Project Summary Distinct histone modifications, on N or C-terminal tails, act as a “histone code” to elicit downstream events. Histone subunit post-translational modification (PTM), such as methylation, acetylation and phosphorylation regulates epigenetic regulation and gene expression in diseases such as cancer. Here we identified a new PTM on H3 called hydroxylation on proline 16, which is catalyzed by proline hydroxylase EglN2. Our preliminary data show that EglN2-mediated H3 Pro16-OH leads to increased Lysine (K)-Specific Demethylase 5A (KDM5A) binding corresponding with decreased H3K4me3 in breast cancer. We hypothesize that H3 prolyl hydroxylation mediated by EglN2 recruits KDM5A therefore controlling gene expression important in breast cancer. This is the first study reporting H3 Prolyl hydroxylation and its potential role in epigenetic regulation and gene expression in cancer. In Specific Aim 1, we will determine the effet of H3 prolyl hydroxylation on epigenetic regulation and gene expression on a genome wide scale in breast cancer cells. In Specific Aim 2, we will elucidate the molecular mechanism by which H3 prolyl hydroxylation regulates epigenetic reprogramming by recruiting KDM5A. In Specific Aim 3, we will elucidate the molecular mechanism by which H3 prolyl hydroxylation regulates Wnt/b-Catenin signaling in breast cancer cells. Successful completion of this proposal will characterize a new H3 post-translational modification in its epigenetic regulation and gene expression important in breast cancer.