ABSTRACT
Fusion oncoproteins (FOs) that are formed by chromosomal translocations are the primary drivers of many
treatment-resistant cancers that afflict children in particular. A subset of FOs contain intrinsically disordered
regions (IDRs) from transcription factors and other transcriptional co-regulators. Recent work has demonstrated
that a number of transcription factors and co-regulators undergo liquid-liquid phase separation (PS) through
interactions mediated by IDRs within their sequences and form transcriptional condensates. Genes at loci
associated with these condensates are significantly upregulated, implicating PS in gene regulation and cell fate.
We hypothesize that a subset of FOs enriched in IDRs form aberrant transcriptional condensates through PS,
which results in aberrant gene expression and oncogenesis. Furthermore, I hypothesize that these FOs that form
aberrant transcriptional condensates are enriched in IDR sequences that were not previously demonstrated to
phase separate, and that these sequence features determine both the conformational and material properties of
the FOs and the resulting condensates. Understanding the biophysical properties of FO-derived IDRs is
important so that others can design therapeutics targeting the resulting condensates. To identify FOs that form
condensates under physiological conditions, I selected 100 FOs based on patient-derived transcripts to transfect
into mammalian cells as eGFP-tagged proteins. I will screen these 100 FOs for the formation of puncta that are
indicative of PS using a semi-automated fluorescence confocal microscopy assay. To determine the IDR
sequences responsible for PS, I will subclone IDRs derived from puncta-forming FOs into Escherichia coli
expression vectors for overexpression and purification. I will use turbidity assays and confocal microscopy to
assess whether these IDRs undergo PS and under what conditions. To probe the specific intermolecular
interactions and identify potentially novel motifs, or “stickers”, that drive PS, I will use nuclear magnetic resonance
(NMR) spectroscopy to measure changes in dynamics and intermolecular contacts for the IDRs upon PS.
Additionally, I will relate sequence/sticker motif composition with the conformational and material properties of
FO-derived IDRs. I will use NMR and fluorescence correlation spectroscopy to derive diffusion coefficients that
report on the hydrodynamic radius of the IDRs inside and outside of condensates. I will further use SAXS to
measure the radius of gyration outside of condensates and the intermolecular spacing within condensates.
Finally, I will use fluorescence recovery after photobleaching to assess the fluid dynamics and material state of
condensates. These complementary, multi-disciplinary studies will enable me to test the hypotheses discussed
above and advance our understanding of the role of PS in the in vitro and cellular behavior of cancer-driving
fusion oncoproteins.