At least half of the human genome is derived from transposable elements (TEs). While some investigations regard TEs as ?parasitic? DNA, other studies suggest that TEs play a more constructive role in genome evolution by providing raw material for new biological functions. TEs commonly harbor active cis- regulatory elements that are occasionally co-opted during evolution to wire new gene regulatory networks. TEs remain under-analyzed in high-throughput data because of methodological hurdles associated with their repetitive nature. Thus, the impact of TEs on the regulation of the human genome, both in normal development and disease, remains largely uncharacterized. We propose to develop advanced genomics approaches to assess and clarify the impact of TEs in regulatory innovation, conservation, and in human diseases. In Aim 1 we combine a novel statistical framework with massively parallel reporter gene assays to understand TE sequence features that contribute to gene regulation. We will take advantage of the repetitive nature of TEs to link sequence changes in different copies of TEs to epigenetic and functional differences, and test their regulatory activities using a new genome integrated massive parallel reporter gene assay. In Aim 2 we will extend the models developed in Aim 1 to understand the role of TEs in shaping the 3D topology of the genome, which is intimately connected to genome function. We will quantify the extent to which TEs underlie the conservation and/or divergence of genome topology across mammalian species. In Aim 3 we will develop technologies to detect TE-gene fusions linked to disease. We aim to detect cases where epigenetically de-repressed TEs initiate transcripts that splice into downstream genes, resulting in TE-gene fusion chimeric RNA and protein products. We will develop tools to detect such TE-gene fusion transcripts, and will adapt CRISPR-based genetic and epigenetic tools in order to manipulate TEs, which will allow us to establish whether TEs play a causal role in this type of abnormal gene activity. In Aim 4 we will test the hypothesis that epigenetic inhibitors commonly used for therapeutics alter TEs? epigenetic regulation. Through the aims of this proposal we hope to develop an understanding of what sequence features drive the regulatory potential of TEs, and the modes of evolution followed by different families of TEs during regulatory network evolution. Such an understanding will improve our picture of regulatory network evolution by including the effects of TEs, a major class of fast evolving sequences that have been largely ignored in functional genomics studies. The methods developed in this proposal will have a high impact on the utility of data produced by consortia such as ENCODE, Roadmap, TCGA, and other large-scale projects, which currently discard most TE derived sequences from their data. Such improvement will in turn accelerate research into understanding the impact of TEs? on normal gene regulation and in human diseases.