Final Activity Report Summary - RHVME (The Role of Histone Variant macroH2A in Epigenetics) The human genome contains approximately 35,000 genes, of which, only a fraction are actively transcribed at any given cell type and time or in response to environmental stresses. The inheritability, plasticity, and regulation of gene expression is essential for cell function. Loss of proper gene expression can lead to a large number of human diseases and developmental abnormalities. The human genome contains approximately two meters of DNA packaged into the cell nucleus. The folding and compacting of the DNA into the nucleus is a highly ordered and dynamic protein-mediated process. DNA is packaged into units called nucleosomes consisting of approximately 150 bp of DNA wrapped twice around a histone octamer containing two molecules of each of the four core histone proteins (H2A, H2B, H3, and H4). How the chromatin is folded and packaged regulates the accessibility to other chromatin binding proteins such as transcription factors and thus plays a central role in gene regulation. In this project we investigated how a histone variant protein called macroH2A1 marks genes for repression. Interestingly, macroH2A1 has two isoforms called macroH2A1.1 and macroH2A1.2. The macroH2A1.1 isoform but not the macroH2A1.2 isoform binds O-acetyl-ADP-ribose (OAADPR) in vitro. OAADPR is an especially interesting small molecule as it is produced by sirtuin proteins, which have been shown to regulate the ageing process, cancer, and several other human conditions. Surprisingly, we found that the macroH2A1.1 and macroH2A1.2 isoforms of macroH2A1 function identically with regards to the regulation of gene expression. These observations suggest that metabolic components such as OAADRP do not directly affect gene expression via macroH2A. We have determined the effect deleting and over-expressing the macroH2A1 gene has on the gene expression profile of the cell. Furthermore, to gain an understanding on how macoH2A1 regulates the immune response to virus infection we have also determined the effect deletion and over-expression of macroH2A1 has on cells infected with virus. We are currently combining this data with a genome wide mapping of where macroH2A1 binds on the DNA to generate a function map of how macroH2A1 regulates gene expression on a genome-wide scale.