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

ABSTRACT: 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.

ORGANISM(S): Candida albicans

PROVIDER: GSE150063 | GEO | 2020/05/08

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

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.

2023-11-07 | GSE246951 | GEO

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

Project description: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.

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

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2023-08-01 | GSE222268 | GEO

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.

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Chromosome-wide mapping of interactions of yeast nucleoporin Nup2 on chromosome VI.

Project description:This series is a complementary data set for the manuscript entitled "Nup-PI: The Nucleopore-Promoter Interactions of Genes in Yeast." (Mol. Cell, 2006). Experimental Background: Genomic interaction sites of nuclear proteins were mapped by the in vivo ChEC technique described in Schmid et al. (2004) Mol. Cell 16, 147-157. Genome-wide probes that are suitable for hybridization of microarrays were prepared. In brief, MboI restriction fragments that were internally cleaved by the MN moiety of the fusion proteins were amplified and labelled. The procedure is explained in detail in the manuscript. "Nup-PI: The Nucleopore-Promoter Interactions of Genes in Yeast." (Schmid et al., Mol. Cell 2006). Experiments: Genome-wide probes were prepared from control, uncleaved chromatin, as well as chromatin cleaved by H2b-MN and Nup2-MN. All samples were from raffinose grown cells induced for 1 hour by galactose in logarithmic growth phase. Keywords: Genome-wide ChEC analysis, Mapping of Nuclear Pore Proteins 3 independent genome-wide probes were prepared for each Sample. That is, the ChEC experiments as well as preparation of genome-wide probes and hybridization of microarrays were carried independently (on different days). RMA values were calculated from the original .CEL files of each array and Average and standard deviation calculated.

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

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

| PRJNA631012 | ENA

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

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Peptide gene sequences of substrate phage for different proteases

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Swi/Snf dynamics on stress responsive genes is governed by competitive bromodomain interactions.

Project description:Acetylation of Snf2 regulates Swi/Snf recruitment during stress ChIP was done for Snf2 in unstressed and stressed condition fro WT, Snf2 K1493R K1497R, Wt delta bromodomain, Snf2 K1493R K1497R delta bromodomain using Snf2 antibodies, Mnase-seq was done for Snf2 in unstressed and stressed condition fro WT, Snf2 K1493R K1497R

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