Biology subjects

Currie, S. L.

Publications and source records attributed to Currie, S. L..

4 recordsLinked to original sources

bioRxiv2023

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

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.

cell biology↗

bioRxiv2022

Quantitative reconstitution of yeast RNA processing bodies

Many biomolecular condensates appear to form through liquid-liquid phase separation (LLPS). Individual condensate components can often undergo LLPS in vitro, capturing some features of the native structures. However, natural condensates contain dozens of components with different concentrations, dynamics, and contributions to compartment formation. Most biochemical reconstitutions of condensates have not benefitted from quantitative knowledge of these cellular features nor attempted to capture natural complexity. Here, we build on prior quantitative cellular studies to reconstitute yeast RNA processing bodies (P bodies) from purified components. Individually, five of the seven highly-concentrated P-body proteins form homotypic condensates at cellular protein and salt concentrations, using both structured domains and intrinsically disordered regions. Combining the seven proteins together at their cellular concentrations with RNA, yields phase separated droplets with partition coefficients and dynamics of most proteins in reasonable agreement with cellular values. The dynamics of most proteins in the reconstitution are also comparable to cellular values. RNA delays the maturation of proteins within, and promotes reversibility of, P bodies. Our ability to quantitatively recapitulate the composition and dynamics of a condensate from its most concentrated components suggests that simple interactions between these components carry much of the information that defines the physical properties of the cellular structure.

biophysics↗

bioRxiv2018

Electrostatic repulsion causes anticooperative DNA binding between tumor suppressor ETS transcription factors and JUN-FOS at composite DNA sites

Many transcription factors regulate gene expression in a combinatorial fashion often by binding in close proximity on composite cis-regulatory DNA elements. Here we investigate the molecular basis by which ETS transcription factors bind with AP1 transcription factors JUN-FOS at composite DNA-binding sites. The ability to bind to DNA with JUN-FOS correlates with the phenotype of these proteins in prostate cancer: the oncogenic ERG and ETV1/4/5 subfamilies co-occupy ETS-AP1 sites with JUN-FOS in vitro, whereas JUN-FOS robustly inhibits DNA binding by the tumor suppressors EHF and SPDEF. EHF binds to ETS-AP1 DNA with tighter affinity than ERG in the absence of JUN-FOS, which may enable EHF to compete with ERG and JUN-FOS for binding to ETS-AP1 sites. Genome-wide mapping of EHF and ERG binding sites in a prostate epithelial cell line reveal that EHF is preferentially excluded from closely spaced ETS-AP1 DNA sequences. Structural modeling and mutational analyses indicate that adjacent positively-charged surfaces from EHF and JUN-FOS disfavor simultaneous DNA binding due to electrostatic repulsion. The conservation of positively charged residues on the JUN-FOS interface identified ELF1 as an additional ETS factor that exhibits anticooperative DNA binding, and we present evidence that ELF1 is frequently downregulated in prostate cancer. In summary, the divergence of electrostatic features of ETS factors at their JUN-FOS interface enables distinct binding events at ETS-AP1 DNA sequences. We propose that this mechanism can drive unique targeting of ETS transcription factors, thereby facilitating distinct transcriptional programs.

biochemistry↗

bioRxiv2017

ETV4 And AP1 Transcription Factors Form Multivalent Interactions With Three Sites On The MED25 Activator-Interacting Domain

The recruitment of transcriptional cofactors by sequence-specific transcription factors challenges the basis of high affinity and selective interactions. Extending previous studies that the N-terminal activation domain (AD) of ETV5 interacts with Mediator subunit 25 (MED25), we establish that similar, aromatic-rich motifs located both in the AD and in the DNA-binding domain (DBD) of the related ETS factor ETV4 interact with MED25. These ETV4 regions bind MED25 independently, display distinct kinetics, and combine to contribute to a high-affinity interaction of full-length ETV4 with MED25. Within the ETS family, high-affinity interactions with MED25 are specific for the ETV1/4/5 subfamily as other ETS factors display weaker or no detectable binding. The AD binds to a single site on MED25 and the DBD interacts with three MED25 sites, allowing for simultaneous binding of both domains in full-length ETV4. MED25 also stimulates the in vitro DNA binding activity of ETV4 by relieving autoinhibition. ETV1/4/5 factors are often overexpressed in prostate cancer and genome-wide studies in a prostate cancer cell line indicate that ETV4 and MED25 occupy enhancers that are enriched for ETS-binding sequences and are both functionally important for the transcription of genes regulated by these enhancers. AP1-binding sequences were observed in MED25-occupied regions and JUN/FOS also contact MED25; FOS strongly binds to the same MED25 site as ETV4 AD and JUN interacts with the other two MED25 sites. In summary, we describe features of the multivalent ETV4- and AP1-MED25 interactions, thereby implicating these factors in the recruitment of MED25 to transcriptional control elements.

biochemistry↗