Single cell RNA sequencing of P7 cortex from Top1 knockout mice
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE146672 | GEO | 2020/03/10
REPOSITORIES: GEO
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Project description:DNA topoisomerases solve topological problems during chromosome metabolism. We investigated where and when Top1 and Top2 are recruited on replicating chromosomes and how their inactivation affects fork integrity and DNA damage checkpoint activation. We show that, in the context of replicating chromatin, Top1 and Top2 act within a 600 bp region spanning the moving forks. Top2 exhibits additional S-phase clusters at specific intergenic loci, mostly containing promoters. TOP1 ablation does not affect fork progression and stability and does not cause activation of the Rad53 checkpoint kinase. top2 mutants accumulate sister chromatid junctions in S phase without affecting fork progression and activate Rad53 at the M/G1 transition. top1 top2 double mutants exhibit fork block and processing, and phosphorylation of Rad53 and γH2A in S phase. The exonuclease Exo1 influences fork processing and DNA damage checkpoint activation in top1 top2 mutants. Our data are consistent with a coordinated action of Top1 and Top2 in counteracting the accumulation of torsional stress and sister chromatid entanglement at replication forks, thus preventing the diffusion of topological changes along large chromosomal regions. A failure in resolving fork-related topological constrains during S phase may therefore result in abnormal chromosome transitions, DNA damage checkpoint activation and chromosome breakage during segregation. Keywords: ChIP-chip analysis
2007-08-01 | GSE8305 | GEO
Project description:Topoisomerase 1 (TOP1) inhibitors, including camptothecin and topotecan, covalently trap TOP1 on DNA, creating cleavage complexes (cc’s) that must be resolved before gene transcription and DNA replication can proceed. We previously found that topotecan reduces the expression of long (>100 kb) genes and unsilences the paternal allele of Ube3a in neurons. Here, we sought to evaluate overlap between TOP1cc-dependent and -independent gene regulation in neurons. To do this, we utilized Top1 conditional knockout mice, Top1 knockdown, the CRISPR-Cas9 system to delete Top1, TOP1 catalytic inhibitors that do not generate TOP1cc’s, and a TOP1 mutation (T718A) that stabilizes TOP1cc’s. We found that topotecan treatment significantly alters the expression of many more genes, including long neuronal genes, immediate early genes, and paternal Ube3a, when compared to Top1 deletion. Our data show that topotecan has a stronger effect on neuronal transcription than Top1 deletion, and identifies TOP1cc-dependent and -independent contributions to gene expression.
2016-05-27 | GSE79951 | GEO
Project description:DNA topoisomerases solve topological problems during chromosome metabolism. We investigated where and when Top1 and Top2 are recruited on replicating chromosomes and how their inactivation affects fork integrity and DNA damage checkpoint activation. We show that, in the context of replicating chromatin, Top1 and Top2 act within a 600 bp region spanning the moving forks. Top2 exhibits additional S-phase clusters at specific intergenic loci, mostly containing promoters. TOP1 ablation does not affect fork progression and stability and does not cause activation of the Rad53 checkpoint kinase. top2 mutants accumulate sister chromatid junctions in S phase without affecting fork progression and activate Rad53 at the M/G1 transition. top1 top2 double mutants exhibit fork block and processing, and phosphorylation of Rad53 and γH2A in S phase. The exonuclease Exo1 influences fork processing and DNA damage checkpoint activation in top1 top2 mutants. Our data are consistent with a coordinated action of Top1 and Top2 in counteracting the accumulation of torsional stress and sister chromatid entanglement at replication forks, thus preventing the diffusion of topological changes along large chromosomal regions. A failure in resolving fork-related topological constrains during S phase may therefore result in abnormal chromosome transitions, DNA damage checkpoint activation and chromosome breakage during segregation. Keywords: ChIP-chip analysis S. cerevisiae chromosomes IIIâV, and chromosome VI highdensity oligonucleotide microarrays were provided by Affymetrix Custom Express Service (SC3456a520015F, P/N 520015; rikDACF, P/N 510636, respectively). Sequence and position of oligonucleotides on the microarrays are available from Affymetrix. ChIP was carried out as previously described (Katou et al. 2003; Katou et al. 2006): we disrupted 1.5 x 108 cells byMulti-beads shocker (MB400U, Yasui Kikai) using glass beads. Anti-HA monoclonal antibody HA.11 (16B12) (CRP Inc.) and anti-Flag monoclonal antibody M2 (Sigma-Aldrich) were used for chromatin immunoprecipitation. ChIPed DNA was purified and amplified by random priming as described (Katou et al. 2003): a total of 10 μg of amplified DNA was digested with DNaseI to a mean size of 100 bp, purified, and the fragments were end-labelled with biotin-N6-ddATP23. Hybridization, washing, staining and scanning were performed according to the manufacturerâs instruction (Affymetrix). Primary data analyses were carried out using the Affymetrix microarray Suite version 5.0 software to obtain hybridization intensity, fold change value, change P-value and detection P-value for each locus. For the discrimination of positive and negative signals for the binding, we compared ChIPed fraction with supernatant fraction by using three criteria. First, the reliability of strength of signal was judged by detection P-value of each locus (P â¥0.025). Second, reliability of binding ratio was judged by change P-value (P ⥠0.025). Third, clusters consisting of at least three contiguous loci that filled the above Bermejo et al. 26 two criteria were selected, because it was known that a single site of proteinâDNA interaction will result in immunoprecipitation of DNA fragments that hybridized not only to the locus of the actual binding site but also to its neighbours. For the analyses of BrdU incorporation, cells were fixed by ice-cold buffer containing 0.1% azide, and then total DNA from 3 x108 cells was purified. DNA was sheared to 300 bp by sonication, denatured, and mixed with 2μg anti-BrdU monoclonal antibody (2B1D5F5H4E2; MBL). Antibody-bound and unbound fractions were subsequently purified, amplified, labelled and hybridized to the DNA chip.
2010-06-05 | E-GEOD-8305 | biostudies-arrayexpress
Project description:Inhibitors of DNA topoisomerase I (TOP1), an enzyme relieving torsional stress of DNA by generating transient single-strand breaks, are clinically used to treat ovarian cancer, small cell lung cancer and cervical cancer. As torsional stress is generated by replication and transcription we tested the effects of clinically used TOP1 inhibitors Topotecan and SN-38 on TNF-induced gene expression. RNA-seq experiments showed that inhibition of TOP1 activity interfered with the vast majority of TNF-triggered genes, while interference with TOP2 activity had only a minor impact. TOP1 inhibition affected the expression of early and late induced genes and the relative strength of gene induction played no role in the sensitivity to TOP1 interfering drugs. These data raise the possibility that the inhibition of inflammatory gene expression contributes to the increased risk of infection seen in some patients treated with TOP1 inhibtors.
2019-06-22 | GSE128798 | GEO
Project description:Single cell RNA sequencing of P7 cortex from Top1 knockout mice
| PRJNA611603 | ENA
Project description:Topoisomerase I (TOP1) is an essential enzyme that relaxes DNA to prevent and dissipate torsional stress during transcription. However, the mechanisms underlying the regulation of TOP1 activity remain elusive. Using eCLIP and UV-RIP-seq we identify TOP1 as a RNA binding protein (RBP). Using a TOP1 RNA binding mutant and TOP1cc-seq to map TOP1 catalytic activity, we reveal that RNA opposes TOP1 activity as RNAPII commences transcription of active genes. These findings support new insights into the coordinated actions of RNA and TOP1 in regulating DNA topological stress intrinsic to RNAPII-dependent transcription.
2024-08-21 | GSE213403 | GEO
Project description:Microarray analysis of control and Lhx2 cKO somatosensory cortices to determine differentially expressed genes at P7 resulting from the loss of Lhx2 in post-mitotic neurons.
2016-12-15 | GSE92372 | GEO
Project description:Physiological and pathogen-induced oscillations in the plant cells' redox environment trigger response mechanisms that involve redox-sensitive cysteines and consequent changes in proteins' biochemical and functional characteristics. Arabidopsis thimet oligopeptidases (TOPs) TOP1 and TOP2 are components of the immune signaling and oxidative stress response. Here, TOP1 and TOP2 were evaluated comparatively to understand their role as redox sensors. We show that TOPs catalytic activity is augmented in plants undergoing an immune response and in mutant lines with altered expression of the chloroplastic NTRC impaired in the chloroplast thiol-disulfide regulatory system; ntrc was unable to mount a systemic immune response. Oxidation promoted TOP1 self-interaction to dimers and oligomers; Cys-to-Ala mutagenesis of multiple Cys residues inhibited TOP1 oxidative self-interaction. In contrast, TOP1 lacking the signal peptide (ΔSPTOP1) and TOP2 were detected solely as monomers. Treatment with oxidized glutathione increased the catalytic activities of TOPs; Ala substitution of the unique Cys405 of TOP2 abolished its oxidative activation. The immune regulator ROC1 was identified as a candidate TOPs substrate. TOPs cleaved a ROC1 peptide (ROC1p) at similar and unique cleavage sites; ROC1p concentration helped determine TOPs cleavage specificity. We propose that TOP1 and TOP2 form a proteolysis couple with divergent redox-sensing mechanisms and specificity.
2021-04-19 | PXD024059 | Pride
Project description:Autism spectrum disorders (ASD) represent neurodevelopmental disorders characterized by social deficits, repetitive behaviors, and various comorbidities, including epilepsy. ANK2, which encodes a neuronal scaffolding protein, is frequently mutated in ASD, but its in vivo functions and disease-related mechanisms are largely unknown. Here, we report that mice with Ank2 knockout restricted to cortical and hippocampal excitatory neurons (Ank2-cKO mice) show ASD-related behavioral abnormalities and juvenile seizure-related death. Ank2-cKO cortical neurons show abnormally increased excitability and firing rate. These changes accompanied decreases in the total level and function of the Kv7.2/KCNQ2 and Kv7.3/KCNQ3 potassium channels and the density of these channels in the enlengthened axon initial segment. Importantly, the Kv7 agonist, retigabine, rescued neuronal excitability, juvenile seizure-related death, and hyperactivity in Ank2-cKO mice. These results suggest that Ank2 regulates neuronal excitability by regulating the length of and Kv7 density in the AIS and that Kv7 channelopathy is involved in Ank2-related brain dysfunctions.
2023-03-19 | MSV000091510 | MassIVE
Project description:Topoisomerases are necessary for the expression of neurodevelopmental genes, and are mutated in some patients with autism spectrum disorder (ASD). We have studied the effects of inhibitors of Topoisomerase 1 (Top1) and Topoisomerase 2 (Top2) enzymes on mouse cortical neurons. We find that topoisomerases selectively inhibit long genes (>100kb), with little effect on all other gene expression. Using ChIPseq against RNA Polymerase II (Pol2) we show that the Top1 inhibitor topotecan blocks transcriptional elongation of long genes specifically. Many of the genes inhibited by topotecan are candidate ASD genes, leading us to propose that topoisomerase inhibition might contribute to ASD pathology. [Mouse] 5 biological replicates of transcriptome sequencing (RNAseq) from topotecan treated neurons and vehicle treated controls; Pol2 ChIPseq of topotecan and vehicle treated neurons [Human] Transcriptome sequencing (RNAseq) from topotecan treated neurons and vehicle treated control.
2013-08-30 | E-GEOD-43526 | biostudies-arrayexpress