The synthesis of pre-mRNA by RNA polymerase II (Pol II) involves the formation of a transcription initiation complex, and a transition to an elongation complex
. The large subunit of Pol II contains ...an intrinsically disordered C-terminal domain that is phosphorylated by cyclin-dependent kinases during the transition from initiation to elongation, thus influencing the interaction of the C-terminal domain with different components of the initiation or the RNA-splicing apparatus
. Recent observations suggest that this model provides only a partial picture of the effects of phosphorylation of the C-terminal domain
. Both the transcription-initiation machinery and the splicing machinery can form phase-separated condensates that contain large numbers of component molecules: hundreds of molecules of Pol II and mediator are concentrated in condensates at super-enhancers
, and large numbers of splicing factors are concentrated in nuclear speckles, some of which occur at highly active transcription sites
. Here we investigate whether the phosphorylation of the Pol II C-terminal domain regulates the incorporation of Pol II into phase-separated condensates that are associated with transcription initiation and splicing. We find that the hypophosphorylated C-terminal domain of Pol II is incorporated into mediator condensates and that phosphorylation by regulatory cyclin-dependent kinases reduces this incorporation. We also find that the hyperphosphorylated C-terminal domain is preferentially incorporated into condensates that are formed by splicing factors. These results suggest that phosphorylation of the Pol II C-terminal domain drives an exchange from condensates that are involved in transcription initiation to those that are involved in RNA processing, and implicates phosphorylation as a mechanism that regulates condensate preference.
The nucleus contains diverse phase-separated condensates that compartmentalize and concentrate biomolecules with distinct physicochemical properties. Here, we investigated whether condensates ...concentrate small-molecule cancer therapeutics such that their pharmacodynamic properties are altered. We found that antineoplastic drugs become concentrated in specific protein condensates in vitro and that this occurs through physicochemical properties independent of the drug target. This behavior was also observed in tumor cells, where drug partitioning influenced drug activity. Altering the properties of the condensate was found to affect the concentration and activity of drugs. These results suggest that selective partitioning and concentration of small molecules within condensates contributes to drug pharmacodynamics and that further understanding of this phenomenon may facilitate advances in disease therapy.
Gene expression is controlled by transcription factors (TFs) that consist of DNA-binding domains (DBDs) and activation domains (ADs). The DBDs have been well characterized, but little is known about ...the mechanisms by which ADs effect gene activation. Here, we report that diverse ADs form phase-separated condensates with the Mediator coactivator. For the OCT4 and GCN4 TFs, we show that the ability to form phase-separated droplets with Mediator in vitro and the ability to activate genes in vivo are dependent on the same amino acid residues. For the estrogen receptor (ER), a ligand-dependent activator, we show that estrogen enhances phase separation with Mediator, again linking phase separation with gene activation. These results suggest that diverse TFs can interact with Mediator through the phase-separating capacity of their ADs and that formation of condensates with Mediator is involved in gene activation.
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•Transcription factors (TFs) form phase-separated condensates with Mediator•Phase separation of activation domains is a general property of TFs•Phase-separation capacity of TFs is associated with gene activation•TF condensates incorporate dynamic and structured interactions
Activation domains from a diverse array of mammalian and yeast transcription factors form phase-separated condensates with Mediator to activate gene expression.
The gene expression programs that define the identity of each cell are controlled by master transcription factors (TFs) that bind cell-type-specific enhancers, as well as signaling factors, which ...bring extracellular stimuli to these enhancers. Recent studies have revealed that master TFs form phase-separated condensates with the Mediator coactivator at super-enhancers. Here, we present evidence that signaling factors for the WNT, TGF-β, and JAK/STAT pathways use their intrinsically disordered regions (IDRs) to enter and concentrate in Mediator condensates at super-enhancers. We show that the WNT coactivator β-catenin interacts both with components of condensates and DNA-binding factors to selectively occupy super-enhancer-associated genes. We propose that the cell-type specificity of the response to signaling is mediated in part by the IDRs of the signaling factors, which cause these factors to partition into condensates established by the master TFs and Mediator at genes with prominent roles in cell identity.
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•Signaling factors incorporate into Mediator condensates at super-enhancers•β-Catenin IDRs are required for both phase separation and target gene activation•Both condensate interactions and TF interactions contribute to β-catenin localization
Zamudio et al. demonstrate that components of the WNT, TGF-β, and JAK/STAT signaling pathways use their intrinsically disordered regions to condense with Mediator and to target specific genes. These findings provide a model for how context-dependent transcriptional responses can be achieved in cell signaling.
A multitude of cellular processes involve biomolecular condensates, which has led to the suggestion that diverse pathogenic mutations may dysregulate condensates. Although proof-of-concept studies ...have identified specific mutations that cause condensate dysregulation, the full scope of the pathological genetic variation that affects condensates is not yet known. Here, we comprehensively map pathogenic mutations to condensate-promoting protein features in putative condensate-forming proteins and find over 36,000 pathogenic mutations that plausibly contribute to condensate dysregulation in over 1,200 Mendelian diseases and 550 cancers. This resource captures mutations presently known to dysregulate condensates, and experimental tests confirm that additional pathological mutations do indeed affect condensate properties in cells. These findings suggest that condensate dysregulation may be a pervasive pathogenic mechanism underlying a broad spectrum of human diseases, provide a strategy to identify proteins and mutations involved in pathologically altered condensates, and serve as a foundation for mechanistic insights into disease and therapeutic hypotheses.
The development of site-specific recombinases (SSRs) as genome editing agents is limited by the difficulty of altering their native DNA specificities. Here we describe Rec-seq, a method for revealing ...the DNA specificity determinants and potential off-target substrates of SSRs in a comprehensive and unbiased manner. We applied Rec-seq to characterize the DNA specificity determinants of several natural and evolved SSRs including Cre, evolved variants of Cre, and other SSR family members. Rec-seq profiling of these enzymes and mutants thereof revealed previously uncharacterized SSR interactions, including specificity determinants not evident from SSR:DNA structures. Finally, we used Rec-seq specificity profiles to predict off-target substrates of Tre and Brec1 recombinases, including endogenous human genomic sequences, and confirmed their ability to recombine these off-target sequences in human cells. These findings establish Rec-seq as a high-resolution method for rapidly characterizing the DNA specificity of recombinases with single-nucleotide resolution, and for informing their further development.
Critical developmental “master transcription factors” (MTFs) can be subverted during tumorigenesis to control oncogenic transcriptional programs. Current approaches to identifying MTFs rely on ...ChIP-seq data, which is unavailable for many cancers. We developed the CaCTS (Cancer Core Transcription factor Specificity) algorithm to prioritize candidate MTFs using pan-cancer RNA sequencing data. CaCTS identified candidate MTFs across 34 tumor types and 140 subtypes including predictions for cancer types/subtypes for which MTFs are unknown, including e.g. PAX8, SOX17, and MECOM as candidates in ovarian cancer (OvCa). In OvCa cells, consistent with known MTF properties, these factors are required for viability, lie proximal to superenhancers, co-occupy regulatory elements globally, co-bind loci encoding OvCa biomarkers, and are sensitive to pharmacologic inhibition of transcription. Our predictions of MTFs, especially for tumor types with limited understanding of transcriptional drivers, pave the way to therapeutic targeting of MTFs in a broad spectrum of cancers.
The cell concentrates and compartmentalizes proteins and nucleic acids into diverse phase-separated biomolecular condensates. The study of condensates has yielded myriad fundamental insights into ...cell biology, ranging from a better understanding of cellular organization, to the exploration of novel mesoscale functions resulting from the emergent properties of these liquid-like compartments. A consideration of the role of condensates in disease and drug development could facilitate similar paradigm shifts, with early evidence suggesting that condensate dysregulation may be a feature of many diseases, and that therapeutics can modulate condensates. In the studies presented in this thesis, we developed and validated a strategy for nominating patient mutations across the spectrum of disease that may cause condensate dysregulation, providing nominated mutations as a resource to the biomedical community for the acceleration of the study of condensates in disease. Further, we tested the hypothesis that small molecule therapeutics can concentrate into condensates, showing that clinically important cancer therapeutics display differential partitioning and that condensate partitioning can affect therapeutic activity (Klein et al., 2020). Lastly, we have expanded upon this condensate partitioning work to show that antisense oligonucleotides (ASOs), nucleic acid-based therapeutics targeting RNA, partition into and modulate certain condensates, and that specific chemical modifications can alter this partitioning behavior. Ultimately, by considering the implications of a condensate model in disease and drug development, this thesis aims to leverage recent insights into biomolecular condensates to facilitate the development of novel disease mechanistic and therapeutic hypotheses.
Ph.D.
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