RESUMEN
Transcription-coupled DNA repair (TCR) removes bulky DNA lesions impeding RNA polymerase II (RNAPII) transcription. Recent studies have outlined the stepwise assembly of TCR factors CSB, CSA, UVSSA, and TFIIH around lesion-stalled RNAPII. However, the mechanism and factors required for the transition to downstream repair steps, including RNAPII removal to provide repair proteins access to the DNA lesion, remain unclear. Here, we identify STK19 as a new TCR factor facilitating this transition. Loss of STK19 does not impact initial TCR complex assembly or RNAPII ubiquitylation but delays lesion-stalled RNAPII clearance, thereby interfering with the downstream repair reaction. Cryo-EM and mutational analysis reveal that STK19 associates with the TCR complex, positioning itself between RNAPII, UVSSA, and CSA. The structural insights and molecular modeling suggest that STK19 positions the ATPase subunits of TFIIH onto DNA in front of RNAPII. Together, these findings provide new insights into the factors and mechanisms required for TCR.
RESUMEN
Cyclin-dependent kinase 7 (CDK7), part of the general transcription factor TFIIH, promotes gene transcription by phosphorylating the C-terminal domain of RNA polymerase II (RNA Pol II). Here, we combine rapid CDK7 kinase inhibition with multi-omics analysis to unravel the direct functions of CDK7 in human cells. CDK7 inhibition causes RNA Pol II retention at promoters, leading to decreased RNA Pol II initiation and immediate global downregulation of transcript synthesis. Elongation, termination, and recruitment of co-transcriptional factors are not directly affected. Although RNA Pol II, initiation factors, and Mediator accumulate at promoters, RNA Pol II complexes can also proceed into gene bodies without promoter-proximal pausing while retaining initiation factors and Mediator. Further downstream, RNA Pol II phosphorylation increases and initiation factors and Mediator are released, allowing recruitment of elongation factors and an increase in RNA Pol II elongation velocity. Collectively, CDK7 kinase activity promotes the release of initiation factors and Mediator from RNA Pol II, facilitating RNA Pol II escape from the promoter.
Asunto(s)
Quinasa Activadora de Quinasas Ciclina-Dependientes , Quinasas Ciclina-Dependientes , Regiones Promotoras Genéticas , ARN Polimerasa II , Iniciación de la Transcripción Genética , Humanos , Quinasas Ciclina-Dependientes/metabolismo , Quinasas Ciclina-Dependientes/genética , ARN Polimerasa II/metabolismo , ARN Polimerasa II/genética , Fosforilación , Inhibidores de Proteínas Quinasas/farmacología , Complejo Mediador/metabolismo , Complejo Mediador/genética , Células HeLa , Factor de Transcripción TFIIH/metabolismo , Factor de Transcripción TFIIH/genética , Células HEK293RESUMEN
Facilitates chromatin transcription (FACT) is a histone chaperone that supports transcription through chromatin in vitro, but its functional roles in vivo remain unclear. Here, we analyze the in vivo functions of FACT with the use of multi-omics analysis after rapid FACT depletion from human cells. We show that FACT depletion destabilizes chromatin and leads to transcriptional defects, including defective promoter-proximal pausing and elongation, and increased premature termination of RNA polymerase II. Unexpectedly, our analysis revealed that promoter-proximal pausing depends not only on the negative elongation factor (NELF) but also on the +1 nucleosome, which is maintained by FACT.
Asunto(s)
Cromatina , Proteínas del Grupo de Alta Movilidad , Nucleosomas , Regiones Promotoras Genéticas , ARN Polimerasa II , Transcripción Genética , Factores de Elongación Transcripcional , ARN Polimerasa II/metabolismo , ARN Polimerasa II/genética , Humanos , Factores de Elongación Transcripcional/metabolismo , Factores de Elongación Transcripcional/genética , Cromatina/metabolismo , Cromatina/genética , Nucleosomas/metabolismo , Nucleosomas/genética , Proteínas del Grupo de Alta Movilidad/metabolismo , Proteínas del Grupo de Alta Movilidad/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/genética , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Células HeLa , Ensamble y Desensamble de Cromatina , Células HEK293 , Elongación de la Transcripción Genética , Terminación de la Transcripción GenéticaRESUMEN
Phenotypic assays detect small-molecule bioactivity at functionally relevant cellular sites, and inherently cover a variety of targets and mechanisms of action. They can uncover new small molecule-target pairs and may give rise to novel biological insights. By means of an osteoblast differentiation assay which employs a Hedgehog (Hh) signaling agonist as stimulus and which monitors an endogenous marker for osteoblasts, we identified a pyrrolo[3,4-g]quinoline (PQ) pseudo-natural product (PNP) class of osteogenesis inhibitors. The most potent PQ, termed Tafbromin, impairs canonical Hh signaling and modulates osteoblast differentiation through binding to the bromodomain 2 of the TATA-box binding protein-associated factor 1 (TAF1). Tafbromin is the most selective TAF1 bromodomain 2 ligand and promises to be an invaluable tool for the study of biological processes mediated by TAF1(2) bromodomains.
Asunto(s)
Factores Asociados con la Proteína de Unión a TATA , Factor de Transcripción TFIID , Factores Asociados con la Proteína de Unión a TATA/metabolismo , Factores Asociados con la Proteína de Unión a TATA/química , Factor de Transcripción TFIID/metabolismo , Factor de Transcripción TFIID/química , Factor de Transcripción TFIID/antagonistas & inhibidores , Humanos , Histona Acetiltransferasas/metabolismo , Histona Acetiltransferasas/antagonistas & inhibidores , Productos Biológicos/química , Productos Biológicos/farmacología , Osteoblastos/efectos de los fármacos , Osteoblastos/metabolismo , Osteoblastos/citología , Diferenciación Celular/efectos de los fármacos , Quinolinas/química , Quinolinas/farmacología , Estructura MolecularRESUMEN
The transition from transcription initiation to elongation is highly regulated in human cells but remains incompletely understood at the structural level. In particular, it is unclear how interactions between RNA polymerase II (RNA Pol II) and initiation factors are broken to enable promoter escape. Here, we reconstitute RNA Pol II promoter escape in vitro and determine high-resolution structures of initially transcribing complexes containing 8-, 10-, and 12-nt ordered RNAs and two elongation complexes containing 14-nt RNAs. We suggest that promoter escape occurs in three major steps. First, the growing RNA displaces the B-reader element of the initiation factor TFIIB without evicting TFIIB. Second, the rewinding of the transcription bubble coincides with the eviction of TFIIA, TFIIB, and TBP. Third, the binding of DSIF and NELF facilitates TFIIE and TFIIH dissociation, establishing the paused elongation complex. This three-step model for promoter escape fills a gap in our understanding of the initiation-elongation transition of RNA Pol II transcription.
Asunto(s)
Fosfoproteínas , Regiones Promotoras Genéticas , ARN Polimerasa II , Proteína de Unión a TATA-Box , Factor de Transcripción TFIIB , Factores de Transcripción , ARN Polimerasa II/metabolismo , ARN Polimerasa II/genética , Humanos , Factor de Transcripción TFIIB/metabolismo , Factor de Transcripción TFIIB/genética , Proteína de Unión a TATA-Box/metabolismo , Proteína de Unión a TATA-Box/genética , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Iniciación de la Transcripción Genética , Factor de Transcripción TFIIH/metabolismo , Factor de Transcripción TFIIH/genética , Factor de Transcripción TFIIH/química , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Unión Proteica , Factor de Transcripción TFIIA/metabolismo , Factor de Transcripción TFIIA/genética , Transcripción Genética , Elongación de la Transcripción Genética , ARN/metabolismo , ARN/genética , Factores de Transcripción TFII/metabolismo , Factores de Transcripción TFII/genéticaRESUMEN
The Integrator complex can terminate RNA polymerase II (Pol II) in the promoter-proximal region of genes. Previous work has shed light on how Integrator binds to the paused elongation complex consisting of Pol II, the DRB sensitivity-inducing factor (DSIF) and the negative elongation factor (NELF) and how it cleaves the nascent RNA transcript1, but has not explained how Integrator removes Pol II from the DNA template. Here we present three cryo-electron microscopy structures of the complete Integrator-PP2A complex in different functional states. The structure of the pre-termination complex reveals a previously unresolved, scorpion-tail-shaped INTS10-INTS13-INTS14-INTS15 module that may use its 'sting' to open the DSIF DNA clamp and facilitate termination. The structure of the post-termination complex shows that the previously unresolved subunit INTS3 and associated sensor of single-stranded DNA complex (SOSS) factors prevent Pol II rebinding to Integrator after termination. The structure of the free Integrator-PP2A complex in an inactive closed conformation2 reveals that INTS6 blocks the PP2A phosphatase active site. These results lead to a model for how Integrator terminates Pol II transcription in three steps that involve major rearrangements.
Asunto(s)
Microscopía por Crioelectrón , Modelos Moleculares , Proteína Fosfatasa 2 , ARN Polimerasa II , ARN Polimerasa II/metabolismo , ARN Polimerasa II/química , ARN Polimerasa II/ultraestructura , Proteína Fosfatasa 2/metabolismo , Proteína Fosfatasa 2/química , Proteína Fosfatasa 2/ultraestructura , Terminación de la Transcripción Genética , Humanos , Factores de Transcripción/metabolismo , Factores de Transcripción/química , Unión Proteica , Factores de Elongación Transcripcional/metabolismo , Factores de Elongación Transcripcional/química , Proteínas Nucleares/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/ultraestructura , Subunidades de Proteína/metabolismo , Subunidades de Proteína/químicaRESUMEN
During transcription-coupled DNA repair (TCR), RNA polymerase II (Pol II) transitions from a transcriptionally active state to an arrested state that allows for removal of DNA lesions. This transition requires site-specific ubiquitylation of Pol II by the CRL4CSA ubiquitin ligase, a process that is facilitated by ELOF1 in an unknown way. Using cryogenic electron microscopy, biochemical assays and cell biology approaches, we found that ELOF1 serves as an adaptor to stably position UVSSA and CRL4CSA on arrested Pol II, leading to ligase neddylation and activation of Pol II ubiquitylation. In the presence of ELOF1, a transcription factor IIS (TFIIS)-like element in UVSSA gets ordered and extends through the Pol II pore, thus preventing reactivation of Pol II by TFIIS. Our results provide the structural basis for Pol II ubiquitylation and inactivation in TCR.
Asunto(s)
ARN Polimerasa II , Transcripción Genética , ARN Polimerasa II/metabolismo , Reparación por Escisión , Reparación del ADN , ADN/metabolismo , Ubiquitinación , Ligasas , Receptores de Antígenos de Linfocitos TRESUMEN
Eukaryotic genomes are organized into chromatin domains. The molecular mechanisms driving the formation of these domains are difficult to dissect in vivo and remain poorly understood. Here we reconstitute Saccharomyces cerevisiae chromatin in vitro and determine its 3D organization at subnucleosome resolution by micrococcal nuclease-based chromosome conformation capture and molecular dynamics simulations. We show that regularly spaced and phased nucleosome arrays form chromatin domains in vitro that resemble domains in vivo. This demonstrates that neither loop extrusion nor transcription is required for basic domain formation in yeast. In addition, we find that the boundaries of reconstituted domains correspond to nucleosome-free regions and that insulation strength scales with their width. Finally, we show that domain compaction depends on nucleosome linker length, with longer linkers forming more compact structures. Together, our results demonstrate that regular nucleosome positioning is important for the formation of chromatin domains and provide a proof-of-principle for bottom-up 3D genome studies.
Asunto(s)
Cromatina , Nucleosomas , Nucleosomas/genética , Cromatina/genética , ADN , Saccharomyces cerevisiae/genéticaRESUMEN
RNA polymerase II (Pol II) transcription is regulated by many elongation factors. Among these factors, TFIIF, PAF-RTF1, ELL and Elongin stimulate mRNA chain elongation by Pol II. Cryo-EM structures of Pol II complexes with these elongation factors now reveal some general principles on how elongation factors bind Pol II and how they stimulate transcription. All four elongation factors contact Pol II at domains external 2 and protrusion, whereas TFIIF and ELL additionally bind the Pol II lobe. All factors apparently stabilize cleft-flanking elements, whereas RTF1 and Elongin additionally approach the active site with a latch element and may influence catalysis or translocation. Due to the shared binding sites on Pol II, factor binding is mutually exclusive, and thus it remains to be studied what determines which elongation factors bind at a certain gene and under which condition.