PROJECT SUMMARY Transcription is an essential and tightly regulated process that requires the coordination of many factors to ensure proper gene expression. Current models of transcription are predicated on stable, hierarchical interactions. These models have been challenged through recent developments in in vivo imaging, which have revealed that many transcriptional regulatory proteins interact transiently with chromatin. Instead of relying on stability, occupancy at target loci is achieved through more frequent interactions resulting from the formation of high local-concentration assemblies within nuclei, called hubs. Little is known about the functional impacts of hub formation on transcription, how hubs alter the kinetics of regulatory proteins and how hubs function in cancer, human expansion repeat disease, and other diseases. Previous studies largely rely on the ectopic overexpression of proteins of interest and qualitative assays to study hub function and there is a of lack of both specific strategies to perturb hub formation/properties with a measurable functional output and application of suitable technologies to look at protein kinetics in vivo. The goal of this project is to use oncogenic mutations found in the chromatin reader protein, ENL, to elucidate the mechanisms by which hubs impact transcription. ENL mutations are among the first examples of pathogenic mutations that result in aberrant hub formation. Importantly, such hub formation is functionally required for hyper-activation of target genes. The high specificity and gain-of-function nature of ENL mutations make them a powerful system to study both the mechanisms of hub formation as well as how aberrant hubs contribute to human disease. I hypothesize that ENL mutant proteins promote the clustering of multiple elements, both genomic and proteomic, to alter transcription at target loci. In Aim 1, I will combine advanced imaging techniques, including single molecule tracking and live imaging of transcription, to determine the effect of hub formation on the molecular kinetics of incorporated proteins and transcription dynamics. In Aim 2, I will investigate the effect of hub formation on the spatial proximity of target genes using DNA-FISH and live imaging to determine if hubs drive genome reorganization for coordinated expression of target loci. Completion of this project will offer novel insights as to how pathogenic mutations result in aberrant hub formation and affect transcriptional dynamics to drive disease. More broadly, this work will advance our understanding of hub-mediated gene regulation, revealing the potential for novel therapeutic strategies to target gene dysregulation in disease.