Promoter-specific dynamics of TATA-binding protein association with the human genome
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE133729 | GEO | 2019/07/03
REPOSITORIES: GEO
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Project description:The Swi2/Snf2-family ATPase Mot1 displaces TBP from DNA in vitro, but the global relationship between Mot1 and TBP in vivo has been unclear. We therefore mapped the distribution of Mot1 and TBP on native chromatin at base-pair resolution. Mot1 and TBP binding sites coincide throughout the genome, and depletion of TBP results in a global decrease in Mot1 binding. Using midpoint-versus-length mapping to assess the spatial relationship of Mot1 and TBP on chromatin, we find evidence that Mot1 approaches TBP from the upstream direction, consistent with its in vitro mode of action. Strikingly, inactivation of Mot1 leads to both increases and decreases in TBP-genome association. Sites of TBP gain tend to contain robust TATA boxes, while sites of TBP loss contain poly(dA:dT) tracts that may contribute to nucleosome exclusion. We propose that the action of Mot1 is required to clear TBP from intrinsically preferred (TATA-containing) binding sites, ensuring sufficient soluble TBP to bind intrinsically disfavored (TATA-less) sites.
2013-10-21 | GSE44200 | GEO
Project description:The conserved core domain of the TATA binding protein (TBP) interacts with multiple partners forming the complexes required for transcription by RNA Polymerases I, II and III. We use genetically modified mouse embryonic fibroblasts to show that many TBP core domain mutants complement loss of endogenous TBP, but this often results in a slow growth phenotype. Two TBP mutations, R188E and K243E, disrupt the TBP-BTAF1 interaction and B-TFIID complex formation. Transcriptome and ChIP-seq analyses show that loss of B-TFIID does not affect global genomic distribution of TBP, but positively or negatively affects TBP and/or RNA Polymerase II (Pol II) recruitment to a selected set of promoters. We identify a set of promoters where wild-type TBP assembles a partial inactive preinitiation complex lacking Pol II and TAF1. Our results suggest that an exchange of the B-TFIID complex in wild-type cells for TFIID in R188E and K243E mutant cells at these primed promoters recruits Pol II to activate their expression. We also observe that both Wt and mutant TBP can occupy promoters without concurrent Pol II recruitment and active transcription. Our data reveal a novel regulatory mechanism involving the formation of a partial preinitiation complex that primes the promoter for productive preinitiation complex formation in mammalian cells. Using homologous recombination, we generated ES cells and mice harbouring a null allele in the Tbp gene by insertion of a hygromycin resistance cassette in exon III and a floxed allele, in which exon III is surrounded by LoxP sites. From these mice, we generated an embryonic fibroblast Tbplox/- cell line in which TBP expression can be inactivated by expression of the Cre recombinase leading to cell death. We adopted a two-step strategy to generate Tbp-/- cell lines expressing human (h)TBP. Cells were first infected with pBABE retrovirus vectors expressing Wt hTBP or a series of mutants in the TBP core region, all of which carry a Flag-HA tag on their N-terminus. Cells expressing endogenous mTBP and exogenous hTBP were then infected with a second retrovirus expressing the 4 hydroxy-tamoxifen (OHT) inducible Cre-ERT2. Subsequently, multiple clonal populations from the OHT treated cells were isolated and genotyped by PCR to identify the Tbp-/-clones. At least two independent clones where expression of the endogenous mTBP was lost and replaced by the exogenous hTBP were isolated.
2012-03-01 | E-GEOD-27908 | biostudies-arrayexpress
Project description:The Swi2/Snf2-family ATPase Mot1 displaces TBP from DNA in vitro, but the global relationship between Mot1 and TBP in vivo has been unclear. We therefore mapped the distribution of Mot1 and TBP on native chromatin at base-pair resolution. Mot1 and TBP binding sites coincide throughout the genome, and depletion of TBP results in a global decrease in Mot1 binding. Using midpoint-versus-length mapping to assess the spatial relationship of Mot1 and TBP on chromatin, we find evidence that Mot1 approaches TBP from the upstream direction, consistent with its in vitro mode of action. Strikingly, inactivation of Mot1 leads to both increases and decreases in TBP-genome association. Sites of TBP gain tend to contain robust TATA boxes, while sites of TBP loss contain poly(dA:dT) tracts that may contribute to nucleosome exclusion. We propose that the action of Mot1 is required to clear TBP from intrinsically preferred (TATA-containing) binding sites, ensuring sufficient soluble TBP to bind intrinsically disfavored (TATA-less) sites. We have analyzed the genomic distributions of yeast TBP and Mot1 using Occupied Regions of Genomes from Affinity-purified Naturally Isolated Chromatin and sequencing (ORGANIC-seq).
2013-10-21 | E-GEOD-44200 | biostudies-arrayexpress
Project description:Background: Eukaryotic genes are controlled by proteins that assemble stepwise into a transcription complex. How the individual biochemically-defined assembly steps are coordinated and applied throughout a genome is largely unknown. Here, we model and experimentally test a portion of the assembly process involving the regulation of the TATA binding protein (TBP) throughout the yeast genome. Results: Biochemical knowledge is used to formulate a series of coupled TBP regulatory reactions involving TFIID, SAGA, NC2, Mot1, and promoter DNA. The reactions are then linked to basic segments of the transcription cycle and modeled computationally. A single framework is employed, allowing the contribution of specific steps to vary from gene to gene. Promoter binding and transcriptional output are measured genome-wide using ChIP-chip and expression microarray assays. Mutagenesis is used to test the framework by shutting down specific parts of the network. Conclusion: The model accounts for the regulation of TBP at most transcriptionally active promoters and provides a conceptual tool for interpreting genome-wide data sets. The findings further demonstrate the interconnections of TBP regulation on a genome-wide scale. Keywords: genetic mutation analysis
2007-04-01 | GSE7385 | GEO
Project description:Transcription factor binding to target sites in vivo is a dynamic process that involves cycles of association and dissociation, with individual proteins differing in their binding dynamics. The dynamics at individual sites on a genomic scale have been investigated in yeast cells, but comparable experiments have not been done in multicellular eukaryotes. Here, we describe a tamoxifen-inducible, time-course ChIP-seq approach to measure transcription factor binding dynamics at target sites throughout the human genome. As observed in yeast cells, the TATA-binding protein (TBP) typically displays rapid turnover at RNA polymerase (Pol) II-transcribed promoters, slow turnover at Pol III promoters, and very slow turnover at the Pol I promoter. Turnover rates vary widely among Pol II promoters in a manner that does not correlate with the level of TBP occupancy. Human Pol II promoters with slow TBP dissociation preferentially contain a TATA consensus motif, support high transcriptional activity of downstream genes, and are linked with specific activators and chromatin remodelers. These properties of human promoters with slow TBP turnover differ from those of yeast promoters with slow turnover. These observations suggest that TBP binding dynamics differentially affect promoter function and gene expression, possibly at the level of transcriptional reinitiation/bursting.
| S-EPMC6886507 | biostudies-literature
Project description:The conserved core domain of the TATA binding protein (TBP) interacts with multiple partners forming the complexes required for transcription by RNA Polymerases I, II and III. We use genetically modified mouse embryonic fibroblasts to show that many TBP core domain mutants complement loss of endogenous TBP, but this often results in a slow growth phenotype. Two TBP mutations, R188E and K243E, disrupt the TBP-BTAF1 interaction and B-TFIID complex formation. Transcriptome and ChIP-seq analyses show that loss of B-TFIID does not affect global genomic distribution of TBP, but positively or negatively affects TBP and/or RNA Polymerase II (Pol II) recruitment to a selected set of promoters. We identify a set of promoters where wild-type TBP assembles a partial inactive preinitiation complex lacking Pol II and TAF1. Our results suggest that an exchange of the B-TFIID complex in wild-type cells for TFIID in R188E and K243E mutant cells at these primed promoters recruits Pol II to activate their expression. We also observe that both Wt and mutant TBP can occupy promoters without concurrent Pol II recruitment and active transcription. Our data reveal a novel regulatory mechanism involving the formation of a partial preinitiation complex that primes the promoter for productive preinitiation complex formation in mammalian cells.
2012-03-01 | GSE27908 | GEO
Project description:Mot1 is an essential TATA-binding protein (TBP)-associated factor and Snf2/Swi2 ATPase that both represses and activates transcription. Biochemical and structural results support a model in which ATP binding and hydrolysis induce a conformational change in Mot1 that drives local translocation along DNA, thus removing TBP. While this activity explains transcriptional repression, it does not as easily explain Mot1-mediated transcriptional activation, and several different models have been proposed to explain how Mot1 activates transcription. To better understand the function of Mot1 in yeast cells in vivo, particularly with regard to gene activation, TBP mutants were identified that bypass the requirement for Mot1 in vivo. Although TBP has been extensively mutated and analyzed previously, this screen uncovered two novel TBP variants that are unique in their ability to bypass the requirement for Mot1. Surprisingly, in vitro analyses reveal that rather than having acquired an improved biochemical activity, one of the TBPs was defective for interaction with Pol II preinitiation complex (PIC) components and other regulators of TBP function. The other mutant was defective for DNA binding in vitro, yet was still recruited to chromatin in vivo. These results suggest that Mot1-mediated dissociation of TBP (or TBP-containing complexes) from chromatin can explain the Mot1 activation mechanism at some promoters. The results also suggest that PICs can be dynamically unstable, and that appropriate PIC instability is critical for the regulation of transcription in vivo.
2008-12-17 | GSE12371 | GEO
Project description:Mot1 redistributes TBP from TATA-containing to TATA-less promoters
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Project description:Despite being one of the first eukaryotic transcriptional regulatory elements identified, the sequence of a native TATA box and its significance remain elusive. Applying criteria associated with TATA boxes we queried several Saccharomyces genomes and arrived at the consensus TATA(A/T)A(A/T)(A/G). Approximately 20% of yeast genes contain a TATA box. Strikingly, TATA box-containing genes are associated with responses to stress, are highly regulated, and preferentially utilize SAGA rather than TFIID when compared to TATA-less promoters. Transcriptional regulation in yeast appears to be mechanistically bipolar, possibly reflecting a need to balance inducible stress-related responses with constitutive housekeeping functions. A strain containing amino terminal HA-tagged TBP and its parental untagged counterpart BY4741 (Resgen) were grown at 23?C in CSM to OD600 = 0.8-1.0. Cultures were shifted to 37?C for 5 minutes (to mimic heat treatments used elsewhere (Huisinga and Pugh, 2004)), then crosslinked at 23?C with 1% formaldehyde. ChIP was performed as described previously (Strahl-Bolsinger et al., 1997) with the some modification. Following cell disruption with glass beads, the chromatin was partially purified by centrifugation (Kurdistani and Grunstein, 2003). HA-TBP was immunopurified with 12CA5 antibody. After elution and reversal of the crosslinks, samples were subjected to non-specific amplification, labeling, and array hybridization as described (Bohlander et al., 1992; Chitikila et al., 2002). The amplification procedure was modified as follows. Following 15 round B amplification cycles, DNA was purified via QIAquick PCR Purification kit. Ten cycles were used in round C. Intergenic microarrays were generated by PCR amplification of entire intergenic regions of all yeast genes, then spotted onto glass slides. Spot intensities were filtered to remove any signal that was less then one standard deviation above local background. Three replicates were performed and their log2 ratios (enriched/input) were averaged. Keywords = Chromatin Immunoprecipitation Keywords = genome-wide binding
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