DNA flow cytometry for detection of genomic instability as a cancer precursor in the gastrointestinal tract.

One of the earliest applications of flow cytometry was the measurement of DNA content in cells. This method is based on the ability to stain DNA in a stoichiometric manner (i.e., the amount of stain is directly proportional to the amount of DNA within the cell). For more than 40years, a number of studies have consistently demonstrated the utility of DNA flow cytometry as a potential diagnostic and/or prognostic tool in patients with most epithelial tumors, including pre-invasive lesions (such as dysplasia) in the gastrointestinal tract. However, its availability as a clinical test has been limited to few medical centers due to the requirement for fresh tissue in earlier studies and perceived technical demands. However, more recent studies have successfully utilized formalin-fixed paraffin-embedded (FFPE) tissue to generate high-quality DNA content histograms, demonstrating the feasibility of this methodology. This review summarizes step-by-step methods on how to perform DNA flow cytometry using FFPE tissue and analyze DNA content histograms based on the published consensus guidelines in order to assist in the diagnosis and/or risk stratification of many different epithelial tumors, with particular emphasis on dysplasia associated with Barrett's esophagus and inflammatory bowel disease.

DNA Damage, Genome Stability, and Adaptation: A Question of Chance or Necessity?

DNA damage causes the mutations that are the principal source of genetic variation. DNA damage detection and repair mechanisms therefore play a determining role in generating the genetic diversity on which natural selection acts. Speciation, it is commonly assumed, occurs at a rate set by the level of standing allelic diversity in a population. The process of speciation is driven by a combination of two evolutionary forces: genetic drift and ecological selection. Genetic drift takes place under the conditions of relaxed selection, and results in a balance between the rates of mutation and the rates of genetic substitution. These two processes, drift and selection, are necessarily mediated by a variety of mechanisms guaranteeing genome stability in any given species. One of the outstanding questions in evolutionary biology concerns the origin of the widely varying phylogenetic distribution of biodiversity across the Tree of Life and how the forces of drift and selection contribute to shaping that distribution. The following examines some of the molecular mechanisms underlying genome stability and the adaptive radiations that are associated with biodiversity and the widely varying species richness and evenness in the different eukaryotic lineages.

Wiskott Aldrich syndrome protein (WASp)-deficient Th1 cells promote R-loop-driven transcriptional insufficiency and transcription-coupled nucleotide excision repair factor (TC-NER)-driven genome-instability in the pathogenesis of T cell acute lymphoblastic leukemia.

BACKGROUND: T-ALL is an aggressive hematological tumor that develops as the result of a multi-step oncogenic process which causes expansion of hematopoietic progenitors that are primed for T cell development to undergo malignant transformation and growth. Even though first-line therapy has a significant response rate, 40% of adult patients and 20% of pediatric patients will relapse. Therefore, there is an unmet need for treatment for relapsed/refractory T-ALL to develop potential targeted therapies. METHODS: Pediatric T-ALL patient derived T cells were grown under either nonskewingTh0 or Th1-skewing conditions to further process for ChIP-qPCR, RDIP-qPCR and other RT-PCR assays. Endogenous WASp was knocked out using CRISPR-Cas9 and was confirmed using flow cytometry and western blotting. LC-MS/MS was performed to find out proteomic dataset of WASp-interactors generated from Th1-skewed, human primary Th-cells. DNA-damage was assessed by immunofluorescence confocal-imaging and single-cell gel electrophoresis (comet assay). Overexpression of RNaseH1 was also done to restore normal Th1-transcription in WASp-deficient Th1-skewed cells. RESULTS: We discovered that nuclear-WASp is required for suppressing R-loop production (RNA/DNA-hybrids) at Th1-network genes by ribonucleaseH2 (RNH2) and topoisomerase1. Nuclear-WASp is associated with the factors involved in preventing and dissolving R-loops in Th1 cells. In nuclear- WASp-reduced malignant Th1-cells, R-loops accumulate in vivo and are processed into DNA-breaks by transcription-coupled-nucleotide-excision repair (TC-NER). Several epigenetic modifications were also found to be involved at Th1 gene locus which are responsible for active/repressive marks of particular genes. By demonstrating WASp as a physiologic regulator of programmed versus unprogrammed R-loops, we suggest that the transcriptional role of WASp in vivo extends also to prevent transcription-linked DNA damage during malignancy and through modification of epigenetic dysregulations. CONCLUSION: Our findings present a provocative possibility of resetting R-loops as a therapeutic intervention to correct both immune deficiency and malignancy in T-cell acute lymphoblastic leukemia patients and a novel role of WASp in the epigenetic regulation of T helper cell differentiation in T-ALL patients, anticipating WASp's requirement for the suppression of T-ALL progression.

Recombination, admixture and genome instability shape the genomic landscape of Saccharomyces cerevisiae derived from spontaneous grape ferments.

Cultural exchange of fermentation techniques has driven the spread of Saccharomyces cerevisiae across the globe, establishing natural populations in many countries. Despite this, Oceania is thought to lack native populations of S. cerevisiae, only being introduced after colonisation. Here we investigate the genomic landscape of 411 S. cerevisiae isolated from spontaneous grape fermentations in Australia across multiple locations, years, and grape cultivars. Spontaneous fermentations contained highly recombined mosaic strains that exhibited high levels of genome instability. Assigning genomic windows to putative ancestral origin revealed that few closely related starter lineages have come to dominate the genetic landscape, contributing most of the genetic variation. Fine-scale phylogenetic analysis of loci not observed in strains of commercial wine origin identified widespread admixture with European derived beer yeast along with three independent admixture events from potentially endemic Oceanic lineages that was associated with genome instability. Finally, we investigated Australian ecological niches for basal isolates, identifying phylogenetically distinct S. cerevisiae of non-European, non-domesticated origin associated with admixture loci. Our results illustrate the effect commercial use of microbes may have on local microorganism genetic diversity and demonstrates the presence of non-domesticated, potentially endemic lineages of S. cerevisiae in Australian niches that are actively admixing.

High Concordance of Different Assays in the Determination of Homologous Recombination Deficiency-Associated Genomic Instability in Ovarian Cancer.

PURPOSE: Poly(ADP-ribose) polymerase inhibitors (PARPi) have shown promising clinical results in the treatment of ovarian cancer. Analysis of biomarker subgroups consistently revealed higher benefits for patients with homologous recombination deficiency (HRD). The test that is most often used for the detection of HRD in clinical studies is the Myriad myChoice assay. However, other assays can also be used to assess biomarkers, which are indicative of HRD, genomic instability (GI), and BRCA1/2 mutation status. Many of these assays have high potential to be broadly applied in clinical routine diagnostics in a time-effective decentralized manner. Here, we compare the performance of a multitude of alternative assays in comparison with Myriad myChoice in high-grade serous ovarian cancer (HGSOC). METHODS: DNA from HGSOC samples was extracted from formalin-fixed paraffin-embedded tissue blocks of cases previously run with the Myriad myChoice assay, and GI was measured by multiple molecular assays (CytoSNP, AmoyDx, Illumina TSO500 HRD, OncoScan, NOGGO GISv1, QIAseq HRD Panel and whole genome sequencing), applying different bioinformatics algorithms. RESULTS: Application of different assays to assess GI, including Myriad myChoice, revealed high concordance of the generated scores ranging from very substantial to nearly perfect fit, depending on the assay and bioinformatics pipelines applied. Interlaboratory comparison of assays also showed high concordance of GI scores. CONCLUSION: Assays for GI assessment not only show a high concordance with each other but also in correlation with Myriad myChoice. Thus, almost all of the assays included here can be used effectively to assess HRD-associated GI in the clinical setting. This is important as PARPi treatment on the basis of these tests is compliant with European Medicines Agency approvals, which are methodologically not test-bound.

Formation of memory assemblies through the DNA-sensing TLR9 pathway.

As hippocampal neurons respond to diverse types of information1, a subset assembles into microcircuits representing a memory2. Those neurons typically undergo energy-intensive molecular adaptations, occasionally resulting in transient DNA damage3-5. Here we found discrete clusters of excitatory hippocampal CA1 neurons with persistent double-stranded DNA (dsDNA) breaks, nuclear envelope ruptures and perinuclear release of histone and dsDNA fragments hours after learning. Following these early events, some neurons acquired an inflammatory phenotype involving activation of TLR9 signalling and accumulation of centrosomal DNA damage repair complexes6. Neuron-specific knockdown of Tlr9 impaired memory while blunting contextual fear conditioning-induced changes of gene expression in specific clusters of excitatory CA1 neurons. Notably, TLR9 had an essential role in centrosome function, including DNA damage repair, ciliogenesis and build-up of perineuronal nets. We demonstrate a novel cascade of learning-induced molecular events in discrete neuronal clusters undergoing dsDNA damage and TLR9-mediated repair, resulting in their recruitment to memory circuits. With compromised TLR9 function, this fundamental memory mechanism becomes a gateway to genomic instability and cognitive impairments implicated in accelerated senescence, psychiatric disorders and neurodegenerative disorders. Maintaining the integrity of TLR9 inflammatory signalling thus emerges as a promising preventive strategy for neurocognitive deficits.

NFATC2IP is a mediator of SUMO-dependent genome integrity.

The post-translational modification of proteins by SUMO is crucial for cellular viability and mammalian development in part due to the contribution of SUMOylation to genome duplication and repair. To investigate the mechanisms underpinning the essential function of SUMO, we undertook a genome-scale CRISPR/Cas9 screen probing the response to SUMOylation inhibition. This effort identified 130 genes whose disruption reduces or enhances the toxicity of TAK-981, a clinical-stage inhibitor of the SUMO E1-activating enzyme. Among the strongest hits, we validated and characterized NFATC2IP, an evolutionarily conserved protein related to the fungal Esc2 and Rad60 proteins that harbors tandem SUMO-like domains. Cells lacking NFATC2IP are viable but are hypersensitive to SUMO E1 inhibition, likely due to the accumulation of mitotic chromosome bridges and micronuclei. NFATC2IP primarily acts in interphase and associates with nascent DNA, suggesting a role in the postreplicative resolution of replication or recombination intermediates. Mechanistically, NFATC2IP interacts with the SMC5/6 complex and UBC9, the SUMO E2, via its first and second SUMO-like domains, respectively. AlphaFold-Multimer modeling suggests that NFATC2IP positions and activates the UBC9-NSMCE2 complex, the SUMO E3 ligase associated with SMC5/SMC6. We conclude that NFATC2IP is a key mediator of SUMO-dependent genomic integrity that collaborates with the SMC5/6 complex.

Genetic determinants of micronucleus formation in vivo.

Genomic instability arising from defective responses to DNA damage1 or mitotic chromosomal imbalances2 can lead to the sequestration of DNA in aberrant extranuclear structures called micronuclei (MN). Although MN are a hallmark of ageing and diseases associated with genomic instability, the catalogue of genetic players that regulate the generation of MN remains to be determined. Here we analyse 997 mouse mutant lines, revealing 145 genes whose loss significantly increases (n = 71) or decreases (n = 74) MN formation, including many genes whose orthologues are linked to human disease. We found that mice null for Dscc1, which showed the most significant increase in MN, also displayed a range of phenotypes characteristic of patients with cohesinopathy disorders. After validating the DSCC1-associated MN instability phenotype in human cells, we used genome-wide CRISPR-Cas9 screening to define synthetic lethal and synthetic rescue interactors. We found that the loss of SIRT1 can rescue phenotypes associated with DSCC1 loss in a manner paralleling restoration of protein acetylation of SMC3. Our study reveals factors involved in maintaining genomic stability and shows how this information can be used to identify mechanisms that are relevant to human disease biology1.

DNA damage remodels the MITF interactome to increase melanoma genomic instability.

Since genome instability can drive cancer initiation and progression, cells have evolved highly effective and ubiquitous DNA damage response (DDR) programs. However, some cells (for example, in skin) are normally exposed to high levels of DNA-damaging agents. Whether such high-risk cells possess lineage-specific mechanisms that tailor DNA repair to the tissue remains largely unknown. Using melanoma as a model, we show here that the microphthalmia-associated transcription factor MITF, a lineage addition oncogene that coordinates many aspects of melanocyte and melanoma biology, plays a nontranscriptional role in shaping the DDR. On exposure to DNA-damaging agents, MITF is phosphorylated at S325, and its interactome is dramatically remodeled; most transcription cofactors dissociate, and instead MITF interacts with the MRE11-RAD50-NBS1 (MRN) complex. Consequently, cells with high MITF levels accumulate stalled replication forks and display defects in homologous recombination-mediated repair associated with impaired MRN recruitment to DNA damage. In agreement with this, high MITF levels are associated with increased single-nucleotide and copy number variant burdens in melanoma. Significantly, the SUMOylation-defective MITF-E318K melanoma predisposition mutation recapitulates the effects of DNA-PKcs-phosphorylated MITF. Our data suggest that a nontranscriptional function of a lineage-restricted transcription factor contributes to a tissue-specialized modulation of the DDR that can impact cancer initiation.

G-quadruplex-mediated genomic instability drives SNVs in cancer.

G-quadruplex (G4s) DNA structures have been implicated in inducing genomic instability and contributing to cancer development. However, the relationship between G4s and cancer-related single nucleotide variants (cSNVs) in clinical settings remains unclear. In this large-scale study, we integrated experimentally validated G4s with genomic cSNVs from 13480 cancer patients to investigate the spatial association of G4s with the cellular cSNV landscape. Our findings demonstrate an increase in local genomic instability with increasing local G4 content in cancer patients, suggesting a potential role for G4s in driving cSNVs. Notably, we observed distinct spatial patterns of cSNVs and common single nucleotide variants (dbSNVs) in relation to G4s, implying different mechanisms for their generation and accumulation. We further demonstrate large, cancer-specific differences in the relationship of G4s and cSNVs, which could have important implications for a new class of G4-stabilizing cancer therapeutics. Moreover, we show that high G4-content can serve as a prognostic marker for local cSNV density and patient survival rates. Our findings underscore the importance of considering G4s in cancer research and highlight the need for further investigation into the underlying molecular mechanisms of G4-mediated genomic instability, especially in the context of cancer.

Methylation of histone H3 lysine 36 is a barrier for therapeutic interventions of head and neck squamous cell carcinoma.

Approximately 20% of head and neck squamous cell carcinomas (HNSCCs) exhibit reduced methylation on lysine 36 of histone H3 (H3K36me) due to mutations in histone methylase NSD1 or a lysine-to-methionine mutation in histone H3 (H3K36M). Whether such alterations of H3K36me can be exploited for therapeutic interventions is still unknown. Here, we show that HNSCC models expressing H3K36M can be divided into two groups: those that display aberrant accumulation of H3K27me3 and those that maintain steady levels of H3K27me3. The former group exhibits reduced proliferation, genome instability, and heightened sensitivity to genotoxic agents like PARP1/2 inhibitors. Conversely, H3K36M HNSCC models with constant H3K27me3 levels lack these characteristics unless H3K27me3 is elevated by DNA hypomethylating agents or inhibiting H3K27me3 demethylases KDM6A/B. Mechanistically, H3K36M reduces H3K36me by directly impeding the activities of the histone methyltransferase NSD3 and the histone demethylase LSD2. Notably, aberrant H3K27me3 levels induced by H3K36M expression are not a bona fide epigenetic mark because they require continuous expression of H3K36M to be inherited. Moreover, increased sensitivity to PARP1/2 inhibitors in H3K36M HNSCC models depends solely on elevated H3K27me3 levels and diminishing BRCA1- and FANCD2-dependent DNA repair. Finally, a PARP1/2 inhibitor alone reduces tumor burden in a H3K36M HNSCC xenograft model with elevated H3K27me3, whereas in a model with consistent H3K27me3, a combination of PARP1/2 inhibitors and agents that up-regulate H3K27me3 proves to be successful. These findings underscore the crucial balance between H3K36 and H3K27 methylation in maintaining genome instability, offering new therapeutic options for patients with H3K36me-deficient tumors.

rDNA transcription, replication and stability in Saccharomyces cerevisiae.

The ribosomal DNA locus (rDNA) is central for the functioning of cells because it encodes ribosomal RNAs, key components of ribosomes, and also because of its links to fundamental metabolic processes, with significant impact on genome integrity and aging. The repetitive nature of the rDNA gene units forces the locus to maintain sequence homogeneity through recombination processes that are closely related to genomic stability. The co-presence of basic DNA transactions, such as replication, transcription by major RNA polymerases, and recombination, in a defined and restricted area of the genome is of particular relevance as it affects the stability of the rDNA locus by both direct and indirect mechanisms. This condition is well exemplified by the rDNA of Saccharomyces cerevisiae. In this review we summarize essential knowledge on how the complexity and overlap of different processes contribute to the control of rDNA and genomic stability in this model organism.

On the edge: how nuclear pore complexes rule genome stability.

Nuclear organization has emerged as a critical layer in the coordination of DNA repair activities. Distinct types of DNA lesions have notably been shown to relocate at the vicinity of nuclear pore complexes (NPCs), where specific repair pathways are favored, ultimately safeguarding genome integrity. Here, we review the most recent progress in this field, notably highlighting the increasingly diverse types of DNA structures undergoing repositioning, and the signaling pathways involved. We further discuss our growing knowledge of the molecular mechanisms underlying the choice of repair pathways at NPCs, and their conservation - or divergences. Intriguingly, a series of recent findings suggest that DNA metabolism may be coupled to NPC biogenesis and specialization, challenging our initial vision of these processes.

Chromatin bridges: stochastic breakage or regulated resolution?

Genetic material is organized in the form of chromosomes, which need to be segregated accurately into two daughter cells in each cell cycle. However, chromosome fusion or the presence of unresolved interchromosomal linkages lead to the formation of chromatin bridges, which can induce DNA lesions and genome instability. Persistent chromatin bridges are trapped in the cleavage furrow and are broken at or after abscission, the final step of cytokinesis. In this review, we focus on recent progress in understanding the mechanism of bridge breakage and resolution. We discuss the molecular machinery and enzymes that have been implicated in the breakage and processing of bridge DNA. In addition, we outline both the immediate outcomes and genomic consequences induced by bridge breakage.

Effects of genome instability of parental CHO cell clones on chromosome number distribution and recombinant protein production in parent-derived subclones.

Chinese hamster ovary (CHO) cells are the de facto standard host cells for biopharmaceuticals, and there is great interest in developing methods for constructing stable production cell lines. In this study, clones with a wide chromosome number distribution were selected from isolated antibody-producing strains, and subclones obtained from these clones were evaluated. The transgene copy number varied between the subclones. Even among subclones with similar copy numbers of antibody genes and maintained insertion sites, clones with different productivity were generated. Although the chromosome number distribution differed between these subclones, there was no correlation between the variability in chromosome number after cloning (genome instability) and productivity. Most of the subclones obtained from a parental strain with a wide chromosome number had the same wide chromosome number distribution as the parental strain. Less frequently, cells with less variation (remaining in one distribution) in chromosome number were isolated from cells with a wide chromosome number distribution, from which subclones with less variation in chromosome number were obtained when subcloning was performed again. These results imply that the characteristics of clones with chromosomal instability are inherited by subclones, and thus provide a better understanding of cell line stability/instability.

Genomic consequences associated with Agrobacterium-mediated transformation of plants.

Attenuated strains of the naturally occurring plant pathogen Agrobacterium tumefaciens can transfer virtually any DNA sequence of interest to model plants and crops. This has made Agrobacterium-mediated transformation (AMT) one of the most commonly used tools in agricultural biotechnology. Understanding AMT, and its functional consequences, is of fundamental importance given that it sits at the intersection of many fundamental fields of study, including plant-microbe interactions, DNA repair/genome stability, and epigenetic regulation of gene expression. Despite extensive research and use of AMT over the last 40 years, the extent of genomic disruption associated with integrating exogenous DNA into plant genomes using this method remains underappreciated. However, new technologies like long-read sequencing make this disruption more apparent, complementing previous findings from multiple research groups that have tackled this question in the past. In this review, we cover progress on the molecular mechanisms involved in Agrobacterium-mediated DNA integration into plant genomes. We also discuss localized mutations at the site of insertion and describe the structure of these DNA insertions, which can range from single copy insertions to large concatemers, consisting of complex DNA originating from different sources. Finally, we discuss the prevalence of large-scale genomic rearrangements associated with the integration of DNA during AMT with examples. Understanding the intended and unintended effects of AMT on genome stability is critical to all plant researchers who use this methodology to generate new genetic variants.

Emerging roles of DNA repair factors in the stability of centromeres.

Satellite DNA sequences are an integral part of centromeres, regions critical for faithful segregation of chromosomes during cell division. Because of their complex repetitive structure, satellite DNA may act as a barrier to DNA replication and other DNA based transactions ultimately resulting in chromosome breakage. Over the past two decades, several DNA repair proteins have been shown to bind and function at centromeres. While the importance of these repair factors is highlighted by various structural and numerical chromosome aberrations resulting from their inactivation, their roles in helping to maintain genome stability by solving the intrinsic difficulties of satellite DNA replication or promoting their repair are just starting to emerge. In this review, we summarize the current knowledge on the role of DNA repair and DNA damage response proteins in maintaining the structure and function of centromeres in different contexts. We also report the recent connection between the roles of specific DNA repair factors at these genomic loci with age-related increase of chromosomal instability under physiological and pathological conditions.

Highly genomic instability of super-polyploid strains of Saccharomyces cerevisiae.

Polyploid (2n, 3n, and 4n) genomes are known to be unstable in Saccharomyces cerevisiae. Here, we attempted construction of super-polypoid strains (defined as having higher ploidy than tetraploidy) up to 32n by using the matα2-PBT method that we newly developed and investigated their genomic stability. It is known that cell size increases as ploidy increases up to tetraploid. However, unexpectedly, there was no change in the average cell size of the super-polyploid strains compared with tetraploid or pentaploid strains. Smaller sized cells were observed at a rather higher frequency in super-polyploid cell populations compared with those of diploid, triploid and tetraploid strains, suggesting that ploidy reduction in super-polyploid strains occurs quickly at a relatively high frequency. Assuming that ploidy reduction occurs through chromosome loss (or non-disjunction) during mitotic growth, we also estimated the frequency of chromosome loss (or non-disjunction) in various polyploid strains. Our results indicated that the frequency of chromosome loss (or non-disjunction) is drastically increased (10-2-10-3/cells plated) in super-polyploid strains compared with that (10-4-10-5/cells plated) of conventional polyploid (2n-4n) strains. This is the first attempt of construction of super-polyploid strains and investigation of their genomic stability in S. cerevisiae. We believe that the matα2-PBT method will be an invaluable tool for investigating a variety of interesting issues regarding polyploidy and their genomic characterization in eukaryotes.

DNA polymerase ε harmonizes topological states and R-loops formation to maintain genome integrity in Arabidopsis.

Genome topology is tied to R-loop formation and genome stability. However, the regulatory mechanism remains to be elucidated. By establishing a system to sense the connections between R-loops and genome topology states, we show that inhibiting DNA topoisomerase 1 (TOP1i) triggers the global increase of R-loops (called topoR-loops) and DNA damages, which are exacerbated in the DNA damage repair-compromised mutant atm. A suppressor screen identifies a mutation in POL2A, the catalytic subunit of DNA polymerase ε, rescuing the TOP1i-induced topoR-loop accumulation and genome instability in atm. Importantly we find that a highly conserved junction domain between the exonuclease and polymerase domains in POL2A is required for modulating topoR-loops near DNA replication origins and facilitating faithful DNA replication. Our results suggest that DNA replication acts in concert with genome topological states to fine-tune R-loops and thereby maintain genome integrity, revealing a likely conserved regulatory mechanism of TOP1i resistance in chemotherapy for ATM-deficient cancers.