Project summary Endogenous retroviruses (ERVs) are ubiquitous genomic parasites originating from ancient infectious retroviruses, and constitute 8% of the human genome. Large-scale genomic studies have revealed ERVs as major source of transcriptional regulatory elements in mammalian cells, suggestive of an extensive role in driving the evolution of gene regulatory networks in both health and disease. However, the specific consequences and biological significance of ERV regulatory activity remains controversial and not well understood. The proposed research seeks to investigate emerging roles for ERVs in (1) the evolution of immune regulatory networks and (2) gene dysregulation in cancer. First, we will investigate a potentially fundamental role for ERVs driving regulatory evolution of innate immune responses in mammals, through genome-wide epigenetic and transcriptional profiling of the interferon response in cells derived from diverse mammalian species. This work is positioned to uncover a recurrent role for ERVs in regulating immune pathways, which may reveal pervasive underappreciated differences in immune responses across species. In a second line of research, we seek to investigate how the epigenetic derepression of ERVs in cancer contributes to pathological gene expression. The transcriptional reactivation of ERVs is a hallmark of many cancers, but their potential contribution to disease remains poorly understood. We will investigate how reactivated ERVs might cause widespread regulatory dysfunction as a global source of cancer-specific promoters, enhancers, and noncoding RNAs. Focusing on colorectal cancer as a model, we will repurpose pooled CRISPR screening to investigate how reactivated ERVs regulate phenotypes contributing to oncogenesis. Our interdisciplinary approach synthesizes functional genomics, genome engineering, evolution, and disease to form a comprehensive platform for deciphering the consequences of ERVs on mammalian biology.