RESUMO
Transcription-coupled nucleotide excision repair (TC-NER) is initiated by the stalling of elongating RNA polymerase II (RNAPIIo) at DNA lesions. The ubiquitination of RNAPIIo in response to DNA damage is an evolutionarily conserved event, but its function in mammals is unknown. Here, we identified a single DNA damage-induced ubiquitination site in RNAPII at RPB1-K1268, which regulates transcription recovery and DNA damage resistance. Mechanistically, RPB1-K1268 ubiquitination stimulates the association of the core-TFIIH complex with stalled RNAPIIo through a transfer mechanism that also involves UVSSA-K414 ubiquitination. We developed a strand-specific ChIP-seq method, which revealed RPB1-K1268 ubiquitination is important for repair and the resolution of transcriptional bottlenecks at DNA lesions. Finally, RPB1-K1268R knockin mice displayed a short life-span, premature aging, and neurodegeneration. Our results reveal RNAPII ubiquitination provides a two-tier protection mechanism by activating TC-NER and, in parallel, the processing of DNA damage-stalled RNAPIIo, which together prevent prolonged transcription arrest and protect against neurodegeneration.
Assuntos
Reparo do DNA/fisiologia , RNA Polimerase II/metabolismo , Animais , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , DNA/metabolismo , Dano ao DNA/fisiologia , DNA Helicases/metabolismo , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , Feminino , Células HCT116 , Células HEK293 , Células HeLa , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , RNA Polimerase II/genética , UbiquitinaçãoRESUMO
Transcription factor IIH (TFIIH) is a multiprotein complex involved in both transcription and DNA repair, revealing a striking functional link between these two processes. Some of its subunits also belong to complexes involved in other cellular processes, such as chromosome segregation and cell cycle regulation, emphasizing the multitasking capabilities of this factor. This review aims to depict the structure of TFIIH and to dissect the roles of its subunits in different cellular mechanisms. Our understanding of the biochemistry of TFIIH has greatly benefited from studies focused on diseases related to TFIIH mutations. We address the etiology of these disorders and underline the fact that TFIIH can be considered a promising target for therapeutic strategies.
Assuntos
Reparo do DNA/efeitos dos fármacos , Fator de Transcrição TFIIH/genética , Transcrição Gênica/efeitos dos fármacos , Síndromes de Tricotiodistrofia/genética , Xeroderma Pigmentoso/genética , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Segregação de Cromossomos , DNA/genética , DNA/metabolismo , Dano ao DNA , Humanos , Modelos Moleculares , Terapia de Alvo Molecular , Mutação , Fenilenodiaminas/uso terapêutico , Subunidades Proteicas/antagonistas & inibidores , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Pirimidinas/uso terapêutico , Espironolactona/uso terapêutico , Fator de Transcrição TFIIH/antagonistas & inibidores , Fator de Transcrição TFIIH/metabolismo , Síndromes de Tricotiodistrofia/tratamento farmacológico , Síndromes de Tricotiodistrofia/metabolismo , Síndromes de Tricotiodistrofia/patologia , Xeroderma Pigmentoso/tratamento farmacológico , Xeroderma Pigmentoso/metabolismo , Xeroderma Pigmentoso/patologiaRESUMO
A complete, 52-protein, 2.5 million dalton, Mediator-RNA polymerase II pre-initiation complex (Med-PIC) was assembled and analyzed by cryo-electron microscopy and by chemical cross-linking and mass spectrometry. The resulting complete Med-PIC structure reveals two components of functional significance, absent from previous structures, a protein kinase complex and the Mediator-activator interaction region. It thereby shows how the kinase and its target, the C-terminal domain of the polymerase, control Med-PIC interaction and transcription.
Assuntos
Complexo Mediador/química , Complexo Mediador/metabolismo , Modelos Moleculares , RNA Polimerase II/química , RNA Polimerase II/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Microscopia Crioeletrônica , Regulação da Expressão Gênica , Espectrometria de Massas , Fosforilação , Estrutura Terciária de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Previous structural studies of the initiation-elongation transition of RNA polymerase II (pol II) transcription have relied on the use of synthetic oligonucleotides, often artificially discontinuous to capture pol II in the initiating state. Here, we report multiple structures of initiation complexes converted de novo from a 33-subunit yeast pre-initiation complex (PIC) through catalytic activities and subsequently stalled at different template positions. We determine that PICs in the initially transcribing complex (ITC) can synthesize a transcript of â¼26 nucleotides before transitioning to an elongation complex (EC) as determined by the loss of general transcription factors (GTFs). Unexpectedly, transition to an EC was greatly accelerated when an ITC encountered a downstream EC stalled at promoter proximal regions and resulted in a collided head-to-end dimeric EC complex. Our structural analysis reveals a dynamic state of TFIIH, the largest of GTFs, in PIC/ITC with distinct functional consequences at multiple steps on the pathway to elongation.
Assuntos
RNA Polimerase II/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Iniciação da Transcrição Genética , Microscopia Crioeletrônica , Regulação Fúngica da Expressão Gênica , Modelos Moleculares , Regiões Promotoras Genéticas , Conformação Proteica , RNA Polimerase II/genética , RNA Polimerase II/ultraestrutura , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/ultraestrutura , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/ultraestrutura , Relação Estrutura-Atividade , Fatores de Tempo , Elongação da Transcrição Genética , Fatores de Transcrição TFII/genética , Fatores de Transcrição TFII/metabolismoRESUMO
Nucleotide excision repair (NER) counteracts the onset of cancer and aging by removing helix-distorting DNA lesions via a "cut-and-patch"-type reaction. The regulatory mechanisms that drive NER through its successive damage recognition, verification, incision, and gap restoration reaction steps remain elusive. Here, we show that the RAD5-related translocase HLTF facilitates repair through active eviction of incised damaged DNA together with associated repair proteins. Our data show a dual-incision-dependent recruitment of HLTF to the NER incision complex, which is mediated by HLTF's HIRAN domain that binds 3'-OH single-stranded DNA ends. HLTF's translocase motor subsequently promotes the dissociation of the stably damage-bound incision complex together with the incised oligonucleotide, allowing for an efficient PCNA loading and initiation of repair synthesis. Our findings uncover HLTF as an important NER factor that actively evicts DNA damage, thereby providing additional quality control by coordinating the transition between the excision and DNA synthesis steps to safeguard genome integrity.
Assuntos
Reparo do DNA , Proteínas de Ligação a DNA , DNA/genética , DNA/metabolismo , Dano ao DNA , Replicação do DNA , Proteínas de Ligação a DNA/genéticaRESUMO
Although telomeres are essential for chromosome stability, they represent fragile structures in our genome. Telomere shortening occurs during aging in cells lacking telomerase due to the end replication problem. In addition, recent work uncovered that the bulk of telomeric DNA poses severe hurdles for the semiconservative DNA replication machinery, requiring the assistance of an increasing number of specialized factors that prevent accidental telomere loss or damage events. In this issue of Genes & Development, Yang and colleagues (pp. 956-969) discover that TFIIH, a basic component of the PolII transcription initiation and nucleotide excision repair machinery, facilitates telomere replication. TFIIH is recruited to telomeres by the shelterin component TRF1, taking on at telomeres a moonlighting function.
Assuntos
Telomerase , Proteína 1 de Ligação a Repetições Teloméricas , Telômero/genética , Telômero/metabolismo , Encurtamento do Telômero , Proteínas de Ligação a Telômeros/metabolismo , Telomerase/metabolismo , Complexo ShelterinaRESUMO
Telomeric DNA challenges the replisome and requires TRF1 for efficient duplication. TRF1 recruits the BLM helicase, but BLM loss does not explain the extensive telomere fragility, ATR signaling, and sister telomere associations (STAs) induced by TRF1 deletion. Here, we document that Helix2 of the TRFH domain and Helix1 of the Myb domain of TRF1 are required for efficient telomere replication. Mutation of both helices generated a TRF1 separation-of-function mutant (TRF1-E83K/LW-TI) that induced severe telomere replication defects but no ATR signaling or STAs. We identified the transcription and nucleotide excision repair (NER) factor TFIIH as a critical effector of TRF1. Loss of TFIIH subunits, but no other NER factors, caused the same telomere replication phenotypes as the TRF1-E83K/LW-TI mutant independent of the effects on TRF1 expression. TFIIH subunits coimmunoprecipitated with wild-type TRF1 but not with TRF1-E83K/LW-TI. These results establish that the major mechanism by which TRF1 ensures telomere replication involves a noncanonical function of TFIIH.
Assuntos
Telômero , Proteína 1 de Ligação a Repetições Teloméricas , Telômero/genética , Telômero/metabolismo , Replicação do DNA/genética , DNA Helicases/genética , DNA Helicases/metabolismo , DNA/metabolismoRESUMO
RNA polymerase II (RNA Pol II) transcription reconstituted from purified factors suggests pre-initiation complexes (PICs) can assemble by sequential incorporation of factors at the TATA box. However, these basal transcription reactions are generally independent of activators and co-activators. To study PIC assembly under more realistic conditions, we used single-molecule microscopy to visualize factor dynamics during activator-dependent reactions in nuclear extracts. Surprisingly, RNA Pol II, TFIIF, and TFIIE can pre-assemble on enhancer-bound activators before loading into PICs, and multiple RNA Pol II complexes can bind simultaneously to create a localized cluster. Unlike TFIIF and TFIIE, TFIIH binding is singular and dependent on the basal promoter. Activator-tethered factors exhibit dwell times on the order of seconds. In contrast, PICs can persist on the order of minutes in the absence of nucleotide triphosphates, although TFIIE remains unexpectedly dynamic even after TFIIH incorporation. Our kinetic measurements lead to a new branched model for activator-dependent PIC assembly.
Assuntos
Complexo Mediador/metabolismo , RNA Polimerase II/metabolismo , Iniciação da Transcrição Genética/fisiologia , Núcleo Celular/metabolismo , Complexo Mediador/genética , Regiões Promotoras Genéticas , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Imagem Individual de Molécula , TATA Box/genética , Proteína de Ligação a TATA-Box/genética , Fator de Transcrição TFIIH/metabolismo , Fatores de Transcrição TFII/metabolismo , Transcrição Gênica/genéticaRESUMO
Eukaryotic transcription starts with the assembly of a preinitiation complex (PIC) on core promoters. Flanking this region is the +1 nucleosome, the first nucleosome downstream of the core promoter. While this nucleosome is rich in epigenetic marks and plays a key role in transcription regulation, how the +1 nucleosome interacts with the transcription machinery has been a long-standing question. Here, we summarize recent structural and functional studies of the +1 nucleosome in complex with the PIC. We specifically focus on how differently organized promoter-nucleosome templates affect the assembly of the PIC and PIC-Mediator on chromatin and result in distinct transcription initiation.
Assuntos
Cromatina , Nucleossomos , Nucleossomos/genética , Cromatina/genética , Regiões Promotoras Genéticas , Transcrição Gênica , RNA Polimerase II/metabolismoRESUMO
RNA polymerase II (Pol II) transcribes all protein-coding genes and many noncoding RNAs in eukaryotic genomes. Although Pol II is a complex, 12-subunit enzyme, it lacks the ability to initiate transcription and cannot consistently transcribe through long DNA sequences. To execute these essential functions, an array of proteins and protein complexes interact with Pol II to regulate its activity. In this review, we detail the structure and mechanism of over a dozen factors that govern Pol II initiation (e.g., TFIID, TFIIH, and Mediator), pausing, and elongation (e.g., DSIF, NELF, PAF, and P-TEFb). The structural basis for Pol II transcription regulation has advanced rapidly in the past decade, largely due to technological innovations in cryoelectron microscopy. Here, we summarize a wealth of structural and functional data that have enabled a deeper understanding of Pol II transcription mechanisms; we also highlight mechanistic questions that remain unanswered or controversial.
Assuntos
RNA Polimerase II/química , RNA Polimerase II/metabolismo , Transcrição Gênica/genética , Animais , Ativação Enzimática , Humanos , Ligação Proteica , Estrutura Quaternária de Proteína , Pesquisa/tendênciasRESUMO
CDK7 associates with the 10-subunit TFIIH complex and regulates transcription by phosphorylating the C-terminal domain (CTD) of RNA polymerase II (RNAPII). Few additional CDK7 substrates are known. Here, using the covalent inhibitor SY-351 and quantitative phosphoproteomics, we identified CDK7 kinase substrates in human cells. Among hundreds of high-confidence targets, the vast majority are unique to CDK7 (i.e., distinct from other transcription-associated kinases), with a subset that suggest novel cellular functions. Transcription-associated factors were predominant CDK7 substrates, including SF3B1, U2AF2, and other splicing components. Accordingly, widespread and diverse splicing defects, such as alternative exon inclusion and intron retention, were characterized in CDK7-inhibited cells. Combined with biochemical assays, we establish that CDK7 directly activates other transcription-associated kinases CDK9, CDK12, and CDK13, invoking a "master regulator" role in transcription. We further demonstrate that TFIIH restricts CDK7 kinase function to the RNAPII CTD, whereas other substrates (e.g., SPT5 and SF3B1) are phosphorylated by the three-subunit CDK-activating kinase (CAK; CCNH, MAT1, and CDK7). These results suggest new models for CDK7 function in transcription and implicate CAK dissociation from TFIIH as essential for kinase activation. This straightforward regulatory strategy ensures CDK7 activation is spatially and temporally linked to transcription, and may apply toward other transcription-associated kinases.
Assuntos
Quinases Ciclina-Dependentes/metabolismo , Modelos Biológicos , Fator de Transcrição TFIIH/metabolismo , Transcrição Gênica/genética , Processamento Alternativo/genética , Sobrevivência Celular/efeitos dos fármacos , Quinases Ciclina-Dependentes/antagonistas & inibidores , Quinases Ciclina-Dependentes/genética , Ativação Enzimática/genética , Células HL-60 , Humanos , Quinase Ativadora de Quinase Dependente de CiclinaRESUMO
Hutchinson-Gilford progeria syndrome (HGPS) is a lethal premature aging disorder without an effective therapeutic regimen. Because of their targetability and influence on gene expression, microRNAs (miRNAs) are attractive therapeutic tools to treat diseases. Here we identified that hsa-miR-59 (miR-59) was markedly upregulated in HGPS patient cells and in multiple tissues of an HGPS mouse model (LmnaG609G/G609G ), which disturbed the interaction between RNAPII and TFIIH, resulting in abnormal expression of cell cycle genes by targeting high-mobility group A family HMGA1 and HMGA2. Functional inhibition of miR-59 alleviated the cellular senescence phenotype of HGPS cells. Treatment with AAV9-mediated anti-miR-59 reduced fibrosis in the quadriceps muscle, heart, and aorta, suppressed epidermal thinning and dermal fat loss, and yielded a 25.5% increase in longevity of LmnaG609G/G609G mice. These results identify a new strategy for the treatment of HGPS and provide insight into the etiology of HGPS disease.
Assuntos
MicroRNAs , Progéria , Camundongos , Animais , Progéria/genética , Antagomirs/uso terapêutico , Senescência Celular/genética , MicroRNAs/genética , FenótipoRESUMO
Current antiretroviral therapy for HIV-1 infection does not represent a cure for infection as viral rebound inevitably occurs following discontinuation of treatment. The "block and lock" therapeutic strategy is intended to enforce proviral latency and durably suppress viremic reemergence in the absence of other intervention. The transcription-associated cyclin-dependent protein kinases (tCDKs) are required for expression from the 5´ HIV-1 long-terminal repeat, but the therapeutic potential of inhibiting these kinases for enforcing HIV-1 latency has not been characterized. Here, we expanded previous observations to directly compare the effect of highly selective small molecule inhibitors of CDK7 (YKL-5-124), CDK9 (LDC000067), and CDK8/19 (Senexin A), and found each of these prevented HIV-1 provirus expression at concentrations that did not cause cell toxicity. Inhibition of CDK7 caused cell cycle arrest, whereas CDK9 and CDK8/19 inhibitors did not, and could be continuously administered to establish proviral latency. Upon discontinuation of drug administration, HIV immediately rebounded in cells that had been treated with the CDK9 inhibitor, while proviral latency persisted for several days in cells that had been treated with CDK8/19 inhibitors. These results identify the mediator kinases CDK8/CDK19 as potential "block and lock" targets for therapeutic suppression of HIV-1 provirus expression.
Assuntos
HIV-1 , Quinases Ciclina-Dependentes/genética , Quinases Ciclina-Dependentes/metabolismo , Quinases Ciclina-Dependentes/farmacologia , Quinase 9 Dependente de Ciclina/genética , Quinase 9 Dependente de Ciclina/metabolismo , Ciclinas/metabolismo , Ciclinas/farmacologiaRESUMO
Mediator is a highly conserved transcriptional coactivator organized into four modules, namely Tail, Middle, Head, and Kinase (CKM). Previous work suggests regulatory roles for Tail and CKM, but an integrated model for these activities is lacking. Here, we analyzed the genome-wide distribution of Mediator subunits in wild-type and mutant yeast cells in which RNA polymerase II promoter escape is blocked, allowing detection of transient Mediator forms. We found that although all modules are recruited to upstream activated regions (UAS), assembly of Mediator within the pre-initiation complex is accompanied by the release of CKM. Interestingly, our data show that CKM regulates Mediator-UAS interaction rather than Mediator-promoter association. In addition, although Tail is required for Mediator recruitment to UAS, Tailless Mediator nevertheless interacts with core promoters. Collectively, our data suggest that the essential function of Mediator is mediated by Head and Middle at core promoters, while Tail and CKM play regulatory roles.
Assuntos
Regulação Fúngica da Expressão Gênica , Complexo Mediador/genética , RNA Polimerase II/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Fator de Transcrição TFIIB/genética , Sítios de Ligação , Complexo Mediador/metabolismo , Modelos Moleculares , Regiões Promotoras Genéticas , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , RNA Polimerase II/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Fator de Transcrição TFIIB/metabolismo , Iniciação da Transcrição Genética , Ativação TranscricionalRESUMO
Mediator is a transcriptional co-activator recruited to enhancers by DNA-binding activators, and it also interacts with RNA polymerase (Pol) II as part of the preinitiation complex (PIC). We demonstrate that a single Mediator complex associates with the enhancer and core promoter in vivo, indicating that it can physically bridge these transcriptional elements. However, the Mediator kinase module associates strongly with the enhancer, but not with the core promoter, and it dissociates from the enhancer upon depletion of the TFIIH kinase. Severing the kinase module from Mediator by removing the connecting subunit Med13 does not affect Mediator association at the core promoter but increases occupancy at enhancers. Thus, Mediator undergoes a compositional change in which the kinase module, recruited via Mediator to the enhancer, dissociates from Mediator to permit association with Pol II and the PIC. As such, Mediator acts as a dynamic bridge between the enhancer and core promoter.
Assuntos
Regulação Fúngica da Expressão Gênica , Complexo Mediador/genética , RNA Polimerase II/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Sítios de Ligação , Elementos Facilitadores Genéticos , Complexo Mediador/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , RNA Polimerase II/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Fator de Transcrição TFIIH/genética , Fator de Transcrição TFIIH/metabolismo , Iniciação da Transcrição Genética , Ativação TranscricionalRESUMO
The cancer-free photosensitive trichothiodystrophy (PS-TTD) and the cancer-prone xeroderma pigmentosum (XP) are rare monogenic disorders that can arise from mutations in the same genes, namely ERCC2/XPD or ERCC3/XPB Both XPD and XPB proteins belong to the 10-subunit complex transcription factor IIH (TFIIH) that plays a key role in transcription and nucleotide excision repair, the DNA repair pathway devoted to the removal of ultraviolet-induced DNA lesions. Compelling evidence suggests that mutations affecting the DNA repair activity of TFIIH are responsible for the pathological features of XP, whereas those also impairing transcription give rise to TTD. By adopting a relatives-based whole transcriptome sequencing approach followed by specific gene expression profiling in primary fibroblasts from a large cohort of TTD or XP cases with mutations in ERCC2/XPD gene, we identify the expression alterations specific for TTD primary dermal fibroblasts. While most of these transcription deregulations do not impact on the protein level, very low amounts of prostaglandin I2 synthase (PTGIS) are found in TTD cells. PTGIS catalyzes the last step of prostaglandin I2 synthesis, a potent vasodilator and inhibitor of platelet aggregation. Its reduction characterizes all TTD cases so far investigated, both the PS-TTD with mutations in TFIIH coding genes as well as the nonphotosensitive (NPS)-TTD. A severe impairment of TFIIH and RNA polymerase II recruitment on the PTGIS promoter is found in TTD but not in XP cells. Thus, PTGIS represents a biomarker that combines all PS- and NPS-TTD cases and distinguishes them from XP.
Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Neoplasias/patologia , Síndromes de Tricotiodistrofia/enzimologia , Animais , Células Cultivadas , Sistema Enzimático do Citocromo P-450/genética , Epoprostenol , Fibroblastos/metabolismo , Fibroblastos/patologia , Fibroblastos/efeitos da radiação , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos da radiação , Camundongos , Pele/patologia , Transcrição Gênica , Síndromes de Tricotiodistrofia/genética , Raios Ultravioleta , Xeroderma Pigmentoso/genéticaRESUMO
TFIIH is an evolutionarily conserved complex that plays central roles in both RNA polymerase II (pol II) transcription and DNA repair. As an integral component of the pol II preinitiation complex, TFIIH regulates pol II enzyme activity in numerous ways. The TFIIH subunit XPB/Ssl2 is an ATP-dependent DNA translocase that stimulates promoter opening prior to transcription initiation. Crosslinking-mass spectrometry and cryo-EM results have shown a conserved interaction network involving XPB/Ssl2 and the C-terminal Hub region of the TFIIH p52/Tfb2 subunit, but the functional significance of specific residues is unclear. Here, we systematically mutagenized the HubA region of Tfb2 and screened for growth phenotypes in a TFB6 deletion background in Saccharomyces cerevisiae. We identified six lethal and 12 conditional mutants. Slow growth phenotypes of all but three conditional mutants were relieved in the presence of TFB6, thus identifying a functional interaction between Tfb2 HubA mutants and Tfb6, a protein that dissociates Ssl2 from TFIIH. Our biochemical analysis of Tfb2 mutants with severe growth phenotypes revealed defects in Ssl2 association, with similar results in human cells. Further characterization of these tfb2 mutant cells revealed defects in GAL gene induction, and reduced occupancy of TFIIH and pol II at GAL gene promoters, suggesting that functionally competent TFIIH is required for proper pol II recruitment to preinitiation complexes in vivo. Consistent with recent structural models of TFIIH, our results identify key residues in the p52/Tfb2 HubA domain that are required for stable incorporation of XPB/Ssl2 into TFIIH and for pol II transcription.
Assuntos
DNA Helicases , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Fator de Transcrição TFIIH , Humanos , DNA Helicases/genética , DNA Helicases/metabolismo , Reparo do DNA , Mutagênese , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Fator de Transcrição TFIIH/genética , Fator de Transcrição TFIIH/metabolismo , Transcrição GênicaRESUMO
Melanoma cell phenotype switching between differentiated melanocytic and undifferentiated mesenchymal-like states drives metastasis and drug resistance. CDK7 is the serine/threonine kinase of the basal transcription factor TFIIH. We show that dedifferentiation of melanocytic-type melanoma cells into mesenchymal-like cells and acquisition of tolerance to targeted therapies is achieved through chronic inhibition of CDK7. In addition to emergence of a mesenchymal-type signature, we identify a GATA6-dependent gene expression program comprising genes such as AMIGO2 or ABCG2 involved in melanoma survival or targeted drug tolerance, respectively. Mechanistically, we show that CDK7 drives expression of the melanocyte lineage transcription factor MITF that in turn binds to an intronic region of GATA6 to repress its expression in melanocytic-type cells. We show that GATA6 expression is activated in MITF-low melanoma cells of patient-derived xenografts. Taken together, our data show how the poorly characterized repressive function of MITF in melanoma participates in a molecular cascade regulating activation of a transcriptional program involved in survival and drug resistance in melanoma.
Assuntos
Melanoma , Fator de Transcrição Associado à Microftalmia , Linhagem Celular Tumoral , Tolerância a Medicamentos , Regulação Neoplásica da Expressão Gênica , Humanos , Melanoma/tratamento farmacológico , Melanoma/genética , Fator de Transcrição Associado à Microftalmia/genética , Fator de Transcrição Associado à Microftalmia/metabolismoRESUMO
Transcription-coupled nucleotide excision repair (TC-NER) is an important DNA repair mechanism that removes RNA polymerase (RNAP)-stalling DNA damage from the transcribed strand (TS) of active genes. TC-NER deficiency in humans is associated with the severe neurological disorder Cockayne syndrome. Initiation of TC-NER is mediated by specific factors such as the human Cockayne syndrome group B (CSB) protein or its yeast homolog Rad26. However, the genome-wide role of CSB/Rad26 in TC-NER, particularly in the context of the chromatin organization, is unclear. Here, we used single-nucleotide resolution UV damage mapping data to show that Rad26 and its ATPase activity is critical for TC-NER downstream of the first (+1) nucleosome in gene coding regions. However, TC-NER on the transcription start site (TSS)-proximal half of the +1 nucleosome is largely independent of Rad26, likely due to high occupancy of the transcription initiation/repair factor TFIIH in this nucleosome. Downstream of the +1 nucleosome, the combination of low TFIIH occupancy and high occupancy of the transcription elongation factor Spt4/Spt5 suppresses TC-NER in Rad26-deficient cells. We show that deletion of SPT4 significantly restores TC-NER across the genome in a rad26∆ mutant, particularly in the downstream nucleosomes. These data demonstrate that the requirement for Rad26 in TC-NER is modulated by the distribution of TFIIH and Spt4/Spt5 in transcribed chromatin and Rad26 mainly functions downstream of the +1 nucleosome to remove TC-NER suppression by Spt4/Spt5.
Assuntos
Adenosina Trifosfatases , Reparo do DNA/genética , Nucleossomos/genética , Proteínas de Saccharomyces cerevisiae , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , DNA Helicases , Enzimas Reparadoras do DNA , Genoma Fúngico/genética , Humanos , Nucleossomos/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
The interaction between mRNA and ribosomal RNA (rRNA) transcription in cancer remains unclear. RNAP I and II possess a common N-terminal tail (NTT), RNA polymerase subunit RPB6, which interacts with P62 of transcription factor (TF) IIH, and is a common target for the link between mRNA and rRNA transcription. The mRNAs and rRNAs affected by FUBP1-interacting repressor (FIR) were assessed via RNA sequencing and qRT-PCR analysis. An FIR, a c-myc transcriptional repressor, and its splicing form FIRΔexon2 were examined to interact with P62. Protein interaction was investigated via isothermal titration calorimetry measurements. FIR was found to contain a highly conserved region homologous to RPB6 that interacts with P62. FIRΔexon2 competed with FIR for P62 binding and coactivated transcription of mRNAs and rRNAs. Low-molecular-weight chemical compounds that bind to FIR and FIRΔexon2 were screened for cancer treatment. A low-molecular-weight chemical, BK697, which interacts with FIRΔexon2, inhibited tumor cell growth with rRNA suppression. In this study, a novel coactivation pathway for cancer-related mRNA and rRNA transcription through TFIIH/P62 by FIRΔexon2 was proposed. Direct evidence in X-ray crystallography is required in further studies to show the conformational difference between FIR and FIRΔexon2 that affects the P62-RBP6 interaction.