RESUMEN
Transcription factors (TFs) control specificity and activity of gene transcription, but whether a relationship between these two features exists is unclear. Here we provide evidence for an evolutionary trade-off between the activity and specificity in human TFs encoded as submaximal dispersion of aromatic residues in their intrinsically disordered protein regions. We identified approximately 500 human TFs that encode short periodic blocks of aromatic residues in their intrinsically disordered regions, resembling imperfect prion-like sequences. Mutation of periodic aromatic residues reduced transcriptional activity, whereas increasing the aromatic dispersion of multiple human TFs enhanced transcriptional activity and reprogramming efficiency, promoted liquid-liquid phase separation in vitro and more promiscuous DNA binding in cells. Together with recent work on enhancer elements, these results suggest an important evolutionary role of suboptimal features in transcriptional control. We propose that rational engineering of amino acid features that alter phase separation may be a strategy to optimize TF-dependent processes, including cellular reprogramming.
Asunto(s)
Factores de Transcripción , Humanos , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Proteínas Intrínsecamente Desordenadas/metabolismo , Proteínas Intrínsecamente Desordenadas/genética , Proteínas Intrínsecamente Desordenadas/química , Mutación , Unión Proteica , Transcripción Genética , ADN/metabolismo , ADN/genética , Células HEK293 , Reprogramación Celular/genética , Regulación de la Expresión GénicaRESUMEN
Transcription factors are among the most attractive therapeutic targets but are considered largely 'undruggable' in part due to the intrinsically disordered nature of their activation domains. Here we show that the aromatic character of the activation domain of the androgen receptor, a therapeutic target for castration-resistant prostate cancer, is key for its activity as transcription factor, allowing it to translocate to the nucleus and partition into transcriptional condensates upon activation by androgens. On the basis of our understanding of the interactions stabilizing such condensates and of the structure that the domain adopts upon condensation, we optimized the structure of a small-molecule inhibitor previously identified by phenotypic screening. The optimized compounds had more affinity for their target, inhibited androgen-receptor-dependent transcriptional programs, and had an antitumorigenic effect in models of castration-resistant prostate cancer in cells and in vivo. These results suggest that it is possible to rationally optimize, and potentially even to design, small molecules that target the activation domains of oncogenic transcription factors.
Asunto(s)
Neoplasias de la Próstata Resistentes a la Castración , Neoplasias de la Próstata , Masculino , Humanos , Neoplasias de la Próstata Resistentes a la Castración/tratamiento farmacológico , Neoplasias de la Próstata Resistentes a la Castración/genética , Receptores Androgénicos/genética , Receptores Androgénicos/química , Andrógenos/uso terapéutico , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/genética , Dominios Proteicos , Factores de Transcripción , Línea Celular TumoralRESUMEN
Gene transcription by RNA polymerase II (Pol II) is under control of promoters and distal regulatory elements known as enhancers. Enhancers are themselves transcribed by Pol II correlating with their activity. How enhancer transcription is regulated and coordinated with transcription at target genes has remained unclear. Here, we developed a high-sensitive native elongating transcript sequencing approach, called HiS-NET-seq, to provide an extended high-resolution view on transcription, especially at lowly transcribed regions such as enhancers. HiS-NET-seq uncovers new transcribed enhancers in human cells. A multi-omics analysis shows that genome-wide enhancer transcription depends on the BET family protein BRD4. Specifically, BRD4 co-localizes to enhancer and promoter-proximal gene regions, and is required for elongation activation at enhancers and their genes. BRD4 keeps a set of enhancers and genes in proximity through long-range contacts. From these studies BRD4 emerges as a general regulator of enhancer transcription that may link transcription at enhancers and genes.
Asunto(s)
Proteínas Nucleares , Factores de Transcripción , Humanos , Proteínas Nucleares/genética , Factores de Transcripción/genética , Secuencias Reguladoras de Ácidos Nucleicos , ARN Polimerasa II/genética , Transcripción Genética , Proteínas de Ciclo Celular/genéticaRESUMEN
Cell identity is orchestrated through an interplay between transcription factor (TF) action and genome architecture. The mechanisms used by TFs to shape three-dimensional (3D) genome organization remain incompletely understood. Here we present evidence that the lineage-instructive TF CEBPA drives extensive chromatin compartment switching and promotes the formation of long-range chromatin hubs during induced B cell-to-macrophage transdifferentiation. Mechanistically, we find that the intrinsically disordered region (IDR) of CEBPA undergoes in vitro phase separation (PS) dependent on aromatic residues. Both overexpressing B cells and native CEBPA-expressing cell types such as primary granulocyte-macrophage progenitors, liver cells, and trophectoderm cells reveal nuclear CEBPA foci and long-range 3D chromatin hubs at CEBPA-bound regions. In short, we show that CEBPA can undergo PS through its IDR, which may underlie in vivo foci formation and suggest a potential role of PS in regulating CEBPA function.
Asunto(s)
Cromatina , Regulación de la Expresión Génica , Núcleo Celular , MacrófagosRESUMEN
Genes that are key to cell identity are generally regulated by cell-type-specific enhancer elements bound by transcription factors, some of which facilitate looping to distant gene promoters. In contrast, genes that encode housekeeping functions, whose regulation is essential for normal cell metabolism and growth, generally lack interactions with distal enhancers. We find that Ronin (Thap11) assembles multiple promoters of housekeeping and metabolic genes to regulate gene expression. This behavior is analogous to how enhancers are brought together with promoters to regulate cell identity genes. Thus, Ronin-dependent promoter assemblies provide a mechanism to explain why housekeeping genes can forgo distal enhancer elements and why Ronin is important for cellular metabolism and growth control. We propose that clustering of regulatory elements is a mechanism common to cell identity and housekeeping genes but is accomplished by different factors binding distinct control elements to establish enhancer-promoter or promoter-promoter interactions, respectively.
Asunto(s)
Elementos de Facilitación Genéticos , Genes Esenciales , Genes Esenciales/genética , Elementos de Facilitación Genéticos/genética , Factores de Transcripción/metabolismo , Regiones Promotoras Genéticas/genéticaRESUMEN
Thousands of genetic variants in protein-coding genes have been linked to disease. However, the functional impact of most variants is unknown as they occur within intrinsically disordered protein regions that have poorly defined functions1-3. Intrinsically disordered regions can mediate phase separation and the formation of biomolecular condensates, such as the nucleolus4,5. This suggests that mutations in disordered proteins may alter condensate properties and function6-8. Here we show that a subset of disease-associated variants in disordered regions alter phase separation, cause mispartitioning into the nucleolus and disrupt nucleolar function. We discover de novo frameshift variants in HMGB1 that cause brachyphalangy, polydactyly and tibial aplasia syndrome, a rare complex malformation syndrome. The frameshifts replace the intrinsically disordered acidic tail of HMGB1 with an arginine-rich basic tail. The mutant tail alters HMGB1 phase separation, enhances its partitioning into the nucleolus and causes nucleolar dysfunction. We built a catalogue of more than 200,000 variants in disordered carboxy-terminal tails and identified more than 600 frameshifts that create arginine-rich basic tails in transcription factors and other proteins. For 12 out of the 13 disease-associated variants tested, the mutation enhanced partitioning into the nucleolus, and several variants altered rRNA biogenesis. These data identify the cause of a rare complex syndrome and suggest that a large number of genetic variants may dysregulate nucleoli and other biomolecular condensates in humans.
Asunto(s)
Nucléolo Celular , Proteína HMGB1 , Humanos , Arginina/genética , Arginina/metabolismo , Nucléolo Celular/genética , Nucléolo Celular/metabolismo , Nucléolo Celular/patología , Proteína HMGB1/química , Proteína HMGB1/genética , Proteína HMGB1/metabolismo , Proteínas Intrínsecamente Desordenadas/química , Proteínas Intrínsecamente Desordenadas/genética , Proteínas Intrínsecamente Desordenadas/metabolismo , Síndrome , Mutación del Sistema de Lectura , Transición de FaseRESUMEN
Long non-coding RNAs (lncRNAs) have emerged as fundamental regulators in various biological processes, including embryonic development and cellular differentiation. Despite much progress over the past decade, the genome-wide annotation of lncRNAs remains incomplete and many known non-coding loci are still poorly characterized. Here, we report the discovery of a previously unannotated lncRNA that is transcribed 230 kb upstream of the SOX17 gene and located within the same topologically associating domain. We termed it T-REX17 (Transcript Regulating Endoderm and activated by soX17) and show that it is induced following SOX17 activation but its expression is more tightly restricted to early definitive endoderm. Loss of T-REX17 affects crucial functions independent of SOX17 and leads to an aberrant endodermal transcriptome, signaling pathway deregulation and epithelial to mesenchymal transition defects. Consequently, cells lacking the lncRNA cannot further differentiate into more mature endodermal cell types. Taken together, our study identified and characterized T-REX17 as a transiently expressed and essential non-coding regulator in early human endoderm differentiation.
Asunto(s)
ARN Largo no Codificante , Embarazo , Femenino , Humanos , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Transición Epitelial-Mesenquimal , Endodermo , Regulación del Desarrollo de la Expresión Génica , Factores de Transcripción SOXF/genética , Factores de Transcripción SOXF/metabolismo , Diferenciación Celular/genéticaRESUMEN
Most endogenous retroviruses (ERVs) in mammals are incapable of retrotransposition; therefore, why ERV derepression is associated with lethality during early development has been a mystery. Here, we report that rapid and selective degradation of the heterochromatin adapter protein TRIM28 triggers dissociation of transcriptional condensates from loci encoding super-enhancer (SE)-driven pluripotency genes and their association with transcribed ERV loci in murine embryonic stem cells. Knockdown of ERV RNAs or forced expression of SE-enriched transcription factors rescued condensate localization at SEs in TRIM28-degraded cells. In a biochemical reconstitution system, ERV RNA facilitated partitioning of RNA polymerase II and the Mediator coactivator into phase-separated droplets. In TRIM28 knockout mouse embryos, single-cell RNA-seq analysis revealed specific depletion of pluripotent lineages. We propose that coding and noncoding nascent RNAs, including those produced by retrotransposons, may facilitate 'hijacking' of transcriptional condensates in various developmental and disease contexts.
Asunto(s)
Retrovirus Endógenos , Animales , Células Madre Embrionarias , Retrovirus Endógenos/genética , Heterocromatina , Mamíferos/genética , Ratones , Cuerpos Nucleares , RetroelementosRESUMEN
The core cohesin subunit STAG2 is recurrently mutated in Ewing sarcoma but its biological role is less clear. Here, we demonstrate that cohesin complexes containing STAG2 occupy enhancer and polycomb repressive complex (PRC2)-marked regulatory regions. Genetic suppression of STAG2 leads to a compensatory increase in cohesin-STAG1 complexes, but not in enhancer-rich regions, and results in reprogramming of cis-chromatin interactions. Strikingly, in STAG2 knockout cells the oncogenic genetic program driven by the fusion transcription factor EWS/FLI1 was highly perturbed, in part due to altered enhancer-promoter contacts. Moreover, loss of STAG2 also disrupted PRC2-mediated regulation of gene expression. Combined, these transcriptional changes converged to modulate EWS/FLI1, migratory, and neurodevelopmental programs. Finally, consistent with clinical observations, functional studies revealed that loss of STAG2 enhances the metastatic potential of Ewing sarcoma xenografts. Our findings demonstrate that STAG2 mutations can alter chromatin architecture and transcriptional programs to promote an aggressive cancer phenotype.
Asunto(s)
Neoplasias Óseas/genética , Neoplasias Óseas/patología , Proteínas de Ciclo Celular/genética , Sarcoma de Ewing/genética , Sarcoma de Ewing/patología , Animales , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Movimiento Celular/genética , Proteínas Cromosómicas no Histona/metabolismo , Elementos de Facilitación Genéticos , Femenino , Regulación Neoplásica de la Expresión Génica , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Ratones Endogámicos NOD , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas de Fusión Oncogénica/genética , Factores del Dominio POU/genética , Factores del Dominio POU/metabolismo , Complejo Represivo Polycomb 2/genética , Complejo Represivo Polycomb 2/metabolismo , Regiones Promotoras Genéticas , Proteína Proto-Oncogénica c-fli-1/genética , Proteína EWS de Unión a ARN/genética , Ensayos Antitumor por Modelo de Xenoinjerto , Pez Cebra/genética , CohesinasRESUMEN
The Mediator complex directs signals from DNA-binding transcription factors to RNA polymerase II (Pol II). Despite this pivotal position, mechanistic understanding of Mediator in human cells remains incomplete. Here we quantified Mediator-controlled Pol II kinetics by coupling rapid subunit degradation with orthogonal experimental readouts. In agreement with a model of condensate-driven transcription initiation, large clusters of hypophosphorylated Pol II rapidly disassembled upon Mediator degradation. This was accompanied by a selective and pronounced disruption of cell-type-specifying transcriptional circuits, whose constituent genes featured exceptionally high rates of Pol II turnover. Notably, the transcriptional output of most other genes was largely unaffected by acute Mediator ablation. Maintenance of transcriptional activity at these genes was linked to an unexpected CDK9-dependent compensatory feedback loop that elevated Pol II pause release rates across the genome. Collectively, our work positions human Mediator as a globally acting coactivator that selectively safeguards the functionality of cell-type-specifying transcriptional networks.
Asunto(s)
Regulación de la Expresión Génica , Complejo Mediador/fisiología , Transcripción Genética , Animales , Línea Celular Tumoral , Cromatina/fisiología , Drosophila , Perfilación de la Expresión Génica , Técnicas de Sustitución del Gen , Humanos , Complejo Mediador/genética , Factor B de Elongación Transcripcional Positiva/metabolismo , ARN Polimerasa II/metabolismoRESUMEN
Expansions of amino acid repeats occur in >20 inherited human disorders, and many occur in intrinsically disordered regions (IDRs) of transcription factors (TFs). Such diseases are associated with protein aggregation, but the contribution of aggregates to pathology has been controversial. Here, we report that alanine repeat expansions in the HOXD13 TF, which cause hereditary synpolydactyly in humans, alter its phase separation capacity and its capacity to co-condense with transcriptional co-activators. HOXD13 repeat expansions perturb the composition of HOXD13-containing condensates in vitro and in vivo and alter the transcriptional program in a cell-specific manner in a mouse model of synpolydactyly. Disease-associated repeat expansions in other TFs (HOXA13, RUNX2, and TBP) were similarly found to alter their phase separation. These results suggest that unblending of transcriptional condensates may underlie human pathologies. We present a molecular classification of TF IDRs, which provides a framework to dissect TF function in diseases associated with transcriptional dysregulation.
Asunto(s)
Expansión de las Repeticiones de ADN/genética , Proteínas de Homeodominio/genética , Factores de Transcripción/genética , Alanina/genética , Animales , Secuencia de Bases/genética , Expansión de las Repeticiones de ADN/fisiología , Modelos Animales de Enfermedad , Proteínas de Homeodominio/metabolismo , Humanos , Masculino , Ratones , Mutación/genética , Linaje , Sindactilia/genética , Factores de Transcripción/metabolismoRESUMEN
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
Transcriptional dysregulation of the MYC oncogene is among the most frequent events in aggressive tumor cells, and this is generally accomplished by acquisition of a super-enhancer somewhere within the 2.8 Mb TAD where MYC resides. We find that these diverse cancer-specific super-enhancers, differing in size and location, interact with the MYC gene through a common and conserved CTCF binding site located 2 kb upstream of the MYC promoter. Genetic perturbation of this enhancer-docking site in tumor cells reduces CTCF binding, super-enhancer interaction, MYC gene expression, and cell proliferation. CTCF binding is highly sensitive to DNA methylation, and this enhancer-docking site, which is hypomethylated in diverse cancers, can be inactivated through epigenetic editing with dCas9-DNMT. Similar enhancer-docking sites occur at other genes, including genes with prominent roles in multiple cancers, suggesting a mechanism by which tumor cell oncogenes can generally hijack enhancers. These results provide insights into mechanisms that allow a single target gene to be regulated by diverse enhancer elements in different cell types.
Asunto(s)
Elementos de Facilitación Genéticos , Proteínas Proto-Oncogénicas c-myc/metabolismo , Secuencias de Aminoácidos , Sitios de Unión , Factor de Unión a CCCTC/metabolismo , Sistemas CRISPR-Cas/genética , Línea Celular Tumoral , Proliferación Celular , Metilación de ADN , Edición Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Regiones Promotoras Genéticas , Unión Proteica , Proteínas Proto-Oncogénicas c-myc/genéticaRESUMEN
Fragile X syndrome (FXS), the most common genetic form of intellectual disability in males, is caused by silencing of the FMR1 gene associated with hypermethylation of the CGG expansion mutation in the 5' UTR of FMR1 in FXS patients. Here, we applied recently developed DNA methylation editing tools to reverse this hypermethylation event. Targeted demethylation of the CGG expansion by dCas9-Tet1/single guide RNA (sgRNA) switched the heterochromatin status of the upstream FMR1 promoter to an active chromatin state, restoring a persistent expression of FMR1 in FXS iPSCs. Neurons derived from methylation-edited FXS iPSCs rescued the electrophysiological abnormalities and restored a wild-type phenotype upon the mutant neurons. FMR1 expression in edited neurons was maintained in vivo after engrafting into the mouse brain. Finally, demethylation of the CGG repeats in post-mitotic FXS neurons also reactivated FMR1. Our data establish that demethylation of the CGG expansion is sufficient for FMR1 reactivation, suggesting potential therapeutic strategies for FXS.
Asunto(s)
Metilación de ADN/genética , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/genética , Síndrome del Cromosoma X Frágil/genética , Edición Génica , Neuronas/patología , Animales , Proteína 9 Asociada a CRISPR/metabolismo , Epigénesis Genética , Células HEK293 , Heterocromatina/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Cinética , Masculino , Ratones , Neuronas/metabolismo , Fenotipo , Regiones Promotoras Genéticas , ARN Guía de Kinetoplastida/metabolismo , Expansión de Repetición de Trinucleótido/genéticaRESUMEN
There is considerable evidence that chromosome structure plays important roles in gene control, but we have limited understanding of the proteins that contribute to structural interactions between gene promoters and their enhancer elements. Large DNA loops that encompass genes and their regulatory elements depend on CTCF-CTCF interactions, but most enhancer-promoter interactions do not employ this structural protein. Here, we show that the ubiquitously expressed transcription factor Yin Yang 1 (YY1) contributes to enhancer-promoter structural interactions in a manner analogous to DNA interactions mediated by CTCF. YY1 binds to active enhancers and promoter-proximal elements and forms dimers that facilitate the interaction of these DNA elements. Deletion of YY1 binding sites or depletion of YY1 protein disrupts enhancer-promoter looping and gene expression. We propose that YY1-mediated enhancer-promoter interactions are a general feature of mammalian gene control.
Asunto(s)
Elementos de Facilitación Genéticos , Regiones Promotoras Genéticas , Factor de Transcripción YY1/metabolismo , Animales , Factor de Unión a CCCTC/metabolismo , Células Madre Embrionarias/metabolismo , Humanos , RatonesRESUMEN
DNA structuring proteins such as CTCF facilitate DNA loop formation and are presumed to be among the major determinants of eukaryotic genome structure. Recent studies, including Rowley et al. (2017), suggest that gene activation and repression play fundamentally important roles in structuring the genome independently of CTCF.
Asunto(s)
Proteínas de Unión al ADN/genética , Proteínas Represoras/genética , ADN , GenomaRESUMEN
This corrects the article DOI: 10.1038/ncomms14385.
RESUMEN
Phase-separated multi-molecular assemblies provide a general regulatory mechanism to compartmentalize biochemical reactions within cells. We propose that a phase separation model explains established and recently described features of transcriptional control. These features include the formation of super-enhancers, the sensitivity of super-enhancers to perturbation, the transcriptional bursting patterns of enhancers, and the ability of an enhancer to produce simultaneous activation at multiple genes. This model provides a conceptual framework to further explore principles of gene control in mammals.
Asunto(s)
Regulación de la Expresión Génica , Modelos Biológicos , Transcripción Genética , Animales , Elementos de Facilitación Genéticos , Células Eucariotas/metabolismo , Humanos , Factores de Transcripción/metabolismo , Activación TranscripcionalRESUMEN
The non-coding regions of tumour cell genomes harbour a considerable fraction of total DNA sequence variation, but the functional contribution of these variants to tumorigenesis is ill-defined. Among these non-coding variants, somatic insertions are among the least well characterized due to challenges with interpreting short-read DNA sequences. Here, using a combination of Chip-seq to enrich enhancer DNA and a computational approach with multiple DNA alignment procedures, we identify enhancer-associated small insertion variants. Among the 102 tumour cell genomes we analyse, small insertions are frequently observed in enhancer DNA sequences near known oncogenes. Further study of one insertion, somatically acquired in primary leukaemia tumour genomes, reveals that it nucleates formation of an active enhancer that drives expression of the LMO2 oncogene. The approach described here to identify enhancer-associated small insertion variants provides a foundation for further study of these abnormalities across human cancers.
Asunto(s)
Elementos de Facilitación Genéticos , Genoma Humano , Mutagénesis Insercional/genética , Oncogenes , Adolescente , Adulto , Secuencia de Bases , Línea Celular Tumoral , Niño , Preescolar , Regulación Leucémica de la Expresión Génica , Humanos , Lactante , Leucemia-Linfoma de Células T del Adulto/genética , Reproducibilidad de los Resultados , Adulto JovenRESUMEN
Cancer arises from genetic alterations that invariably lead to dysregulated transcriptional programs. These dysregulated programs can cause cancer cells to become highly dependent on certain regulators of gene expression. Here, we discuss how transcriptional control is disrupted by genetic alterations in cancer cells, why transcriptional dependencies can develop as a consequence of dysregulated programs, and how these dependencies provide opportunities for novel therapeutic interventions in cancer.