Osmotic disruption of chromatin induces Topoisomerase 2 activity at sites of transcriptional stress [Mnase-Seq]
Ontology highlight
ABSTRACT: Transcription generates superhelical stress in DNA that poses problems for genome stability, but determining when and where such stress arises within chromosomes is challenging. Here, using G1-arrested S. cerevisiae cells, and employing rapid fixation and ultra-sensitive enrichment, we utilise the physiological activity of endogenous topoisomerase 2 (Top2) as a probe of transcription-induced superhelicity. We demonstrate that Top2 activity is surprisingly uncorrelated with transcriptional activity, suggesting that superhelical stress is obscured from Top2 within chromatin in vivo. We test this idea using osmotic perturbation—a treatment that transiently destabilises chromatin in vivo—revealing that Top2 activity redistributes within sub-minute timescales into broad zones patterned by long genes, convergent gene arrays, and transposon elements—and also by acute transcriptional induction. We propose that latent superhelical stress is normally absorbed by the intrinsic topological buffering capacity of chromatin, helping to avoid spurious topoisomerase activity arising within the essential coding regions of the genome.
Project description:Transcription generates superhelical stress in DNA that poses problems for genome stability, but determining when and where such stress arises within chromosomes is challenging. Here, using G1-arrested S. cerevisiae cells, and employing rapid fixation and ultra-sensitive enrichment, we utilise the physiological activity of endogenous topoisomerase 2 (Top2) as a probe of transcription-induced superhelicity. We demonstrate that Top2 activity is surprisingly uncorrelated with transcriptional activity, suggesting that superhelical stress is obscured from Top2 within chromatin in vivo. We test this idea using osmotic perturbation—a treatment that transiently destabilises chromatin in vivo—revealing that Top2 activity redistributes within sub-minute timescales into broad zones patterned by long genes, convergent gene arrays, and transposon elements—and also by acute transcriptional induction. We propose that latent superhelical stress is normally absorbed by the intrinsic topological buffering capacity of chromatin, helping to avoid spurious topoisomerase activity arising within the essential coding regions of the genome.
Project description:Transcription generates superhelical stress in DNA that poses problems for genome stability, but determining when and where such stress arises within chromosomes is challenging. Here, using G1-arrested S. cerevisiae cells, and employing rapid fixation and ultra-sensitive enrichment, we utilise the physiological activity of endogenous topoisomerase 2 (Top2) as a probe of transcription-induced superhelicity. We demonstrate that Top2 activity is surprisingly uncorrelated with transcriptional activity, suggesting that superhelical stress is obscured from Top2 within chromatin in vivo. We test this idea using osmotic perturbation—a treatment that transiently destabilises chromatin in vivo—revealing that Top2 activity redistributes within sub-minute timescales into broad zones patterned by long genes, convergent gene arrays, and transposon elements—and also by acute transcriptional induction. We propose that latent superhelical stress is normally absorbed by the intrinsic topological buffering capacity of chromatin, helping to avoid spurious topoisomerase activity arising within the essential coding regions of the genome.
Project description:During chromosome duplication the parental DNA molecule becomes over-wound, or positively supercoiled, in the region ahead of the advancing replication fork. To allow fork progression this superhelical tension has to be removed by topoisomerases, which operate by introducing transient DNA breaks. Positive supercoiling can also be diminished if the advancing fork rotates along the DNA helix, but then sister chromatid intertwinings form in its wake. Despite these insights it remains largely unknown how replicationinduced superhelical stress is dealt with on linear, eukaryotic chromosomes. Here we show that this stress increases with the length of budding yeast chromosomes. This opens for the possibility that superhelical tension is handled on a chromosome scale and not only within topologically closed chromosomal domains as the current view predicts. We found that inhibition of type I topoisomerases leads to a late replication delay of longer, but not shorter chromosomes. This phenotype is also displayed by cells expressing mutated versions of the cohesin- and condensin–related Smc5/6 complex. The chromosomal association of the Smc5/6 complex is shown to increase in response to chromosome lengthening, chromosome circularization, or inactivation of Topoisomerase 2, all having the potential to increase the number of sister chromatid intertwinings 3. Furthermore, non-functional Smc6 reduces the accumulation of intertwined sister plasmids after one round of replication in the absence of Topoisomerase 2 function. Our results demonstrate that the length of a chromosome influences the need of superhelical tension release in Saccharomyces cerevisiae, and allow us to propose a model where the Smc5/6 complex facilitates fork rotation by sequestering nascent chromatid intertwinings which form behind the replication machinery. Smc6、Nse4, and Scc2 profiles with or without topological tension was analyzed (in top2 mutant, cells with circular chromosome, and fragmented chromosome) . 17 ChIP samples and corresponding WCE fractions have been analyzed.
Project description:Topological stress can cause replication forks to stall as they converge upon one another during termination of vertebrate DNA synthesis. However, replication forks ultimately overcome topological stress and complete DNA synthesis, suggesting that alternative mechanisms can overcome topological stress. We performed a proteomic analysis of converging replication forks that were stalled by topological stress induced by loss or inhibition of topoisomerase IIα (TOP2α). Plasmid DNA was replicated in mock- or TOP2α-depleted Xenopus egg extracts as previously described (Heintzman et al. 2019). In parallel, replication was performed in the presence of the TOP2 inhibitor ICRF-193 (‘TOP2-i’) as an alternate means of preventing TOP2 activity (Heintzman et al. 2019). Chromatinized plasmid DNA was recovered 18 minutes after the onset of DNA synthesis, when most forks have normally merged but are stalled when TOP2 activity is prevented (Heintzman et al. 2019). Chromatin-bound proteins were recovered (Larsen et al. 2019) then analyzed by chromatin mass spectrometry and quantified by label free quantification.
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:DNA topoisomerases are required to resolve DNA topological stress. Despite this essential role, abortive topoisomerase activity generates aberrant protein-linked DNA breaks, jeopardising genome stability. Here, to understand the genomic distribution and mechanisms underpinning topoisomerase-induced DNA breaks, we map Top2 DNA cleavage with strand-specific nucleotide resolution across the S. cerevisiae and human genomes—and use the meiotic Spo11 protein to validate the broad applicability of this method to explore the role of diverse topoisomerase family members. Our data characterises Mre11-dependent repair in yeast and defines two strikingly different fractions of Top2 activity in humans: tightly localised CTCF-proximal, and broadly distributed transcription-proximal, the latter correlated with gene length and expression. Moreover, single nucleotide accuracy reveals the influence primary DNA sequence has upon Top2 cleavage—distinguishing sites likely to form canonical DNA double-strand breaks (DSBs) from those predisposed to form strand-biased DNA single-strand breaks (SSBs) induced by etoposide (VP16) in vivo. This data set contains maps of Top2 CCs in the S. cerevisiae genome, generated by CC-seq of BY4741 cells -/+ etoposide (VP16).
Project description:The Autoimmune Regulator (AIRE) protein is the key factor in thymic negative selection of autoreactive T-cells by promoting the ectopic expression of tissue-specific genes in thymic medullary epithelium. Mutations in AIRE cause a monogenic autoimmune disease called autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED). AIRE has been shown to promote DNA breaks via its interaction with topoisomerase 2 (TOP2). In this study, we investigated topoisomerase-induced DNA breaks and chromatin structural alterations in conjunction with AIRE-dependent gene expression. Using RNA-seq, we found that the inhibition of TOP2 religation activity by etoposide in AIRE-expressing cells had a synergistic effect on genes with low expression levels. AIRE-mediated transcription was not only enhanced by TOP2 inhibition but also by topoisomerase 1 (TOP1) inhibitor camptothecin. The transcriptional activation was associated with structural rearrangements in chromatin, notably the accumulation of γH2AX and the exchange of histone H1 with HMGB1 at AIRE target gene promoters. In addition, we found the transcriptional upregulation to co-occur with the chromatin structural changes within the genomic cluster of carcino-embryonic antigen-like cellular adhesion molecule (CEACAM) genes. Overall, our results suggest that the presence of AIRE can trigger molecular events leading to an altered chromatin landscape and the enhanced transcription of low-expressed genes.
Project description:The Autoimmune Regulator (AIRE) protein is the key factor in thymic negative selection of autoreactive T-cells by promoting the ectopic expression of tissue-specific genes in thymic medullary epithelium. Mutations in AIRE cause a monogenic autoimmune disease called autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED). AIRE has been shown to promote DNA breaks via its interaction with topoisomerase 2 (TOP2). In this study, we investigated topoisomerase-induced DNA breaks and chromatin structural alterations in conjunction with AIRE-dependent gene expression. Using RNA-seq, we found that the inhibition of TOP2 religation activity by etoposide in AIRE-expressing cells had a synergistic effect on genes with low expression levels. AIRE-mediated transcription was not only enhanced by TOP2 inhibition but also by topoisomerase 1 (TOP1) inhibitor camptothecin. The transcriptional activation was associated with structural rearrangements in chromatin, notably the accumulation of γH2AX and the exchange of histone H1 with HMGB1 at AIRE target gene promoters. In addition, we found the transcriptional upregulation to co-occur with the chromatin structural changes within the genomic cluster of carcino-embryonic antigen-like cellular adhesion molecule (CEACAM) genes. Overall, our results suggest that the presence of AIRE can trigger molecular events leading to an altered chromatin landscape and the enhanced transcription of low-expressed genes.
Project description:DNA topoisomerases are required to resolve DNA topological stress. Despite this essential role, abortive topoisomerase activity generates aberrant protein-linked DNA breaks, jeopardising genome stability. Here, to understand the genomic distribution and mechanisms underpinning topoisomerase-induced DNA breaks, we map Top2 DNA cleavage with strand-specific nucleotide resolution across the S. cerevisiae and human genomes—and use the meiotic Spo11 protein to validate the broad applicability of this method to explore the role of diverse topoisomerase family members. Our data characterises Mre11-dependent repair in yeast and defines two strikingly different fractions of Top2 activity in humans: tightly localised CTCF-proximal, and broadly distributed transcription-proximal, the latter correlated with gene length and expression. Moreover, single nucleotide accuracy reveals the influence primary DNA sequence has upon Top2 cleavage—distinguishing sites likely to form canonical DNA double-strand breaks (DSBs) from those predisposed to form strand-biased DNA single-strand breaks (SSBs) induced by etoposide (VP16) in vivo.
Project description:DNA topoisomerases are required to resolve DNA topological stress. Despite this essential role, abortive topoisomerase activity generates aberrant protein-linked DNA breaks, jeopardising genome stability. Here, to understand the genomic distribution and mechanisms underpinning topoisomerase-induced DNA breaks, we map Top2 DNA cleavage with strand-specific nucleotide resolution across the S. cerevisiae and human genomes - and use the meiotic Spo11 protein to validate the broad applicability of this method to explore the role of diverse topoisomerase family members. Our data characterises Mre11-dependent repair in yeast, and defines two strikingly different fractions of Top2 activity in humans: tightly localised CTCF-proximal, and broadly distributed transcription-proximal, the latter correlated with gene length and expression. Moreover, single nucleotide accuracy enables us to reveal the influence primary DNA sequence has upon Top2 cleavage - distinguishing canonical DNA double-strand breaks (DSBs) from a major population of DNA single-strand breaks (SSBs) induced by etoposide (VP16) in vivo.