RESUMEN
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.
Asunto(s)
Antineoplásicos/farmacología , Núcleo Celular/metabolismo , Resistencia a Antineoplásicos , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Antineoplásicos/uso terapéutico , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Homólogo de la Proteína Chromobox 5 , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Subunidad 1 del Complejo Mediador/genética , Subunidad 1 del Complejo Mediador/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Nucleofosmina , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Factores de Empalme Serina-Arginina/genética , Factores de Empalme Serina-Arginina/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
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-ß, 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 ß-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.
Asunto(s)
Elementos de Facilitación Genéticos/genética , Complejo Mediador/metabolismo , Factores de Transcripción/metabolismo , Animales , Línea Celular , Regulación de la Expresión Génica/fisiología , Humanos , Proteínas Intrínsecamente Desordenadas/metabolismo , Complejo Mediador/fisiología , Factores de Transcripción STAT/metabolismo , Factor de Transcripción STAT3/metabolismo , Transducción de Señal/fisiología , Proteína smad3/metabolismo , Proteínas de la Superfamilia TGF-beta/metabolismo , Transcripción Genética , Vía de Señalización Wnt , beta Catenina/metabolismoRESUMEN
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 complex1-4. 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 apparatus5,6. Recent observations suggest that this model provides only a partial picture of the effects of phosphorylation of the C-terminal domain7-12. 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-enhancers7,8, and large numbers of splicing factors are concentrated in nuclear speckles, some of which occur at highly active transcription sites9-12. 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.
Asunto(s)
Complejo Mediador/química , Complejo Mediador/metabolismo , ARN Polimerasa II/química , ARN Polimerasa II/metabolismo , Empalme del ARN , Transcripción Genética , Animales , Línea Celular , Elementos de Facilitación Genéticos/genética , Regulación de la Expresión Génica/genética , Humanos , Complejo Mediador/genética , Ratones , Fosforilación , Dominios Proteicos , ARN Polimerasa II/genética , Factores de Empalme de ARN/química , Factores de Empalme de ARN/genética , Factores de Empalme de ARN/metabolismoRESUMEN
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.
Asunto(s)
Células Madre Embrionarias de Ratones/metabolismo , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Receptores de Estrógenos/metabolismo , Activación Transcripcional/fisiología , Animales , Células HEK293 , Humanos , Ratones , Células Madre Embrionarias de Ratones/citología , Factor 3 de Transcripción de Unión a Octámeros/genética , Dominios Proteicos , Receptores de Estrógenos/genéticaRESUMEN
Super-enhancers (SEs) are clusters of enhancers that cooperatively assemble a high density of the transcriptional apparatus to drive robust expression of genes with prominent roles in cell identity. Here we demonstrate that the SE-enriched transcriptional coactivators BRD4 and MED1 form nuclear puncta at SEs that exhibit properties of liquid-like condensates and are disrupted by chemicals that perturb condensates. The intrinsically disordered regions (IDRs) of BRD4 and MED1 can form phase-separated droplets, and MED1-IDR droplets can compartmentalize and concentrate the transcription apparatus from nuclear extracts. These results support the idea that coactivators form phase-separated condensates at SEs that compartmentalize and concentrate the transcription apparatus, suggest a role for coactivator IDRs in this process, and offer insights into mechanisms involved in the control of key cell-identity genes.
Asunto(s)
Elementos de Facilitación Genéticos , Regulación de la Expresión Génica , Proteínas Intrínsecamente Desordenadas/metabolismo , Subunidad 1 del Complejo Mediador/metabolismo , Proteínas Nucleares/metabolismo , Transactivadores/metabolismo , Factores de Transcripción/metabolismo , Animales , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Secuencia Conservada , Células Madre Embrionarias/metabolismo , Elementos de Facilitación Genéticos/efectos de los fármacos , Recuperación de Fluorescencia tras Fotoblanqueo , Regulación de la Expresión Génica/efectos de los fármacos , Glicoles/farmacología , Células HEK293 , Humanos , Inmunoprecipitación , Proteínas Intrínsecamente Desordenadas/química , Proteínas Intrínsecamente Desordenadas/genética , Subunidad 1 del Complejo Mediador/química , Subunidad 1 del Complejo Mediador/genética , Ratones , Imagen Molecular , Células 3T3 NIH , Proteínas Nucleares/química , Proteínas Nucleares/genética , Serina/química , Serina/genética , Transactivadores/química , Transactivadores/genética , Factores de Transcripción/química , Factores de Transcripción/genéticaRESUMEN
The RNA-guided endonuclease Cpf1 is a promising tool for genome editing in eukaryotic cells. However, the utility of the commonly used Acidaminococcus sp. BV3L6 Cpf1 (AsCpf1) and Lachnospiraceae bacterium ND2006 Cpf1 (LbCpf1) is limited by their requirement of a TTTV protospacer adjacent motif (PAM) in the DNA substrate. To address this limitation, we performed a structure-guided mutagenesis screen to increase the targeting range of Cpf1. We engineered two AsCpf1 variants carrying the mutations S542R/K607R and S542R/K548V/N552R, which recognize TYCV and TATV PAMs, respectively, with enhanced activities in vitro and in human cells. Genome-wide assessment of off-target activity using BLISS indicated that these variants retain high DNA-targeting specificity, which we further improved by introducing an additional non-PAM-interacting mutation. Introducing the identified PAM-interacting mutations at their corresponding positions in LbCpf1 similarly altered its PAM specificity. Together, these variants increase the targeting range of Cpf1 by approximately threefold in human coding sequences to one cleavage site per â¼11 bp.