Project Summary: Our long-term objective is to define the epigenetic mechanisms that ensure accurate genome maintenance, and to exploit these findings to improve human health. Singe-stranded DNA lesions (SSLs) are among the most abundant genomic aberrations and are the primary source of Poly-(ADP-ribose) Polymerase (PARP) activation, which orchestrates downstream repair events. In somatic cells, aberrant SSL repair promotes mutagenic events that are relevant to tumorigenesis. In cancer, SSL-inducing agents, including topoisomerase 1 inhibitors (TOP1i) and alkylating drugs, are mainstay therapies, which can be potentiated in the presence of PARP inhibitors. SSL repair is thus fundamentally important not only for normal genome maintenance but also as a potential cancer vulnerability. In this proposal, we will investigate the epigenetic control of SSL repair, a feature that remains poorly understood to date. In recent unpublished work, we have identified macroH2A1, an abundant histone H2A variant in mammalian chromatin, as an effector of this process. MacroH2A1 exists as two alternative splice isoforms, macroH2A1.1 and macroH2A1.2, and aberrant macroH2A1 splicing is a shared albeit poorly understood feature of many cancers. MacroH2A1.1, but not the other isoform, binds poly-(ADP-ribose) derivatives, and our preliminary data point to a macroH2A1.1-specific role in SSL removal. We further find that macroH2A1.1 levels are inversely correlated with sensitivity to SSL-inducing agents in cancer cell lines. We hypothesize that changes in SSL repair proficiency due to altered macroH2A1 splicing have a direct impact on mutation burden, tumor development and cancer treatment response. In Aim 1, we will investigate the role of alternatively spliced macroH2A1 isoforms in SSL repair, identify specific repair effectors and assess the role of the damage-proximal chromatin environment on repair kinetics using innovative cell-based reporters. We will then investigate how macroH2A1 isoform manipulation affects cancer cell sensitivity to SSL-inducing agents. Aim 2 will determine the impact of macroH2A1 on genome integrity specifically in the context of TOP1-related lesions, which result in increased mutation burden that is relevant to human cancer. What protects the genome from excessive TOP1 activity remains unknown. We hypothesize that macroH2A1 isoforms control TOP1-associated DNA damage and mutagenesis. We will test these predictions using genomic approaches and cell-based models. Aim 3 will establish a proof of principle for macroH2A1 function in cancer in the context of impaired SSL repair. First, we will determine the dependency of TOP1-associated tumorigenesis on macroH2A1.1 in a mouse model. Then, we will test whether macroH2A1 splicing imbalance in human tumor organoids predicts sensitivity to SSL- inducing drugs and PARP inhibitors. Collectively, these studies promise to transform current models of SSL repair and advance our understanding of the epigenetic ...