Genome-wide cytosine methylation analysis

DESCRIPTION (provided by applicant): We propose to develop the techniques that allow cytosine methylation patterns to be quantified genome-wide. To achieve these goals, a combination of resources have to be assembled: a technique to isolate a distinctively-methylated fraction of the genome, and a reagent that allows the genomic source of this DNA to be identified quantitatively. We have successfully developed a ligation-mediated polymerase chain reaction (LM-PCR) technique to isolate the hypomethylated fraction of the mouse genome, and are using this to hybridize to bacterial artificial chromosome (BAC) microarrays developed by collaborators at the Roswell Park Cancer Institute (RPCI). Cytosine methylation represents the known intersection between the influence of diet on cancer predisposition and on genetic regulation. Single carbon donor molecules in the diet (such as folate, choline and methionine) have been found to influence global genomic cytosine methylation, and are implicated in mouse models of intestinal neoplasia. We need a tool to identify the loci that are most influenced by dietary or other influences on cytosine methylation, to identify the loci that mediate these environmental effects in neoplastic transformation. We propose a project to validate and optimize the LMPCR technique, and to develop the BAC microarray resources and bioinformatic analysis tools that are necessary for analyzing the genomic distribution of methylation. We have also created a unique model system for testing de novo methylation, using a mouse X;autosomal translocation, which will be used to test the performance of our assay system. The successful development of the ability to perform genome-wide cytosine methylation studies will allow insights into this critically-important mechanistic mediator of neoplasia, and the foundation for studies of genomic methylation changes in response to dietary influences. We will make the developed assay broadly available to this institution's Cancer Center researchers through our fluorescence in situ hybridisation (FISH) core facility, for which the P.I. is scientific director.