Imaging and manipulating oncoprotein phase separation and compartmentalization

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Abstract MYC oncoproteins including c-Myc and MYCN are major drivers of human tumorigenesis. Expression of MYC is deregulated and enhanced in many types of human cancer. For instance, MYCN is a major driver of childhood cancers. While upregulated MYC expression induces tumor development in many tissues, depletion of MYC abolishes tumorigenesis and results in tumor regression in various tumor models. Despite significant progress in understanding the link between MYC upregulation and tumorigenesis, many key questions remain regarding how upregulated expression of MYC leads to tumorigenesis. The relationship between MYC upregulation and its transforming activity has not been investigated in a quantitative manner, which is important because one potential consequence of elevated expression is protein phase separation that occurs when protein concentration is above critical concentration. Recently, many transcription factors that contain intrinsically disordered region (IDR) have been reported to undergo concentration-dependent liquid-liquid phase separation (LLPS), forming biomolecular condensates (also known as membraneless compartments, liquid droplets). MYC oncoproteins are transcription factors and contain IDR and our preliminary study reveals punctate structures of MYCN in the nucleus of Kelly MYCN-amplified neuroblastoma cells, suggesting that MYCN forms condensates in the Kelly cells where MYCN is highly expressed. Functions of biological phase- separated condensates include compartmentalized signaling. Condensates of transcriptional factors have been proposed to compartmentalize transcriptional machineries and remodel gene transcription. MYC oncogenic signaling relies on its interaction with the key proteins including MAX and TRRAP for the transcriptional activity and transforming, and inhibition of these interactions results in MYC inactivation. To understand upregulated MYC oncoproteins and their link to tumorigenesis, several basic molecular biological questions need to be addressed regarding biophysical/chemical mechanisms and functional roles of MYC condensates, including: 1) Whether and how MYC undergoes phase separation and forms condensates in biochemical and cellular systems; 2) Whether MYC condensates compartmentalize transcriptional machineries; 3) Whether MYC phase separation remodels downstream gene expression and whether condensates of MYC is necessary for the transforming activity. Because LLPS is dependent on protein concentration, biological functions of phase separation are often entangled with effects from changes of expression levels. To dissect MYC LLPS and functional consequences, there is a need for chemogenetic tools that can manipulate MYC LLPS without changing protein levels, and ideally such manipulation can be performed in two opposite directions: driving condensate formation and dissolving existing condensates. Here we propose to develop such chemogenetic tools and combine them with fluorescent protein labeling technology and multicolor fluorescence imaging to dissect biochemical mechanisms and functional roles of MYCN condensates.