Research Project

Summary

Over the last decade a series of new methods have been developed to investigate DNA methylation profiles across large DNA regions - chromosomes and even entire genomes. Unfortunately, all these methods exhibit significant limitations as they require large amounts of DNA, interrogate only a small fraction of methylatable nucleotides or are able to scan only short DNA fragments.  This Genome Canada project was dedicated to development of a new technology for genome- wide DNA methylation, or methylome, analysis.

Fine mapping of 5-methyl- and 5-hydroxymethyl-cytosines may significantly contribute to our understanding of development, tissue differentiation, aging, and numerous other important questions of the life sciences. Even more importantly, the genome-wide DNA modification studies may allow for the identification of unique and informative signatures of common human diseases, such as cancer, diabetes, schizophrenia, multiple sclerosis, among numerous others. This effort may be of critical importance for the developing of new early diagnostic tests and individualized treatments.
 
The first part of the project focused on the development of novel synthetic AdoMet cofactors and mutant DNA methyltransferases (MTases) for reporter-labeling of unmethylated cytosines via DNA methyltransferase-directed transfer of activated groups (mTAG).  Development of cofactors for use in a 2-step labeling protocol was successful, as well as the engineering of multiple MTases variants with enhanced transalkylation activity compared to the wild type enzymes.  The second part of the project integrated this methylome labeling protocol with affinity capture for subsequent analysis using tiling and SNP microarrays.  This approach is now in the final stages of testing on complex epigenomes.