Molecular features driving condensate formation and gene expression by the BRD4-NUT fusion oncoprotein are overlapping but distinct [ChIP-seq]

Aberrant formation of biomolecular condensates has been proposed to play a role in several cancers. The oncogenic fusion protein BRD4-NUT forms condensates and drives changes in gene expression in Nut Carcinoma (NC). Here we sought to understand the molecular elements of BRD4-NUT and its associated histone acetyltransferase (HAT), p300, that promote these activities. We determined that a minimal fragment of NUT (MIN) in fusion with BRD4 is necessary and sufficient to bind p300 and form condensates. Furthermore, a BRD4-p300 fusion protein also forms condensates and drives gene expression similarly to BRD4-NUT(MIN), suggesting the p300 fusion may mimic certain features of BRD4-NUT. The intrinsically disordered regions, transcription factor-binding domains, and HAT activity of p300 all collectively contribute to condensate formation by BRD4-p300, suggesting that these elements might contribute to condensate formation by BRD4-NUT. Conversely, only the HAT activity of BRD4-p300 appears necessary to mimic the transcriptional profile of cells expressing BRD4-NUT. Our results suggest a model for condensate formation by the BRD4-NUT:p300 complex involving a combination of positive feedback and phase separation, and show that multiple overlapping, yet distinct, regions of p300 contribute to condensate formation and transcriptional regulation. Overall design: Chromatin immunoprecipitation DNA-sequencing (ChIP-seq) for HEK293 T-Rex Flp-In - based stable cell lines, expressing BRD4-NUT, BRD4-NUT(MIN) or BRD4-p300, in a doxycycline-inducible manner. ChIP-seq performed using anti-NUT antibody for BRD4-NUT and BRD4-NUT(MIN) - expressing cell lines. ChIP-seq performed using anti-histone H3K27 acetyl antibody for BRD4-NUT(MIN) and BRD4-p300 - expressing cell lines.