DNA methylation is intrinsically linked to chromatin structure and gene expression. Changes in DNA methylation during development suggest that it may play a role in development and in tissue-specific differentiation. Restriction Landmark Genomic Scanning (RLGS) will be used to identify sites of developmental and tissue specific differences in DNA methylation. RLGS is a method for the two dimensional display of end-labeled DNA restriction fragments. Using Notl as the restriction landmark, RLGS targets gene rich CpG island regions and detects differences in DNA methylation since cleavage by Notl is methylation sensitive. RLGS will be performed on a collection of different tissues from C57BL6/J mice of different ages. Green Fluorescent Protein (GFP) transgenic mice in conjunction with fluorescence activated cell sorting will be used to mark and purify selected populations of cells within a tissue for RLGS analysis. Magnetic Cell Sorting and Centrifugal Elutriation methods will be used to purify """"""""differentiating"""""""" cell populations within adult mice for RLGS analysis. New advances in development of """"""""virtual"""""""" RLGS software provide unique opportunities to rapidly clone """"""""in silico"""""""" the genomic regions with tissue specific differences in DNA methylation/chromatin structure. Quantitative and semi-quantitative methylation sensitive PCR, along with bisulfite sequencing, will be used to delineate the boundaries and density of methylation in the regions. The tissue expression patterns of genes associated with the region will be accessed using available microarray data to evaluate possible """"""""long distance effects"""""""" of chromatin structure on gene expression. Quantitative RTPCR methods will be used to evaluate expression of genes with apparent CpG island promoter DNA methylation. It is becoming increasing clear that epigenetic alterations have a major role in aging and the development of Cancer and some other diseases. Yet our understanding of the role of epigenetic alterations during normal development is poorly understood. Recent advances in completing the sequence of the mouse genome and the vast array of bioinformatics tools that are now available makes this an opportune time to undertake a systematic, genome-wide investigation of developmental and tissue-specific differences in genome methylation and assess its impact on gene expression and differentiation.
