The major objective of this application is to gain mechanistic insights into the roles of Tet-induced oxidation products of 5-methyl-2'-deoxycytidine (5-mdC) in epigenetic regulation in mammals. Methylation at the C5 position of cytosine at CpG dinucleotide sites constitutes a major mechanism of epigenetic regulation in mammals. It was not clear whether 5-mdC in the mammalian genome can be converted to its unmethylated counterpart through a process that is independent of DNA replication (a.k.a. active cytosine demethylation). Recent discovery of the functions of ten-eleven translocation (Tet) family of enzymes offered important new insights into active cytosine demethylation in mammals. Tet enzymes can oxidize 5-mdC in DNA to yield 5- hydroxymethyl-2'-deoxycytidine (5-HmdC), 5-formyl-2'-deoxycytidine (5-FodC) and 5-carboxyl-2'-deoxycytidine (5-CadC). In addition, 5-FodC and 5-CadC can be efficiently cleaved from DNA by thymine DNA glycosylase, and subsequent action by the base excision repair machinery can result in the ultimate replacement of 5-mdC with an unmethylated dC. In this R21 application, we propose experiments to explore the novel mechanisms of Tet-mediated oxidation products in epigenetic regulation and the proposed research is organized according to the following two specific aims: (1) To exploit the cellular roles of de novo DNA cytosine methyltransferases, DNMT3a and DNMT3b, in the direct conversions of 5-HmdC, 5-FodC and 5-CadC to unmethylated dC. We will assess the functions of DNMT3a and DNMT3b in the transformations of 5-HmdC, 5-FodC and 5-CadC to unmethylated dC in human cells and how this process is modulated by histone epigenetic marks. (2) To examine the occurrence, biosynthesis and transcriptional perturbation of potential glucose-conjugated derivatives of 5-HmdC in human cells. Based on our newly developed method for the quantification of base J in trypanosome DNA, we will examine the formation of the analogous glycosylated 5-HmdC in human cells, identify the potential enzyme(s) involved in this conversion, and assess the impact of the glycosylated 5-HmdC on transcription in human cells. The outcome of the proposed research will yield important new knowledge for understanding the role of Tet-mediated oxidation products of 5-mdC in epigenetic regulation in mammals. Exposure to many environmental agents is known to stimulate the generation of reactive oxygen species, which could also result in the inadvertent oxidation of 5-mdC to 5-HmdC, 5-FodC and 5-CadC. Thus, the proposed research may also provide new knowledge for understanding how environmental exposure perturbs epigenetic mechanisms of gene regulation.
Oxidation of 5-methylcytosine in DNA by the ten-eleven translocation proteins is important in the epigenetic regulation of gene expression, and exposure to various environmental toxicants can also stimulate the generation of reactive oxygen species and induce the same oxidation products of 5-methylcytosine. The emphasis of the present application is placed on assessing the novel enzyme-mediated transformations of these oxidation products and their implications in epigenetic regulation. The proposed research may provide mechanistic insights about how environmental exposure leads to aberrant epigenetic regulation.
