Title
DNA methyltransferase I, found in all cells, is a protein which methylates DNA strands during DNA replication to maintain patterns of DNA methylation from one cell generation to the next. The pattern of DNA methylation influences gene expression and can be directly inherited through successive cell divisions.
Reduced methylation is generally correlated with increased gene expression while increased methylation is associated with decreased gene expression. We are testing whether the absence of the Dnmt 1 gene has an effect on a T cell's ability to produce certain cytokines. Removing the Dnmt 1 gene in mice will allow us to examine whether demethylation is required to permit cytokine production or whether cytokine production results in demethylation merely as a marker of gene expression.
The cytokines to be studied include IL 2, IL4, IL3, and IFNg. Interleukin 2, (or IL2), is produced by all T cells upon activation and drives them to proliferate. IL4 is produced by the T helper 2 cell CD4+ T cells to trigger the humoral response. IFNg (interferon gamma) is produced by T helper 1 cells, and it promotes cell-mediated immunity. IFNg and IL 3 both have sites in the promoter which are demethylated in cells that express these genes. Additionally, previous studies indicate a correlation of increased IFNg and IL3 production upon demethylation.
The project involves three parts. First, the mice will be genotyped for either the 2 lox or WT alleles to confirm that the correct mice are used in these experiments. Second, the DNA must be tested for either the 2 lox or 1 lox allele, to indicate whether the Dnmt 1 gene has been fully deleted in the T cells being studied. Lastly, Real Time PCR will be used to determine the relative expression of the cytokines IL 2, IL 3, IL 4, and IFNg in naove T cells from these mice. The hypothesis is that if demethylation causes increased gene expression, then the T cells lacking the Dnmt 1 gene will produce more of these cytokines compared to the T cells in which the Dnmt 1 gene is undeleted
