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High-resolution, genome-wide mapping of positive supercoiling in chromosomes [ecChIP]

ABSTRACT: Supercoiling impacts DNA replication, transcription, protein binding to DNA, and the three- dimensional organization of chromosomes. However, there are currently no methods to directly interrogate or map positive supercoils, so their distribution in genomes remains unknown. Here, we describe a method, GapR-seq, based on the chromatin immunoprecipitation of GapR, a bacterial protein that preferentially recognizes overtwisted DNA, for generating high-resolution maps of positive supercoiling. Applying this method to E. coli and S. cerevisiae, we find that positive supercoiling is widespread, associated with transcription, and particularly enriched between convergently-oriented genes, consistent with the “twin-domain” model of supercoiling. In yeast, we also find positive supercoils associated with centromeres, cohesin binding sites, autonomously replicating sites, and the borders of R-loops (DNA-RNA hybrids). Our results suggest that GapR-seq is a powerful approach, likely applicable in any organism, to investigate aspects of chromosome structure and organization not accessible by Hi-C or other existing methods.

ORGANISM(S): Escherichia coli str. K-12 substr. MG1655

PROVIDER: GSE152880 | GEO | 2021/06/25

REPOSITORIES: GEO

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High-resolution, genome-wide mapping of positive supercoiling in chromosomes [RNA-seq]

Project description:Supercoiling impacts DNA replication, transcription, protein binding to DNA, and the three- dimensional organization of chromosomes. However, there are currently no methods to directly interrogate or map positive supercoils, so their distribution in genomes remains unknown. Here, we describe a method, GapR-seq, based on the chromatin immunoprecipitation of GapR, a bacterial protein that preferentially recognizes overtwisted DNA, for generating high-resolution maps of positive supercoiling. Applying this method to E. coli and S. cerevisiae, we find that positive supercoiling is widespread, associated with transcription, and particularly enriched between convergently-oriented genes, consistent with the “twin-domain” model of supercoiling. In yeast, we also find positive supercoils associated with centromeres, cohesin binding sites, autonomously replicating sites, and the borders of R-loops (DNA-RNA hybrids). Our results suggest that GapR-seq is a powerful approach, likely applicable in any organism, to investigate aspects of chromosome structure and organization not accessible by Hi-C or other existing methods.

2021-06-25 | GSE152879 | GEO

High-resolution, genome-wide mapping of positive supercoiling in chromosomes [yChIP]

Project description:Supercoiling impacts DNA replication, transcription, protein binding to DNA, and the three-dimensional organization of chromosomes. However, there are currently no methods to directly interrogate or map positive supercoils, so their distribution in genomes remains unknown. Here, we describe a method based on the chromatin immunoprecipitation of GapR, a bacterial protein that preferentially recognizes overtwisted DNA, for generating high-resolution maps of positive supercoiling. Applying this method to E. coli and S. cerevisiae, we find that positive supercoiling is widespread, associated with transcription, and enriched between convergently-oriented genes, consistent with the ?twin-domain? model of supercoiling. In yeast, we also find positive supercoils associated with centromeres, cohesin binding sites, replication-transcription encounters, and the borders of R-loops (DNA-RNA hybrids). Our results suggest that GapR-seq is a powerful approach that can be applied in any organism to investigate aspects of chromosome structure and organization not accessible by Hi-C or other existing methods.

2021-06-25 | GSE152881 | GEO

High-resolution, genome-wide mapping of positive supercoiling in chromosomes [RNA-Seq 2]

Project description:Supercoiling impacts DNA replication, transcription, protein binding to DNA, and the three-dimensional organization of chromosomes. However, there are currently no methods to directly interrogate or map positive supercoils, so their distribution in genomes remains unknown. Here, we describe a method based on the chromatin immunoprecipitation of GapR, a bacterial protein that preferentially recognizes overtwisted DNA, for generating high-resolution maps of positive supercoiling. Applying this method to E. coli and S. cerevisiae, we find that positive supercoiling is widespread, associated with transcription, and enriched between convergently-oriented genes, consistent with the “twin-domain” model of supercoiling. In yeast, we also find positive supercoils associated with centromeres, cohesin binding sites, replication-transcription encounters, and the borders of R-loops (DNA-RNA hybrids). Our results suggest that GapR-seq is a powerful approach that can be applied in any organism to investigate aspects of chromosome structure and organization not accessible by Hi-C or other existing methods.

2021-06-25 | GSE164186 | GEO

A bacterial chromosome structuring protein binds overtwisted DNA to stimulate type II topoisomerases and enable DNA replication

Project description:When DNA is unwound during replication, it becomes overtwisted and forms positive supercoils in front of the translocating DNA polymerase. Unless removed or dissipated, this superhelical tension can impede replication elongation. Topoisomerases, including gyrase and topoisomerase IV in bacteria, are required to relax positive supercoils ahead of DNA polymerase, but may not be sufficient for replication. Here, we find that GapR, a chromosome structuring protein in Caulobacter crescentus, is required to complete DNA replication. GapR associates in vivo with positively supercoiled chromosomal DNA, and our biochemical and structural studies demonstrate that GapR forms a dimer-of-dimers that fully encircles overtwisted DNA. Further, we show that GapR stimulates gyrase and topo IV to relax positive supercoils, thereby enabling DNA replication. Analogous chromosome structuring proteins that locate to the overtwisted DNA in front of replication forks may be present in other organisms, similarly helping to recruit and stimulate topoisomerases during DNA replication.

2018-09-13 | GSE100657 | GEO

Differential roles of positive and negative supercoiling in organizing the E. coli genome

Project description:This study aims to explore whether and how positive and negative supercoiling contribute to the three-dimensional (3D) organization of the bacterial genome. We used recently published Escherichia coli GapR ChIP-seq and TopoI ChIP-seq (also called EcTopoI-seq) data, which marks positive and negative supercoiling sites, respectively, to study how supercoiling correlates with the spatial contact maps obtained from chromosome conformation capture sequencing (Hi-C and 5C). We find that supercoiled chromosomal loci have overall higher Hi-C contact frequencies than sites that are not supercoiled. Surprisingly, positive supercoiling corresponds to higher spatial contact than negative supercoiling. Additionally, positive, but not negative, supercoiling could be identified from Hi-C data with high accuracy. We further find that the majority of positive and negative supercoils coincide with highly active transcription units, with a minor group associated with replication and other genomic processes. Our results show that both positive and negative supercoiling enhance spatial contact, with positive supercoiling playing a larger role in bringing genomic loci closer in space. Based on our results, we propose new physical models of how the E. coli chromosome is organized by positive and negative supercoils.

2023-11-03 | GSE214856 | GEO

Novel fluorescence-based high-throughput method uncovers single-cell DNA supercoiling heterogeneity

Project description:DNA supercoiling is essential for life because it controls critical processes, including transcription, replication and recombination. Current methods to measure DNA supercoiling in vivo are laborious and unable to examine single cells. Here we report a method for high-throughput measurement of bacterial DNA supercoiling in vivo. Fluorescent Evaluation of DNA Supercoiling (FEDS) utilizes a plasmid harboring the gene for a green fluorescent protein transcribed by a discovered promoter that responds exclusively to DNA supercoiling, and the gene for a red fluorescent protein transcribed by a constitutive promoter as internal standard. Using FEDS, we uncovered single cell heterogeneity in DNA supercoiling and established that, surprisingly, population-level decreases in DNA supercoiling result from a low mean/high variance DNA supercoiling subpopulation, rather than a homogeneous shift in supercoiling of the whole population. In addition, we identified a regulatory loop in which a gene that decreases DNA supercoiling is transcriptionally repressed when DNA supercoiling increases.

2020-07-02 | GSE137586 | GEO

Transcription dependent dynamic supercoiling in Raji human B cells in vivo

Project description:Transcription has the capacity to modify mechanically DNA topology, DNA structure, and nucleosome arrangement. Resulting from ongoing transcription, these modifications in turn, may provide instant feedback to the transcription machinery. To substantiate the connection between transcription and DNA dynamics, we charted an ENCODE map of transcription-dependent dynamic supercoiling in human Burkitt lymphoma cells using psoralen photobinding to probe DNA topology in vivo. Dynamic supercoils spread ~1.5 kb upstream of the start sites of active genes. Low and high output promoters handle this torsional stress differently as shown using inhibitors of transcription and topoisomerases, and by chromatin immunoprecipation of RNA polymerase and topoisomerases I and II. Whereas lower outputs are managed adequately by topoisomerase I, high output promoters additionally require topoisomerase II. The genome-wide coupling between transcription and DNA topology emphasizes the importance of dynamic supercoiling for gene regulation.

2013-02-16 | GSE43751 | GEO

Transcription dependent dynamic supercoiling in Raji human B cells in vivo

2013-02-16 | E-GEOD-43751 | biostudies-arrayexpress

Alteration of DNA supercoiling serves as a trigger of short-term cold shock repressed genes of E. coli

Project description:Cold shock adaptability is a key survival skill of gut bacteria of warm-blooded animals. Escherichia coli cold shock responses are controlled by a complex multi-gene, timely-ordered transcriptional program. We investigated its underlying mechanisms. Having identified short-term, cold shock repressed genes, we show that their responsiveness is unrelated to their transcription factors or global regulators, while their single-cell protein numbers’ variability increases after cold shock. We hypothesized that some cold shock repressed genes could be triggered by high propensity for transcription locking due to changes in DNA supercoiling (likely due to DNA relaxation caused by an overall reduction in negative supercoiling). Concomitantly, we found that nearly half of cold shock repressed genes are also highly responsive to gyrase inhibition (albeit most genes responsive to gyrase inhibition are not cold shock responsive). Further, their response strengths to cold shock and gyrase inhibition correlate. Meanwhile, under cold shock, nucleoid density increases, and gyrases and nucleoid become more colocalized. Moreover, the cellular energy decreases, which may hinder positive supercoils resolution. Overall, we conclude that sensitivity to diminished negative supercoiling is a core feature of E. coli’s short-term, cold shock transcriptional program, and could be used to regulate the temperature sensitivity of synthetic circuits.

2022-07-20 | GSE194037 | GEO

RNA Polymerase-II-generated DNA supercoils destabilize nucleosomes

Project description:The twin supercoiled domain model posits that, as RNA Polymerase II (Pol II) transcribes a gene, it generates negative and positive supercoils upstream and downstream respectively, but little is known about the functional consequence in vivo of the resulting torsional strain. Here we provide a method for high resolution mapping of DNA supercoils using next-generation sequencing, and show that the level of supercoiling is correlated with gene expression in Drosophila cells. Inhibition of topoisomerases, enzymes that relieve torsional strain, leads to accumulation of supercoils surrounding gene bodies and of Pol II at the transcription start sites. Topoisomerase I inhibition results in increased nascent RNA transcripts with Topoisomerase II inhibition shows little change in nascent RNA levels. Despite these different effects on transcription, inhibition of either enzyme results in increased nucleosome turnover within gene bodies, suggesting that torsional stress contributes to destabilizing nucleosomes ahead of Pol II. 12 paired-end samples and 8 single-end samples were sequenced and analyzed.

2013-10-31 | E-GEOD-47795 | biostudies-arrayexpress

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