Regulation of T-cell DNA Methylation

T 细胞 DNA 甲基化的调节

• 批准号: 7062419
• 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/7062419
• 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(由申请者提供)：这项资助计划的长期目标是让申请者进一步了解DNA甲基转移酶在T细胞中的作用。这笔赠款还将允许申请者在T细胞生物学领域从事研究科学家的职业生涯，该提案的具体目标和实验设计将支持他作为一名独立研究人员的职业发展。这项研究的结果将促进对自身免疫性疾病中发生的失调DNA甲基化的更好理解。具体目的1：验证DNA甲基转移酶调节静息和活化T细胞基因表达的假说，以及参与DNA甲基化的蛋白质在T细胞亚群中的差异表达，如下：(I)DNA甲基转移酶在静息和活化T细胞中的位置；(Ii)DNMT1和DNMT3A在调节静息T细胞基因功能中的作用，通过吗啉反义寡核苷酸处理、T细胞增殖测定和基因芯片分析确定；(Iii)通过免疫共沉淀鉴定体内与DNMT1和DNMT3A络合的蛋白质；以及(Iv)实时定量RT-PCR和免疫印迹检测到的CD4+和CD8+T细胞亚群中脱甲基酶和甲基胞嘧啶结合蛋白的差异表达。具体目的2：验证DNA甲基转移酶1的表达调节发生在涉及RNA结合蛋白的转录稳定水平的假设如下：(I)蛋白质水平上的T细胞Dnmt1上调的动力学；(Ii)经放线菌素D和实时定量RT-PCR处理后，静息和激活T细胞中Dnmt1mRNA稳定性的变化；(Iii)通过实时RT-PCR测量静息和激活T细胞的初级转录产物来确定在T细胞激活过程中Dnmt1mRNA的转录上调；(Iv)通过紫外光交联分析确定RNA结合蛋白是否介导活化T细胞中Dnmt1转录稳定性的变化；(V)通过亲和层析、聚丙烯酰胺凝胶电泳法和蛋白质显微测序鉴定RNA蛋白；以及(Vi)通过用特定的信号通路抑制剂处理来确定参与Dnmt1基因表达上调的细胞内信号通路。