PROJECT 1 | SUMMARY Mechanisms underlying the epithelial to mesenchymal transition (EMT)—a normal developmental process and an early event at the onset of epithelium-derived cancers—are minimally understood. Our findings, corroborated by others in multiple biologically relevant models, have established mitotic gene bookmarking—occupancy of target genes by transcription factors during mitosis—as a novel dimension to epigenetic control of lineage commitment and cell identity. Because EMT results from loss of the epithelial phenotype, Project 1 will address whether disruption of mitotic gene bookmarking constitutes a key mechanistic component of EMT. Recently, we have shown that depletion of the RUNX1 transcription factor in normal mammary epithelial (NME) cells results in EMT in both in vivo and cell-based models, indicating that RUNX1 stabilizes the normal ME phenotype. Our preliminary data show that in ME cells undergoing mitosis, RUNX1 bookmarks genes involved in cell proliferation, growth and maintenance of the epithelial phenotype. These findings provide the rationale for our hypothesis that mitotic bookmarking of target genes by RUNX1 is a key epigenetic mechanism for maintenance of the normal mammary epithelial phenotype, and perturbation of this mechanism leads to EMT. Innovative molecular and cellular experimental strategies will be combined with state-of-the-art genomics approaches to investigate how the RUNX1 mitotic epigenome contributes to initiation of EMT, an early event that leads to development of breast cancer. Aim 1 will use candidate and unbiased CRISPR screens to determine how RUNX1 bookmarked genes are physiologically linked to the mammary epithelial phenotype and whether estrogen receptor signaling—a key determinant of NME phenotype—plays a role in regulation of RUNX1- bookmarked genes. Aim 2 will employ Hi-ChIP and HIPMap to establish whether RUNX1 bookmarked genes reside in same genomic neighborhoods for coordinate control. We will assess whether disruption of these genomic regions confers EMT. Aim 3 will take advantage of degron-based RUNX1 depletion to investigate the regulatory impact of RUNX1 mitotic bookmarking on target gene expression and maintenance of the NME phenotype. Project 1 studies will address significant gaps in current knowledge of the epigenetic mechanisms that regulate EMT. Our findings will establish RUNX1 mitotic bookmarking as a unique dimension to epigenetic control of the normal mammary epithelial phenotype, which when disrupted, results in EMT, a key early event in the development and progression of breast cancer.