Mot1 redistributes TBP from TATA-containing to TATA-less promoters
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PROVIDER: PRJNA189052 | ENA |
REPOSITORIES: ENA
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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 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: Not available
| S-EPMC3889552 | biostudies-literature
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: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
2010-06-09 | E-GEOD-886 | biostudies-arrayexpress
Project description:An important distinction is frequently made between constitutively expressed housekeeping genes versus regulated genes. Although generally characterized by different DNA elements, chromatin architecture and cofactors, it is not known to what degree promoter classes strictly follow regulatability rules and which molecular mechanisms dictate such differences. We show that SAGA-dominated/TATA-box promoters are more responsive to changes in the amount of activator, even compared to TFIID/TATA-like promoters that depend on the same activator Hsf1. Regulatability is therefore an inherent property of promoter class. Further analyses show that SAGA/TATA-box promoters are more dynamic because TBP recruitment through SAGA is susceptible to removal by Mot1. In addition, the nucleosome configuration upon activator depletion shifts on SAGA/TATA-box promoters and seems less amenable to preinitiation complex formation. The results explain the fundamental difference between housekeeping and regulatable genes, revealing an additional facet of combinatorial control: an activator can elicit a different response dependent on core promoter class.
2016-11-11 | GSE81479 | GEO
Project description:An important distinction is frequently made between constitutively expressed housekeeping genes versus regulated genes. Although generally characterized by different DNA elements, chromatin architecture and cofactors, it is not known to what degree promoter classes strictly follow regulatability rules and which molecular mechanisms dictate such differences. We show that SAGA-dominated/TATA-box promoters are more responsive to changes in the amount of activator, even compared to TFIID/TATA-like promoters that depend on the same activator Hsf1. Regulatability is therefore an inherent property of promoter class. Further analyses show that SAGA/TATA-box promoters are more dynamic because TBP recruitment through SAGA is susceptible to removal by Mot1. In addition, the nucleosome configuration upon activator depletion shifts on SAGA/TATA-box promoters and seems less amenable to preinitiation complex formation. The results explain the fundamental difference between housekeeping and regulatable genes, revealing an additional facet of combinatorial control: an activator can elicit a different response dependent on core promoter class.
2016-11-11 | GSE81480 | GEO
Project description:An important distinction is frequently made between constitutively expressed housekeeping genes versus regulated genes. Although generally characterized by different DNA elements, chromatin architecture and cofactors, it is not known to what degree promoter classes strictly follow regulatability rules and which molecular mechanisms dictate such differences. We show that SAGA-dominated/TATA-box promoters are more responsive to changes in the amount of activator, even compared to TFIID/TATA-like promoters that depend on the same activator Hsf1. Regulatability is therefore an inherent property of promoter class. Further analyses show that SAGA/TATA-box promoters are more dynamic because TBP recruitment through SAGA is susceptible to removal by Mot1. In addition, the nucleosome configuration upon activator depletion shifts on SAGA/TATA-box promoters and seems less amenable to preinitiation complex formation. The results explain the fundamental difference between housekeeping and regulatable genes, revealing an additional facet of combinatorial control: an activator can elicit a different response dependent on core promoter class.
2016-11-11 | GSE81787 | GEO
Project description:An important distinction is frequently made between constitutively expressed housekeeping genes versus regulated genes. Although generally characterized by different DNA elements, chromatin architecture and cofactors, it is not known to what degree promoter classes strictly follow regulatability rules and which molecular mechanisms dictate such differences. We show that SAGA-dominated/TATA-box promoters are more responsive to changes in the amount of activator, even compared to TFIID/TATA-like promoters that depend on the same activator Hsf1. Regulatability is therefore an inherent property of promoter class. Further analyses show that SAGA/TATA-box promoters are more dynamic because TBP recruitment through SAGA is susceptible to removal by Mot1. In addition, the nucleosome configuration upon activator depletion shifts on SAGA/TATA-box promoters and seems less amenable to preinitiation complex formation. The results explain the fundamental difference between housekeeping and regulatable genes, revealing an additional facet of combinatorial control: an activator can elicit a different response dependent on core promoter class.
2016-11-11 | GSE81987 | GEO
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 Keywords: other
2004-03-05 | GSE886 | GEO