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Genome-wide Localization of Mot1, Spt16, and Nucleosomes in mot1, spt16, and mot1 spt16 cells

ABSTRACT: Mot1 is a conserved and essential Swi2/Snf2 ATPase that can remove TATA-binding protein (TBP) from DNA using ATP hydrolysis, and in so doing exerts global effects on transcription. Spt16 is also essential and functions globally in transcriptional regulation as a component of the FACT histone chaperone complex. Here we demonstrate that Mot1 and Spt16 regulate a largely overlapping set of genes in Saccharomyces cerevisiae. As expected, Mot1 was found to control TBP levels at co-regulated promoters. In contrast, Spt16 did not affect TBP recruitment. On a global scale, Spt16 was required for Mot1 promoter localization, and Mot1 also affected Spt16 localization to genes. Interestingly, we find that Mot1 has an unanticipated role in establishing or maintaining the occupancy and positioning of nucleosomes at the 5’ ends of genes. Spt16 has a broad role in regulating chromatin organization in gene bodies, including those nucleosomes affected by Mot1. These results suggest that the large-scale overlap in Mot1 and Spt16 function arises from a combination of both their unique and shared functions in transcription complex assembly and chromatin structure regulation.

ORGANISM(S): Saccharomyces cerevisiae

PROVIDER: GSE80234 | GEO | 2016/05/16

SECONDARY ACCESSION(S): PRJNA318371

REPOSITORIES: GEO

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Genome-wide Localization of Mot1, Spt16, and Nucleosomes in mot1, spt16, and mot1 spt16 cells

Project description:Mot1 is a conserved and essential Swi2/Snf2 ATPase that can remove TATA-binding protein (TBP) from DNA using ATP hydrolysis, and in so doing exerts global effects on transcription. Spt16 is also essential and functions globally in transcriptional regulation as a component of the FACT histone chaperone complex. Here we demonstrate that Mot1 and Spt16 regulate a largely overlapping set of genes in Saccharomyces cerevisiae. As expected, Mot1 was found to control TBP levels at co-regulated promoters. In contrast, Spt16 did not affect TBP recruitment. On a global scale, Spt16 was required for Mot1 promoter localization, and Mot1 also affected Spt16 localization to genes. Interestingly, we find that Mot1 has an unanticipated role in establishing or maintaining the occupancy and positioning of nucleosomes at the 5â ends of genes. Spt16 has a broad role in regulating chromatin organization in gene bodies, including those nucleosomes affected by Mot1. These results suggest that the large-scale overlap in Mot1 and Spt16 function arises from a combination of both their unique and shared functions in transcription complex assembly and chromatin structure regulation. ChIP was performed for Spt16-myc in WT cells and mot1-42 cells in duplicate with input DNA from WT as control. ChIP was performed for Mot1-myc in WT cells and spt16-197 cells in dublicate with input DNA from WT as control. Micrococcal nuclease digested chromatin from WT, mot1-42, spt16-197, and mot1-42 spt16-197 cells were immunoprecipitated with H3 antibody in duplicate. All samples were sequenced by Illumina MiSeq.

2016-05-16 | E-GEOD-80234 | biostudies-arrayexpress

The Mot1 ATPase and Spt16 Histone Chaperone Cooperatively Regulate Transcription

2016-05-16 | GSE72117 | GEO

The Mot1 ATPase and Spt16 Histone Chaperone Cooperatively Regulate Transcription

Project description:Mot1 is a conserved and essential Swi2/Snf2 ATPase that can remove TATA-binding protein (TBP) from DNA using ATP hydrolysis, and in so doing exerts global effects on transcription. Spt16 is also essential and functions globally in transcriptional regulation as a component of the FACT histone chaperone complex. Here we demonstrate that Mot1 and Spt16 regulate a largely overlapping set of genes in Saccharomyces cerevisiae. As expected, Mot1 was found to control TBP levels at co-regulated promoters. In contrast, Spt16 did not affect TBP recruitment. Interestingly, Mot1 was required for Spt16 recruitment to co-activated promoters. In contrast, Spt16 levels in gene coding regions were unaffected by Mot1 as well as RNA polymerase II density. The co-localization of Mot1 and Spt16 at promoters and the broad overlap in the sets of genes they control is consistent with physical and genetic interactions between them. The data support a model in which these factors participate in a regulatory pathway in which Mot1 acts upstream of Spt16. Tiling arrays covering the entirety of the S.cerevisiae genome were used to identify the effects of Mot1 and Spt16 on RNA expression genome-wide. All samples were done in biological duplicates. The average signal from the spt16-197 samples was compared to the SPT16-WT to determine changes in expression. The average signal from the double mutant mot1-42 spt16-197 was compared to both SPT16-WT and MOT1-WT. The MOT1-WT data was previously published by our lab and is available at GEO accession GSM456548. Comparisons were made from our Spt16 dataset to the previously published MOT1-WT and mot1-42 data, and the entire study is available at GEO accession GSE18283. Differential RNA (spt16-197/SPT16): spt16-197_over_SPT16-WT.bar Differential RNA (mot1-42 spt16-197/SPT16): dbl_mut_over_SPT16-WT.bar Differential RNA (mot1-42 spt16-197/MOT1): dbl_mut_over_MOT1-WT.bar

2016-05-16 | E-GEOD-72117 | biostudies-arrayexpress

Mot1 redistributes TBP from TATA-containing to TATA-less promoters

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

Mot1 redistributes TBP from TATA-containing to TATA-less promoters

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TATA-Binding Protein Variants that Bypass the Requirement for Mot1 In Vivo

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

Saccharomyces cerevisiae

Project description:Genome-wide Localization of Mot1, Spt16, and Nucleosomes in mot1, spt16, and mot1 spt16 cells

| PRJNA318371 | ENA

RNA Synthesis Precision is Regulated by Preinitiation Complex Turnover

Project description:TATA-binding protein (TBP) nucleates the assembly of the transcription preinitiation complex (PIC), and although TBP can bind promoters with high stability in vitro, recent results establish that virtually the entire TBP population is highly dynamic in yeast nuclei in vivo. This dynamic behavior is surprising in light of models which posit that a stable TBP-containing scaffold facilitates transcription reinitiation at active promoters. The dynamic behavior of TBP is a consequence of the enzymatic activity of the essential Snf2/Swi2 ATPase Mot1, suggesting that ensuring a highly mobile TBP population is critical for transcriptional regulation on a global scale. Here high-resolution tiling arrays were used to define how perturbed TBP dynamics impact the precision of RNA synthesis in Saccharomyces cerevisiae. We find that Mot1 plays a broad role in establishing the precision and efficiency of RNA synthesis: in mot1-42 cells, RNA length changes were observed for 713 genes, about twice the number observed in set2∆ cells, which display a previously reported propensity for spurious initiation within open reading frames. Loss of Mot1 led to both aberrant transcription initiation and termination, with prematurely terminated transcripts representing the largest class of events. Genetic and genomic analyses support the conclusion that these effects on RNA length are mechanistically tied to dynamic TBP occupancies at certain types of promoters. These results suggest a new model whereby dynamic disassembly of the PIC can influence productive RNA synthesis.

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Cryptic transcription is the primary driving force for nucleosome instability in Spt16 mutant cells

Project description:FACT consists of two essential subunits Spt16 and Pob3 and is a histone chaperone. Depletion of Spt16 using a an spt16 mutant results in a global alteration of nucleosome positions as well as aberrant transcription. Here we show that the majority of nucleosomal changes at gene body upon Spt16 depletion are independent of gene activity, but correlates with cryptic gene transcription and are suppressed by inhibition of RNA Polymerase II activities. In addition, A small fraction of nucleosomal changes is resistant to Pol II inhibition, and Spt16 is enriched at this subgroup of nucleosomes. Moreover, nucleosomes surrounding the initiation sites of cryptic transcription in the spt16 mutant cells are more dynamic than other regions. These results support a model that Spt16 maintains nucleosome stability locally to inhibit the initiation from cryptic transcription, which that once initiated drives additional nucleosome loss upon Spt16 depletion.

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Mot1 redistributes TBP from TATA-containing to TATA-less promoters

Project description:Mot1 redistributes TBP from TATA-containing to TATA-less promoters

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