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Tissue-clearing methods allow every cell in the mouse brain to be imaged without physical sectioning. However, the computational tools currently available for cell quantification in cleared tissue images have been limited to counting sparse cell populations in stereotypical mice. Here, we introduce NuMorph, a group of analysis tools to quantify all nuclei and nuclear markers within the mouse cortex after clearing and imaging by light-sheet microscopy. We apply NuMorph to investigate two distinct mouse models: a Topoisomerase 1 (Top1) model with severe neurodegenerative deficits and a Neurofibromin 1 (Nf1) model with a more subtle brain overgrowth phenotype. In each case, we identify differential effects of gene deletion on individual cell-type counts and distribution across cortical regions that manifest as alterations of gross brain morphology. These results underline the value of whole-brain imaging approaches, and the tools are widely applicable for studying brain structure phenotypes at cellular resolution., Competing Interests: Declaration of interests O.K. is currently employed at Scribe Therapeutics., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Topoisomerase 1 (TOP1) relieves torsional stress in DNA during transcription and facilitates the expression of long (>100 kb) genes, many of which are important for neuronal functions. To evaluate how loss of Top1 affected neurons in vivo, we conditionally deleted (cKO) Top1 in postmitotic excitatory neurons in the mouse cerebral cortex and hippocampus. Top1 cKO neurons develop properly, but then show biased transcriptional downregulation of long genes, signs of DNA damage, neuroinflammation, increased poly(ADP-ribose) polymerase-1 (PARP1) activity, single-cell somatic mutations, and ultimately degeneration. Supplementation of nicotinamide adenine dinucleotide (NAD + ) with nicotinamide riboside partially blocked neurodegeneration, and increased the lifespan of Top1 cKO mice by 30%. A reduction of p53 also partially rescued cortical neuron loss. While neurodegeneration was partially rescued, behavioral decline was not prevented. These data indicate that reducing neuronal loss is not sufficient to limit behavioral decline when TOP1 function is disrupted.

Topoisomerase III beta (TOP3B) is one of the least understood members of the topoisomerase family of proteins and remains enigmatic. Our recent data shed light on the function and relevance of TOP3B to disease. A homozygous deletion for the TOP3B gene was identified in a patient with bilateral renal cancer. Analyses in both patient and modelled human cells show the disruption of TOP3B causes genome instability with a rise in DNA damage and chromosome bridging (mis-segregation). The primary molecular defect underlying this pathology is a significant increase in R-loop formation. Our data show that TOP3B is necessary to prevent the accumulation of excessive R-loops and identify TOP3B as a putative cancer gene, and support recent data showing that R-loops are involved in cancer aetiology.

G-quadruplex or G4 DNA is a non-B secondary DNA structure consisting of a stacked array of guanine-quartets that can disrupt critical cellular functions such as replication and transcription. When sequences that can adopt Non-B structures including G4 DNA are located within actively transcribed genes, the reshaping of DNA topology necessary for transcription process stimulates secondary structure-formation thereby amplifying the potential for genome instability. Using a reporter assay designed to study G4-induced recombination in the context of an actively transcribed locus in Saccharomyces cerevisiae, we tested whether co-transcriptional activator Sub1, recently identified as a G4-binding factor, contributes to genome maintenance at G4-forming sequences. Our data indicate that, upon Sub1-disruption, genome instability linked to co-transcriptionally formed G4 DNA in Top1-deficient cells is significantly augmented and that its highly conserved DNA binding domain or the human homolog PC4 is sufficient to suppress G4-associated genome instability. We also show that Sub1 interacts specifically with co-transcriptionally formed G4 DNA in vivo and that yeast cells become highly sensitivity to G4-stabilizing chemical ligands by the loss of Sub1. Finally, we demonstrate the physical and genetic interaction of Sub1 with the G4-resolving helicase Pif1, suggesting a possible mechanism by which Sub1 suppresses instability at G4 DNA., (© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.)

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.

Topoisomerases are expressed throughout the developing and adult brain and are mutated in some individuals with autism spectrum disorder (ASD). However, how topoisomerases are mechanistically connected to ASD is unknown. Here we find that topotecan, a topoisomerase 1 (TOP1) inhibitor, dose-dependently reduces the expression of extremely long genes in mouse and human neurons, including nearly all genes that are longer than 200 kilobases. Expression of long genes is also reduced after knockdown of Top1 or Top2b in neurons, highlighting that both enzymes are required for full expression of long genes. By mapping RNA polymerase II density genome-wide in neurons, we found that this length-dependent effect on gene expression was due to impaired transcription elongation. Interestingly, many high-confidence ASD candidate genes are exceptionally long and were reduced in expression after TOP1 inhibition. Our findings suggest that chemicals and genetic mutations that impair topoisomerases could commonly contribute to ASD and other neurodevelopmental disorders.

The SUMO-dependent ubiquitin ligase Slx8 plays key roles in promoting genome stability, including the processing of trapped Topoisomerase I (Top1) cleavage complexes and removal of toxic SUMO conjugates. We show that it is the latter function that constitutes Slx8's primary role in fission yeast. The SUMO conjugates in question are formed by the SUMO ligase Pli1, which is necessary for limiting spontaneous homologous recombination when Top1 is present. Surprisingly there is no requirement for Pli1 to limit recombination in the vicinity of a replication fork blocked at the programmed barrier RTS1. Notably, once committed to Pli1-mediated SUMOylation Slx8 becomes essential for genotoxin resistance, limiting both spontaneous and RTS1 induced recombination, and promoting normal chromosome segregation. We show that Slx8 removes Pli1-dependent Top1-SUMO conjugates and in doing so helps to constrain recombination at RTS1. Overall our data highlight how SUMOylation and SUMO-dependent ubiquitylation by the Pli1-Slx8 axis contribute in different ways to maintain genome stability.

Background: Manipulations of the DNA double helix during replication, transcription and other nucleic acid processing cause a change of DNA topology, which results in torsional stress. This stress is relaxed by DNA topoisomerases, a class of enzymes present in all domains of life. Negatively supercoiled DNA is relaxed by type IA topoisomerases that are widespread in bacteria, archaea and eukaryotes. In Escherichia coli there is conflicting data about viability of ΔtopA cells lacking topoisomerase I., Results: In this study we sought to clarify whether E. coli cells lacking topoisomerase I are viable by using a plasmid-based lethality assay that allowed us to investigate the phenotype of ΔtopA cells without the presence of any compensatory mutations. Our results show that cells lacking topoisomerase I show an extreme growth defect and cannot be cultured without the accumulation of compensatory mutations. This growth defect can be partially suppressed by overexpression of topoisomerase III, the other type IA topoisomerase in E. coli, suggesting that the accumulation of torsional stress is, at least partially, responsible for the lethality of ΔtopA cells. The absence of RNase HI strongly exacerbates the phenotype of cells lacking topoisomerase I, which supports the idea that the processing of RNA:DNA hybrids is vitally important in ΔtopA cells. However, we did not observe suppression of the ΔtopA phenotype by increasing the level of R-loop processing enzymes, such as RNase HI or RecG., Conclusions: Our data show unambiguously that E. coli cells are not viable in the absence of DNA topoisomerase I without the presence of compensatory mutations. Furthermore, our data suggest that the accumulation of R-loops is not the primary reason for the severe growth defect of cells lacking topoisomerase I, which is in contrast to the current literature. Potential reasons for this discrepancy are discussed.

Human topoisomerase I has been suggested to be implicated in the maintenance of genomic stability via its ability to regulate genome topology during transcription and replication. In the present study, we demonstrate by whole-genome array comparative genomic hybridization (aCGH) and fluorescence in situ hybridisation (FISH) analysis that topoisomerase I deficiency results in chromosome 5p gain in the cervical cancer cell line, HeLa-CCL2. In contrast, chromosome 5p copy number remained unaffected by topoisomerase I down-regulation in the non-cancer cell line HEK293T, as demonstrated by FISH analysis. Chromosome 5p gain is the most frequent genetic alteration in invasive cervical cancer, which leads to overexpression of genes involved in proliferation and occurs primarily at late stages in cancer development. The amplification of this region upon topoisomerase I down-regulation specifically in HeLa-CCL2 cells may indicate an important role of topoisomerase I in preventing malignant progression of precancerous lesions in the cervix.

In animal cells, recent studies have emphasized the role played by DNA topoisomerase I (topo I) both as a cofactor of DNA repair complexes and/or as a damage sensor. All these functions are still unexplored in plant cells, where information concerning the relationships between DNA damage, PCD induction, and topo I are also limited. The main goal of this study was to investigate the possible responses activated in topo I-depleted plant cells under oxidative stress conditions which induce DNA damage. The carrot (Daucus carota L.) AT1-beta/22 cell line analysed in this study (characterized by an antisense-mediated reduction of top1beta gene expression of approximately 46% in association with a low ascorbate content) was more sensitive to UV-C radiation than the control line, showing consistent cell death and high levels of 8-oxo-dG accumulation. The topo I-depleted cells were also highly susceptible to the cross-linking agent mitomycin C. The death response was associated with a lack of oxidative burst and there were no changes in ascorbate metabolism in response to UV-C treatment. Electron and fluorescence microscopy suggested the presence of three forms of cell death in the UV-C-treated AT1-beta/22 population: necrosis, apoptotic-like PCD, and autophagy. Taken together, the data reported here support a reduced DNA repair capability in carrot topo I-deficient cells while the putative relationship between topo I-depletion and ascorbate impairment is also discussed.

The type IA topoisomerases have been implicated in the repair of dsDNA breaks by homologous recombination and in the resolution of stalled or damaged DNA replication forks; thus, these proteins play important roles in the maintenance of genomic stability. We studied the functions of one of the two mammalian type IA enzymes, Top3beta, using murine embryonic fibroblasts (MEFs) derived from top3beta(-/-) embryos. top3beta(-/-) MEFs proliferated more slowly than TOP3beta(+/+) control MEFs, demonstrated increased sensitivity to DNA-damaging agents such as ionizing and UV radiation, and had increased DNA double-strand breaks as manifested by increased gamma-H2-AX phosphorylation. However, incomplete enforcement of the G(1)-S cell cycle checkpoint was observed in top3beta(-/-) MEFs. Notably, ataxia-telangiectasia, mutated (ATM)/ATM and Rad3-related (ATR)-dependent substrate phosphorylation after UV-B and ionizing radiation was impaired in top3beta(-/-) versus TOP3beta(+/+) control MEFs, and impaired up-regulation of total and Ser-18-phosphorylated p53 was observed in top3beta(-/-) cells. Taken together, these results suggest an unanticipated role for Top3beta beyond DNA repair in the activation of cellular responses to DNA damage.

Mice lacking DNA topoisomerase 3beta are predisposed to a shortened lifespan, infertility, and lesions in multiple organs resulting from inflammatory responses. Examination of the immune system of 6- and 52-week-old top3beta(-/-) mice revealed no significant aberrations in their central and peripheral tolerance or in T lymphocyte activation. However, the older but not the younger cohort shows a high incidence of serum autoantibodies relative to their TOP3beta(+/+) age-mates. The mutant mice also show an increase in numerical aberrations of chromosomes in splenocytes and bone marrow cells, as well as an increase in apoptotic cells in the thymus. Thus, it appears plausible that the inflammatory lesions in top3beta(-/-) mice are caused by the development of autoimmunity as they age: Chromosomal abnormalities in top3beta(-/-) mice might lead to a persistent increase in apoptotic cells, which might in turn lead to the progression of autoimmunity.

Bloom's syndrome (BS) is an autosomal disorder characterized by predisposition to a wide variety of cancers. The gene product whose mutation leads to BS is the RecQ family helicase BLM, which forms a complex with DNA topoisomerase IIIalpha (Top3alpha). However, the physiological relevance of the interaction between BLM and Top3alpha within the cell remains unclear. We show here that Top3alpha depletion causes accumulation of cells in G2 phase, enlargement of nuclei, and chromosome gaps and breaks that occur at the same position in sister chromatids. The transition from metaphase to anaphase is also inhibited. All of these phenomena except cell lethality are suppressed by BLM gene disruption. Taken together with the biochemical properties of BLM and Top3alpha, these data indicate that BLM and Top3alpha execute the dissolution of sister chromatids.

RecQ helicase BLM-deficient cells are characteristically hypersensitive to 4-nitroquinoline-1-oxide (4NQO). We recently reported that isogenic BLM-deficient cells (PNSG13) are more sensitive than BLM-complemented cells (PNSF5) to camptothecin, which specifically traps topoisomerase I cleavage complexes (Top1cc). We now report that PNSG13 are also 3.5-fold more sensitive to 4NQO compared with PNSF5 and that 4NQO induces higher levels of Top1cc and reduced histone gamma-H2AX in PSNG13 than in PNSF5. Similarly, 4NQO induces more Top1cc in primary fibroblasts from a patient with Bloom syndrome than in normal human fibroblasts. 4NQO also induces Top1cc in colon cancer HCT116 and HT29 cells in a time- and concentration-dependent fashion. Of note, distinct from camptothecin, the Top1cc produced by 4NQO accumulate progressively after 4NQO addition and persist following 4NQO removal. The Top1cc induced by 4NQO are detectable by alkaline elution. To examine the functional relevance of the Top1cc induced by 4NQO, we used two stable topoisomerase I small interfering RNA (siRNA) cell lines derived from HCT116 and MCF7 cells. Both topoisomerase I siRNA cell lines are resistant to 4NQO, indicating that Top1cc contribute to the cellular activity of 4NQO. Collectively, these data show that 4NQO is an effective inducer of cellular Top1cc. Because 4NQO does not directly trap Top1cc in biochemical assays, we propose that active metabolites of 4NQO trap Top1cc by forming DNA adducts. Induction of Top1cc and histone gamma-H2AX by 4NQO may contribute to the cellular effects of 4NQO, including its selective activity toward RecQ helicase BLM-deficient cells.

Acid resistance (AR) in Escherichia coli is important for its survival in the human gastrointestinal tract and involves three systems. The first AR system is dependent on the sigma factor RpoS. The second system (the GAD system) requires the glutamate decarboxylase isoforms encoded by the gadA and gadB genes. The third system (the ARG system) requires the arginine decarboxylase encoded by adiA. Loss of topoisomerase I function from topA deletion or Tn10 insertion mutations lowered the resistance to killing by pH 2 or 2.5 treatment by 10-fold to >100-fold. The RpoS and GAD systems were both affected by the topA mutation, but the ARG system of AR was not affected. Northern blot analysis showed that induction of gadA and gadB transcription in stationary phase and at pH 5.5 was decreased in the topA mutant. Western blot analysis showed that the topA mutation did not affect accumulation of RpoS, GadX or GadW proteins. Topoisomerase I might have a direct influence on the transcription of AR genes. This influence does not involve R-loop formation as the overexpression of RNase H did not alleviate the decrease of AR caused by the topA mutation. The effect of the topA mutation could be suppressed by an hns mutation, so topoisomerase I might be required to counteract the effect of H-NS protein on gene expression, in addition to its influence on RpoS-dependent transcription.

DNA topoisomerase I (Topo I) specifically phosphorylates arginine-serine-rich (SR proteins) splicing factors and is potentially involved in pre-mRNA-splicing regulation. Using a Topo I-deficient murine B lymphoma-derived subclone (P388-45/C) selected for its resistance to high dosage of the antitumor drug camptothecin, we show that Topo I depletion results in the hypophosphorylation of SR proteins and impairs exonic splicing enhancer (ESE)-dependent but not constitutive splicing. The Affymetrix GeneChip system analysis revealed that several alternatively spliced genes, characterized by small exons and large introns, are down-regulated in Topo I-deficient cells. Given that ectopic expression of green fluorescent protein-Topo I fusion in Topo I-deficient cells restores both wild-type phosphorylation of SR proteins and ESE-dependent splicing, we conclude that Topo I-mediated phosphorylation plays a specific role in ESE-regulated splicing.

Type I DNA topoisomerases are ubiquitous enzymes involved in many aspects of DNA metabolism. Escherichia coli possesses two type I topoisomerase activities, DNA topoisomerase I (Topo I) and III (Topo III). The gene encoding Topo III (topB) can be deleted without affecting cell viability. Cells possessing a deletion of the gene encoding Topo I (topA) are only viable in the presence of an additional compensatory mutation. In the presence of compensatory mutations, Topo I deletion strains grow normally; however, if Topo III activity is repressed in these cells, they filament extensively and possess an abnormal nucleoid structure. These defects can be suppressed by the deletion of the recA gene, suggesting that these enzymes may be involved in RecA-mediated recombination and may specifically resolve recombination intermediates before partitioning.

Previous biochemical studies have suggested a role for bacterial DNA topoisomerase (TOPO) I in the suppression of R-loop formation during transcription. In this report, we present several pieces of genetic evidence to support a model in which R-loop formation is dynamically regulated during transcription by activities of multiple DNA TOPOs and RNase H. In addition, our results suggest that events leading to the serious growth problems in the absence of DNA TOPO I are linked to R-loop formation. We show that the overexpression of RNase H, an enzyme that degrades the RNA moiety of an R loop, can partially compensate for the absence of DNA TOPO I. We also note that a defect in DNA gyrase can correct several phenotypes associated with a mutation in the rnhA gene, which encodes the major RNase H activity. In addition, we found that a combination of topA and rnhA mutations is lethal.

Introducing the Escherichia coli topA20::Tn10 allele to Shigella flexneri results in osmotic sensitivity, a reduced growth rate, an increase in reporter plasmid supercoiling (all common to the E. coli mutants), an inability to grow on MacConkey agar and a loss of virulence gene expression. E. coli mutants harbouring this topA allele often compensate for the loss of DNA topoisomerase I by amplifying the genes coding for topoisomerase IV. Unlike the E. coli topA mutants, derivatives of S. flexneri harbouring this topA allele did not appear to acquire any compensatory mutations. We investigated the possibility that this was due in part to an inability of the S. flexneri topoisomerase IV genes to compensate for loss of DNA topoisomerase I when overexpressed. The S. flexneri genes encoding the alpha- and beta subunits of topoisomerase IV were detected and cloned in separate multicopy plasmids. These plasmids complemented well-characterized Salmonella typhimurium temperature-sensitive topoisomerase IV mutations, showing that the S. flexneri and S. typhimurium proteins are capable of combining to form active complexes. When the S. flexneri topoisomerase IV genes were cloned in the same multicopy plasmid and introduced into a S. flexneri topA mutant, the plasmid restored osmotic tolerance, improved the growth rate, allowed growth on MacConkey indicator plates and resulted in a relaxation of reporter plasmid supercoiling. The same plasmid also partially restored S. flexneri virulence gene transcription. These data show that overexpression of the S. flexneri topoisomerase IV genes can compensate for the loss of topoisomerase I in terms of general viability of the cell, DNA supercoiling, and (partially) virulence gene expression. The fact that S. flexneri does not exploit topoisomerase IV gene amplification as E. coli does points to a significant difference in the abilities of these species to adapt to the loss of topoisomerase I.