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.