Regulated patterns of chromatin structure and of DNA methylation on cytosines and CpG dinucleotides have been suggested to provide the basis for a stable and heritable program in mammals, whereby developmental decisions regarding expression of tissue-specific genes within individual cells are pre-programmed and passed from one cell generation to the next in an epigenetic manner. Correlative and in vitro data suggest that DNA methylation contributes to the regulation of T cell development and function, including T cell receptor gene arrangement, allelic exclusion and differential cytokine gene expression. However, the role of DNA methylation in the development and function of T cells in vivo has not been tested directly. We have used the Cre/loxP recombination system to create mice in which the major DNA methyltransferase gene (Dnmt1) is selected and efficiently ablated during T cell development either at the CD4-CD8- (DN) stage (in lckCreDnmt/2lox mice) or at the subsequent CD4+CD8+ (DP) stage (CD4CreDnmt/2lox mice). The phenotypes of these mice differ markedly. LckCreDnmt/2lox mice have a marked reduction in the numbers of thymocytes and mature CD4 and CD8 T cells, aberrant CD4 and CD8 TCRbeta/CD3/low/- SP thymocytes and T cells, and increased numbers of TCRgammadelta thymocytes. In contrast, T cell development in CD4CREDnmt/2lox mice is essentially normal. We propose to use these unique mice to address:
Aim 1 : Determine the role of DNA methylation in the function of mature CD4 and CD8 T cells. Hypotheses: Dnmt1-deficient naive CD4 and CD8 T cells from CD4CreDnmt/2lox mice will produce effector cytokines more readily after primary activation than naive T cells from controls; the ability of CD4 and CD8 T cells to develop and maintain a heritable, polarized type 1 versus type 2 pattern of cytokine gene expression will be reduced in CD4CreDnmt/2lox mice; CD4CreDnmt/2lox mice will develop memory/effector T cells following challenge in vivo, but their ability to maintain a polarized and protective pattern of cytokine production or cytolytic activity will be compromised.
Aim 2 : Determine if DNA methylation contributes to allelic exclusion of TCRbeta through the use of lckCreDnmt/2lox mice. Hypothesis: Allelic exclusion will be less complete in lckCreDnmt/2lox.
Aim 3 : Determine the basis for the reduced surface expression of TCRbeta/CD3 on CD4 and CD8 single- positive (SP) thymocytes and mature T cells and increased numbers of TCRbetagamma thymocytes in lckCre Dnmt mice. Hypothesis: 1) Decreased expression of TCRbeta/CD3 on SP thymocytes and T cells will reflect the emergence of these cells in the absence of positive-selection. 2) The increased numbers of TCRgammabeta thymocytes will reflect either increased locus accessibility and rearrangement of the relevant gammabeta TCR genes or reduced Notch signaling.

National Institute of Health (NIH)
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Research Project (R01)
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Immunobiology Study Section (IMB)
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Lock, Allan
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University of Washington
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