Understanding condensate formation by fusion oncoprotein-derived intrinsically disordered regions.

Abstract

Fusion oncoproteins (FOs), which arise from chromosomal rearrangements that fuse two genes, are drivers of many aggressive pediatric cancers. FOs often are comprised of intrinsically disordered regions (IDRs) derived from one parent transcription factor protein that are fused to a DNA- or chromatin-binding domain of another parent. Several FOs with these structural features have been shown to undergo liquid-liquid phase separation (LLPS) to form nuclear condensates that promote aberrant gene expression and transform cells. To investigate the generality of this mechanism, we screened 166 GFP-tagged FOs in HeLa cells using confocal fluorescence microscopy and observed that 96/166 formed condensates suggestive of LLPS [termed puncta(+)]. We hypothesized that IDRs within puncta(+) FOs promote their condensate formation. To test this idea, we used an in-house sequence analysis pipeline to identify >200 LLPS-prone IDRs within both puncta(+) and puncta(-) FOs and established an experimental platform for testing His6, MBP and GFP-tagged IDRs for condensate formation in E. coli bacterial lysates. Though experimental testing is still underway, preliminary results indicate that 39/96 tested IDRs form round condensates indicative of LLPS. My project seeks to understand relationships between multivalent patterns of physicochemical features within IDRs and their condensate formation propensity. I expressed and purified His6 and MBP-tagged IDR constructs using metal affinity and reverse-phase high-performance liquid chromatography to obtain, after cleavage of the tags, pure IDRs for biophysical characterization. Next, I will confirm disorder with circular dichroism, assay droplet formation and determine saturation concentrations with turbidity assays, and construct phase diagrams. These experiments will reveal links between physicochemical features and condensation behavior in vitro and, more broadly, provide insight into the biophysical basis of FO-driven oncogenesis.