RESUMO
Viruses encode strategies to degrade cellular proteins to promote infection and pathogenesis. Here, we revealed that the non-structural protein NSs of Rift Valley fever virus forms a filamentous E3 ligase to trigger efficient degradation of targeted proteins. Reconstitution in vitro and cryoelectron microscopy analysis with the 2.9-Å resolution revealed that NSs forms right-handed helical fibrils. The NSs filamentous oligomers associate with the cellular FBXO3 to form a remodeled E3 ligase. The NSs-FBXO3 E3 ligase targets the cellular TFIIH complex through the NSs-P62 interaction, leading to ubiquitination and proteasome-dependent degradation of the TFIIH complex. NSs-FBXO3-triggered TFIIH complex degradation resulted in robust inhibition of antiviral immunity and promoted viral pathogenesis in vivo. Furthermore, it is demonstrated that NSs can be programmed to target additional proteins for proteasome-dependent degradation, serving as a versatile targeted protein degrader. These results showed that a virulence factor forms a filamentous and programmable degradation machinery to induce organized degradation of cellular proteins to promote viral infection.
RESUMO
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
A characteristic of the cellular response to stress is the production of RNAs generated from a readthrough transcription of genes, called downstream-of-gene-(DoG)-containing transcripts. Additionally, transcription inhibitor drugs are candidates for fighting cancer. In this work, we report the results of a bioinformatic analysis showing that one of the responses to transcription inhibition is the generation of DoGs in cancer cells. Although some genes that form DoGs were shared between the two cancer lines, there did not appear to be a functional correlation between them. However, our findings show that DoGs are generated as part of the cellular response to transcription inhibition like other types of cellular stress, suggesting that they may be part of the defense against transcriptional stress.
RESUMO
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
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.
Assuntos
Neoplasias , Proteínas Repressoras , Humanos , Fatores de Processamento de RNA/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Repressoras/genética , Processamento Alternativo , Neoplasias/tratamento farmacológico , Neoplasias/genética , Fator de Transcrição TFIIH/genética , Fator de Transcrição TFIIH/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a RNA/metabolismoRESUMO
The heterodecameric transcription factor IIH (TFIIH) functions in multiple cellular processes, foremost in nucleotide excision repair (NER) and transcription initiation by RNA polymerase II. TFIIH is essential for life and hereditary mutations in TFIIH cause the devastating human syndromes xeroderma pigmentosum, Cockayne syndrome or trichothiodystrophy, or combinations of these. In NER, TFIIH binds to DNA after DNA damage is detected and, using its translocase and helicase subunits XPB and XPD, opens up the DNA and checks for the presence of DNA damage. This central activity leads to dual incision and removal of the DNA strand containing the damage, after which the resulting DNA gap is restored. In this review, we discuss new structural and mechanistic insights into the central function of TFIIH in NER. Moreover, we provide an elaborate overview of all currently known patients and diseases associated with inherited TFIIH mutations and describe how our understanding of TFIIH function in NER and transcription can explain the different disease features caused by TFIIH deficiency.
Assuntos
Proteína Grupo D do Xeroderma Pigmentoso , Xeroderma Pigmentoso , Humanos , Fator de Transcrição TFIIH/genética , Fator de Transcrição TFIIH/metabolismo , Proteína Grupo D do Xeroderma Pigmentoso/genética , Proteína Grupo D do Xeroderma Pigmentoso/metabolismo , Reparo do DNA , Xeroderma Pigmentoso/genética , DNA/genéticaRESUMO
The general transcription factor TFIIH is a multi-subunit complex involved in transcription, DNA repair, and cell cycle in eukaryotes. In the human p62 subunit and the budding yeast Saccharomyces cerevisiae Tfb1 subunit of TFIIH, the pleckstrin homology (PH) domain (hPH/scPH) recruits TFIIH to transcription-start and DNA-damage sites by interacting with an acidic intrinsically disordered region in transcription and repair factors. Whereas metazoan PH domains are highly conserved and adopt a similar structure, fungal PH domains are divergent and only the scPH structure is available. Here, we have determined the structure of the PH domain from Tfb1 of fission yeast Schizosaccharomyces pombe (spPH) by NMR. spPH holds an architecture, including the core and external backbone structures, that is closer to hPH than to scPH despite having higher amino acid sequence identity to scPH. In addition, the predicted target-binding site of spPH shares more amino acid similarity with scPH, but spPH contains several key residues identified in hPH as required for specific binding. Using chemical shift perturbation, we have identified binding modes of spPH to spTfa1, a homologue of hTFIIEα, and to spRhp41, a homologue of the repair factors hXPC and scRad4. Both spTfa1 and spRhp41 bind to a similar but distinct surface of spPH by modes that differ from those of target proteins binding to hPH and scPH, revealing that the PH domain of TFIIH interacts with its target proteins in a polymorphic manner in Metazoa, and budding and fission yeasts.
Assuntos
Domínios de Homologia à Plecstrina , Proteínas de Saccharomyces cerevisiae , Animais , Humanos , Estrutura Terciária de Proteína , Fator de Transcrição TFIIH/genética , Fator de Transcrição TFIIH/química , Fator de Transcrição TFIIH/metabolismo , Sítios de Ligação , Domínios Proteicos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
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
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
Trichothiodystrophy (TTD) is a rare hereditary disease whose prominent feature is brittle hair. Additional clinical signs are physical and neurodevelopmental abnormalities and in about half of the cases hypersensitivity to UV radiation. The photosensitive form of TTD (PS-TTD) is most commonly caused by mutations in the ERCC2/XPD gene encoding a subunit of the transcription/DNA repair complex TFIIH. Here we report novel ERCC2/XPD mutations affecting proper protein folding, which generate thermo-labile forms of XPD associated with thermo-sensitive phenotypes characterized by reversible aggravation of TTD clinical signs during episodes of fever. In patient cells, the newly identified XPD variants result in thermo-instability of the whole TFIIH complex and consequent temperature-dependent defects in DNA repair and transcription. Improving the protein folding process by exposing patient cells to low temperature or to the chemical chaperone glycerol allowed rescue of TFIIH thermo-instability and a concomitant recovery of the complex activities. Besides providing a rationale for the peculiar thermo-sensitive clinical features of these new cases, the present findings demonstrate how variations in the cellular concentration of mutated TFIIH impact the cellular functions of the complex and underlie how both quantitative and qualitative TFIIH alterations contribute to TTD clinical features.
Assuntos
Doenças do Cabelo , Dermatopatias , Síndromes de Tricotiodistrofia , Xeroderma Pigmentoso , Humanos , Fator de Transcrição TFIIH/genética , Fator de Transcrição TFIIH/metabolismo , Síndromes de Tricotiodistrofia/genética , Síndromes de Tricotiodistrofia/complicações , Reparo do DNA , Doenças do Cabelo/genética , Transcrição Gênica , Xeroderma Pigmentoso/genética , Proteína Grupo D do Xeroderma Pigmentoso/genética , Proteína Grupo D do Xeroderma Pigmentoso/metabolismoRESUMO
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
Condensins compact chromosomes to promote their equal segregation during mitosis, but the mechanism of condensin engagement with and action on chromatin is incompletely understood. Here, we show that the general transcription factor TFIIH complex is continuously required to establish and maintain a compacted chromosome structure in transcriptionally silent Xenopus egg extracts. Inhibiting the DNA-dependent ATPase activity of the TFIIH complex subunit XPB rapidly and reversibly induces a complete loss of chromosome structure and prevents the enrichment of condensins I and II, but not topoisomerase II, on chromatin. In addition, inhibiting TFIIH prevents condensation of both mouse and Xenopus nuclei in Xenopus egg extracts, which suggests an evolutionarily conserved mechanism of TFIIH action. Reducing nucleosome density through partial histone depletion restores chromosome structure and condensin enrichment in the absence of TFIIH activity. We propose that the TFIIH complex promotes mitotic chromosome condensation by dynamically altering the chromatin environment to facilitate condensin loading and condensin-dependent loop extrusion.
Assuntos
Cromossomos , DNA Topoisomerases Tipo II , Animais , Cromatina , Cromossomos/metabolismo , DNA Topoisomerases Tipo II/metabolismo , Histonas , Camundongos , Mitose , Nucleossomos , Xenopus laevis/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
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
Advances in structural analysis by cryogenic electron microscopy (cryo-EM) and X-ray crystallography have revealed the tertiary structures of various chromatin-related proteins, including transcription factors, RNA polymerases, nucleosomes, and histone chaperones; however, the dynamic structures of intrinsically disordered regions (IDRs) in these proteins remain elusive. Recent studies using nuclear magnetic resonance (NMR), together with molecular dynamics (MD) simulations, are beginning to reveal dynamic structures of the general transcription factor TFIIH complexed with target proteins including the general transcription factor TFIIE, the tumor suppressor p53, the cell cycle protein DP1, the DNA repair factors XPC and UVSSA, and three RNA polymerases, in addition to the dynamics of histone tails in nucleosomes and histone chaperones. In complexes of TFIIH, the PH domain of the p62 subunit binds to an acidic string formed by the IDR in TFIIE, p53, XPC, UVSSA, DP1, and the RPB6 subunit of three RNA polymerases by a common interaction mode, namely extended string-like binding of the IDR on the positively charged surface of the PH domain. In the nucleosome, the dynamic conformations of the N-tails of histones H2A and H2B are correlated, while the dynamic conformations of the N-tails of H3 and H4 form a histone tail network dependent on their modifications and linker DNA. The acidic IDRs of the histone chaperones of FACT and NAP1 play important roles in regulating the accessibility to histone proteins in the nucleosome.
RESUMO
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
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
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
The general transcription factor II H (TFIIH) plays an essential role in transcription and nucleotide excision DNA repair (NER). TFIIH is a complex 10 subunit containing molecular machine that harbors three enzymatic activities while the remaining subunits assume regulatory and/or structural functions. Intriguingly, the three enzymatic activities of the CDK7 kinase, the XPB translocase, and the XPD helicase exert different impacts on the overall activities of TFIIH. While the enzymatic function of the XPD helicase is exclusively required in NER, the CDK7 kinase is deeply involved in transcription, whereas XPB is essential to both processes. Recent structural and biochemical endeavors enabled unprecedented details towards the molecular basis of these different TFIIH functions and how the enzymatic activities are regulated within the entire complex. Due to its involvement in two fundamental processes, TFIIH has become increasingly important as a target in cancer therapy and two of the three enzymes have already been addressed successfully. Here we explore the possibilities of recent high resolution structures in the context of TFIIH druggability and shed light on the functional consequences of the different approaches towards TFIIH inhibition.