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Protein–protein interactions (PPIs) in the nucleus play key roles in transcriptional regulation and ensure genomic stability. Critical to this are histone-mediated PPI networks, which are further fine-tuned through dynamic post-translational modification. Perturbation to these networks leads to genomic instability and disease, presenting epigenetic proteins as key therapeutic targets. This mini-review will describe progress in mapping the combinatorial histone PTM landscape, and recent chemical biology approaches to map histone interactors. Recent advances in mapping direct interactors of histone PTMs as well as local chromatin interactomes will be highlighted, with a focus on mass-spectrometry based workflows that continue to illuminate histone-mediated PPIs in unprecedented detail.

The incidence of cancer globally is increasing, partly due to lifestyle factors. Despite a better understanding of cancer biology and advancement in cancer management and therapies, current strategies in cancer treatment remain costly and cause socioeconomic burden especially in Asian countries. Hence, instead of putting more efforts in searches for new cancer cures, attention has now shifted to understanding how to mitigate cancer risk by modulating lifestyle factors. It has been established that carcinogenesis is multifactorial, and the important detrimental role of oxidative stress, chronic inflammation, and genomic instability is evident. To date, there is no study linking dietary pattern and genomic stability in cancer risk in the Asian food landscape. Thus, this present review article discusses recent literature on dietary pattern and genomic stability and its relationship with cancer risk in Asia.

Neurodegeneration is a complex neurological phenomenon characterized by disturbed coherence in neuronal efflux. Progressive neuronal loss and brain damage due to various age-related pathological hallmarks perturb the behavioral balance and quality of life. Sirtuins have been widely investigated for their neuroprotective role, with SIRT1 being the most contemplated member of the family. SIRT1 exhibits significant capabilities to enhance neurogenesis and cellular lifespan by regulating various pathways, which makes it an exciting therapeutic target to inhibit neurodegenerative disease progression. SIRT1 mediated neuronal fortification involves modulation of molecular co-factors and biochemical pathways responsible for the induction and sustenance of pro-inflammatory and pro-oxidative environment in the cellular milieu. In this review, we present the major role played by SIRT1 in maintaining cellular strength through the regulation of genomic stability, neuronal growth, energy metabolism, oxidative stress, inhibiting mechanisms and anti-inflammatory responses. The therapeutic significance of SIRT1 has been put into perspective through a comprehensive discussion about its ameliorating potential against neurodegenerative stimuli in a variety of diseases that characteristically impair cognition, memory and motor coordination. This review enhances the acquaintance concerned with the neuroprotective potential of SIRT1 and thus promotes the development of novel SIRT1 regulating therapeutic agents and strategies.

BackgroundMillions of workers worldwide are exposed to diesel engine exhaust (DEE), a known genotoxic carcinogen. Alu retroelements are repetitive DNA sequences that can multiply and compromise genomic stability. There is some evidence linking altered Alu repeats to cancer and elevated mortality risks. However, whether Alu repeats are influenced by environmental pollutants is unexplored. In an occupational setting with high DEE exposure levels, we investigated associations with Alu repeat copy number.MethodsA cross-sectional study of 54 male DEE-exposed workers from an engine testing facility and a comparison group of 55 male unexposed controls was conducted in China. Personal air samples were assessed for elemental carbon, a DEE surrogate, using NIOSH Method 5040. Quantitative PCR (qPCR) was used to measure Alu repeat copy number relative to albumin (Alb) single-gene copy number in leucocyte DNA. The unitless Alu/Alb ratio reflects the average quantity of Alu repeats per cell. Linear regression models adjusted for age and smoking status were used to estimate relations between DEE-exposed workers versus unexposed controls, DEE tertiles (6.1–39.0, 39.1–54.5 and 54.6–107.7 µg/m3) and Alu/Alb ratio.ResultsDEE-exposed workers had a higher average Alu/Alb ratio than the unexposed controls (p=0.03). Further, we found a positive exposure–response relationship (p=0.02). The Alu/Alb ratio was highest among workers exposed to the top tertile of DEE versus the unexposed controls (1.12±0.08 SD vs 1.06±0.07 SD, p=0.01).ConclusionOur findings suggest that DEE exposure may contribute to genomic instability. Further investigations of environmental pollutants, Alu copy number and carcinogenesis are warranted.

The collapse of DNA replication forks leads to the formation of single-ended DNA double-strand breaks. One would have assumed that these breaks would be repaired as soon as possible. However, a recent study suggests that a delay in DNA polymerase θ-mediated end joining until mitosis, orchestrated by BRCA2 and RAD52, favors genomic stability.

Study of the effects of low background radiation on biological systems is a new field in radiation biology. Due to the availability of new experimental methods, studies of the problem of adaptation to low background radiation conditions entered an even more dynamic stage in 2015–2019. Genomic stability/variability during prolonged exposure to low background radiation conditions and the positive/negative effects of the absence of natural background radiation on living organisms are the most debated issues. The data obtained by different scientific teams do not allow obtaining a universal model explaining the observed events. This report discusses currently accumulated experimental data, requirements for work under conditions of low background radiation, and prospects of cooperation work between JINR and BNO INR RAS in carrying out biological experiments under unique conditions of the low-background laboratory DULB-4900.

Folic acid (FA) is a synthetic form of vitamin B9, generally used as a nutritional supplement and an adjunctive medication in cancer therapy. FA is involved in genetic and epigenetic regulation; therefore, it has a dual modulatory role in established neoplasms. We aimed to investigate the effect of short-term (72 h) FA supplementation on colorectal cancer; hence, HT-29 and SW480 cells were exposed to different FA concentrations (0, 100, 10,000 ng/mL). HT-29 cell proliferation and viability levels elevated after 100 ng/mL but decreased for 10,000 ng/mL FA. Additionally, a significant (p ≤ 0.05) improvement of genomic stability was detected in HT-29 cells with micronucleus scoring and comet assay. Conversely, the FA treatment did not alter these parameters in SW480 samples. RRBS results highlighted that DNA methylation changes were bidirectional in both cells, mainly affecting carcinogenesis-related pathways. Based on the microarray analysis, promoter methylation status was in accordance with FA-induced expression alterations of 27 genes. Our study demonstrates that the FA effect was highly dependent on the cell type, which can be attributed to the distinct molecular background and the different expression of proliferation- and DNA-repair-associated genes (YWHAZ, HES1, STAT3, CCL2). Moreover, new aspects of FA-regulated DNA methylation and consecutive gene expression were revealed.

Sumoylation is an essential post-translational modification intimately involved in a diverse range of eukaryotic cellular mechanisms. Small ubiquitin-like modifier (SUMO) protein isoforms can be reversibly linked to lysine residues that reside within specific motifs on thousands of target substrates, leading to modulations in stability, solubility, localization, and interactor profile. Since its initial discovery almost 25 years ago, SUMO has been described as a key regulator of genomic stability, cell proliferation, and infection among other processes. In this review, we trace the exciting developments in the history of this critical modifier, highlighting SUMO's roles in pathogenesis as well as its potential for the development of targeted therapies for numerous diseases.

Telomere attrition and mitochondrial DNA variations are implicated in the biological aging process and genomic stability can be influenced by nutritional factors. This study aims to analyze the relative telomere length (rTL) and mitochondrial DNA copy number (mtCN) in aged individuals and their association with plasma folate and vitamin B12 levels. This community-based cross-sectional study involves 428 subjects (60 years: 242≥60 years: 186). Quantitative real-time PCR was used to measure rTL and mtCN variation, and radioimmunoassay to measure plasma folate and vitamin B12 levels. The subjects in the ≥60 years age group have significantly shorter telomeres and lower mtCN compared to the60 years age group. A significant positive correlation was observed between the rTL and mtCN, and both of them were positively associated with plasma folate and vitamin B12 levels. In the ≥60 age group; folate and vitamin B12 positively correlated with rTL and vitamin B12 with mtCN. The study revealed a decline of rTL and mtCN with age in the Indian population and their association suggests that they may co-regulate each other with age. In conclusion, folate and vitamin B12 may delay aging by preventing the reduction in rTL length and mtCN.

Fanconi anemia is a genetically and phenotypically heterogenous disorder characterized by DNA repair deficiency and telomeric erosion. Different antioxidants have been assessed to regain the chromosomal stability in Fanconi anemia, among those is the Resveratrol. In Vitro Micronucleus assay provides a convenient and reliable index of both chromosome breakage and chromosome loss assessment. This study aims to assess the effect of Resveratrol on the stability of the chromosomes in Fanconi anemia patients using in vitro micronucleus assay as a parameter of improvement. The present study was conducted on 10 patients and 10 normal controls matching in age and sex. Induction of breakage by 1,3-Butadiene diepoxide and evaluation of the antioxidant effect of Resveratrol on genomic stability using In Vitro Micronucleus assay technique were done. Significant reduction in the level of micronuclei in cultures treated with Resveratrol was found. Resveratrol could be an effective adjuvant therapy for patients with FA to improve their hematological condition, chromosomal stability and it might protect from cancer development.

Pluripotent stem cells (PSCs) are capable of generating all types of cells in the body and have promising applications in basic research and cell-based regenerative medicine. Compared to the differentiated cells, PSCs are superior in maintaining genomic stability. However, the underlying molecular mechanisms are far from clear. Here, we summarized the understandings on the molecules and pathways that PSCs specifically utilize to cope with DNA replication-associated stress, to repair DNA damages and to determine cell fates.

Telomeres are genetically conserved nucleoprotein complexes located at the ends of chromosomes that preserve genomic stability. In large mammals, somatic cell telomeres shorten with age, owing to the end replication problem and lack of telomere-lengthening events (e.g. telomerase and ALT activity). Therefore, telomere length reflects cellular replicative reserve and mitotic potential. Environmental insults can accelerate telomere attrition in response to cell division and DNA damage. As such, telomere shortening is considered one of the major hallmarks of ageing. Much effort has been dedicated to understanding the environmental perturbations that accelerate telomere attrition and therapeutic strategies to preserve or extend telomeres. As telomere dynamics seem to reflect cumulative cellular stress, telomere length could serve as a biomarker of animal welfare. The assessment of telomere dynamics (i.e. rate of shortening) in conjunction with telomere-regulating genes and telomerase activity in racehorses could monitor long-term animal health, yet it could also provide some unique opportunities to address particular limitations with the use of other animal models in telomere research. Considering the ongoing efforts to optimise the health and welfare of equine athletes, the purpose of this review is to discuss the potential utility of assessing telomere length in Thoroughbred racehorses. A brief review of telomere biology in large and small mammals will be provided, followed by discussion on the biological implications of telomere length and environmental (e.g. lifestyle) factors that accelerate or attenuate telomere attrition. Finally, the utility of quantifying telomere dynamics in horses will be offered with directions for future research.

The proteins within the Poly-ADP Ribose Polymerase (PARP) family encompass a diverse and integral set of cellular functions. PARP1 and PARP2 have been extensively studied for their roles in DNA repair and as targets for cancer therapeutics. Several PARP inhibitors (PARPi) have been approved for clinical use, however, while their efficacy is promising, tumours readily develop PARPi resistance. Many other members of the PARP protein family share catalytic domain homology with PARP1/2, however, these proteins are comparatively understudied, particularly in the context of DNA damage repair and tumourigenesis. This review explores the functions of PARP4,6-16 and discusses the current knowledge of the potential roles these proteins may play in DNA damage repair and as targets for cancer therapeutics.

Breast Cancer gene 1 (BRCA1) is a large, multi-functional protein that regulates a variety of mechanisms in multiple different tissues. Our work established that Brca1 is expressed in skeletal muscle and localizes to the mitochondria and nucleus. Here, we propose BRCA1 expression is critical for the maintenance of force production and mitochondrial respiration in skeletal muscle.

The Knl1-Mis12-Ndc80 (KMN) network is an essential component of the kinetochore–microtubule attachment interface, which is required for genomic stability in eukaryotes. However, little is known about plant Knl1 proteins because of their complex evolutionary history. Here, we cloned the Knl1 homolog from maize (Zea mays) and confirmed it as a constitutive central kinetochore component. Functional assays demonstrated their conserved role in chromosomal congression and segregation during nuclear division, thus causing defective cell division during kernel development when Knl1 transcript was depleted. A 145 aa region in the middle of maize Knl1, that did not involve the MELT repeats, was associated with the interaction of spindle assembly checkpoint (SAC) components Bub1/Mad3 family proteins 1 and 2 (Bmf1/2) but not with the Bmf3 protein. They may form a helical conformation with a hydrophobic interface with the TPR domain of Bmf1/2, which is similar to that of vertebrates. However, this region detected in monocots shows extensive divergence in eudicots, suggesting that distinct modes of the SAC to kinetochore connection are present within plant lineages. These findings elucidate the conserved role of the KMN network in cell division and a striking dynamic of evolutionary patterns in the SAC signaling and kinetochore network.

DNA damage seriously threats the genomic stability and is linked to mutagenesis, carcinogenesis, and cell death. DNA damage includes the isolated damage and the clustered damages, but few approaches are available for efficient detection of the clustered damage due to its spatial distribution. Herein, we present a single-molecule counting approach with the capability of detecting both the isolated and the clustered damages in genomic DNAs. We employed the repair enzymes to remove the DNA damage and used the terminal deoxynucleotidyl transferase (TdT) to incorporate biotinylated nucleotides and fluorescent nucleotides into the damage sites in a template-independent manner. The number of total oxidative damaged bases is quantified to be 7328-7406 in a single HeLa cell treated with 150 μM H

Poly(ADP-ribosyl)ation (PARylation) is a reversible post-translational modification regulating various biological pathways including DNA damage repair (DDR). Rapid turnover of PARylation is critically important for an optimal DNA damage response and maintaining genomic stability. Recent studies show that PARylation is tightly regulated by a group of enzymes that can erase the ADP-ribose (ADPR) groups from target proteins. The aim of this review is to present a comprehensive understanding of dePARylation enzymes, their substrates and roles in DDR. Special attention will be laid on the role of these proteins in the development of cancer and their feasibility in anticancer therapeutics.

Polyploidization, also whole-genome duplication (WGD), is ubiquitous in plants but in vertebrates it occurs relatively rarely. The mechanisms that allow polyploid plants to overcome “genome shock” and achieve genomic stability, and how doubled genomes evolve had been well characterized. These studies yield several models that serve as theoretical references for analyses of polyploid vertebrates. Herein, we review the development of theories in WGD studies, summarize the research models and progress in vertebrates, and discuss the challenges of “omics” research and effective technical strategies. We hope to discover feasible means of unravelling the genome evolution of polyploid vertebrates.

Space travel since the 1960s has led to a number of physiological alterations to homeostasis in astronauts. Extensive variation in the pattern of responses observed has led a concerted effort to develop countermeasures to overcome such changes and restore homeostasis, and thus “health” is defined as more “Earth-like”. These adaptations to a space environment by a species which evolved and normally exists in the 1 g environment, the geomagnetic field, and background radiation of Earth are viewed as threats to health as defined by the conditions of Earth. Exposure to space can lead to alterations in genomic stability and epigenetic signatures, alterations which could redefine “health” and responses to risks for loss of health for those who will return to Earth. In contrast, in the future individuals born in non-Earth space environments will likely develop an integrated metabolic set point defined by those conditions. They will thus be shaped by both the local environments, and space-associated genomic/epigenomic alterations to their parents. Therefore, such an altered set point for those born and raised in non-Earth space environments will potentially have physiological and molecular consequences which may lead to either new evolutionary adaptation, or to compromise of long term health due to drastically altered set points for integrated physiologic function which is at odds with the evolutionary history of humans. The implications of the two options will be critical for defining “health” in altered environments encountered during space ventures, as well as providing insights into the regulation of human integrity at the physiological level. Therefore, the definition of “health” is dependent on the boundary conditions surrounding development and maturation, and is a dynamic concept.

One of the most important environmental pollutants is cement dust. In this study, RAPD technology was used todefine the potential genotoxic impact of cement dust on plants. For this purpose, the DNA comparison of 12 plant species(Convolvulus sepium, Astragalus christianus, Taraxacum androssovii Medicago varia, Alyssum murale, Artemisiaspisigera, Falcaria vulgaris, Anchusa strigose, Glaucium leiocarpum, Salvia syriaca, Cryciata taurica Tragopogonalbinervis)collected from the areas of 10000 m (Control area) or 0-100 m away from Askale cement factory in Erzurum;was performed. 16 primers were used, and 390 bands were obtained. Important differences were observed in plant RAPDprofiles collected from 0-100 m areas when compared to their respective controls (10000 m away from the factory). Somebands in control plants got lost, and new band formations were observed. The Genomic template stability (GTS) valuedecreased significantly in plants collected from 0-100 m area as compared to the controls (%56.20). Soil samples from 0-100 m and control area were collected and examined for their heavy metal contents. According to the analysis results,Nickel, Cadmium, Zinc, Lead and Copper concentrations were found to be dramatically high in the soil collected from 0-100 m as compared to the control area. These results indicate that the high heavy metal content in the soil causes thechanges in RAPD profiles and GTS in plants. The data also indicated that RAPD and GTS assays are basic, effective andreproducible means of the genotoxicity control of environmental pollution. En önemli çevre kirleticilerinden biri çimento tozudur. Bu çalışmada, çimento tozunun bitkiler üzerindekipotansiyel genotoksik etkisini tanımlamak için RAPD teknolojisi kullanılmıştır. Bu amaçla, Erzurum'da Askale çimentofabrikasına 10000 m (Kontrol alanı) ve 0-100 m uzaklıkta toplanan 12 bitki türünün ( Convolvulus sepium, Astragaluschristianus, Taraxacum androssovii Medicago varia, Alyssum murale, Artemisia spisigera, Falcaria vulgaris, Anchusastrigose, Glaucium leiocarpum, Salvia syriaca, Cryciata taurica Tragopogon albinervis)DNA karşılaştırması yapıldı.Çalışmada 16 RAPD primeri kullanılmış ve 390 bant elde edilmiştir. 0-100m alanlardan toplanan bitki RAPDprofillerinde, kontrollerine göre (fabrikadan 10000 metre uzakta) önemli farklılıklar gözlenmiştir. Kontrol bitkilerindebazı bantların kaybolduğu ve yeni bantların oluştuğu görülmüştür. Ayrıca, GTS (Genomic template stability) değeri, 0-100 m alandan toplanan bitkilerde, kontrollerinkilerle karşılaştırıldığında önemli ölçüde azaldığı görülmüştür( %56.20).0-100m ve kontrol alanından toprak örnekleri toplanmış ve ağır metal içerikleri bakımından incelenmiştir. Analizsonuçlarına göre, 0-100 m'den toplanan topraklarda Nikel, Kadmiyum, Çinko, Kurşun ve Bakır konsantrasyonlarının,çarpıcı şekilde yüksek olduğu bulunmuştur. Bu sonuçlar topraktaki yüksek ağır metal içeriğinin bitkilerde RAPDprofillerinde ve GTS’de değişikliklere neden olduğunu göstermektedir. Sonuçlar RAPD ve GTS testlerinin çevre kirliliğinin bitkiler üzerindeki genotoksik etkisini tanımlamak için temel, etkili ve tekrarlanabilir yöntemler olduğunugöstermiştir.

The aim of the present work was to determine proliferation capacity, immunophenotype and genome integrity of mesenchymal stromal cells (MSCs) from horse umbilical cord blood (UCB) at passage stage 5 and 10. Passage 4 cryopreserved UCB-MSCs from six unrelated donors were evaluated. Immunophenotypic analysis of UCB-MSC revealed a cell identity consistent with equine MSC phenotype by high expression of CD90, CD44, CD29, and very low expression of CD4, CD11a/18, CD73, and MHC class I and II antigens. Proliferative differences were noted among the UCB-MSC cultures. UCB-MSCs karyotype characteristics at passage 5 (eg, 2n = 64; XY, or XX) included 20% polyploidy and 62% aneuploidy. At passage 10, the proportion of polyploidy and aneuploidy was 21% and 82%, respectively, with the increase in aneuploidy being significant compared with passage 5. Furthermore, conventional GTG-banded karyotyping revealed several structural chromosome abnormalities at both passage 5 and 10. The clinical relevance of such chromosome instability is unknown, but determination of MSC cytogenetic status and monitoring of patient response to MSC therapies would help address this question.

Apart from its well-documented role in long-term promoter silencing, the genome-wide distribution patterns of ~ 28 million methylated or unmethylated CpG dinucleotides, e. g. in the human genome, is in search of genetic functions. We have set out to study changes in the cellular CpG methylation profile upon introducing foreign DNA into mammalian cells. As stress factors served the genomic integration of foreign (viral or bacterial plasmid) DNA, virus infections or the immortalization of cells with Epstein Barr Virus (EBV). In all instances investigated, alterations in cellular CpG methylation and transcription profiles were observed to different degrees. In the case of adenovirus DNA integration in adenovirus type 12 (Ad12)-transformed hamster cells, the extensive changes in cellular CpG methylation persisted even after the complete loss of all transgenomic Ad12 DNA. Hence, stress-induced alterations in CpG methylation can be inherited independent of the continued presence of the transgenome. Upon virus infections, changes in cellular CpG methylation appear early after infection. In EBV immortalized as compared to control cells, CpG hypermethylation in the far-upstream region of the human FMR1 promoter decreased four-fold. We conclude that in the wake of cellular stress due to foreign DNA entry, preexisting CpG methylation patterns were altered, possibly at specific CpG dinucleotides. Frequently, transcription patterns were also affected. As a working concept, we view CpG methylation profiles in mammalian genomes as a guarding sensor for genomic stability under epigenetic control. As a caveat towards manipulations of cells with foreign DNA, such cells can no longer be considered identical to their un-manipulated counterparts.

Most cells in the human body, including human mesenchymal stem cells (hMSCs), have evolved to survive and function in a low physiological oxygen (O2) environment. Investigators have become increasingly aware of the effects of O2 levels on hMSC biology and culture and are mimicking the natural niche of these cells in vitro to improve cell culture yields. This presents many challenges in relation to hMSC identity and function and in the maintenance of a controlled O2 environment for cell culture. The aim of this review was to discuss an "hMSC checklist" as a guide to establishing which identity and potency assays to implement when studying hMSCs. The checklist includes markers, differentiation potential, proliferation and growth, attachment and migration, genomic stability, and paracrine activity. Evidence drawn from the current literature demonstrates that low O2 environments could improve most "hMSC checklist" attributes. However, there are substantial inconsistencies around both the terminology and the equipment used in low O2 studies. Therefore, "hypoxia" as a term and as a culture condition is discussed. The biology of short-term (acute) versus long-term (chronic) hypoxia is considered, and a nascent hypothesis to explain the behavior of hMSCs in long-term hypoxia is presented. It is hoped that by establishing an ongoing discourse and driving toward a regulatory recognizable "hMSC checklist," we may be better able to provide the patient population with safe and efficacious regenerative treatments.

Recent studies have revealed the prevalence and biological significance of guanidine metabolism in nature. However, the metabolic pathways used by microbes to degrade guanidine or mitigate its toxicity have not been widely studied. Here we report a novel guanidine-degrading enzyme, Sll1077, identified in the model cyanobacterium Synechocystis sp. PCC 6803 through comparative proteomics and subsequent experimental validation. Although previously annotated as an agmatinase enzyme, Sll1077 is more likely a “guanidinase”, because it degrades guanidine rather than agmatine to urea. We demonstrate that the model cyanobacterium Synechococcus elongatus PCC 7942 strain engineered to express the bacterial ethylene-forming enzyme (EFE) exhibits unstable ethylene production due to toxicity and genomic instability induced by accumulation of the EFE-byproduct guanidine. Co-expression of EFE and Sll1077 significantly enhanced genomic stability and enabled the resulting strain to achieve sustained high-level ethylene production. These findings expand our knowledge of natural guanidine degradation pathways and demonstrate their biotechnological application to support ethylene bioproduction.

The dual-specificity phosphatase 3 (DUSP3), an atypical protein tyrosine phosphatase (PTP), regulates cell cycle checkpoints and DNA repair pathways under conditions of genotoxic stress. DUSP3 interacts with the nucleophosmin protein (NPM) in the cell nucleus after UV-radiation, implying a potential role for this interaction in mechanisms of genomic stability. Here, we show a high-affinity binding between DUSP3-NPM and NPM tyrosine phosphorylation after UV stress, which is increased in DUSP3 knockdown cells. Specific antibodies designed to the four phosphorylated NPM’s tyrosines revealed that DUSP3 dephosphorylates Y29, Y67, and Y271 after UV-radiation. DUSP3 knockdown causes early nucleolus exit of NPM and ARF proteins allowing them to disrupt the HDM2-p53 interaction in the nucleoplasm after UV-stress. The anticipated p53 release from proteasome degradation increased p53-Ser15 phosphorylation, prolonged p53 half-life, and enhanced p53 transcriptional activity. The regular dephosphorylation of NPM’s tyrosines by DUSP3 balances the p53 functioning and favors the repair of UV-promoted DNA lesions needed for the maintenance of genomic stability.

Proper functioning of cells—their ability to divide, differentiate, and regenerate—is dictated by genomic stability. The main factors contributing to this stability are the telomeric ends that cap chromosomes. Telomere biology and telomerase activity have been of interest to scientists in various medical science fields for years, including the study of both cancer and of senescence and aging. All these processes are accompanied by telomere-length modulation. Maintaining the key levels of telomerase component (hTERT) expression and telomerase activity that provide optimal telomere length as well as some nontelomeric functions represents a promising step in advanced anti-aging strategies, especially in dermocosmetics. Some known naturally derived compounds contribute significantly to telomere and telomerase metabolism. However, before they can be safely used, it is necessary to assess their mechanisms of action and potential side effects. This paper focuses on the metabolic potential of natural compounds to modulate telomerase and telomere biology and thus prevent senescence and skin aging.

DNA replication is a fundamental process of life. Any perturbation of this process by endogenous or exogenous factors impacts on genomic stability and thereby on carcinogenesis. More recently, the replication machinery has been discovered as an interesting target for cancer therapeutic strategies. Given its high biological and clinical relevance, technologies for the analysis of DNA replication have attracted major attention. The so-called DNA fiber spreading technique is a powerful tool to directly monitor various aspects of the replication process by sequential incorporation of halogenated nucleotide analogs which later can be fluorescently stained and analyzed. This chapter outlines the use of the DNA fiber spreading technique for the analysis of replication dynamics and replication structures.

Oxidative and alkylating DNA damage occurs under normal physiological conditions and exogenous exposure to DNA damaging agents. To counteract DNA base damage, cells have evolved several defense mechanisms that act at different levels to prevent or repair DNA base damage. Cells combat genomic lesions like these including base modifications, abasic sites, as well as single-strand breaks, via the base excision repair (BER) pathway. In general, the core BER process involves well-coordinated five-step reactions to correct DNA base damage. In this review, we will uncover the current understanding of BER mechanisms to maintain genomic stability and the biological consequences of its failure due to repair gene mutations. The malfunction of BER can often lead to BER intermediate accumulation, which is genotoxic and can lead to different types of human disease. Finally, we will address the use of BER intermediates for targeted cancer therapy.

Endocrine-driven malignancies, including breast and prostate cancer, are among the most common human cancers. The relationship between sex steroid hormones (eg, androgen, estrogen, and progesterone), their cognate receptors, and genomic stability lie at the center of endocrine-driven cancer development, progression, and therapeutic resistance. A variety of direct and indirect mechanisms have been described that link steroid hormone signaling to the loss of genomic integrity that drives early carcinogenesis. These effects are often enriched within endocrine receptor cistromes, accounting for the high proportion of mutations and rearrangements in the region of hormone response elements. In other cases, the effects are generalized and rely on a complex array of genetic, epigenetic, and metabolic interactions. Both androgen and estrogen receptors directly modulate the DNA damage response by trans-activating DNA damage response genes and redirecting the cellular repair machinery in the wake of genotoxic stress. Here we review the key mechanistic underpinnings of the relationship between sex steroid hormone receptors and genomic stability. In addition, we summarize emerging research in this area and discuss important implications for cancer prevention and treatment.

Eukaryotic genomes are compacted into loops and topologically associating domains (TADs), which contribute to transcription, recombination and genomic stability. Cohesin extrudes DNA into loops that are thought to lengthen until CTCF boundaries are encountered. Little is known about whether loop extrusion is impeded by DNA-bound macromolecular machines. We demonstrate that the replicative helicase MCM is a barrier that restricts loop extrusion in G1 phase. Single-nucleus Hi-C of one-cell embryos revealed that MCM loading reduces CTCF-anchored loops and decreases TAD boundary insulation, suggesting loop extrusion is impeded before reaching CTCF. Single-molecule imaging shows that MCMs are physical barriers that frequently constrain cohesin translocationin vitro.Simulations are consistent with MCMs as abundant, random barriers. We conclude that distinct loop extrusion barriers contribute to shaping 3D genomes.One Sentence SummaryMCM complexes are obstacles that impede the formation of CTCF-anchored loops.

Single-stranded DNA-binding proteins (SSBs) play crucial roles in DNA replication, recombination and repair, and serve as key players in the maintenance of genomic stability. While a number of SSBs bind single-stranded DNA (ssDNA) non-specifically, the others recognize and bind specific ssDNA sequences. The mechanisms underlying this binding discrepancy, however, are largely unknown. Here, we present a comparative study of protein–ssDNA interactions by annotating specific and non-specific SSBs and comparing structural features such as DNA-binding propensities and secondary structure types of residues in SSB–ssDNA interactions, protein–ssDNA hydrogen bonding and π–π interactions between specific and non-specific SSBs. Our results suggest that protein side chain-DNA base hydrogen bonds are the major contributors to protein–ssDNA binding specificity, while π–π interactions may mainly contribute to binding affinity. We also found the enrichment of aspartate in the specific SSBs, a key feature in specific protein–double-stranded DNA (dsDNA) interactions as reported in our previous study. In addition, no significant differences between specific and non-specific groups with respect of conformational changes upon ssDNA binding were found, suggesting that the flexibility of SSBs plays a lesser role than that of dsDNA-binding proteins in conferring binding specificity.

TP53 binding protein 1 (53BP1) plays an important role in DNA damage repair and maintaining genomic stability. However, the mutations of 53BP1 in human cancers have not been systematically examined. Here, we have analyzed 541 somatic mutations of 53BP1 across 34 types of human cancer from databases of The Cancer Genome Atlas, International Cancer Genome Consortium and Catalogue of Somatic Mutations in Cancer. Among these cancer-associated 53BP1 mutations, truncation mutations disrupt the nuclear localization of 53BP1 thus abolish its biological functions in DNA damage repair. Moreover, with biochemical analyses and structural modeling, we have examined the detailed molecular mechanism by which missense mutations in the key domains causes the DNA damage repair defects. Taken together, our results reveal the functional defects of a set of cancer-associated 53BP1 mutations.

Rho is a general transcription termination factor playing essential roles in RNA polymerase (RNAP) recycling, gene regulation, and genomic stability in most bacteria. Textbook models of transcription termination postulate that hexameric Rho loads onto RNA prior to contacting RNAP and then translocates along the transcript in pursuit of the moving RNAP to pull RNA from it. Here, we report the cryo-EM structures of two termination process intermediates. Prior to interacting with RNA, Rho forms a specific “pre-termination complex” (PTC) with RNAP and elongation factors NusA and NusG, which stabilize the PTC. RNA exiting RNAP interacts with NusA before entering the central channel of Rho from the distal C-terminal side of the ring. We map the principal interactions in the PTC and demonstrate their critical role in termination. Our results overturn the traditional termination models and support a mechanism in which the formation of a persistent PTC is a prerequisite for termination.

Genomic perturbations due to inaccurate DNA replication, including inappropriate chromosomal segregation often underlie the development of cancer and neurodegenerative diseases. The incidence of these two diseases increases with age and exhibits an inverse association. Therefore, elderly subjects with cancer exhibit a reduced risk of a neurodegenerative disease, and vice versa. Both of these diseases are associated with aging and share several risk factors. Cells have multiple mechanisms to repair DNA damage and inaccurate replication. Previous studies have demonstrated that tumor suppressor proteins serve a critical role in the DNA damage response, which may result in genomic instability and thus induction of cellular apoptosis. Tumor suppressor genes, such as phosphatase and tensin homologue deleted on chromosome 10 (PTEN), breast cancer susceptibility gene 1 (BRCA1) and TP53 reduce genomic susceptibility to cancer by repairing the damaged DNA. In addition, these genes work cooperatively to ensure the inhibition of the development of several types of cancer. PTEN, BRCA1 and TP53 have been recognized as the most frequently deleted and/or mutated genes in various types of human cancer. Recently, tumor suppressor genes have also been shown to be involved in the development of neurodegenerative diseases. The present review summarizes the recent findings of the functions of these tumor suppressors that are associated with genomic stability, and are involved in carcinogenic and neurodegenerative cell signaling. A summary is presented regarding the interactions of these tumor suppressors with their partners which results in transduction of downstream signals. The implications of these functions for cancer and neurodegenerative disease-associated biology are also highlighted.

Objective The alphaproteobacterium Zymomonas mobilis is an efficient ethanol producer, and Z. mobilis-based biorefinery shows great potential for biofuel production. Serial co-cultivation is an emerging approach that promotes inter-species interactions which can improve or rewire the metabolic features in industrially useful microorganisms by inducing frequent mutations. We applied this method to assess if it improves or rewires the desirable physiological features of Z. mobilis, especially ethanol production. Results We performed serial co-culture of Z. mobilis with the baker’s yeast, Saccharomyces cerevisiae. We observed filamentation of Z. mobilis cells in the co-culture, indicating that the Z. mobilis cells were exposed to stress due to the presence of a competitor. After 50 times of serial transfers, we characterized the generated Z. mobilis strains, showing that long term co-culture did not drive significant changes in either the growth or profile of excreted metabolites in the generated strains. In line with this, whole genome sequencing of the generated Z. mobilis strains revealed only minor genetic variations from the parental strain. 50 generations of Z. mobilis monoculture did not induce morphological changes or any significant genetic variations. The result indicates that the method needs to be carefully optimized for Z. mobilis strain improvement. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

RECQ1 (also known as RECQL or RECQL1) belongs to the RecQ family of DNA helicases, members of which are linked with rare genetic diseases of cancer predisposition in humans. RECQ1 is implicated in several cellular processes, including DNA repair, cell cycle and growth, telomere maintenance, and transcription. Earlier studies have demonstrated a unique requirement of RECQ1 in ensuring chromosomal stability and suggested its potential involvement in tumorigenesis. Recent reports have suggested that RECQ1 is a potential breast cancer susceptibility gene, and missense mutations in this gene contribute to familial breast cancer development. Here, we provide a framework for understanding how the genetic or functional loss of RECQ1 might contribute to genomic instability and cancer.

The tremendous variation that exists between bacterial species illustrates the power of evolution, which is the continuous process of mutation and selection over time. Even within a bacterial species, individual members can harbour an impressive degree of genetic variation, depending on the species. The question then arises how similar the offspring of a given bacterial cell over time is, and how long it takes before differences are noticeable? Here we show that on the one hand one can expect random mutations to arise, as a result of various mechanisms. On the other hand, there are forces at play that keep the offspring of a cell genetically relatively constant, unless there is selection for a particular characteristic. The most common mechanisms behind mutations that can appear in a bacterial population are briefly introduced. Next, it is explained why nevertheless such mutations are rarely observed, as long as single colonies are randomly selected, unless selective pressures apply. Since quality control of industrial bacterial cultures is likely to depend heavily on genome sequencing in the near future, the accuracy of whole‐genomic sequencing technologies is also discussed. It can be concluded that the bacteriologists who started picking single colonies from agar plates more than hundred years ago were unknowingly ingeneous, as their practice maintains a bacterial culture stable over time. Significance and Impact of Study The questions addressed here are relevant for industries that depend on live bacteria for (manufacturing of) their products, as they have to guard their bacterial cultures that remain unchanged over time. The explanation why randomly selection of single colonies keeps a population stable can be of use in bacteriology courses. The limitations of whole‐genome sequencing are relevant to legislators to avoid overinterpretation of those data., Significance and Impact of Study: The questions addressed here are relevant for industries that depend on live bacteria for (manufacturing of) their products, as they have to guard their bacterial cultures that remain unchanged over time. The explanation why randomly selection of single colonies keeps a population stable can be of use in bacteriology courses. The limitations of whole‐genome sequencing are relevant to legislators to avoid overinterpretation of those data.

Fondazione Italiana per la Ricerca sul Cancro FIRC 18112 Sina Atashpaz.Fondazione Umberto Veronesi Sina Atashpaz Associazione Italiana per la Ricerca sul Cancro AIRC 5xmille METAMECH program Vincenzo Costanzo Giovanni Armenise-Harvard Foundation Vincenzo Costanzo European Research Council Consolidator grant 614541 Vincenzo Costanzo Associazione Italiana per la Ricerca sul Cancro Fellowship 23961 Negar ArghavanifarDanish Cancer Society KBVU-2014 Andres Joaquin Lopez-Contreras Danish Council for Independent Research Sapere Aude, DFF Starting Grant 2014 Andres Joaquin Lopez-Contreras European Research Council ERC-2015-STG-679068 Andres Joaquin Lopez-Contreras Danish National Research Foundation DNRF115 Andres Joaquin Lopez-Contreras The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. Unrepaired DNA damage during embryonic development can be potentially inherited by a large population of cells. However, the quality control mechanisms that minimize the contribution of damaged cells to developing embryos remain poorly understood. Here, we uncovered an ATR- and CHK1-mediated transcriptional response to replication stress (RS) in mouse embryonic stem cells (ESCs) that induces genes expressed in totipotent two-cell (2C) stage embryos and 2C-like cells. This response is mediated by Dux, a multicopy retrogene defining the cleavage-specific transcriptional program in placental mammals. In response to RS, DUX triggers the transcription of 2C-like markers such as murine endogenous retrovirus-like elements (MERVL) and Zscan4. This response can also be elicited by ETAA1-mediated ATR activation in the absence of RS. ATR-mediated activation of DUX requires GRSF1-dependent post-transcriptional regulation of Dux mRNA. Strikingly, activation of ATR expands ESCs fate potential by extending their contribution to both embryonic and extra-embryonic tissues. These findings define a novel ATR dependent pathway involved in maintaining genome stability in developing embryos by controlling ESCs fate in response to RS. Sí

Pluripotent stem cell (PSC) cultures are subjected to selective pressures that can result in acquisition and expansion of recurrent genetic abnormalities at any time. These recurrent abnormalities enhance the variant cells harboring them with a competitive advantage over wild-type cells. Variant cells can eventually supplant wild-type cells entirely and become fixed in culture. Such variants can impact the efficacy of PSCs in research and clinical applications. Therefore, routine genomic characterization is required for reliable and effective use of PSCs. In this article we describe the capabilities and limitations of several assays commonly used for assessing PSC genomic stability. Based on this analysis, we provide a recommendation for integrating assays into a comprehensive testing regimen that maximizes coverage while minimizing cost. © 2020 by John Wiley & Sons, Inc.

Protein phosphatase 4 (PP4), one of serine/threonine phosphatases, is involved in many critical cellular pathways, including DNA damage response (DNA repair, cell cycle regulation, and apoptosis), tumorigenesis, cell migration, immune response, stem cell development, glucose metabolism, and diabetes. PP4 has been steadily studied over the past decade about wide spectrum of physiological activities in cells. Given the many vital functions in cells, PP4 has great potential to develop into the finding of key working mechanisms and effective treatments for related diseases such as cancer and diabetes. In this review, we provide an overview of the cellular and molecular mechanisms by which PP4 impacts and also discuss the functional significance of it in cell health. [BMB Reports 2020; 53(4): 181-190].

Efficient DNA repair is critical for cell survival following exposure to DNA topoisomerase I (Top1) inhibitors camptothecin, a nature product from which the common chemotherapeutic drugs irinotecan and topotecan are derived. The camptothecin-derived agents exert their antitumor activities by specifically stabilizing the Top1–DNA covalent complexes (Top1cc) and blocking the DNA religation step. When exposed to these DNA damage agents, tumor cells quickly activate DNA damage response. This allows sufficient time to remove the Top1ccs and prevent tumor cells from apoptosis. Several repair pathways have been implicated in this process. One of the most relevant repair modes is DNA single strand break repair (SSBR) pathway. The expression level or mutagenesis of specific repair factors involved in SSBR pathway may play an indispensable role in individual’s capacity of repairing camptothecin induced DNA damage. Therefore, understanding of the tolerance pathways counteracted to camptothecin cytotoxicity is crucial in alleviating chemotherapy resistance. This review focus on the SSBR pathway in repair camptothecin induced DNA damage, aiming to provide insights into the potential molecular determinants of camptothecin chemosensitivity.

A barrier to cost‐efficient biomanufacturing is the instability of engineered genetic elements, such as plasmids. Instability can also manifest at the whole‐genome level, when fungal dikaryons revert to parental species due to nuclear segregation during cell division. Here, we show that by encapsulating Saccharomyces cerevisiae‐Pichia stipitis dikaryons in an alginate matrix, we can limit cell division and preserve their expanded metabolic capabilities. As a proxy to cellulosic ethanol production, we tested the capacity of such cells to carry out ethanologenic fermentation of glucose and xylose, examining substrate use, ploidy, and cell viability in relation to planktonic fusants, as well as in relation to planktonic and encapsulated cell cultures consisting of mixtures of these species. Glucose and xylose consumption and ethanol production by encapsulated dikaryons were greater than planktonic controls. Simultaneous co‐fermentation did not occur; rather the order and kinetics of glucose and xylose catabolism by encapsulated dikaryons were similar to cultures where the two species were encapsulated together. Over repeated cycles of fed‐batch culture, encapsulated S. cerevisiae‐P. stipitis fusants exhibited a dramatic increase in genomic stability, relative to planktonic fusants. Encapsulation also increased the stability of antibiotic‐resistance plasmids used to mark each species and preserved a fixed ratio of S. cerevisiae to P. stipitis cells in mixed cultures. Our data demonstrate how encapsulating cells in an extracellular matrix restricts cell division and, thereby, preserves the stability and biological activity of entities ranging from genomes to plasmids to mixed populations, each of which can be essential to cost‐efficient biomanufacturing., Genetic instability of engineered organisms causes significant losses in productivity and profits for biomanufacturing. Instability typically occurs upon cell division, when genetic material is lost due to chance or nuclear segregation. We encapsulated yeast dikaryons capable of fermenting both hexose and pentose sugars in an alginate matrix. Within this matrix, cells demonstrated high levels of metabolic activity despite near‐zero growth rates. We found that encapsulated cells had similar metabolic capabilities, but significantly higher genetic stability than their planktonic counterparts, likely due to their lower rates of cell division. These results indicate that alginate matrices can be used to improve the genetic stability of both plasmids and dikaryons across multiple use cycles.

Objective: Telomeres are specialized nucleoprotein structures located at the ends of chromosomes, which play a crucial role in genomic stability. Telomere shortening has been proposed as a ...

Background: For many years, researchers have sought to overcome major challenges in the use of nanoparticles as therapeutics, including issues related to intracellular delivery, biocompatibility, and activation. In particular, the genetic stability of cells treated with nanoparticles has become increasingly important in the context of stem cell therapy. Methods: Functional nanoparticles (Sunflower typed nanoparticles; SF-NPs) were fabricated by coating heparin pluronic F127 gels with quantum dot nanoparticles (QDs), and then bound the SOX9 gene to the QD nanogels. The resultant nanoparticles were transferred into stem cells, and the effect on genetic stability was monitored. To determinate gene delivery efficacy and long-term genomic stability of cells transfected with QD nanogels, hMSCs were transfected with nanogels at passage 4 (T1; Transfected cells 1) and then sub-cultured to passage of (T4). Following transplantation of transfected T1-T4 cells, the cells were monitored by in vivo imaging. The genetic stability of cells treated with nanoparticles was confirmed by chromosomal analysis, copy number variation (CNV) analysis, and mRNA profiling. Results: After 21 days of pellet culture after sub-culture from T1 to T4, hMSCs treated with QD nanogels complexed with SOX9 plasmid DNA (pDNA) significantly increased expression of specific extracellular matrix (ECM) polysaccharides and glycoproteins, as determined by Safranin O and Alcian blue staining. Moreover, the T4 hMSCs expressed higher levels of specific proteins, including collagen type II (COLII) and SOX9, than P4 hMSCs, with no evidence of DNA damage or genomic malfunction. Microarray analysis confirmed expression of genes specific to matured chondrocytes. Stem cells that internalized nanoparticles at the early stage retained genetic stability, even after passage. In in vivo studies in rats, neuronal cartilage formation was observed in damaged lesions 6 weeks after transplantation of T1 and T4 cells. The degree of differentiation into chondrocytes in the cartilage defect area, as determined by mRNA and protein expression of COLII and SOX9, was higher in rats treated with SF-NPs. Conclusion: The QD nanogels used in this study, did not affect genome integrity during long-term subculture, and are thus suitable for multiple theranostic applications.

In this issue, Li et al. (2019) report a previously unknown Ca2+-CaMKK2-AMPK signaling cascade that protects stalled forks from degradation by phosphorylating and inhibiting the EXO1 nuclease, revealing a surprising role for Ca2+ influx in the maintenance of genomic stability.

The tumor suppressor p53 is one of the most important proteins for protection of genomic stability and cancer prevention. Cancers often inactivate it by either mutating its gene or disabling its function. Thus, activating p53 becomes an attractive approach for the development of molecule-based anti-cancer therapy. The past decade and half have witnessed tremendous progress in this area. This essay offers readers with a grand review on this progress with updated information about small molecule activators of p53 either still at bench work or in clinical trials.

The chromatin modifier SETD2 often mutated in clear cell renal cell carcinoma (ccRCC), was recently shown to be a dual-function methyltransferase that “writes” methyl marks on both chromatin and microtubules, revealing α-tubulin methylation as a new posttranslational modification of the mitotic spindle. Here, we report that the polybromo protein PBRM1, the 2nd most mutated gene in ccRCC, is a “reader” for this SETD2-dependent methyl mark on α-tubulin. PBRM1 is a component of the PBAF (Polybromo BRG1 associated factor) chromatin remodeler complex. Our western and immunocytochemistry data in multiple kidney-derived cell lines, including HEK293T, HKC and 786-O, revealed that PBRM1 binds to methylated α-tubulin and localizes to the mitotic spindle and spindle pole during cell division. PBRM1 has six bromo domains, two bromo-associated homology (BAH) domains and one HMG domain. While PBRM1 is known to bind acetylated histones via its bromo domains, our GST pull down assays showed that PBRM1 binds methylated α-tubulin via its two BAH domains. Additional western and immunocytochemical experiments following knockout or re-expression of PBRM1 revealed that PBRM1 recruits other PBAF components to the mitotic spindle to maintain genomic stability. Two clinically established ccRCC mutations (P1048R and C1233W) in PBRM1 BAH domains result in loss of microtubule binding, mislocalization of PBAF, and the inability of PBRM1 to maintain genomic stability, as assessed by increased lagging chromosomes, chromosome bridges, multipolar spindles and micronuclei count. A third pathogenic ccRCC mutation (T1202K) in the PBRM1 BAH domain did not affect microtubule binding and consequently was not associated with mitotic spindle defects or genomic instability. Mass spectrometry and RNASeq confirmed BAH domain mutant PBRM1 still assembled a transcriptionally competent PBAF complex, clearly distinguishing the cytoskeletal from the chromatin impact of these mutations. These data reveal a previously unknown function of PBRM1 beyond reading acetylated histones, and expand the repertoire of chromatin remodelers acting on the cytoskeleton to maintain genomic stability. Citation Format: Menuka Karki, Rahul Jangid, Ramakrishnan Anish, Riyad N. Seervai, Jean-Philippe Bertocchio, Takashi Hotta, Pavlos Msaouel, Sung Y. Jung, Sandra L. Grimm, Cristian Coarfa, Bernard E. Weissman, Ryoma Ohi, Kristen J. Verhey, Courtney H. Hodges, Ruhee Dere, In Young Park, B. V. Venkataram Prasad, W. Kimryn Rathmell, Cheryl L. Walker, Durga N. Tripathi. A cytoskeletal function for PBRM1: reading methylated microtubules to maintain genomic stability [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2042.

Background: Genomically instable (GI) chronic lymphocytic leukemia (CLL) is characterized by frequent alterations in DNA-damage response (DDR) genes (e.g. TP53, ATM) and related pathways. Conversely, pathogenic networks in CLL cases which maintain genomic integrity and operate with a functional DDR remain incompletely described. Methods: Molecular profiling was conducted on CD19 sorted samples derived from patients registered on the CLL8 study (1st-line, FC vs. FCR) for gene expression (GEP)(n=337, Exon 1.0 ST arrays, Affymetrix), copy number aberrations (CNAs) (n=309, SNP Arrays 6.0, Affymetrix) and mutation analyses/signature projections (n=171, whole exome sequencing, Illumina). FISH, IGHV and TP53 mutation analysis was conducted at trial enrolment. Results: Unsupervised consensus clustering (k=2-6) on variably expressed genes (SD>0.5) was used for class discovery. Two small, but highly differentiated clusters were identified, characterized through NRIP1 and EBF1/tri12. GSEA also segregated the remaining samples into four major clusters showing signatures of inflammation (I) and without inflammation (NI). These clusters were further segregated into GI-CLL clusters with increased “DNA-repair” or clusters with “epithelial-mesenchymal transition”-like signatures (EMT-L). Variability for del(17p)/TP53 mutation was found across clusters (p