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UVB induces a major genome-wide rearrangement of RNA polymerase II at transcribed human genes

ABSTRACT: Abstract Background: Faithful transcription of DNA is constantly threatened by different endogenous and environmental genotoxic effects. Transcription coupled repair has been described to quickly remove DNA lesions from the transcribed strand of active genes, permitting rapid resumption of blocked transcription. This repair mechanism has been well characterized in the past using individual target genes. However, the precise mechanism by which RNA polymerase II (Pol II) transcription is affected following UV irradiation during the repair processes genome-wide is not well understood. Results: We investigated the effect of a non-lethal dose of UVB on global DNA-bound Pol II distribution in human cells. We find that about 90% of the promoters of expressed genes show reduced Pol II occupancy 2-4 hours following UVB irradiation, and that the presence of Pol II is restored to “normal”, or higher, levels 5-6 hours after irradiation. We also identified a smaller set of genes, where the presence of Pol II at the promoter regions does not decrease after UVB irradiation, but often increases throughout the entire transcription units. Interestingly, at promoters, where Pol II promoter clearance occurs, TFIIH but not TBP follows the behavior of Pol II suggesting that at these genes TFIIH may be sequestered for DNA repair upon UVB treatment. Conclusions: Thus, our study reveals a global negative regulatory mechanism that targets Pol II transcription initiation on the large majority of transcribed genes following sublethal UVB irradiation, and a small subset of genes (including regulators of repair, cell growth and survival), where Pol II escapes this negative regulation.

ORGANISM(S): Homo sapiens

PROVIDER: GSE52789 | GEO | 2014/07/25

SECONDARY ACCESSION(S): PRJNA230028

REPOSITORIES: GEO

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UVB induces a major genome-wide rearrangement of RNA polymerase II at transcribed human genes

Project description:Abstract Background: Faithful transcription of DNA is constantly threatened by different endogenous and environmental genotoxic effects. Transcription coupled repair has been described to quickly remove DNA lesions from the transcribed strand of active genes, permitting rapid resumption of blocked transcription. This repair mechanism has been well characterized in the past using individual target genes. However, the precise mechanism by which RNA polymerase II (Pol II) transcription is affected following UV irradiation during the repair processes genome-wide is not well understood. Results: We investigated the effect of a non-lethal dose of UVB on global DNA-bound Pol II distribution in human cells. We find that about 90% of the promoters of expressed genes show reduced Pol II occupancy 2-4 hours following UVB irradiation, and that the presence of Pol II is restored to “normal”, or higher, levels 5-6 hours after irradiation. We also identified a smaller set of genes, where the presence of Pol II at the promoter regions does not decrease after UVB irradiation, but often increases throughout the entire transcription units. Interestingly, at promoters, where Pol II promoter clearance occurs, TFIIH but not TBP follows the behavior of Pol II suggesting that at these genes TFIIH may be sequestered for DNA repair upon UVB treatment. Conclusions: Thus, our study reveals a global negative regulatory mechanism that targets Pol II transcription initiation on the large majority of transcribed genes following sublethal UVB irradiation, and a small subset of genes (including regulators of repair, cell growth and survival), where Pol II escapes this negative regulation. Following genome-wide RNA Polymerase II redistribution over time upon UVB irradiation

2014-07-25 | E-GEOD-52789 | biostudies-arrayexpress

Structural basis of human transcription-DNA repair coupling

Project description:Transcription-coupled DNA repair (TCR) efficiently removes bulky DNA lesions from transcribed parts of the genome and constitutes a major defence against DNA damage by UV irradiation. TCR begins when RNA polymerase II (Pol II) stalls at a DNA lesion and recruits the Cockayne syndrome protein CsB, the E3 ubiquitin ligase complex CRL4CsA, and the UV-stimulated scaffold protein A (UVSSA). Here we provide five high-resolution structures of Pol II transcription complexes with bound TCR factors and complementary biochemical data. Together with published work, our results converge on a model for the molecular mechanism of transcription-DNA repair coupling. In this model, Pol II stalling triggers the formation of an alternative transcription elongation complex that we call ECact. In ECact, CsB has replaced the elongation factor DSIF, binds the PAF1 complex, and repositions upstream DNA such that it contacts the elongation factor SPT6. The CsB translocase now pulls on the upstream DNA template, thereby pushing Pol II forward. If the lesion cannot be bypassed, Pol II gets stably stalled. CRL4CsA spans over the Pol II clamp to reach the Pol II jaw and direct ubiquitination of RPB1 residue lysine-1268. This triggers the recruitment of TFIIH to UVSSA, which is located at downstream DNA, and enables nucleotide excision repair. Finally, large-scale conformational changes in CRL4CsA enable ubiquitination of CsB and the release of TCR factors, thereby allowing Pol II to resume elongation and continue transcription over repaired DNA.

2022-02-17 | PXD025328 | Pride

Structure of TFIIK for phosphorylation of CTD of RNA polymerase II

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

Systematic mutagenesis of TFIIH subunit p52/Tfb2 identifies residues required for XPB/Ssl2 subunit function and genetic interactions with TFB6

Project description: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 Pre-Initiation Complex (PIC), 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 twelve 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 PICs 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.

2022-09-07 | PXD036107 | Pride

Systematic mutagenesis identifies p52/Tfb2 residues required for XPB/Ssl2 function within TFIIH and genetic interactions with TFB6

Project description: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 Pre-Initiation Complex (PIC), TFIIH regulates the activity of the pol II enzyme 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. 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 twelve 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. 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 PICs 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.

2023-03-11 | PXD035576 | Pride

Structural basis of SNAPc-dependent snRNA transcription intiation by RNA polymerase II

Project description:RNA Polymerase II (Pol II) carries out transcription of both protein-coding and non-coding genes. Whereas Pol II initiation at protein-coding genes has been studied in detail, Pol II initiation at non-coding genes such as small nuclear RNA (snRNA) genes is not understood at the structural level. Here we study Pol II initiation at snRNA gene promoters and show that the snRNA-activating protein complex (SNAPc) enables DNA opening and transcription initiation independent of TFIIE and TFIIH in vitro. We then resolve cryo-EM structures of the SNAPc-containing Pol II preinitiation complex (PIC) assembled on U1 and U5 snRNA promoters. The core of SNAPc binds two turns of DNA and recognizes the snRNA promoter-specific proximal sequence element (PSE) located upstream of the TATA box-binding protein TBP. Two extensions of SNAPc called wing-1 and wing-2 bind TFIIA and TFIIB, respectively, explaining how SNAPc directs Pol II to snRNA promoters. Comparison of structures of closed and open promoter complexes elucidates TFIIH-independent DNA opening. These results provide the structural basis of Pol II initiation at non-coding RNA gene promoters.

2023-03-11 | PXD033638 | Pride

Systematic mutagenesis identifies p52/Tfb2 residues required for XPB/Ssl2 function within TFIIH and genetic interactions with TFB6

Project description: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 Pre-Initiation Complex, TFIIH regulates the activity of the pol II enzyme in numerous ways. One of the essential functions of TFIIH resides in its XPB/Ssl2 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 p52/Tfb2 core subunit. 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 twelve conditional mutants. Growth phenotypes of all but three of the conditional mutants were suppressed in the presence of Tfb6, thus identifying a functional interaction between Tfb2 HubA mutants and Tfb6, a protein that dissociates Ssl2 from TFIIH. Biochemical analysis of p52/Tfb2 mutants with severe growth phenotypes revealed defects in XPB/Ssl2 association. Further characterization of these Tfb2 mutant cells revealed defects in induction of Gal genes, and reduced occupancy TFIIH and pol II at Gal gene promoters, suggesting that functional TFIIH is required for proper Pol II recruitment to PICs 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.

2022-06-30 | GSE196069 | GEO

CX-MS of TFIIH and Def1 complex

Project description:The DNA damage response is an essential process for the survival of living cells. In a subset of stress-responsive genes in humans,Elongin controls transcription in response to multiple stimuli, such as DNA damage, oxidative stress, and heat shock. Yeast Elongin (Ela1-Elc1), along with Def1, is known to facilitate ubiquitylation and degradation of RNA polymerase II (pol II) in response tomultiple stimuli, yet transcription activity has not been examined. We have found that Def1 copurifies from yeast whole-cell extract with TFIIH,the largest general transcription factor required for transcription initiation and nucleotide excision repair. The addition of recombinant Def1 and Ela1-Elc1 enhanced transcription initiation in an in vitro reconstituted system including pol II, the general transcription factors, and TFIIS. Def1 also enhanced transcription restart from TFIIS-induced cleavage in a pol II transcribing complex. In the Δdef1 strain, heat shock genes were misregulated, indicating that Def1 is required for induction of some stress-responsive genes in yeast. Taken together, our results extend the understanding of the molecular mechanism of transcription regulation on cellular stress and reveal functional similarities to the mammalian system.

2018-10-22 | PXD008223 | Pride

Ssl2/TFIIH Function in Transcription Start Site Scanning by RNA Polymerase II in Saccharomyces cerevisiae

Project description:The initial step of RNA polymerase II (Pol II) transcription involves a large number of transcription factors and arises at multiple sites within most promoters. TFIIH is an essential, multi-subunit transcription factor that assembles on promoter DNA with Pol II and five other general transcription factors (GTFs) to form a pre-initiation complex (PIC) for basal transcription. During transcription initiation, TFIIH melts promoter DNA through the ATPase activity of its Ssl2 subunit. In the model eukaryote Saccharomyces cerevisiae, after DNA melting, Pol II scans downstream for usable transcription start sites (TSSs). To understand the function of Ssl2/TFIIH in promoter scanning and TSS selection, we identified novel alleles of SSL2 in genetic screens for mutants defective in TSS distribution that may potentially arise from altered scanning. Consistent with this notion, these ssl2 alleles alter scanning in ways that are distinct from how changes to the Pol II active site alter scanning and this difference is observed genome-wide. Our investigations support two major pathways in controlling promoter scanning and TSS selection, one controlling the efficiency of initiation through Pol II activity or factors regulating Pol II activity; another network appears to control the processivity of scanning by Ssl2/TFIIH.

2021-08-28 | GSE182792 | GEO

Elongation factor ELOF1 drives transcription-coupled repair and prevents genome instability

Project description:Correct transcription is crucial for life. However, DNA damage severely impedes elongating RNA Polymerase II (Pol II), causing transcription inhibition and transcription-replication conflicts. Cells are equipped with intricate mechanisms to counteract the severe consequence of these transcription-blocking lesions (TBLs). However, the exact mechanism and factors involved remain largely unknown. Here, using a genome-wide CRISPR/cas9 screen, we identified elongation factor ELOF1 as an important new factor in the transcription stress response upon DNA damage. We show that ELOF1 has an evolutionary conserved role in Transcription-Coupled Nucleotide Excision Repair (TC-NER), where it promotes recruitment of the TC-NER factors UVSSA and TFIIH to efficiently repair TBLs and resume transcription. Additionally, ELOF1 modulates transcription to protect cells from transcription-mediated replication stress, thereby preserving genome stability. Thus, ELOF1 protects the transcription machinery from DNA damage via two distinct mechanisms.

2021-04-20 | PXD025304 | Pride

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