Novel fluorescence-based high-throughput method uncovers single-cell DNA supercoiling heterogeneity
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ABSTRACT:
PROVIDER: PRJNA565972 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR10133000_1.fastq.gz | Fastqsanger.gz | |||
| SRR10133000_2.fastq.gz | Fastqsanger.gz | |||
| SRR10133001_1.fastq.gz | Fastqsanger.gz | |||
| SRR10133001_2.fastq.gz | Fastqsanger.gz | |||
| SRR10133002_1.fastq.gz | Fastqsanger.gz |
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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
Project description:DNA supercoiling (DS) 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 vivoFluorescent 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 the 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 from 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.IMPORTANCE DNA represents the chemical support of genetic information in all forms of life. In addition to its linear sequence of nucleotides, it bears critical information in its structure. This information, called DNA supercoiling, is central to all fundamental DNA processes, such as transcription and replication, and defines cellular physiology. Unlike reading of a nucleotide sequence, DNA supercoiling determinations have been laborious. We have now developed a method for rapid measurement of DNA supercoiling and established its utility by identifying a novel regulator of DNA supercoiling in the bacterium Salmonella enterica as well as behaviors that could not have been discovered with current methods.
| S-EPMC7387798 | biostudies-literature
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
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 | GSE152880 | GEO
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
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
Project description:Juvenile Fluorescence Raw sequence reads
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Project description:Most genes are transcribed from multiple transcription start sites (TSSs), defined as alternative TSSs, which are highly regulated and can lead to various gene regulatory outcomes including changes in translation efficiency and protein isoforms. Transcription factors and chromatin regulators control alternative TSS selection. DNA supercoiling affects multiple aspects of transcription including transcription initiation. However, its regulatory effect on genes with multiple TSSs is not known. Here we investigated how DNA supercoiling impacts alternative TSS usage in Saccharomyces cerevisiae. We depleted topoisomerases during early meiosis, where alternative TSS usage is prevalent, and applied an improved TSS sequencing protocol. We show that supercoiling affects alternative TSS usage of almost 600 genes. Increased alternative and aberrant TSS usage were observed near and within open reading frames, likely resulting from transcription-induced supercoiling originating from upstream alternative TSSs. DNA supercoiling had the greatest impact on genes with a dominant alternative TSS, significant spacing between alternative TSSs, and greater overall length. Our results establish that DNA supercoiling release during transcription is critical for correct TSS selection.
2025-03-24 | GSE292782 | GEO
Project description: Not available
| S-EPMC10790772 | biostudies-literature
Project description:This study was designed to investigate DNA supercoiling across the human genome and to understand how supercoiling domains impact on higher levels of genome organisation. DNA supercoiling is an inherent consequence of twisting DNA and is critical for regulating gene expression and DNA replication. However, DNA supercoiling at a genomic scale in human cells is uncharacterized. To map supercoiling we used biotinylated-trimethylpsoralen as a DNA structure probe to show the genome is organized into supercoiling domains. Domains are formed and remodeled by RNA polymerase and topoisomerase activities and are flanked by GC-AT boundaries and CTCF binding sites. Under-wound domains are transcriptionally active, enriched in topoisomerase I, “open” chromatin fibers and DNaseI sites, but are depleted of topoisomerase II. Furthermore DNA supercoiling impacts on additional levels of chromatin compaction as under-wound domains are cytologically decondensed, topologically constrained, and decompacted by transcription of short RNAs. We suggest that supercoiling domains create a topological environment that facilitates gene activation providing an evolutionary purpose for clustering genes along chromosomes.
2013-02-17 | GSE43449 | GEO