The molecular basis of immune dysfunction in the elderly remains to be elucidated. Several lines of evidence suggest a role for genomic DNA alterations in this process. There is compelling evidence, based on analysis of total methylcytosine content and of the methylation status of specific genes, that points to altered cytosine methylation in genomic DNA with aging. Changes in DNA methylation have also be implicated in altered gene expression, malignant transformation, sequence-specific strand breaks, translocations, point mutations, and the development of autoimmunity. Of critical importance to the understanding of the relevance of DNA methylation changes to the aging process is: 1) elucidation of the extent to which genomic methylation changes occur with aging in regulatory DNA sequences, and 2) assessment of whether the methylation changes have functional consequences on gene expression. A technology for genome scanning has been implemented by our group that allows detection of methylation changes in CpG islands which occur close to gene coding sequences. This technology uses computerized analysis of two-dimensional (2-D) separations of genomic restriction fragments. The investigators have also developed the means to clone, sequence and map any fragment of interest detected in 2-D patterns. This group has applied this technique to the study of T cell methylation changes with aging. Advantages of T cells include their ready availability, the ability to precisely define their differentiation state and clonality, and the large amount of relevant data and materials available for this population. In preliminary studies they have established the feasibility and relevance of this approach. In an analysis of a subset of CpG islands they have detected a locus in murine T cells which undergoes a methylation change with aging, and have identified a striking heterogeneity in T cell DNA methylation patterns at the clonal level. They now propose to undertake detailed investigations of methylation changes in CpG island-containing genomic fragments, determine the functional significance of the methylation changes that occur with aging, and examine mechanisms contributing these changes. The hypothesis to be tested is that age-dependent DNA methylation changes occur in transcriptionally relevant CpG islands, and commonly affect expression of the associated genes.
The Specific Aims are to: 1) Identify CpG islands that undergo a detectable methylation change with aging, 2) Determine the effect of age-dependent CpG island methylation changes on expression of the corresponding genes, and 3) Determine if clonal heterogeneity in T cell CpG island methylation patterns increases with aging. These studies will thus provide insights into the role of DNA methylation in modifying gene expression with aging. This approach may also prove useful in characterizing genes that have altered expression with aging in lymphocytes and other cell types, and that may contribute to the development of autoimmunity and malignancies in the elderly.
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