Project Summary/Abstract Activation and maintenance of cancer related pathways is associated with re-organization of the genome and chromatin landscape. Chromatin structure is important for genome function and is regulated by epigenetic modulators and architectural proteins. Epigenetic modulators have been shown to promote pro-oncogenic gene expression profiles making these factors the focus of studies to identify biomarkers and druggable targets in cancer cells. However, the mechanism and factors that regulate genome organization during oncogenic processes are largely unknown. The genome is organized into loops and topologically associated domains (TADs), which are formed by the ATP-dependent cohesin complex and maintained by binding of the architectural protein CTCF at domain boundaries. We hypothesize that epigenetic factors contribute to re-organization of key genomic loci during oncogenesis including the MYC locus. The goal of this proposal is to characterize cancer specific genome organization using the MYC locus as a model and gain insight into epigenetic mechanisms that regulate architectural proteins. In Aim 1 we will use single-molecule imaging to map the 3 Mb MYC locus by DNA fluorescence in situ hybridization (FISH) in colorectal cancer cells. We will also characterize the functional role of nuclear 3D positioning for the MYC locus and association with nuclear structures. In Aim 2 we will identify epigenetic modulators that regulate genome organization at the MYC locus in colorectal cancer cells by performing a high-throughput CRISPR based imaging screen using DNA FISH. We will also elucidate the mechanism by which these factors organize the genome and regulate CTCF and cohesins by mass spectrometry, high-resolution mapping, and ChIP-seq. Finally, in Aim 3 we will map organizational changes at the MYC locus in response to the induction of epithelial-to-mesenchymal transition (EMT), a process associated with invasiveness in cancer. Additionally, in Aim 3 we will determine how architectural proteins regulate EMT marker genes and the phenotypes associated with this cellular transition. The studies outlined in this proposal will determine how changes to oncogenic genome organization occur at the single cell level and identify novel epigenetic factors previously uncharacterized for their roles in regulating genome organization in colorectal cancer cells and during EMT.