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Transcription-mediated supercoiling regulates genome folding and loop formation [RNA-Seq]

ABSTRACT: The chromatin fiber folds into loops, however the mechanisms controlling loop extrusion are still poorly understood. Using super-resolution microscopy, we visualized that loops in intact nuclei are formed by a scaffold of cohesin complexes from which the DNA protrudes. RNA polymerase II decorates the top of the loops and is physically segregated from cohesin. Augmented looping upon increased loading of cohesin on chromosomes causes disruption of Lamin at the nuclear rim and chromatin blending, a homogenous distribution of chromatin within the nucleus. Altering supercoiling via either transcription or topoisomerase inhibition counteracts chromatin blending, increases chromatin condensation, disrupts loop formation and leads to altered cohesin distribution and mobility on chromatin. Overall, negative supercoiling generated by transcription is an important regulator of loop formation in vivo.

ORGANISM(S): Homo sapiens

PROVIDER: GSE169476 | GEO | 2021/07/22

REPOSITORIES: GEO

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Transcription-mediated supercoiling regulates genome folding and loop formation [ChIP-Seq]

Project description:The chromatin fiber folds into loops, however the mechanisms controlling loop extrusion are still poorly understood. Using super-resolution microscopy, we visualized that loops in intact nuclei are formed by a scaffold of cohesin complexes from which the DNA protrudes. RNA polymerase II decorates the top of the loops and is physically segregated from cohesin. Augmented looping upon increased loading of cohesin on chromosomes causes disruption of Lamin at the nuclear rim and chromatin blending, a homogenous distribution of chromatin within the nucleus. Altering supercoiling via either transcription or topoisomerase inhibition counteracts chromatin blending, increases chromatin condensation, disrupts loop formation and leads to altered cohesin distribution and mobility on chromatin. Overall, negative supercoiling generated by transcription is an important regulator of loop formation in vivo.

2021-07-22 | GSE169474 | 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 [ecChIP]

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

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 [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

The cohesin acetylation cycle controls chromatin loop length through a PDS5A brake mechanism

Project description:Cohesin structures the genome through the formation of chromatin loops and by holding together the sister chromatids. The acetylation of cohesin’s SMC3 subunit is a dynamic process that involves the acetyltransferase ESCO1 and deacetylase HDAC8. Here we show that this cohesin acetylation cycle controls the 3D genome. ESCO1 restricts the length of chromatin loops and architectural stripes, while HDAC8 promotes the extension of such loops and stripes. This role in controlling loop length turns out to be distinct from the canonical role of cohesin acetylation that protects against WAPL-mediated DNA release. We reveal that acetylation rather controls cohesin’s interaction with PDS5A to restrict chromatin loop length. Our data supports a model in which this PDS5A-bound state acts as a brake that enables the pausing and restart of loop enlargement. The cohesin acetylation cycle hereby provides punctuation in the process of genome folding.

2022-03-14 | PXD032185 | Pride

Cohesin residency determines chromatin loop patterns

Project description:The organization of chromatin into higher-order structures is essential for chromosome segregation, the repair of DNA damage, and the regulation of gene expression. These structures are formed by the evolutionarily conserved SMC (structural maintenance of chromosomes) complexes. By analyzing synchronized populations of budding yeast with Micro-C, we observed that chromatin loops are formed genome-wide, and are dependent upon the SMC complex, cohesin. We correlated the loop signal with the position and intensity of cohesin binding to chromosomes in wild-type and cells depleted for the cohesin regulators Wpl1p and Pds5p. We generate a model to explain how the genomic distribution and frequency of loops are driven by cohesin residency on chromosomes. In this model a dynamic pool of cohesin with loop extrusion activity stops when encounters two regions occupied by stably bound cohesin, forming a loop. Different regions are occupied by cohesin in different cells, defining different patterns of chromatin loops.

2020-11-04 | GSE151553 | GEO

Cohesin residency determines chromatin loop patterns

2020-11-04 | GSE151416 | GEO

Smc3 acetylation, Pds5 and Scc2 control the translocase activity that establishes cohesin dependent chromatin loops

Project description:Cohesin is a DNA translocase instrumental in the folding of the genome into chromatin loops, with functional consequences on DNA-related processes. Chromatin loop length and organization likely depend on cohesin processivity, translocation rate, and stability on DNA. Here we investigate and provide a comprehensive overview of the roles of various cohesin regulators in tuning chromatin loop expansion. We demonstrate that Scc2, which stimulates cohesin ATPase activity, is also essential for cohesin translocation, driving loop expansion in vivo. Smc3 acetylation during S-phase counteracts this activity through the stabilization of Pds5, which finely tunes the size and stability of loops in G2.

2022-02-23 | GSE186987 | 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

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