蛋白质;和（vi）通过用信号传导途径的特异性抑制剂处理来确定参与Dnmt 1 mRNA上调的细胞内信号传导途径。