Chemical Strategies for Epigenome Wide Discrimination of 5-Hydroxymethylcytosine and 5-Methylcytosine

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Methylation of cytosine is a major modification of the mammalian genome generally associated with gene repression and regulation, with alteration of 5-methylcytosine patterns during development contributing to the regulation of gene expression and cell specification. Given its importance methods of so-called bisulfite sequencing have been developed that allow comparative 5-methylcytosine/cytosine analysis of Genomic DNA. However in addition to 5-methyl-C another cytosine modification, 5-hydroxymethylcytosine (5-hydroxymethyl-C or 5-hmC) has recently been identified as an epigenetic modification. In humans, mutations in the enzymes that make hydroxymethyl-C are associated with myeloid malignancies. Evidence also suggests that's that 5-hydroxymethyl-C is an epigenetic marker of "stem-cellness" and neuronal lineage specification. However current DNA sequencing techniques cannot differentiate between 5-methyl-C and 5-hydroxymethyl-C. Indeed it is certain that previous studies have misread 5-hydroxymethyl-C as 5-methyl-C. The presence of 5-hydroxymethyl-C makes it necessary to not only re-evaluate existing DNA methylation data, but also to develop a new method to determine the relative distribution and changes of 5-hydroxymethyl-C in human tissues of healthy and diseased status. The basis of this proposal is to develop chemistry based approaches to allow the genome wide analysis of 5-methyl-C and 5-hydroxymethyl-C.