Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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

ABSTRACT: 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).

ORGANISM(S): Saccharomyces cerevisiae

SUBMITTER: Jorja Henikoff

PROVIDER: E-GEOD-44200 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

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

Zentner Gabriel E GE Henikoff Steven S

Molecular and cellular biology 20131021 24

The Swi2/Snf2 family ATPase Mot1 displaces TATA-binding protein (TBP) from DNA in vitro, but the global relationship between Mot1 and TBP in vivo is unclear. In particular, how Mot1 activates transcription is poorly understood. To address these issues, we 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. We find evidence that Mot1 approa ...[more]

PMID: 24144978

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Project description:Sequence-specific DNA-binding proteins including transcription factors (TFs) are key determinants of gene regulation and chromatin architecture. Formaldehyde cross-linking and sonication followed by Chromatin ImmunoPrecipitation (X-ChIP) and sequencing is widely used for genome-wide profiling of protein binding, but is limited by low resolution and poor specificity and sensitivity. We have implemented a simple genome-wide ChIP protocol that starts with micrococcal nuclease-digested uncross-linked chromatin followed by affinity purification and paired-end sequencing without size-selection. The resulting ORGANIC (Occupied Regions of Genomes from Affinity-purified Naturally Isolated Chromatin) profiles of the budding yeast TFs Abf1 and Reb1 achieved near-perfect accuracy, in contrast to other profiling methods, which were much less sensitive and specific. Unlike profiles produced using X-ChIP methods such as ChIP-exo, ORGANIC profiles are not biased toward identifying sites in accessible chromatin and do not require input normalization. We also demonstrate the high specificity of our method when applied to larger genomes by profiling Drosophila GAGA Factor and Pipsqueak. Taken together, these results suggest that ORGANIC profiling outperforms current X-ChIP methodologies for genome-wide profiling of TF binding sites. Chromatin immunoprecipitation of micrococcal nuclease-digested native chromatin followed by paired-end sequencing (Occupied Regions of Genomes from Affinity-purified Naturally Isolated Chromatin 'ORGANIC' profiling) of DNA-binding proteins Abf1 and Reb1 from S. cerevisiae and GAGA-binding factor (GAF) and Pipsqueak (Psq) from D. melanogaster S2 cells; and, Sono-seq (paired-end sequencing of formaldehyde cross-linked and sonicated chromatin) of yeast nuclei. Reb1 ORGANIC profiling was performed at three different salt (NaCl) concentrations (80, 150, and 600 mM) and Abf1 ORGANIC profiling was done at two different salt concentrations (80 and 600 mM) to achieve varying levels of stringency. GAF and Psq ORGANIC profiles were determined at 80 mM salt. Two replicates each of Reb1 and Abf1 600 mM ORGANIC experiments, mixed Drosophila S2 cell and S. cerevisiae nuclei Reb1 ORGANIC experiments, yeast Sono-seq, and GAF and Psq ORGANIC experiments were performed. Each S. cerevisiae and mixed S2 cell/yeast ORGANIC profiling experiment included separately sequenced input chromatin and ChIP samples. Total of 24 samples.

Dataset Information

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

Publications

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

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