The goal of this proposal is to examine the role of DNA methylation during T cell development utilizing a conditional gene ablation strategy. Changes in DNA methylation have been shown to be correlated with the expression or rearrangement of many lymphoid specific genes. However, many of these observations have arisen from analyses of cultured cells and treatment of these cells with artificial demethylating agents. The precise role methylation plays in vivo in controlling expression of these genes has not been determined. To address these questions, this study will systematically examine the effect of DNA methylation on lymphoid cell development in vivo in which DNA methyltransferase (Dnmt) will be specifically ablated at different stages of thymocyte development using the bacterial phage P1 Cre/loxP recombination system. Restricted ablation of Dnmt alleles in T lineage cells will presumably overcome the embryonic lethality of global Dnmt disruption. Depending on the promoter (CD4 or lck) used to express Cre recombinase, Dnmt ablation could occur at the DN stage, late DP stage, and/or after activation in the periphery. The extent of Dnmt inactivation and DNA methylation in relevant T lineage subsets will be evaluated. Thymic, lymph node and splenic cellularity and cellular profile will be analyzed. The ability of peripheral T cells to develop antigen specific responses and a memory phenotype will be assessed. Significantly, this system provides a powerful tool to examine the role of methylation in tumorigenesis, especially lymphomas and leukemias which may arise at a greater incidence in T cells lacking regulatory methylation. The immediate career goals are to study the molecular mechanisms of T cell development and develop a model system to evaluate the significance of loss of gene regulation via DNA modification. The long term goal will be to use a blend of clinical and basic science investigation in studying both immune system development as well as the interplay between infectious agents and the host immune system as a faculty member of a major university. The Research Career Award would enable me to conduct independent research in basic science in the field of immunology, to gain additional knowledge and skills in the field of developmental immunology by attending immunology meetings and presenting research posters and talks.
