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ChEC-Seq: a robust method to identify protein-DNA interactions genome-wide

ABSTRACT: Here we show that the ChEC-Seq technique is able to differentiate the binding specificities of Esa1 and Gcn5 two chromatin-binding factors displaying widespread genome-wide associations. We also show that the ChEC-Seq technique reveals strong binding of the transcription factor Sfp1 at Ribi gene promoters. Furthermore, our data provide the first evidence that a specific DNA motif previously identified by ChEC-Seq (Albert 2019 PMID: 30804227) is in fact an in vivo binding site for Sfp1.

ORGANISM(S): Saccharomyces cerevisiae

PROVIDER: GSE133645 | GEO | 2020/07/01

REPOSITORIES: GEO

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ChEC-seq kinetics discriminates transcription factor binding sites by DNA sequence and shape in vivo

Project description:Chromatin endogenous cleavage (ChEC) uses fusion of a protein of interest to micrococcal nuclease (MNase) to target calcium-dependent cleavage to specific genomic loci in vivo. Here we report the combination of ChEC with high-throughput sequencing (ChEC-seq) to map budding yeast transcription factor (TF) binding. Temporal analysis of ChEC-seq data reveals two classes of sites for TFs, one displaying rapid cleavage at sites with robust consensus motifs and the second showing slow cleavage at largely unique sites with low-scoring motifs. Sites with high-scoring motifs also display asymmetric cleavage, indicating that ChEC-seq provides information on the directionality of TF-DNA interactions. Strikingly, similar DNA shape patterns are observed regardless of motif strength, indicating that the kinetics of ChEC-seq discriminates DNA recognition through sequence and/or shape. We propose that time-resolved ChEC-seq detects both high-affinity interactions of TFs with consensus motifs and sites preferentially sampled by TFs during diffusion and sliding.

2015-10-22 | GSE67453 | GEO

High resolution genome-wide occupancy in Candida species using ChEC-seq

Project description:Chromatin endogenous cleavage (ChEC) uses fusion of a protein of interest to micrococcal nuclease (MNase) to target calcium-dependent cleavage to specific genomic loci in vivo. Here we report the combination of ChEC with high-throughput sequencing (ChEC-seq) to determine genomic occupancy of the generalist transcription factor Nsi1 and the catalytic subunit of the SWI/SNF chromatin remodelling complex Snf2 in the opportunistic yeast Candida albicans. Time-based analysis of ChEC-seq data reveals two classes of sites for each transcriptional regulator, one exhibiting rapid cleavage during the first 5 min with robust consensus motifs and the second showing slow cleavage at largely unique sites with low-scoring motifs. The ChEC-seq procedure described here will allow a high-resolution genomic location definition which will enable a better understanding of transcriptional regulatory circuits that govern fungal fitness and drug resistance in these medically important fungi.

2020-05-08 | GSE150063 | GEO

Gcn5 and Esa1 function as histone crotonyltransferases to regulate crotonylation-dependent transcription

Project description:Histone post-translational modifications (PTMs) are critical for processes such as transcription. The more notable among these are the non-acetyl histone lysine acylation modifications such as crotonylation, butyrylation and succinylation. However, the biological relevance of these PTMs is not fully understood because their regulation is largely unknown. Here, we set out to investigate whether the main histone acetyltransferases in budding yeast, Gcn5 and Esa1, possess crotonyltransferase activity. In vitro studies revealed that the Gcn5-Ada2-Ada3 (ADA) and Esa1-Yng2-Epl1 (Piccolo NuA4) histone acetyltransferase complexes have the capacity to crotonylate histones. Mass spectrometry analysis revealed that ADA and Piccolo NuA4 crotonylate lysines in the N-terminal tails of histone H3 and H4, respectively. Functionally, we show that crotonylation selectively affects gene transcription in vivo in a manner dependent on Gcn5 and Esa1. Thus, we identify the Gcn5- and Esa1-containing ADA and Piccolo NuA4 complexes as bona fide crotonyltransferases that promote crotonylation-dependent transcription.

2019-11-06 | PXD015488 | Pride

TF binding profiles acquired using ChEC-seq in various TF co-deletion strains

Project description:This data set contains ChEC-seq binding profiles of various TF in yeast strains deleted of other TFs. Each sample has a pair-end sequencing file and a processed file (.out) is a genomic signal track after alignment to S.cerevisiae (R64) reference genome. Mapping was done using the read end. This dataset also contains raw and processed MNase-seq data files for nucleosome occupancy. Data related to manuscript: The architecture of binding cooperativity between densely bound transcription factors.

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ChEC-seq2: an improved chromatin endogenous cleavage sequencing method and bioinformatic analysis pipeline for mapping in vivo protein–DNA interactions

Project description:We validated ChEC-seq2 with MNase-fusions to transcription factors with well-documented motifs and binding sites in S. cerevisiae.

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Simultaneous Mapping of DNA Binding and Nucleosome Positioning with SpLiT-ChEC

Project description:The organization of chromatin – including the positions of nucleosomes and the binding of other proteins to DNA – helps define transcriptional profiles in eukaryotic organisms. While techniques like ChIP-Seq and MNase-Seq can map protein-DNA and nucleosome localization separately, assays designed to simultaneously capture nucleosome positions and protein-DNA interactions can produce a detailed picture of the chromatin landscape. Most assays that monitor chromatin organization and protein binding rely on antibodies, which often exhibit nonspecific binding, and/or the addition of bulky adducts to the DNA- binding protein being studied, which can affect their expression and activity. Here, we describe SpyCatcher Linked Targeting of Chromatin Endogenous Cleavage (SpLiT-ChEC), where a 13-amino acid SpyTag peptide, appended to a protein of interest, serves as a highly-specific targeting moiety for in situ enzymatic digestion. The SpyTag/SpyCatcher system forms a covalent bond, linking the target protein and a co-expressed MNase-SpyCatcher fusion construct. SpyTagged proteins are expressed from endogenous loci, whereas MNase-SpyCatcher expression is induced immediately before harvesting cultures. MNase is activated with high concentrations of calcium, which primarily digests DNA near target protein binding sites. By sequencing the DNA fragments released by targeted MNase digestion, we found that this method recovers information on protein binding and proximal nucleosome positioning. SpLiT-ChEC provides precise temporal control that we anticipate can be used to monitor chromatin under various conditions and at distinct points in the cell cycle.

2023-07-03 | GSE236273 | GEO

Genome-wide relationships between TAF1 and histone acetyltransferases in S. cerevisiae - chIP-chip study

Project description:Evidence suggests that the TAF1 subunit of TFIID is a histone acetyltransferase (HAT) that is functionally redundant with the Gcn5 HAT of the SAGA and ADA complexes. Here we test a number of predictions of this hypothesis by examining the in vivo histone acetylation targets of TAF1 and Gcn5, and re-examining the basis for the reported genome-wide functional redundancy between TAF1 and Gcn5. Our findings do not support a number of basic tenets of the hypothesis, thus bringing into question the physiological presence of any TAF1 HAT function in yeast. We have also conducted genome-wide expression profiles of numerous other HATs (Elp3, Hat1, Hpa2, Sas3) in an effort identify potential functional redundancy between TAF1 and other HATs, and find none. Further investigation of TAF1 and the Esa1 HAT re-affirm a link between histone H4 acetylation by Esa1, and TFIID binding via interactions with acetylated histone H4-binding protein Bdf1. Keywords: ChIP-chip, genetic modification

2006-03-20 | GSE3346 | GEO

Genome-wide relationships between TAF1 and histone acetyltransferases in S. cerevisiae

2006-03-20 | GSE3345 | GEO

Chromatin immunoprecipitation genome wide occupancy of 4 different chromatin regulating complexes encoded in yeast

Project description:We explore the genome-wide occupancy of 4 different chromatin regulating complexes encoded in S. cerevisiae. We provide the data for histone acetyltransferases Gcn5 and Esa1 and histone deacetylases Hst1 and Rpd3/Sin3 under rich growth condition (YPD medium). We also include the occupancy data for RNA polymerase II under the same growth condition.

2005-11-03 | E-WMIT-2 | biostudies-arrayexpress

Glucose Pulse to sfp1delta continuous cultures

Project description:The Saccharomyces cerevisiae SFP1 is required for proper regulation of ribosome biogenesis and cell size in response to nutrients. A mutant deleted for SFP1 shows specific traits among which a slow growth phenotype, which is particularly evident during growth on glucose. To assess the effects of nutrients on the activity of Sfp1 independent by growth rate related feedback we grew an sfp1Δ mutant and its isogenic reference strain in chemostat cultures, at the same specific growth rate, under glucose/ethanol-limitation. Our data show that Sfp1 is involved in the modulation of cell size and RiBi gene expression and that these two functions are differently influenced by nutrients. The continuous cultures were then pulsed with a glucose excess generating a situation of batch growth similar to shake flask cultures. The dynamic analysis of the metabolic and transcriptional response following the glucose addition suggested that Sfp1 plays a role at the crossroads of ribosome biogenesis and central carbon metabolism regulation. Finally, we show that the down-regulation of RP genes, which was observed in an sfp1Δ strain during shake flask growth, cannot be directly ascribed to the absence of Sfp1 but is most probably a secondary effect due to the low growth potential of the mutant strain. Keywords: time-course (Steady-state based anaerobic glucose pulse)

2008-06-09 | GSE9644 | GEO

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