RESUMO
Cyclin-dependent kinase 7 (Cdk7) is required in cell-cycle and transcriptional regulation owing to its function as both a CDK-activating kinase (CAK) and part of transcription factor TFIIH. Cdk7 forms active complexes by associating with Cyclin H and Mat1, and is regulated by two phosphorylations in the activation segment (T loop): the canonical activating modification at T170 and another at S164. Here we report the crystal structure of the human Cdk7/Cyclin H/Mat1 complex containing both T-loop phosphorylations. Whereas pT170 coordinates basic residues conserved in other CDKs, pS164 nucleates an arginine network unique to the ternary Cdk7 complex, involving all three subunits. We identify differential dependencies of kinase activity and substrate recognition on the individual phosphorylations. CAK function is unaffected by T-loop phosphorylation, whereas activity towards non-CDK substrates is increased several-fold by T170 phosphorylation. Moreover, dual T-loop phosphorylation stimulates multisite phosphorylation of the RNA polymerase II (RNAPII) carboxy-terminal domain (CTD) and SPT5 carboxy-terminal repeat (CTR) region. In human cells, Cdk7 activation is a two-step process wherein S164 phosphorylation precedes, and may prime, T170 phosphorylation. Thus, dual T-loop phosphorylation can regulate Cdk7 through multiple mechanisms, with pS164 supporting tripartite complex formation and possibly influencing processivity, while pT170 enhances activity towards key transcriptional substrates.
Assuntos
Quinase Ativadora de Quinase Dependente de Ciclina , Quinases Ciclina-Dependentes , Fosforilação , Humanos , Quinases Ciclina-Dependentes/metabolismo , Quinases Ciclina-Dependentes/química , Quinases Ciclina-Dependentes/genética , Ciclina H/metabolismo , Ciclina H/química , Ciclina H/genética , Cristalografia por Raios X , RNA Polimerase II/metabolismo , RNA Polimerase II/química , Fator de Transcrição TFIIH/metabolismo , Fator de Transcrição TFIIH/química , Fator de Transcrição TFIIH/genética , Modelos Moleculares , Fatores de Transcrição/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/genética , Domínios Proteicos , Proteínas de Ciclo CelularRESUMO
Transcription coupled-nucleotide excision repair (TC-NER) removes DNA lesions that block RNA polymerase II (Pol II) transcription. A key step in TC-NER is the recruitment of the TFIIH complex, which initiates DNA unwinding and damage verification; however, the mechanism by which TFIIH is recruited during TC-NER, particularly in yeast, remains unclear. Here, we show that the C-terminal domain (CTD) of elongation factor-1 (Elf1) plays a critical role in TC-NER in yeast by binding TFIIH. Analysis of genome-wide repair of UV-induced cyclobutane pyrimidine dimers (CPDs) using CPD-seq indicates that the Elf1 CTD in yeast is required for efficient TC-NER. We show that the Elf1 CTD binds to the pleckstrin homology (PH) domain of the p62 subunit of TFIIH in vitro, and identify a putative TFIIH-interaction region (TIR) in the Elf1 CTD that is important for PH binding and TC-NER. The Elf1 TIR shows functional, structural, and sequence similarities to a conserved TIR in the mammalian UV sensitivity syndrome A (UVSSA) protein, which recruits TFIIH during TC-NER in mammalian cells. These findings suggest that the Elf1 CTD acts as a functional counterpart to mammalian UVSSA in TC-NER by recruiting TFIIH in response to Pol II stalling at DNA lesions.
Assuntos
Reparo do DNA , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Fator de Transcrição TFIIH , Fator de Transcrição TFIIH/metabolismo , Fator de Transcrição TFIIH/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Ligação Proteica , Transcrição Gênica , Raios Ultravioleta , Domínios Proteicos , RNA Polimerase II/metabolismo , RNA Polimerase II/genética , Dano ao DNA , Dímeros de Pirimidina/metabolismo , Reparo por ExcisãoRESUMO
Transcription factor (TF) IIH is a factor involved in transcription, DNA repair, mitosis, and telomere stability. These functions stem from its helicase/ATPase and kinase activities. Recent reports on the structure and function of the transcription machinery, as well as chromosome compaction during mitosis, suggest that TFIIH also influences nucleosome movement, are explored here.
Assuntos
Nucleossomos , Fator de Transcrição TFIIH , Nucleossomos/genética , Nucleossomos/metabolismo , Fator de Transcrição TFIIH/metabolismo , Fator de Transcrição TFIIH/genética , Humanos , Transcrição Gênica , Reparo do DNA/genética , Mitose/genética , AnimaisRESUMO
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.
Assuntos
Quinase Ativadora de Quinase Dependente de Ciclina , Quinases Ciclina-Dependentes , Regiões Promotoras Genéticas , RNA Polimerase II , Iniciação da Transcrição Genética , Humanos , Quinases Ciclina-Dependentes/metabolismo , Quinases Ciclina-Dependentes/genética , RNA Polimerase II/metabolismo , RNA Polimerase II/genética , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Complexo Mediador/metabolismo , Complexo Mediador/genética , Células HeLa , Fator de Transcrição TFIIH/metabolismo , Fator de Transcrição TFIIH/genética , Células HEK293RESUMO
Congenital nucleotide excision repair (NER) deficiency gives rise to several cancer-prone and/or progeroid disorders. It is not understood how defects in the same DNA repair pathway cause different disease features and severity. Here, we show that the absence of functional ERCC1-XPF or XPG endonucleases leads to stable and prolonged binding of the transcription/DNA repair factor TFIIH to DNA damage, which correlates with disease severity and induces senescence features in human cells. In vivo, in C. elegans, this prolonged TFIIH binding to non-excised DNA damage causes developmental arrest and neuronal dysfunction, in a manner dependent on transcription-coupled NER. NER factors XPA and TTDA both promote stable TFIIH DNA binding and their depletion therefore suppresses these severe phenotypical consequences. These results identify stalled NER intermediates as pathogenic to cell functionality and organismal development, which can in part explain why mutations in XPF or XPG cause different disease features than mutations in XPA or TTDA.
Assuntos
Caenorhabditis elegans , Dano ao DNA , Reparo do DNA , Proteínas de Ligação a DNA , Endonucleases , Fator de Transcrição TFIIH , Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/genética , Humanos , Animais , Fator de Transcrição TFIIH/metabolismo , Fator de Transcrição TFIIH/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Endonucleases/metabolismo , Endonucleases/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteína de Xeroderma Pigmentoso Grupo A/metabolismo , Proteína de Xeroderma Pigmentoso Grupo A/genética , Ligação Proteica , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Mutação , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genéticaRESUMO
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.
Assuntos
Fosfoproteínas , Regiões Promotoras Genéticas , RNA Polimerase II , Proteína de Ligação a TATA-Box , Fator de Transcrição TFIIB , Fatores de Transcrição , RNA Polimerase II/metabolismo , RNA Polimerase II/genética , Humanos , Fator de Transcrição TFIIB/metabolismo , Fator de Transcrição TFIIB/genética , Proteína de Ligação a TATA-Box/metabolismo , Proteína de Ligação a TATA-Box/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Iniciação da Transcrição Genética , Fator de Transcrição TFIIH/metabolismo , Fator de Transcrição TFIIH/genética , Fator de Transcrição TFIIH/química , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Ligação Proteica , Fator de Transcrição TFIIA/metabolismo , Fator de Transcrição TFIIA/genética , Transcrição Gênica , Elongação da Transcrição Genética , RNA/metabolismo , RNA/genética , Fatores de Transcrição TFII/metabolismo , Fatores de Transcrição TFII/genéticaRESUMO
Transcription-coupled repair (TCR) and global genomic repair (GGR) are two subpathways of nucleotide excision repair (NER). The TFIIH subunit Tfb1 contains a Pleckstrin homology domain (PHD), which was shown to interact with one PHD-binding segment (PB) of Rad4 and two PHD-binding segments (PB1 and PB2) of Rad2 in vitro. Whether and how the different Rad2 and Rad4 PBs interact with the same Tfb1 PHD, and whether and how they affect TCR and GGR within the cell remain mysterious. We found that Rad4 PB constitutively interacts with Tfb1 PHD, and the two proteins may function within one module for damage recognition in TCR and GGR. Rad2 PB1 protects Tfb1 from degradation and interacts with Tfb1 PHD at a basal level, presumably within transcription preinitiation complexes when NER is inactive. During a late step of NER, the interaction between Rad2 PB1 and Tfb1 PHD augments, enabling efficient TCR and GGR. Rather than interacting with Tfb1 PHD, Rad2 PB2 constrains the basal interaction between Rad2 PB1 and Tfb1 PHD, thereby weakening the protection of Tfb1 from degradation and enabling rapid augmentation of their interactions within TCR and GGR complexes. Our results shed new light on NER mechanisms.
Assuntos
Reparo do DNA , Proteínas de Saccharomyces cerevisiae , Transcrição Gênica , Dano ao DNA , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Endodesoxirribonucleases , Reparo por Excisão , Ligação Proteica , Domínios Proteicos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Fator de Transcrição TFIIH/metabolismo , Fator de Transcrição TFIIH/genéticaRESUMO
DNA damage occurs throughout life from a variety of sources, and it is imperative to repair damage in a timely manner to maintain genome stability. Thus, DNA repair mechanisms are a fundamental part of life. Nucleotide excision repair (NER) plays an important role in the removal of bulky DNA adducts, such as cyclobutane pyrimidine dimers from ultraviolet light or DNA crosslinking damage from platinum-based chemotherapeutics, such as cisplatin. A main component for the NER pathway is transcription factor IIH (TFIIH), a multifunctional, 10-subunit protein complex with crucial roles in both transcription and NER. In transcription, TFIIH is a component of the pre-initiation complex and is important for promoter opening and the phosphorylation of RNA Polymerase II (RNA Pol II). During repair, TFIIH is important for DNA unwinding, recruitment of downstream repair factors, and verification of the bulky lesion. Several different disease states can arise from mutations within subunits of the TFIIH complex. Most strikingly are xeroderma pigmentosum (XP), XP combined with Cockayne syndrome (CS), and trichothiodystrophy (TTD). Here, we summarize the recruitment and functions of TFIIH in the two NER subpathways, global genomic (GG-NER) and transcription-coupled NER (TC-NER). We will also discuss how TFIIH's roles in the two subpathways lead to different genetic disorders.
Assuntos
Reparo por Excisão , Xeroderma Pigmentoso , Humanos , Reparo do DNA/genética , Xeroderma Pigmentoso/genética , Fator de Transcrição TFIIH/genética , Fator de Transcrição TFIIH/metabolismo , Dano ao DNA/genética , DNA/genética , Nucleotídeos , Transcrição GênicaRESUMO
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
In this issue of Molecular Cell, Abril-Garrido et al.1 used cryo-EM to uncover that the +1 nucleosome inhibits transcription by interfering with the function of the TFIIH translocase via mechanisms that depend on its position relative to the transcription start site.
Assuntos
Nucleossomos , Transcrição Gênica , Nucleossomos/genética , Fator de Transcrição TFIIH/genética , Fator de Transcrição TFIIH/metabolismoRESUMO
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
BACKGROUND: Major depressive disorder (MDD) is a type of emotional dysfunction, and its pathogenesis has not been fully elucidated. Specifically, the key molecules in depression-related brain regions involved in this disease and their contributions to this disease are currently unclear. METHODS: GSE53987 and GSE54568 were selected from the Gene Expression Omnibus database. The data were standardized to identify the common differentially expressed genes (DEGs) in the cortex of MDD patients in the 2 datasets. The DEGs were subjected to Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses. The STRING database was used to build protein-protein interaction networks, and the cytoHubba plugin was used to identify hub genes. Furthermore, we selected another blood transcriptome dataset that included 161 MDD and 169 control samples to explore the changes in the screened hub genes. Mice were subjected to 4 weeks of chronic unpredictable mild stress to establish an animal model of depression, and the expression of these hub genes in tissues of the prefrontal cortex was then detected by quantitative real time polymerase chain reaction (qRT-PCR). We subsequently predicted the possible posttranscriptional regulatory networks and traditional Chinese medicine according to the hub genes using a few online databases. RESULTS: The analysis identified 147 upregulated genes and 402 downregulated genes were identified in the cortex of MDD patients compared with that of the controls. Enrichment analyses revealed that DEGs were predominantly enriched in synapse-related cell functions, linoleic acid metabolism, and other pathways. Protein-protein interaction analysis identified 20 hub genes based on the total score. The changes in KDM6B, CUX2, NAAA, PHKB, NFYA, GTF2H1, CRK, CCNG2, ACER3, and SLC4A2 in the peripheral blood of MDD patients were consistent with those in the brain. Furthermore, the prefrontal cortex of mice with depressive-like behaviors showed significantly increased Kdm6b, Aridb1, Scaf11, and Thoc2 expression and decreased Ccng2 expression compared with that of normal mice, which was consistent with the results found for the human brain. Potential therapeutic candidates, such as citron, fructus citri, leaves of Panax Notoginseng, sanchi flower, pseudoginseng, and dan-shen root, were selected via traditional Chinese medicine screening. CONCLUSIONS: This study identified several novel hub genes in specific brain regions involved in the pathogenesis of MDD, which may not only deepen our understanding of depression but may also provide new ideas for its diagnosis and treatment.
Assuntos
Transtorno Depressivo Maior , Humanos , Animais , Camundongos , Transtorno Depressivo Maior/genética , Redes Reguladoras de Genes , Perfilação da Expressão Gênica/métodos , Mapas de Interação de Proteínas , Encéfalo , Biologia Computacional/métodos , Fator de Transcrição TFIIH/genética , Histona Desmetilases com o Domínio Jumonji/genética , Antiportadores de Cloreto-Bicarbonato/genéticaRESUMO
Transcription factor IIH (TFIIH) is a protein assembly essential for transcription initiation and nucleotide excision repair (NER). Yet, understanding of the conformational switching underpinning these diverse TFIIH functions remains fragmentary. TFIIH mechanisms critically depend on two translocase subunits, XPB and XPD. To unravel their functions and regulation, we build cryo-EM based TFIIH models in transcription- and NER-competent states. Using simulations and graph-theoretical analysis methods, we reveal TFIIH's global motions, define TFIIH partitioning into dynamic communities and show how TFIIH reshapes itself and self-regulates depending on functional context. Our study uncovers an internal regulatory mechanism that switches XPB and XPD activities making them mutually exclusive between NER and transcription initiation. By sequentially coordinating the XPB and XPD DNA-unwinding activities, the switch ensures precise DNA incision in NER. Mapping TFIIH disease mutations onto network models reveals clustering into distinct mechanistic classes, affecting translocase functions, protein interactions and interface dynamics.
Assuntos
DNA Helicases , Reparo do DNA , Fator de Transcrição TFIIH/genética , Fator de Transcrição TFIIH/metabolismo , DNA Helicases/genética , DNA Helicases/metabolismo , Conformação Molecular , DNA/metabolismo , Transcrição GênicaRESUMO
XPA is a central scaffold protein that coordinates the assembly of repair complexes in the global genome (GG-NER) and transcription-coupled nucleotide excision repair (TC-NER) subpathways. Inactivating mutations in XPA cause xeroderma pigmentosum (XP), which is characterized by extreme UV sensitivity and a highly elevated skin cancer risk. Here, we describe two Dutch siblings in their late forties carrying a homozygous H244R substitution in the C-terminus of XPA. They present with mild cutaneous manifestations of XP without skin cancer but suffer from marked neurological features, including cerebellar ataxia. We show that the mutant XPA protein has a severely weakened interaction with the transcription factor IIH (TFIIH) complex leading to an impaired association of the mutant XPA and the downstream endonuclease ERCC1-XPF with NER complexes. Despite these defects, the patient-derived fibroblasts and reconstituted knockout cells carrying the XPA-H244R substitution show intermediate UV sensitivity and considerable levels of residual GG-NER (~50%), in line with the intrinsic properties and activities of the purified protein. By contrast, XPA-H244R cells are exquisitely sensitive to transcription-blocking DNA damage, show no detectable recovery of transcription after UV irradiation, and display a severe deficiency in TC-NER-associated unscheduled DNA synthesis. Our characterization of a new case of XPA deficiency that interferes with TFIIH binding and primarily affects the transcription-coupled subpathway of nucleotide excision repair, provides an explanation of the dominant neurological features in these patients, and reveals a specific role for the C-terminus of XPA in TC-NER.
Assuntos
Neoplasias Cutâneas , Xeroderma Pigmentoso , Humanos , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Alelos , Proteína de Xeroderma Pigmentoso Grupo A/genética , Proteína de Xeroderma Pigmentoso Grupo A/metabolismo , Reparo do DNA/genética , Dano ao DNA/genética , Xeroderma Pigmentoso/genética , Xeroderma Pigmentoso/metabolismo , Neoplasias Cutâneas/genética , Fator de Transcrição TFIIH/genética , Fator de Transcrição TFIIH/metabolismoRESUMO
MicroRNAs (miRNAs) are small non coding RNAs responsible for posttranscriptional regulation of gene expression. Even though almost 2000 precursors have been described so far, additional miRNAs are still being discovered in normal as well as malignant cells. Alike protein coding genes, miRNAs may acquire oncogenic properties in consequence of altered expression or presence of gain or loss of function mutations. In this study we mined datasets from miRNA expression profiling (miRNA-seq) of 7 classic Hodgkin Lymphoma (cHL) cell lines, 10 non-Hodgkin lymphoma (NHL) cell lines and 56 samples of germinal center derived B-cell lymphomas. Our aim was to discover potential novel cHL oncomiRs not reported in miRBase (release 22.1) and expressed in cHL cell lines but no other B-cell lymphomas. We identified six such miRNA candidates in cHL cell lines and verified the expression of two of them encoded at chr2:212678788-212678849 and chr5:168090507-168090561 (GRCh38). Interestingly, we showed that one of the validated miRNAs (located in an intron of the TENM2 gene) is expressed together with its host gene. TENM2 is characterized by hypomethylation and open chromatin around its TSS in cHL cell lines in contrast to NHL cell lines and germinal centre B-cells respectively. It indicates an epigenetic mechanism responsible for aberrant expression of both, the TENM2 gene and the novel miRNA in cHL cell lines. Despite the GO analysis performed with the input of the in silico predicted novel miRNA target genes did not reveal ontologies typically associated with cHL pathogenesis, it pointed to several interesting candidates involved in i.e. lymphopoiesis. These include the lymphoma related BCL11A gene, the IKZF2 gene involved in lymphocyte development or the transcription initiator GTF2H1.
Assuntos
Doença de Hodgkin , Linfoma de Células B , Linfoma não Hodgkin , MicroRNAs , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Doença de Hodgkin/patologia , Linhagem Celular , Centro Germinativo/patologia , Linfoma de Células B/genética , Linfoma não Hodgkin/genética , Regulação Neoplásica da Expressão Gênica , Fator de Transcrição TFIIH/genética , Fator de Transcrição TFIIH/metabolismoRESUMO
TFIIH is a complex essential for transcription of protein-coding genes by RNA polymerase II, DNA repair of UV-lesions and transcription of rRNA by RNA polymerase I. Mutations in TFIIH cause the cancer prone DNA-repair disorder xeroderma pigmentosum (XP) and the developmental and premature aging disorders trichothiodystrophy (TTD) and Cockayne syndrome. A total of 50% of the TTD cases are caused by TFIIH mutations. Using TFIIH mutant patient cells from TTD and XP subjects we can show that the stress-sensitivity of the proteome is reduced in TTD, but not in XP. Using three different methods to investigate the accuracy of protein synthesis by the ribosome, we demonstrate that translational fidelity of the ribosomes of TTD, but not XP cells, is decreased. The process of ribosomal synthesis and maturation is affected in TTD cells and can lead to instable ribosomes. Isolated ribosomes from TTD patients show an elevated error rate when challenged with oxidized mRNA, explaining the oxidative hypersensitivity of TTD cells. Treatment of TTD cells with N-acetyl cysteine normalized the increased translational error-rate and restored translational fidelity. Here we describe a pathomechanism that might be relevant for our understanding of impaired development and aging-associated neurodegeneration.
Assuntos
Síndromes de Tricotiodistrofia , Xeroderma Pigmentoso , Humanos , Fator de Transcrição TFIIH/genética , Fator de Transcrição TFIIH/metabolismo , Reparo do DNA/genética , Xeroderma Pigmentoso/genética , Xeroderma Pigmentoso/patologia , Mutação , Síndromes de Tricotiodistrofia/genética , Síndromes de Tricotiodistrofia/patologia , Ribossomos/genética , Ribossomos/metabolismoRESUMO
BACKGROUND: Repair pathway genes play an important role in the development of lung cancer. The study aimed to assess the correlation between single nucleotide polymorphisms (SNPs) in DNA repair gene (GTF2H1 and RAD54L2) and the risk of lung cancer. METHODS: Five SNPs in GTF2H1 and four SNPs in RAD54L2 in 506 patients with lung cancer and 510 age-and gender-matched healthy controls were genotyped via the Agena MassARRAY platform. The influence of GTF2H1 and RAD54L2 polymorphisms on lung cancer susceptibility was assessed using logistic regression analysis by calculating odds ratios (ORs) and their corresponding 95% confidence intervals (CIs). RESULTS: RAD54L2 rs9864693 GC genotype increased the risk of lung cancer (OR = 1.33, 95%CI: 1.01-1.77, p = 0.045). Stratified analysis found that associations of RAD54L2 rs11720298, RAD54L2 rs4687592, RAD54L2 rs9864693 and GTF2H1 rs4150667 with lung cancer risk were found in subjects aged ≤ 59 years. Precisely, a protective effect of RAD54L2 rs11720298 on the occurrence of lung cancer was observed in non-smokers and drinkers. GTF2H1 rs4150667 was associated with a decreased risk of lung cancer in subjects with BMI ≤ 24 kg/m2. RAD54L2 rs4687592 was associated with an increased risk of lung cancer in drinkers. In addition, GTF2H1 rs3802967 was associated with a reduced risk of lung squamous cell carcinoma. CONCLUSION: Our study first revealed that RAD54L2 rs9864693 was associated with an increased risk of lung cancer in the Chinese Han population. This study may increase the understanding of the effect of RAD54L2 and GTF2H1 polymorphisms on lung cancer occurrence.
Assuntos
DNA Helicases , Predisposição Genética para Doença , Neoplasias Pulmonares , Fator de Transcrição TFIIH , Humanos , Povo Asiático/genética , China/epidemiologia , Neoplasias Pulmonares/epidemiologia , Neoplasias Pulmonares/genética , Polimorfismo de Nucleotídeo Único , Fator de Transcrição TFIIH/genética , DNA Helicases/genéticaRESUMO
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.