Chemo-Enzymatic Labeling of Epigenetics Markers

Gil Nifker, Tel Aviv University, Tel Aviv, Israel

The field of Epigenetics focuses on DNA and chromatin modifications not encoded in the DNA sequence. While the genetic sequence map is identical in all cells within a given organism, the epigenetic information varies between cells and regulates cellular phenotype and function. 5-mehylcytosine (5mc) DNA methylation is known to play a key role in diseases and differentiation mechanisms. It recently has been shown that 5mc is oxidized to 5-hydroxymethylcytosine (5hmc) in an endogenous enzymatic reaction. This new modification, 5hmc, displays tissue specific distribution and has been related to gene expression. Mapping of 5hmc patterns in the mammalian genome holds great importance for the future of epigenetic and genetic research, and in the field of personal medicine. Thus, the need for tagging and analysis tools of this modification is vital. The common procedure for tagging 5hmc is based on the use of the T4 phage β-glucosyltransferase enzyme, by the incorporation of UDP-glucose-6-N3and tagging with fluorescent dyes via click chemistry. As the synthesis of this sugar nucleotide is complex and became the bottleneck for exploring epigenetic modifications. Through the combination of organic chemistry and microbiology we are able to design sugar nucleotides and use a variety of enzymes, in order to attack this problem through parallel routs that will lead to easy and effective labeling of 5hmc. Currently we are working on a chemoenzymatic approach for the synthesis of UDP-glucose-6-N3. In addition, we have synthesized an additional sugar nocleotide and expressed a second glycosylation enzyme, T4 α-glucosyltransferase.