Systematic mutagenesis identifies p52/Tfb2 residues required for XPB/Ssl2 function within TFIIH and genetic interactions with TFB6
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ABSTRACT:
PROVIDER: PRJNA803201 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR17873670.fastq.gz | Fastqsanger.gz | |||
| SRR17873671.fastq.gz | Fastqsanger.gz | |||
| SRR17873672.fastq.gz | Fastqsanger.gz | |||
| SRR17873673.fastq.gz | Fastqsanger.gz | |||
| SRR17873674.fastq.gz | Fastqsanger.gz |
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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
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
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
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 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.
| S-EPMC9557730 | biostudies-literature
Project description:It is well-established that the five transcription factors of the NFκB family form homo/hetero dimers amongst themselves to regulate gene expression by binding DNA. Our study challenges this paradigm by showing that p52 activates transcription without directly binding DNA but as part of a hetero-tetrameric partnership with ETS1, a transcription factor outside the NFκB family. By generating a knock-in mouse model (p52ki/ki) with three mutated residues on p52 required for its interactions with ETS1 but not with RelB, we demonstrate that the p52:ETS1 complex is the hitherto undiscovered regulator of transcription factors, OCT1 and OBF1, known to be critical for the germinal centre (GC) program. Consequently B cell-intrinsic expression of p52:ETS1 complex is indispensable for splenic GC B cell formation and T cell-dependent antibody responses. Functionally, loss of p52:ETS1 interaction led to diminished antigen-specific IgE thereby protecting mice from allergic responses. Collectively, our expand our current knowledge of NFkB signalling and provide new therapeutic targets for the treatment of allergic diseases.
2025-07-26 | GSE300336 | GEO
Project description:It is well-established that the five transcription factors of the NFκB family form homo/hetero dimers amongst themselves to regulate gene expression by binding DNA. Our study challenges this paradigm by showing that p52 activates transcription without directly binding DNA but as part of a hetero-tetrameric partnership with ETS1, a transcription factor outside the NFκB family. By generating a knock-in mouse model (p52ki/ki) with three mutated residues on p52 required for its interactions with ETS1 but not with RelB, we demonstrate that the p52:ETS1 complex is the hitherto undiscovered regulator of transcription factors, OCT1 and OBF1, known to be critical for the germinal centre (GC) program. Consequently B cell-intrinsic expression of p52:ETS1 complex is indispensable for splenic GC B cell formation and T cell-dependent antibody responses. Functionally, loss of p52:ETS1 interaction led to diminished antigen-specific IgE thereby protecting mice from allergic responses. Collectively, our expand our current knowledge of NFkB signalling and provide new therapeutic targets for the treatment of allergic diseases.
2025-07-26 | GSE300335 | GEO
Project description:It is well-established that the five transcription factors of the NFκB family form homo/hetero dimers amongst themselves to regulate gene expression by binding DNA. Our study challenges this paradigm by showing that p52 activates transcription without directly binding DNA but as part of a hetero-tetrameric partnership with ETS1, a transcription factor outside the NFκB family. By generating a knock-in mouse model (p52ki/ki) with three mutated residues on p52 required for its interactions with ETS1 but not with RelB, we demonstrate that the p52:ETS1 complex is the hitherto undiscovered regulator of transcription factors, OCT1 and OBF1, known to be critical for the germinal centre (GC) program. Consequently B cell-intrinsic expression of p52:ETS1 complex is indispensable for splenic GC B cell formation and T cell-dependent antibody responses. Functionally, loss of p52:ETS1 interaction led to diminished antigen-specific IgE thereby protecting mice from allergic responses. Collectively, our expand our current knowledge of NFkB signalling and provide new therapeutic targets for the treatment of allergic diseases.
2025-07-26 | GSE300334 | GEO
Project description:Dense, systematic mutagenesis to identify the features required for CSEs
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Project description:To understand the mechanism by which p52 expression augments tumorigenesis, gene expression microarray analysis was performed using mRNA isolated from lungs of CCSP-p52 and WT mice on dox for 1 week to identify genes with altered expression in CCSP-p52 mice. The microarray dataset includes eight samples, with two replicates for each combination of two factors (p52 and LPS).Since samples with and without LPS stimulation were processed and microarray profiled at the same time, we assumed the random data variations were similar across all samples and built a linear model across the eight samples accounting for both p52 and LPS experimental factors. The coefficient (estimated log fold change) and p-value associated with the p52 factor were retrieved for the selection of differentially expressed genes. When selecting top-ranking p52-induced genes, only probe sets mapping to known genes and having positive expression changes were considered. While the used microarrays were Affymetrix Mouse Gene 1.0 ST arrays, in the later step of probe set annotation we used annotation table associated with Mouse Gene 1.1 ST (GPL11533 table). The Robust Multichip Average method implemented in R package “oligo” (v1.28.3) was employed to normalize imported raw data. Probe sets interrogating control, unmapped, or intron sequences were ignored, leaving 79% of probe sets for a differential expression analysis. Linear models and empirical Bayes methods implemented in R package “limma” (v3.20.8) were applied to estimate log fold changes and p-values for the p52-vs-WT effect.
2017-11-22 | GSE71648 | GEO