Entangling and disentangling mitotic chromosomes.

The DNA topological challenges generated by cellular manipulation of extremely long DNA fibers remain poorly understood. In this issue of Molecular Cell, Hildebrand et al.1 describe how mitotic chromosomes are self entangled and that disentanglement requires TOP2 activity in late mitosis.

TOP2A modulates signaling via the AKT/mTOR pathway to promote ovarian cancer cell proliferation.

Ovarian cancer (OC) is a form of gynecological malignancy that is associated with worse patient outcomes than any other cancer of the female reproductive tract. Topoisomerase II α (TOP2A) is commonly regarded as an oncogene that is associated with malignant disease progression in a variety of cancers, its mechanistic functions in OC have yet to be firmly established. We explored the role of TOP2A in OC through online databases, clinical samples, in vitro and in vivo experiments. And initial analyses of public databases revealed high OC-related TOP2A expression in patient samples that was related to poorer prognosis. This was confirmed by clinical samples in which TOP2A expression was elevated in OC relative to healthy tissue. Kaplan-Meier analyses further suggested that higher TOP2A expression levels were correlated with worse prognosis in OC patients. In vitro, TOP2A knockdown resulted in the inhibition of OC cell proliferation, with cells entering G1 phase arrest and undergoing consequent apoptotic death. In rescue assays, TOP2A was confirmed to regulate cell proliferation and cell cycle through AKT/mTOR pathway activity. Mouse model experiments further affirmed the key role that TOP2A plays as a driver of OC cell proliferation. These data provide strong evidence supporting TOP2A as an oncogenic mediator and prognostic biomarker related to OC progression and poor outcomes. At the mechanistic level, TOP2A can control tumor cell growth via AKT/mTOR pathway modulation. These preliminary results provide a foundation for future research seeking to explore the utility of TOP2A inhibitor-based combination treatment regimens in platinum-resistant recurrent OC patients.

Use of CRISPR/Cas9 with Homology-Directed Repair to Gene-Edit Topoisomerase IIß in Human Leukemia K562 Cells: Generation of a Resistance Phenotype.

DNA topoisomerase IIß (TOP2ß/180; 180 kDa) is a nuclear enzyme that regulates DNA topology by generation of short-lived DNA double-strand breaks, primarily during transcription. TOP2ß/180 can be a target for DNA damage-stabilizing anticancer drugs, whose efficacy is often limited by chemoresistance. Our laboratory previously demonstrated reduced levels of TOP2ß/180 (and the paralog TOP2α/170) in an acquired etoposide-resistant human leukemia (K562) clonal cell line, K/VP.5, in part due to overexpression of microRNA-9-3p/5p impacting post-transcriptional events. To evaluate the effect on drug sensitivity upon reduction/elimination of TOP2ß/180, a premature stop codon was generated at the TOP2ß/180 gene exon 19/intron 19 boundary (AGAA//GTAA→ATAG//GTAA) in parental K562 cells (which contain four TOP2ß/180 alleles) by CRISPR/Cas9 editing with homology-directed repair to disrupt production of full-length TOP2ß/180. Gene-edited clones were identified and verified by quantitative polymerase chain reaction and Sanger sequencing, respectively. Characterization of TOP2ß/180 gene-edited clones, with one or all four TOP2ß/180 alleles mutated, revealed partial or complete loss of TOP2ß mRNA/protein, respectively. The loss of TOP2ß/180 protein correlated with decreased (2-{4-[(7-chloro-2-quinoxalinyl)oxy]phenoxy}propionic acid)-induced DNA damage and partial resistance in growth inhibition assays. Partial resistance to mitoxantrone was also noted in the gene-edited clone with all four TOP2ß/180 alleles modified. No cross-resistance to etoposide or mAMSA was noted in the gene-edited clones. Results demonstrated the role of TOP2ß/180 in drug sensitivity/resistance in K562 cells and revealed differential paralog activity of TOP2-targeted agents. SIGNIFICANCE STATEMENT: Data indicated that CRISPR/Cas9 editing of the exon 19/intron 19 boundary in the TOP2ß/180 gene to introduce a premature stop codon resulted in partial to complete disruption of TOP2ß/180 expression in human leukemia (K562) cells depending on the number of edited alleles. Edited clones were partially resistant to mitoxantrone and XK469, while lacking resistance to etoposide and mAMSA. Results demonstrated the import of TOP2ß/180 in drug sensitivity/resistance in K562 cells and revealed differential paralog activity of TOP2-targeted agents.

Mitotic chromosomes are self-entangled and disentangle through a topoisomerase-II-dependent two-stage exit from mitosis.

The topological state of chromosomes determines their mechanical properties, dynamics, and function. Recent work indicated that interphase chromosomes are largely free of entanglements. Here, we use Hi-C, polymer simulations, and multi-contact 3C and find that, by contrast, mitotic chromosomes are self-entangled. We explore how a mitotic self-entangled state is converted into an unentangled interphase state during mitotic exit. Most mitotic entanglements are removed during anaphase/telophase, with remaining ones removed during early G1, in a topoisomerase-II-dependent process. Polymer models suggest a two-stage disentanglement pathway: first, decondensation of mitotic chromosomes with remaining condensin loops produces entropic forces that bias topoisomerase II activity toward decatenation. At the second stage, the loops are released, and the formation of new entanglements is prevented by lower topoisomerase II activity, allowing the establishment of unentangled and territorial G1 chromosomes. When mitotic entanglements are not removed in experiments and models, a normal interphase state cannot be acquired.

Localization and quantitative distribution of a chromatin structural protein Topoisomerase II on plant chromosome using HVTEM and UHVTEM.

Topoisomerase II (TopoII) is an essential structural protein of the metaphase chromosome. It maintains the axial compaction of chromosomes during metaphase. It is localized at the axial region of chromosomes and accumulates at the centromeric region in metaphase chromosomes. However, little is known about TopoII localization and distribution in plant chromosomes, except for several publications. We used high voltage transmission electron microscopy (HVTEM) and ultra-high voltage transmission electron microscopy (UHVTEM) in conjunction with immunogold labeling and visualization techniques to detect TopoII and investigate its localization, alignment, and density on the barley chromosome at 1.4 nm scale. We found that HVTEM and UHVTEM combined with immunogold labeling is suitable for the detection of structural proteins, including a single molecule of TopoII. This is because the average size of the gold particles for TopoII visualization after silver enhancement is 8.9 ± 3.9 nm, which is well detected. We found that 31,005 TopoII molecules are distributed along the barley chromosomes in an unspecific pattern at the chromosome arms and accumulate specifically at the nucleolus organizer regions (NORs) and centromeric region. The TopoII density were 1.32-fold, 1.58-fold, and 1.36-fold at the terminal region, at the NORs, and the centromeric region, respectively. The findings of TopoII localization in this study support the multiple reported functions of TopoII in the barley metaphase chromosome.

Structure-function analysis of the ATPase domain of African swine fever virus topoisomerase.

Type II topoisomerase utilizes the energy from ATP hydrolysis to alter DNA topology during genome replication and transcription. The ATPase domain of this enzyme is required for ATP hydrolysis and plays a crucial role in coupling DNA binding and ATP turnover with the DNA strand passage reaction. The African swine fever virus (ASFV) specifically encodes a topoisomerase II (topo II), which is critical for viral replication and an attractive target for antiviral development. Here, we present a high-resolution crystal structure of the ASFV topo II ATPase domain complexed with the substrate analog AMPPNP. Structural comparison reveals that the ASFV topo II ATPase domain shares a conserved overall structure with its homologs from eukaryotes and prokaryotes but also has three characteristic regions, including the intra-molecular interface formed by the ATP-lid and QTK loop as well as helix α9, the K-loop in the transducer domain, and the antennae-like α-helix at the ATP binding domain. Mutating the key residues within these three regions impairs or abolishes the basal and DNA-stimulated ATPase activities and reduces or eliminates the relaxation activity of the holoenzyme. Our data indicate that all three regions are functionally important for the ATPase and relaxation activities and strongly suggest that ATP hydrolysis, DNA binding, and strand passage are highly coupled and managed by the allosteric coordination of multiple domains of the type II topoisomerase. Moreover, we find a promising druggable pocket in the dimeric interface of the ASFV topo II ATPase domain, which will benefit future anti-ASFV drug development. IMPORTANCE: The ATPase domain of type II topoisomerase provides energy by hydrolyzing ATP and coordinates with the DNA-binding/cleavage domain to drive and control DNA transport. The precise molecular mechanisms of how these domains respond to DNA binding and ATP hydrolysis signals and communicate with each other remain elusive. We determine the first high-resolution crystal structure of the ATPase domain of African swine fever virus (ASFV) topo II in complex with AMPPNP and biochemically investigate its function in ATPase and DNA relaxation activities. Importantly, we find that mutations at three characteristic regions of the ASFV ATPase domain produce parallel effects on the basal/DNA-stimulated ATPase and relaxation activities, implying the tight coupling of the ATP hydrolysis and strand passage process. Therefore, our data provide important implications for understanding the strand passage mechanism of the type II topoisomerase and the structural basis for developing ATPase domain-targeting antivirals against ASFV.

Anthracyclines induce cardiotoxicity through a shared gene expression response signature.

TOP2 inhibitors (TOP2i) are effective drugs for breast cancer treatment. However, they can cause cardiotoxicity in some women. The most widely used TOP2i include anthracyclines (AC) Doxorubicin (DOX), Daunorubicin (DNR), Epirubicin (EPI), and the anthraquinone Mitoxantrone (MTX). It is unclear whether women would experience the same adverse effects from all drugs in this class, or if specific drugs would be preferable for certain individuals based on their cardiotoxicity risk profile. To investigate this, we studied the effects of treatment of DOX, DNR, EPI, MTX, and an unrelated monoclonal antibody Trastuzumab (TRZ) on iPSC-derived cardiomyocytes (iPSC-CMs) from six healthy females. All TOP2i induce cell death at concentrations observed in cancer patient serum, while TRZ does not. A sub-lethal dose of all TOP2i induces limited cellular stress but affects calcium handling, a function critical for cardiomyocyte contraction. TOP2i induce thousands of gene expression changes over time, giving rise to four distinct gene expression response signatures, denoted as TOP2i early-acute, early-sustained, and late response genes, and non-response genes. There is no drug- or AC-specific signature. TOP2i early response genes are enriched in chromatin regulators, which mediate AC sensitivity across breast cancer patients. However, there is increased transcriptional variability between individuals following AC treatments. To investigate potential genetic effects on response variability, we first identified a reported set of expression quantitative trait loci (eQTLs) uncovered following DOX treatment in iPSC-CMs. Indeed, DOX response eQTLs are enriched in genes that respond to all TOP2i. Next, we identified 38 genes in loci associated with AC toxicity by GWAS or TWAS. Two thirds of the genes that respond to at least one TOP2i, respond to all ACs with the same direction of effect. Our data demonstrate that TOP2i induce thousands of shared gene expression changes in cardiomyocytes, including genes near SNPs associated with inter-individual variation in response to DOX treatment and AC-induced cardiotoxicity.

DNA-PKcs-mediated transcriptional regulation of TOP2B drives chemoresistance in acute myeloid leukemia.

Anthracyclines, topoisomerase II enzyme poisons that cause DNA damage, are the mainstay of acute myeloid leukemia (AML) treatment. However, acquired resistance to anthracyclines leads to relapse, which currently lacks effective treatment and is the cause of poor survival in individuals with AML. Therefore, the identification of the mechanisms underlying anthracycline resistance remains an unmet clinical need. Here, using patient-derived primary cultures and clinically relevant cellular models that recapitulate acquired anthracycline resistance in AML, we have found that GCN5 (also known as KAT2A) mediates transcriptional upregulation of DNA-dependent protein kinase catalytic subunit (DNA-PKcs) in AML relapse, independently of the DNA-damage response. We demonstrate that anthracyclines fail to induce DNA damage in resistant cells, owing to the loss of expression of their target enzyme, TOP2B; this was caused by DNA-PKcs directly binding to its promoter upstream region as a transcriptional repressor. Importantly, DNA-PKcs kinase activity inhibition re-sensitized AML relapse primary cultures and cells resistant to mitoxantrone, and abrogated their tumorigenic potential in a xenograft mouse model. Taken together, our findings identify a GCN5-DNA-PKcs-TOP2B transcriptional regulatory axis as the mechanism underlying anthracycline resistance, and demonstrate the therapeutic potential of DNA-PKcs inhibition to re-sensitize resistant AML relapse cells to anthracycline.

Using energy to go downhill-a genoprotective role for ATPase activity in DNA topoisomerase II.

Type II topoisomerases effect topological changes in DNA by cutting a single duplex, passing a second duplex through the break, and resealing the broken strand in an ATP-coupled reaction cycle. Curiously, most type II topoisomerases (topos II, IV and VI) catalyze DNA transformations that are energetically favorable, such as the removal of superhelical strain; why ATP is required for such reactions is unknown. Here, using human topoisomerase IIß (hTOP2ß) as a model, we show that the ATPase domains of the enzyme are not required for DNA strand passage, but that their loss elevates the enzyme's propensity for DNA damage. The unstructured C-terminal domains (CTDs) of hTOP2ß strongly potentiate strand passage activity in ATPase-less enzymes, as do cleavage-prone mutations that confer hypersensitivity to the chemotherapeutic agent etoposide. The presence of either the CTD or the mutations lead ATPase-less enzymes to promote even greater levels of DNA cleavage in vitro, as well as in vivo. By contrast, aberrant cleavage phenotypes of these topo II variants is significantly repressed when the ATPase domains are present. Our findings are consistent with the proposal that type II topoisomerases acquired ATPase function to maintain high levels of catalytic activity while minimizing inappropriate DNA damage.

Cancer sensitizing effect of deazaflavin analogs is associated with increased intracellular drug accumulation.

As part of our efforts geared towards developing mechanism-based cancer sensitizing agents, we have previously synthesized and characterized novel deazaflavin analogs as potent tyrosyl DNA phosphodiesterase 2 (TDP2) inhibitors for combination treatments with topoisomerase II (TOP2) poisons. Interestingly, the sensitizing effect of a few analogs toward TOP2 poison etoposide (ETP) was associated with a significant increase in intracellular drug accumulation, which could be an alternative mechanism to boost the clinical efficacy of ETP in cancer chemotherapies. Hence, we evaluated more deazaflavin TDP2 inhibitors for their impact on drug retention in cancer cells. We found that all but one tested TDP2 inhibitors substantially increased the ETP retention in DT40 cells. Particularly, we identified an exceptionally potent analog, ZW-1226, which at 3 nM increased the intracellular ETP by 13-fold. Significantly, ZW-1226 also stimulated cellular accumulation of two other anticancer drugs, TOP2 poison teniposide and antifolate pemetrexed, and produced an effect more pronounced than those of ABC transporter inhibitors verapamil and elacridar in human leukemic CCRF-CEM cells toward ETP. Lastly, ZW-1226 potentiated the action of ETP in the sensitive human CCRF-CEM cells and a few resistant non-small-cell lung cancer (NSCLC) cells, including H460 and H838 cells. Collectively, the results of this study strongly suggest that deazaflavin analog ZW-1226 could be an effective cancer sensitizing agent which warrants further investigation.

RAD54L2-mediated DNA damage avoidance pathway specifically preserves genome integrity in response to topoisomerase 2 poisons.

Type II topoisomerases (TOP2) form transient TOP2 cleavage complexes (TOP2ccs) during their catalytic cycle to relieve topological stress. TOP2ccs are covalently linked TOP2-DNA intermediates that are reversible but can be trapped by TOP2 poisons. Trapped TOP2ccs block transactions on DNA and generate genotoxic stress, which are the mechanisms of action of TOP2 poisons. How cells avoid TOP2cc accumulation remains largely unknown. In this study, we uncovered RAD54 like 2 (RAD54L2) as a key factor that mediates a TOP2-specific DNA damage avoidance pathway. RAD54L2 deficiency conferred unique sensitivity to treatment with TOP2 poisons. RAD54L2 interacted with TOP2A/TOP2B and ZATT/ZNF451 and promoted the turnover of TOP2 from DNA with or without TOP2 poisons. Additionally, inhibition of proteasome activity enhanced the chromatin binding of RAD54L2, which in turn led to the removal of TOP2 from chromatin. In conclusion, we propose that RAD54L2-mediated TOP2 turnover is critically important for the avoidance of potential TOP2-linked DNA damage under physiological conditions and in response to TOP2 poisons.

RAD54L2 counters TOP2-DNA adducts to promote genome stability.

The catalytic cycle of topoisomerase 2 (TOP2) enzymes proceeds via a transient DNA double-strand break (DSB) intermediate termed the TOP2 cleavage complex (TOP2cc), in which the TOP2 protein is covalently bound to DNA. Anticancer agents such as etoposide operate by stabilizing TOP2ccs, ultimately generating genotoxic TOP2-DNA protein cross-links that require processing and repair. Here, we identify RAD54 like 2 (RAD54L2) as a factor promoting TOP2cc resolution. We demonstrate that RAD54L2 acts through a novel mechanism together with zinc finger protein associated with tyrosyl-DNA phosphodiesterase 2 (TDP2) and TOP2 (ZATT/ZNF451) and independent of TDP2. Our work suggests a model wherein RAD54L2 recognizes sumoylated TOP2 and, using its ATPase activity, promotes TOP2cc resolution and prevents DSB exposure. These findings suggest RAD54L2-mediated TOP2cc resolution as a potential mechanism for cancer therapy resistance and highlight RAD54L2 as an attractive candidate for drug discovery.

ERK2-topoisomerase II regulatory axis is important for gene activation in immediate early genes.

The function of the mitogen-activated protein kinase signaling pathway is required for the activation of immediate early genes (IEGs), including EGR1 and FOS, for cell growth and proliferation. Recent studies have identified topoisomerase II (TOP2) as one of the important regulators of the transcriptional activation of IEGs. However, the mechanism underlying transcriptional regulation involving TOP2 in IEG activation has remained unknown. Here, we demonstrate that ERK2, but not ERK1, is important for IEG transcriptional activation and report a critical ELK1 binding sequence for ERK2 function at the EGR1 gene. Our data indicate that both ERK1 and ERK2 extensively phosphorylate the C-terminal domain of TOP2B at mutual and distinctive residues. Although both ERK1 and ERK2 enhance the catalytic rate of TOP2B required to relax positive DNA supercoiling, ERK2 delays TOP2B catalysis of negative DNA supercoiling. In addition, ERK1 may relax DNA supercoiling by itself. ERK2 catalytic inhibition or knock-down interferes with transcription and deregulates TOP2B in IEGs. Furthermore, we present the first cryo-EM structure of the human cell-purified TOP2B and etoposide together with the EGR1 transcriptional start site (-30 to +20) that has the strongest affinity to TOP2B within -423 to +332. The structure shows TOP2B-mediated breakage and dramatic bending of the DNA. Transcription is activated by etoposide, while it is inhibited by ICRF193 at EGR1 and FOS, suggesting that TOP2B-mediated DNA break to favor transcriptional activation. Taken together, this study suggests that activated ERK2 phosphorylates TOP2B to regulate TOP2-DNA interactions and favor transcriptional activation in IEGs. We propose that TOP2B association, catalysis, and dissociation on its substrate DNA are important processes for regulating transcription and that ERK2-mediated TOP2B phosphorylation may be key for the catalysis and dissociation steps.

Cell cycle responses to Topoisomerase II inhibition: Molecular mechanisms and clinical implications.

DNA Topoisomerase IIA (Topo IIA) is an enzyme that alters the topological state of DNA and is essential for the separation of replicated sister chromatids and the integrity of cell division. Topo IIA dysfunction activates cell cycle checkpoints, resulting in arrest in either the G2-phase or metaphase of mitosis, ultimately triggering the abscission checkpoint if non-disjunction persists. These events, which directly or indirectly monitor the activity of Topo IIA, have become of major interest as many cancers have deficiencies in Topoisomerase checkpoints, leading to genome instability. Recent studies into how cells sense Topo IIA dysfunction and respond by regulating cell cycle progression demonstrate that the Topo IIA G2 checkpoint is distinct from the G2-DNA damage checkpoint. Likewise, in mitosis, the metaphase Topo IIA checkpoint is separate from the spindle assembly checkpoint. Here, we integrate mechanistic knowledge of Topo IIA checkpoints with the current understanding of how cells regulate progression through the cell cycle to accomplish faithful genome transmission and discuss the opportunities this offers for therapy.

Association of TOP2A and ADH1B with lipid levels and prognosis in patients with lung adenocarcinoma and squamous cell carcinoma.

BACKGROUND: Although lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC) have different pathological and clinical features, they may share common driver genes. It was found that lipid levels can be used for early diagnosis of NSCLC; however, the relationship between driver genes and genes regulating lipid metabolism and their relationship with patient prognosis needs further investigation. METHODS: Genes whose expression was up- or down-regulated in both LUAD and LUSC were identified using the GEO database. Online tools like GEPIA 2, PrognoScan, UALCAN, and TIMER2.0 were used to investigate the association of these gene expressions with the patient's prognosis and lipid regulatory genes. The association between clinical lipid levels and the risk of LUAD and LUSC was analyzed by using a multiple logistic regression model. RESULTS: Topoisomerase II alpha (TOP2A) and alcohol dehydrogenase 1B (ADH1B) were identified as the only genes up- and down-regulated in both LUAD and LUSC. TOP2A and ADH1B expression levels significantly correlated with the patient's gender, age, individual cancer stage, histological subtype, nodal metastasis status, and TP53 mutation status. Additionally, only LUAD patients with higher TOP2A or lower ADH1B expressions displayed poor overall and relapse-free survival rates. Moreover, TOP2A levels exhibited a negative correlation with adipose triglyceride lipase (ATGL) and ATP-binding cassette transporter A1 (ABCA1) in both LUAD and LUSC. However, ADH1B showed inverse associations with the above-mentioned genes when compared to TOP2A expressions in both LUAD and LUSC. Furthermore, elevated triglyceride (OR = 1.59; 95% CI = 1.01 to 2.49; P < 0.05) and total cholesterol (OR = 2.45; 95% CI = 1.08 to 5.57; P < 0.05) levels might increase the risk of LUAD. CONCLUSIONS: TOP2A and ADH1B can be used as diagnostic markers for LUAD and LUSC, but only as independent prognostic factors for LUAD, and may be involved in lipid metabolism in LUAD patients but not in LUSC. Thus, combining genetic diagnostics with lipid panel tests might be an effective method for an early diagnosis and improved prognosis of LUAD.

EZH2-H3K27me3-mediated silencing of mir-139-5p inhibits cellular senescence in hepatocellular carcinoma by activating TOP2A.

BACKGROUND: Epigenetic alterations play an important role in hepatocellular carcinoma (HCC) development. Enhancer of zeste homolog 2 (EZH2) is a well-known epigenetic modifier that functions as an oncogene in tumors by promoting the H3K27me3-mediated transcriptional repression of tumor suppressor genes. "Senescent cells" has been proposed as a possible core component of the hallmarks of cancer conceptualization. Induction of cell senescence and targeted elimination of these senescent tumor cells are new strategies for tumor therapy. However, the role of EZH2 in regulating cellular senescence remains poorly understood. METHODS: Bioinformatics analyses suggested that EZH2 and DNA topoisomerase II alpha (TOP2A) are coexpressed in tumors, including HCC. Kyoto Encyclopedia of Genes and Genome (KEGG) pathway enrichment analyses and gene set enrichment analyses (GSEA) suggests a correlation of EZH2 and TOP2A expression with cellular senescence in HCC. MicroRNA (miRNA) inhibitor and mimics, siRNA, PLKO-shRNA, and plenti6.3-miR-139 were used to upregulate or downregulate the expression of target genes. CCK8, EdU, clone formation, and senescence-associated ß-galactosidase (SA-ß-gal) staining assays were performed to assess cell proliferation and cellular senescence phenotypes. Dual-luciferase reporter and chromatin immunoprecipitation assays were performed to investigate the targeted binding and inhibition of TOP2A 3' untranslated region (UTR) by miR-139-5p and the DNA enrichment of miR139-5p by EZH2 and H3K27me3. BALB/c nude mice were used to establish a xenograft tumor model and verify the phenotypes upon EZH2 and TOP2A silencing and miR-139 overexpression in vivo. In addition, tissue microarrays were used to analyze the expression patterns and correlations among EZH2, TOP2A, and miR-139-5p expression in HCC. RESULTS: Bioinformatics analysis revealed that EZH2 and TOP2A are coexpressed in HCC. In vitro gain- and loss-of-function experiments showed that inhibition of EZH2 and TOP2A induces cellular senescence and inhibits proliferation of HCC cells. In vivo tumorigenesis assays indicated that EZH2 and TOP2A knockdown inhibits tumorigenesis by inducing cellular senescence. Mechanistically, EZH2 promotes TOP2A expression by regulating the H3K27me3-mediated epigenetic silencing of miR-139-5p. TOP2A is a direct target of miR-139-5p, and inhibition of miR-139-5p can reverse the promotion by EZH2 of TOP2A expression. The overexpression of miR-139-5p induces cellular senescence and inhibits proliferation of HCC cells both in vitro and in vivo. Clinically, expression of EZH2 and TOP2A are higher in HCC tissues than in normal tissues, and this high coexpression indicates a worse outcome of patients with HCC. Moreover, expression of EZH2 and TOP2A is significantly correlated with tumor differentiation grade, tumor invasion, and TNM stage in HCC. miR-139-5p expression is lower in HCC tumors than in normal tissues and is correlated with better prognosis of HCC patients. CONCLUSIONS: Our study revealed the role of the EZH2/miR-139-5p/TOP2A axis in regulating cellular senescence and cell proliferation in HCC, enriching the molecular mechanisms of EZH2-mediated epigenetic regulation in HCC. Therefore, our results provide insight into the therapeutic potential of targeting EZH2 to induce cellular senescence and then destroy senescent cells for HCC.

To Break or Not to Break: The Role of TOP2B in Transcription.

Transcription and its regulation pose challenges related to DNA torsion and supercoiling of the DNA template. RNA polymerase tracking the helical groove of the DNA introduces positive helical torsion and supercoiling upstream and negative torsion and supercoiling behind its direction of travel. This can inhibit transcriptional elongation and other processes essential to transcription. In addition, chromatin remodeling associated with gene activation can generate or be hindered by excess DNA torsional stress in gene regulatory regions. These topological challenges are solved by DNA topoisomerases via a strand-passage reaction which involves transiently breaking and re-joining of one (type I topoisomerases) or both (type II topoisomerases) strands of the phosphodiester backbone. This review will focus on one of the two mammalian type II DNA topoisomerase enzymes, DNA topoisomerase II beta (TOP2B), that have been implicated in correct execution of developmental transcriptional programs and in signal-induced transcription, including transcriptional activation by nuclear hormone ligands. Surprisingly, several lines of evidence indicate that TOP2B-mediated protein-free DNA double-strand breaks are involved in signal-induced transcription. We discuss the possible significance and origins of these DSBs along with a network of protein interaction data supporting a variety of roles for TOP2B in transcriptional regulation.

Cryo-EM structures of African swine fever virus topoisomerase.

IMPORTANCE: African swine fever virus (ASFV) is a highly contagious virus that causes lethal hemorrhagic diseases known as African swine fever (ASF) with a case fatality rate of 100%. There is an urgent need to develop anti-ASFV drugs. We determine the first high-resolution structures of viral topoisomerase ASFV P1192R in both the closed and open C-gate forms. P1192R shows a similar overall architecture with eukaryotic and prokaryotic type II topoisomerases, which have been successful targets of many antimicrobials and anticancer drugs, with the most similarity to yeast topo II. P1192R also exhibits differences in the details of active site configuration, which are important to enzyme activity. These two structures offer useful structural information for antiviral drug design and provide structural evidence to support that eukaryotic type IIA topoisomerase likely originated from horizontal gene transfer from the virus.

The abscission checkpoint senses chromatin bridges through Top2α recruitment to DNA knots.

In response to chromatin bridges, the abscission checkpoint delays completion of cytokinesis to prevent chromosome breakage or tetraploidization. Here, we show that spontaneous or replication stress-induced chromatin bridges exhibit "knots" of catenated and overtwisted DNA next to the midbody. Topoisomerase IIα (Top2α) forms abortive Top2-DNA cleavage complexes (Top2ccs) on DNA knots; furthermore, impaired Top2α-DNA cleavage activity correlates with chromatin bridge breakage in cytokinesis. Proteasomal degradation of Top2ccs is required for Rad17 localization to Top2-generated double-strand DNA ends on DNA knots; in turn, Rad17 promotes local recruitment of the MRN complex and downstream ATM-Chk2-INCENP signaling to delay abscission and prevent chromatin breakage. In contrast, dicentric chromosomes that do not exhibit knotted DNA fail to activate the abscission checkpoint in human cells. These findings are the first to describe a mechanism by which the abscission checkpoint detects chromatin bridges, through generation of abortive Top2ccs on DNA knots, to preserve genome integrity.

Inactivating TDP2 missense mutation in siblings with congenital abnormalities reminiscent of fanconi anemia.

Mutations in TDP2, encoding tyrosyl-DNA phosphodiesterase 2, have been associated with a syndromal form of autosomal recessive spinocerebellar ataxia, type 23 (SCAR23). This is a very rare and progressive neurodegenerative disorder described in only nine patients to date, and caused by splice site or nonsense mutations that result in greatly reduced or absent TDP2 protein. TDP2 is required for the rapid repair of DNA double-strand breaks induced by abortive DNA topoisomerase II (TOP2) activity, important for genetic stability in post-mitotic cells such as neurons. Here, we describe a sibship that is homozygous for the first TDP2 missense mutation (p.Glu152Lys) and which presents with clinical features overlapping both SCAR23 and Fanconi anemia (FA). We show that in contrast to previously reported SCAR23 patients, fibroblasts derived from the current patient retain significant levels of TDP2 protein. However, this protein is catalytically inactive, resulting in reduced rates of repair of TOP2-induced DNA double-strand breaks and cellular hypersensitivity to the TOP2 poison, etoposide. The TDP2-mutated patient-derived fibroblasts do not display increased chromosome breakage following treatment with DNA crosslinking agents, but both TDP2-mutated and FA cells exhibit increased chromosome breakage in response to etoposide. This suggests that the FA pathway is required in response to TOP2-induced DNA lesions, providing a possible explanation for the clinical overlap between FA and the current TDP2-mutated patients. When reviewing the relatively small number of patients with SCAR23 that have been reported, it is clear that the phenotype of such patients can extend beyond neurological features, indicating that the TDP2 protein influences not only neural homeostasis but also other tissues as well.