The overall goal of this project is to understand how two related mammalian base excision repair enzymes, MED1 and TDG, maintain genomic and epigenomic stability at CpG sites, by preventing both mutations and altered methylation patterns. DNA methylation is an important epigenetic modification of the mammalian genome consisting in the formation of 5-methylcytosine from cytosine at CpG sites. DNA methylation increases the risk of mutations, because both methylated and unmethylated cytosines have a tendency - higher for the former - to spontaneously deaminate, generating thymine and uracil, respectively. Indeed, deamination at CpG sites is estimated to cause nearly one-third of all mutations in cancer and human genetic diseases. In order to maintain genomic stability at CpG sites, MED1 (also known as MBD4) and TDG, remove the offending thymine or uracil. Organisms also establish and regulate the proper chromatin states/DNA methylation patterns at CpG sites (epigenomic stability). Alterations in epigenomic stability occur in cloned mammals as well as in cancer and thus have implications for both stem cell biology and cancer diagnosis/prognosis/treatment. We made the unexpected discovery of embryonic lethality associated with TDG nullizygosity and found that TDG is required for DNA demethylation at some CpG-rich promoters and modulation of other epigenetic states, such as histone H3 acetylation. These observations suggest a model in which MED1 and TDG promote both the genomic and epigenomic stability of CpG sites, in order to ward off against developmental defects, mutagenesis and tumorigenesis. The Specific Aims are: 1) Characterize the requirement of TDG during development and its role in DNA demethylation and chromatin modification. We will determine whether TDG catalytic activity is required for normal development using a knock-in strain and assess which TDG function is required for ES cell differentiation in vitro and for phenotypes in mouse embryo fibroblasts. We will also study by ChIP-seq the relationship between promoter occupancy by TDG and methylation patterns on a genomic scale; and determine whether TDG is involved in demethylation of the paternal genome after fertilization. 2) Evaluate the in vivo cooperation between MED1 and TDG in avoidance of mutations and altered methylation, by measuring: G:T and G:U mismatch repair in genetically defined cell lines (deficient in TDG, MED1 or both), mutation frequency and altered methylation of single- and MED1-TDG double-mutant mice. 3) Evaluate the role of MED1 and TDG in tumorigenesis. Using the Min mouse model, we will evaluate the role of MED1 and TDG in intestinal tumorigenesis. We will also analyze TDG alterations in human cancer. These studies will shed light on the emerging link between genomic and epigenomic stability at CpG sites and the employment of the DNA repair machinery to effect DNA demethylation.