Project Summary The development of distant metastases accounts for a significant proportion of breast cancer mortality; as many as 30% of patients initially diagnosed at an early stage will eventually progress to metastatic disease. A key early step in metastatic progression is an increase in cellular plasticity that enables a subset of tumor cells to lose residual epithelial features and gain migratory and invasive behavior, molecularly reflected in the epithelial to mesenchymal transition (EMT). Considerable evidence now points to cancer-associated EMT as a highly dynamic and reversible process with disseminated cancer cells exhibiting many hybrid intermediate states (partial-EMT) proposed to possess the greatest potential for aggressive, stem-like, behavior. However, the epigenetic mechanisms that control such phenotypic plasticity and the role of this process in early invasion remain incompletely understood. Recently we discovered that the local regulation of RNA polymerase (Pol II) pause release by the histone methyltransferase SUV420H2 plays an important role in stabilizing the epithelial ‘identity’ of luminal breast cancer cells and in so doing, suppresses breast cancer cell invasion. Specifically, we find that the local conversion of H4K20me1 to me3 by SUV420H2 enforces RNA polymerase pausing by blocking recruitment of the MOF/MSL complex, which is in turn necessary for the acetylation of H4K16, recruitment of pTEFb and Pol II pause release. We further find that SUV420H2-mediated repression constrains the mesenchymal program in luminal breast epithelial cells, yet is directed to new sites upon TGF-β induced EMT. SUV420H2 is downregulated in triple negative/basal subtype of breast cancers, and its forced downregulation or inhibition promotes collective invasion in breast cell spheroids grown in 3D. These and other findings lead us to propose that the relaxation of SUV420H2-mediated Pol II pausing control is one source of the epigenetic plasticity and transcriptional heterogeneity that underlies breast cancer cell adaptation and the emergence of tumor cells with invasive properties. Using a combination of precision run-on sequencing (Pro-seq) and native chromatin analyses via CUT&Tag technology, we will determine the how the SUV420H2 mediated pause constraints enforces phenotypic stability and how loss of these constraints allows for transcriptional promiscuity. We will explore the role of the HEXIM1 / 7SK snRNP complex as a novel ‘reader’ of histone H4 modifications and its role in SUV420H2-mediated pause control. Lastly, we will determine the impact of dysregulated Pol II pausing dynamics on transcriptional diversity and the emergence of breast cells with invasive “leader” potential in a 3D spheroid model of collective invasion. Over the long term, the results of our studies will provide important insight into the mechanisms underlying epigenetic plasticity and its role in tumor cell adaptation, and a framework for the development of nove...