A disease-associated XPA allele interferes with TFIIH binding and primarily affects the transcription-coupled sub-pathway of nucleotide excision repair
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Q Exactive HF
ORGANISM(S): Homo Sapiens (human)
SUBMITTER: 
Román González-Prieto
LAB HEAD: Alfred Vertegaal
PROVIDER: PXD033520 | Pride | 2023-02-19
REPOSITORIES: Pride
Dataset's files
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| HUMAN.fasta | Fasta | |||
| mqpar.xml | Xml | |||
| q108986a.raw | Raw | |||
| q108987a.raw | Raw | |||
| q108988a.raw | Raw |
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Project description:Nucleotide excision repair (NER) is the major DNA repair pathway that removes UV-induced and bulky DNA lesions. There is currently no structure of NER intermediates, which form around the large multisubunit transcription factor IIH (TFIIH). Here we report the cryo-EM structure of an NER intermediate containing TFIIH and the NER factor XPA. Compared to its transcription conformation, the TFIIH structure is rearranged such that its ATPase subunits XPB and XPD bind double- and single-stranded DNA, consistent with their translocase and helicase activities, respectively. XPA releases the inhibitory kinase module of TFIIH, displaces a ‘plug’ element from the DNA-binding pore in XPD, and together with the NER factor XPG stimulates XPD activity. Our results explain how TFIIH is switched from a transcription to a repair factor, and provide the basis for a mechanistic analysis of the NER pathway.
2019-07-02 | PXD013947 | Pride
Project description:Cockayne syndrome (CS) is a photosensitive, DNA repair disorder associated with progeria caused by a defect in the transcription-coupled repair (TCR) subpathway of nucleotide excision repair (NER). Here, complete inactivation of NER in Csbm/m/Xpa-/- mutants causes a phenotype that reliably mimics the human progeroid CS syndrome. Newborn Csbm/m/Xpa-/- mice display attenuated growth, progressive neurological dysfunction, retinal degeneration, cachexia, kyphosis and die before weaning. To investigate whether a disturbance in growth and metabolism could explain the pronounced accelerated organismal deterioration seen in Csbm/m/Xpa-/- mice, we evaluated the liver transcriptome of 15-day old wt, single and double mutant mice (n=4). At this age, the Csbm/m/Xpa-/- pups have not yet become cachectic. Mouse liver transcriptome analysis and several physiological endpoints revealed systemic suppression of the GH/IGF1 somatotroph axis and oxidative metabolism, increased antioxidant responses, hypoglycemia together with hepatic glycogen and fat accumulation. Broad genome-wide parallels between Csbm/m/Xpa-/- and naturally aged mouse liver transcriptomes suggested that these changes are intrinsic to natural aging and the DNA repair-deficient mice. Importantly, wild type (wt) mice exposed to a low dose of chronic genotoxic stress and adult Csbm/m mutant mice recapitulated this response, thereby pointing to a novel link between genome instability and the age-related decline of the somatotroph axis.
2007-01-01 | E-MEXP-835 | biostudies-arrayexpress
Project description:Transcription-coupled nucleotide excision repair (TC-NER) is an important DNA repair mechanism that responds to RNA polymerase (RNAP) stalling and removes DNA lesions from transcribed genes. Activation of TC-NER requires specific factors, such as human Cockayne syndrome group B (CSB) protein or its yeast homolog Rad26. Mutations in CSB are associated with the severe neurological disorder Cockayne syndrome. However, the genome-wide role of CSB/Rad26 in TC-NER, particularly in the context of chromatin organization, is not fully understood. Here we used single-nucleotide resolution UV damage mapping data to investigate the genome-wide function of Rad26 in TC-NER. Our data shows that Rad26 is critical for TC-NER in transcribed regions downstream of the first (+1) nucleosome; however, Rad26 is largely dispensable for TC-NER in the +1 nucleosome. We further show that the Rad26-independent TC-NER in the +1 nucleosome is correlated with high occupancy of the transcription initiation/repair factor TFIIH. Downstream of the +1 nucleosome, the combination of low TFIIH occupancy and high occupancy of the transcription elongation factor Spt4/Spt5 suppresses TC-NER when Rad26 is dysfunctional. Deletion of SPT4 significantly restores TC-NER in the downstream nucleosomes in a rad26∆ mutant. Collectively, these data indicate that the requirement for Rad26 in TC-NER is modulated by the distribution of TFIIH and Spt4/Spt5, and Rad26 mainly functions in the downstream nucleosomes to remove TC-NER suppression by Spt4/Spt5.
2020-06-30 | GSE145911 | GEO
Project description:Purpose: Nucleotide excision repair (NER) in mammalian cells requires the xeroderma pigmentosum group A protein (XPA) as a core factor. Remarkably, XPA and other NER proteins have been detected by chromatin immunoprecipitation at some active promoters, and NER deficiency is reported to influence the activated transcription of selected genes. However, the global influence of XPA on transcription in human cells has not been determined. Methods: We analyzed the human transcriptome by RNA sequencing (RNA-Seq). Four genetically matched human cell line pairs deficient or proficient in XPA were analyzed Results: Of the ~14,000 genes transcribed in each cell line, 325 genes (2%) had a significant XPA-dependent directional change in gene expression that was common to all four pairs (with a false discovery rate of 0.05). These genes were enriched in pathways for the maintenance of mitochondria. Only 27 genes were different by more than 1.5-fold. The most significant hits were AKR1C1 and AKR1C2, involved in steroid hormone metabolism. AKR1C2 protein was lower in all of the immortalized XPA-deficient cells. Retinoic acid treatment led to modest XPA-dependent activation of some genes with transcription-related functions. Conclusions: We conclude that XPA status does not globally influence human gene transcription. However, XPA significantly influences expression of a small subset of genes important for mitochondrial functions and steroid hormone metabolism. The results may help explain defects in neurological function and sterility in individuals with xeroderma pigmentosum.
2017-07-07 | GSE100855 | GEO
Project description:Background: The ability of an organism to repair DNA damage is implicated in carcinogenesis and aging. Interestingly expression profiling of Nucleotide Excision Repair (NER) deficient segmental progeroid mice revealed gene expression changes resembling these observed in aged wild type animals. Our previous transcriptional profiling of NER-deficient C. elegans xpa-1 mutant showed overrepresentation of genes involved in lifespan determination and upregulation of several oxidative stress response genes (Fensgard et al. Aging 2010). However, since an independent study performed by Boyd and coworkers (Boyd et al. Mut Res 2010) showed limited number of changes in xpa-1 mutant. Therefore to independently validate that transcriptome modulation does take place in xpa-1 mutants, we performed another global gene expression profiling based on 5 independent biological replicates allowing more stringent statistical analysis. Results: In agreement with what was observed by Boyd and coworkers (Boyd et al. Mut Res 2010) current transcriptomic analysis detected fewer changes in xpa-1 C. elegans mutant with only a few genes regulated more than 4-fold. Nevertheless, Gene Ontology (GO) enrichment analysis performed on statistically significantly regulated unique protein coding genes revealed overrepresentation of aging gene cluster. Moreover, as before, overexpression of several genes involved in oxidative stress responses was detected. Conclusion: More stringent statistical analysis predictably resulted in a smaller number of regulated genes and thus overrepresented GOs comparing to the earlier paper. However, major conclusions of the previous study can be still regarded as valid, as the most important aging GO is still overrepresented. Background: The ability of an organism to repair DNA damage is implicated in carcinogenesis and aging. Interestingly expression profiling of Nucleotide Excision Repair (NER) deficient segmental progeroid mice revealed gene expression changes resembling these observed in aged wild type animals. Our previous transcriptional profiling of NER-deficient C. elegans xpa-1 mutant showed overrepresentation of genes involved in lifespan determination and upregulation of several oxidative stress response genes (Fensgard et al. Aging 2010). However, since an independent study performed by Boyd and coworkers (Boyd et al. Mut Res 2010) showed limited number of changes in xpa-1 mutant. Therefore to independently validate that transcriptome modulation does take place in xpa-1 mutants, we performed another global gene expression profiling based on 5 independent biological replicates allowing more stringent statistical analysis. Results: In agreement with what was observed by Boyd and coworkers (Boyd et al. Mut Res 2010) current transcriptomic analysis detected fewer changes in xpa-1 C. elegans mutant with only a few genes regulated more than 4-fold. Nevertheless, Gene Ontology (GO) enrichment analysis performed on statistically significantly regulated unique protein coding genes revealed overrepresentation of aging gene cluster. Moreover, as before, overexpression of several genes involved in oxidative stress responses was detected. Conclusion: More stringent statistical analysis predictably resulted in a smaller number of regulated genes and thus overrepresented GOs comparing to the earlier paper. However, major conclusions of the previous study can be still regarded as valid, as the most important aging GO is still overrepresented. Background: The ability of an organism to repair DNA damage is implicated in carcinogenesis and aging. Interestingly expression profiling of Nucleotide Excision Repair (NER) deficient segmental progeroid mice revealed gene expression changes resembling these observed in aged wild type animals. Our previous transcriptional profiling of NER-deficient C. elegans xpa-1 mutant showed overrepresentation of genes involved in lifespan determination and upregulation of several oxidative stress response genes (Fensgard et al. Aging 2010). However, since an independent study performed by Boyd and coworkers (Boyd et al. Mut Res 2010) showed limited number of changes in xpa-1 mutant. Therefore to independently validate that transcriptome modulation does take place in xpa-1 mutants, we performed another global gene expression profiling based on 5 independent biological replicates allowing more stringent statistical analysis. Results: In agreement with what was observed by Boyd and coworkers (Boyd et al. Mut Res 2010) current transcriptomic analysis detected fewer changes in xpa-1 C. elegans mutant with only a few genes regulated more than 4-fold. Nevertheless, Gene Ontology (GO) enrichment analysis performed on statistically significantly regulated unique protein coding genes revealed overrepresentation of aging gene cluster. Moreover, as before, overexpression of several genes involved in oxidative stress responses was detected. Conclusion: More stringent statistical analysis predictably resulted in a smaller number of regulated genes and thus overrepresented GOs comparing to the earlier paper. However, major conclusions of the previous study can be still regarded as valid, as the most important aging GO is still overrepresented. Activation of oxidative stress-responses and downregulation of insulin-like signaling (ILS) is seen in Nucleotide Excision Repair (NER) deficient segmental progeroid mice. Evidence suggests that this is a survival response to persistent transcription-blocking DNA damage, although the relevant lesions have not been identified. Here we provide evidence for transcriptional reprogramming in NER-deficient C. elegans xpa-1 by transcriptomic and proteomic approaches. This reprograming is accompanied by increased intracellular ROS and ATP levels and lifespan shortening in xpa-1 mutant. Moreover we show that Base Excision Repair DNA glycosylase NTH-1 is upstream form the signaling events leading to transcriptomic changes, as its downregulation reverses overexpression of sod-3, gst-4 and aqp-1 genes, reduces intracellular ROS and ATP levels and reverses lifespan shortening observed in xpa-1 mutant. Surprisingly, however, these responses appear to not depend on cyclopurine levels, since these lesions are lower in xpa-1 C. elegans mutant than in the wild type. Finally, we also explore here which other upstream factors are necessary for transcriptional reprograming in xpa-1 mutant. Untreated Caenorhabditis elegans mutant deficient in xpa-1 and the wild type N2 strain were subjected to transcriptome analysis using Affymetrix platform. For each sample group five replicates were analyzed.
2013-05-03 | E-GEOD-39252 | biostudies-arrayexpress
Project description:Background: The ability of an organism to repair DNA damage is implicated in carcinogenesis and aging. Interestingly expression profiling of Nucleotide Excision Repair (NER) deficient segmental progeroid mice revealed gene expression changes resembling these observed in aged wild type animals. Our previous transcriptional profiling of NER-deficient C. elegans xpa-1 mutant showed overrepresentation of genes involved in lifespan determination and upregulation of several oxidative stress response genes (Fensgard et al. Aging 2010). However, since an independent study performed by Boyd and coworkers (Boyd et al. Mut Res 2010) showed limited number of changes in xpa-1 mutant. Therefore to independently validate that transcriptome modulation does take place in xpa-1 mutants, we performed another global gene expression profiling based on 5 independent biological replicates allowing more stringent statistical analysis. Results: In agreement with what was observed by Boyd and coworkers (Boyd et al. Mut Res 2010) current transcriptomic analysis detected fewer changes in xpa-1 C. elegans mutant with only a few genes regulated more than 4-fold. Nevertheless, Gene Ontology (GO) enrichment analysis performed on statistically significantly regulated unique protein coding genes revealed overrepresentation of aging gene cluster. Moreover, as before, overexpression of several genes involved in oxidative stress responses was detected. Conclusion: More stringent statistical analysis predictably resulted in a smaller number of regulated genes and thus overrepresented GOs comparing to the earlier paper. However, major conclusions of the previous study can be still regarded as valid, as the most important aging GO is still overrepresented. Activation of oxidative stress-responses and downregulation of insulin-like signaling (ILS) is seen in Nucleotide Excision Repair (NER) deficient segmental progeroid mice. Evidence suggests that this is a survival response to persistent transcription-blocking DNA damage, although the relevant lesions have not been identified. Here we provide evidence for transcriptional reprogramming in NER-deficient C. elegans xpa-1 by transcriptomic and proteomic approaches. This reprograming is accompanied by increased intracellular ROS and ATP levels and lifespan shortening in xpa-1 mutant. Moreover we show that Base Excision Repair DNA glycosylase NTH-1 is upstream form the signaling events leading to transcriptomic changes, as its downregulation reverses overexpression of sod-3, gst-4 and aqp-1 genes, reduces intracellular ROS and ATP levels and reverses lifespan shortening observed in xpa-1 mutant. Surprisingly, however, these responses appear to not depend on cyclopurine levels, since these lesions are lower in xpa-1 C. elegans mutant than in the wild type. Finally, we also explore here which other upstream factors are necessary for transcriptional reprograming in xpa-1 mutant.
2013-05-03 | GSE39252 | GEO
Project description:Background: The ability of an organism to repair damages to DNA is inextricably linked to aging and cancer. We have characterized and compared the transcriptome of C. elegans mutants deficient in DNA base excision repair, nucleotide excision repair or both to elucidate the transcriptional changes incurred by the reduction of these repair pathways. Results: The gene expression signatures from nth-1, xpa-1 and nth-1;xpa-1 are deciphered. We find that the single mutants are more similar in overall transcriptomic activity compared to the double mutant and their corresponding wild-type (N2). Interestingly, of the enriched Gene Ontology terms for respective mutants are aging and proteolysis for nth-1, aging, energy production and unfolded protein response for xpa-1, and DNA repair and regulation of M-phase for nth-1;xpa-1. We also find striking similarities between the biological processes found to be enriched in the single mutants, which show a shared response in genes associated with aging and insulin signaling. Loss of either BER or NER induces an oxidative stress response, but does not confer resistance to heat shock. Conclusion: Taken together, our study provides a general overview about the global transcriptional changes in the absence of nth-1, xpa-1 and nth-1;xpa-1. Our results also point to a two-tiered global response to combat the effects of decreased DNA repair capacity.
2010-04-12 | GSE16405 | GEO
Project description:TFIIH is a 10-protein complex that is conserved throughout eukaryotes. TFIIH has two primary cellular functions: transcription initiation and nucleotide excision repair (NER). NER in eukaryotes begins by recognition of a bulky lesion by the obligate dimer Rad4-Rad23. This is followed closely by the recruitment of TFIIH which is a structural scaffold, opens a bubble around damaged DNA and scans the damaged strand for bulky lesions. This facilitates the recruitment of two exonucleases which excise the damages strand before an undamaged complement is synthesize. In yeast (Saccharomyces cerevisiae), TFIIH is composed of the two helicases Ssl2 and Rad3, the scaffolding subunits Tfb1, Tfb2, Tfb4 and Ssl1 and the kinase subunits Kin28, Ccl1 and Tfb3, though these later 3 are dispensable for NER.
2021-07-07 | PXD021212 | Pride
Project description:DNA damage forms a major obstacle for gene transcription by RNA polymerase II (Pol II). Transcription-coupled nucleotide excision repair (TC-NER) efficiently eliminates transcription-blocking lesions (TBLs), thereby safeguarding accurate transcription, preserving correct cellular function and counteracting aging. TC-NER initiation involves the recognition of lesion-stalled Pol II by CSB, which recruits the CRL4CSA E3 ubiquitin ligase complex and UVSSA. TBL-induced Pol II ubiquitylation at lysine K1268 by CRL4CSA serves as a critical TC-NER checkpoint, governing Pol II stability and initiating TBL excision by TFIIH recruitment. However, the precise regulatory mechanisms of the CRL4CSA E3 ligase activity and TFIIH recruitment remains elusive. Here, we reveal Serine/Threonine Kinase 19 (STK19) as a novel TC-NER factor. Cryo-EM studies revealed that STK19 is an integral part of the Pol II-TC‐NER complex, bridging CSA with UVSSA, RPB1 and downstream DNA. Live-cell imaging and interaction proteomics shows that STK19 stimulates TC-NER complex stability and proper CRL4CSA complex assembly, resulting in Pol II (K1268) ubiquitylation and subsequent UVSSA and TFIIH recruitment. These findings underscore the crucial role of STK19 as a core component of the TC-NER machinery and its key involvement to the cellular responses to genomic injuries that interfere with transcription.
2024-10-16 | PXD051720 | Pride
Project description:TFIIH is a 10-protein complex that is conserved through out eukaryotes. TFIIH has two primary cellular functions: transcription initiation and nucleotide excision repair (NER). In transcription initiation, TFIIH acts as a structural scaffold, phosphorylates the RNA polymerase II (pol II) C-terminal domain (CTD) and translocates promoter DNA through the pol II active site to facilitate start site selection. In NER, again is a structural scaffold, opens a bubble around damaged DNA and scans the damaged strand for bulky lesions. In yeast (Saccharomyces cerevisiae), TFIIH is composed of the two helicases Ssl2 and Rad3, the scaffolding subunits Tfb1, Tfb2, Tfb4 and Ssl1 and the kinase subunits Kin28, Ccl1 and Tfb3.
2021-07-07 | PXD021211 | Pride