PROJECT SUMMARY Gene regulatory networks play a crucial role in determining how genes are expressed in response to cellular or environmental signals. Changes to these networks are important in evolution and disease, but the mechanisms responsible for rewiring these networks are not well understood. Transposable elements (TEs) are genomic parasites that replicate within host DNA and have long been proposed to drive the evolution of gene regulatory networks, yet their actual impact on health and disease remains controversial and poorly understood. My research group uses bioinformatic and experimental approaches to uncover important roles for TEs as components of both beneficial and pathological gene regulatory networks. Over the next five years, we will pursue research in 3 major areas that will investigate how TEs impact biological function across species, organismal, and cellular levels. In one area, we will investigate how TEs shape species-specific immunity by diversifying host transcriptomes. Building on our work establishing the impact of TEs on immune epigenomes, we are expanding our focus to immune transcriptomes. We will use long-read RNA-seq to characterize TE- derived isoforms in multiple species, and experimentally follow-up candidates with potentially significant functions. In a second area, we will study the contribution of TEs to interindividual regulatory variation. We will leverage newly developed genome graph assemblies, which enable accurate read mapping to complex structural variants including TE-derived variants. Using a TE-focused genome graph assembly, we will re-analyze population-scale epigenome studies to identify and characterize TE-derived variants with evidence of regulatory activity. In a third area, we will elucidate the epigenetic mechanisms underlying TE dysregulation in disease. TEs undergo aberrant transcriptional and epigenetic reactivation in many diseases, and this is increasingly recognized to contribute to pathogenesis through a variety of mechanisms. However, the upstream causes of disease-specific TE reactivation are poorly understood. To investigate why certain TEs become reactivated in disease, we will integrate single-cell multi-omics, functional screens, and targeted experimental studies to define the key steps involved in TE reactivation in cancer cells. Our work will integrate several cutting-edge technologies including single-cell epigenomics, long-read functional genomics, pan-genome graph assemblies, and single- cell CRISPR screening. Ultimately, our studies are positioned to demonstrate the profound impact of TEs on the evolution of species-specific immune responses and the development of diseases.