PROJECT SUMMARY/ABSTRACT. Thymine DNA glycosylase (TDG) is a key enzyme in somatic cell reprogramming. TDG catalyzes DNA demethylation and reactivation of specific sets of genes to induce a pluripotent cell fate in somatic cells. However, there is a fundamental lack of understanding of how TDG is directed to its target genes to catalyze dynamic demethylation to facilitate their timely activation and thereby cell fate conversion. Thus, our understanding of epigenetic mechanisms of cell fate change remains incomplete. The long-term goal is to understand fundamental mechanisms by which factors such as TDG control processes requiring widespread epigenetic changes, including normal embryonic development and somatic cell reprogramming. Our recent studies have identified the nucleotide excision repair protein XPC as a potent activator of TDG-dependent DNA demethylation genome-wide and preferentially at gene enhancers bound by transcription factor HIF1a. The overall objective of this project is to determine how XPC and TDG together integrate signals to regulate active DNA demethylation and epigenetic reprogramming. The central hypothesis is that XPC is a novel regulator of dynamic DNA demethylation via TDG and thereby plays an essential role in pluripotent cell fate transitions. The rationale of our work is that by deciphering how XPC regulates TDG-dependent DNA demethylation in response to HIF1a, we will gain a new understanding of how the XPC-TDG-HIF1a axis integrates signals to coordinately regulate epigenetic changes that contribute to normal development and reprogramming. The central hypothesis will be tested by pursuing three Specific Aims: 1) Identify mechanisms by which XPC-TDG-HIF1a axis regulates DNA demethylation and metabolism in somatic cell reprogramming; 2) Identify mechanisms by which XPC promotes recruitment of TDG by HIF1a; and 3) Identify the role of post-translational modifications of TDG via SUMOylation in regulating DNA demethylation dynamics. Under the first aim, genome-wide and functional studies will be employed to identify how XPC and TDG regulate epigenetic changes at HIF1a-regulated gene loci to induce transcriptional and metabolic changes favorable for reprogramming. In the second aim, biochemical and loss-of-function studies will be performed to identify the role of XPC and novel associating factors in directing TDG to specific gene enhancers for demethylation. Under the third aim, the role of XPC in regulating post-translational modifications of TDG and the effect of TDG SUMOylation on demethylation dynamics and specificity will be directly measured in living cells using single particle tracking. The proposed research is innovative because new activities of XPC in DNA demethylation, through post-translational modification of TDG and novel TDG-HIF1a complex assembly mechanism, will be uncovered using new tools. This contribution is significant, because the regulatory components and epigenetic mechanisms we identify are likel...