Project description:This SuperSeries is composed of the following subset Series: GSE18240: Saccharomyces cerevisiae cells: control vs positive supercoiling accumulation after 0, 30 and 120 min GSE18241: S. cerevisiae cells: control vs positive supercoiling accumulation in absence of telomere silencing after 0 and 120 min GSE18605: Saccharomyces cerevisiae cells: effect of Top2 depletion without accumulation of positive superhelical stress Refer to individual Series
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.
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.
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.
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.
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.