Genome Integrity Research Articles

Arabidopsis KNL1 recruits type one protein phosphatase to kinetochores to silence the spindle assembly checkpoint.

Proper chromosome segregation during cell division is essential for genomic integrity and organismal development. This process is monitored by the spindle assembly checkpoint (SAC), which delays anaphase onset until all chromosomes are properly attached to the mitotic spindle. The kinetochore protein KNL1 plays a critical role in recruiting SAC proteins. Here, we reveal that Arabidopsis KNL1 regulates SAC silencing through the direct recruitment of type one protein phosphatase (TOPP) to kinetochores. We show that KNL1 interacts with all nine TOPPs via a conserved RVSF motif in its N terminus, and this interaction is required for the proper localization of TOPPs to kinetochores during mitosis. Disrupting KNL1-TOPP interaction leads to persistent SAC activation, resulting in a severe metaphase arrest and defects in plant growth and development. Our findings highlight the evolutionary conservation of KNL1 in coordinating kinetochore-localized phosphatase to ensure timely SAC silencing and faithful chromosome segregation in Arabidopsis.

PIWI proteins and piRNAs: key regulators of stem cell biology

In this mini review, we discussed the functional roles of PIWI proteins and their associated small RNAs, piRNAs, in regulating gene expression within stem cell biology. Guided by piRNAs, these proteins transcriptionally and post-transcriptionally repress transposons using mechanisms such as the ping-pong amplification cycle and phasing to protect germline genomes. Initially identified in Drosophila melanogaster, the piRNA pathway regulate germline stem cell self-renewal and differentiation via cell-autonomous and non-cell-autonomous mechanisms. Precisely, in GSCs, PIWI proteins and piRNAs regulate gene expression by modulating chromatin states and directly influencing mRNA translation. For instance, the PIWI protein Aubergine loaded with piRNAs promotes and represses translation of certain mRNAs to balance self-renewal and differentiation. Thus, the piRNA pathway exhibits dual regulatory roles in mRNA stability and translation, highlighting its context-dependent functions. Moreover, PIWI proteins are essential in somatic stem cells to support the regenerative capacity of highly regenerative species, such as planarians. Similarly, in Drosophila intestinal stem cells, the PIWI protein Piwi regulates metabolic pathways and genome integrity, impacting longevity and gut homeostasis. In this case, piRNAs appear absent in the gut, suggesting piRNA-independent regulatory mechanisms. Together, PIWI proteins and piRNAs demonstrate evolutionary conservation in stem cell regulation, integrating TE silencing and gene expression regulation at chromatin and mRNA levels in somatic and germline lineages. Beyond their canonical roles, emerging evidence reveal their broader significance in maintaining stem cell properties and organismal health under physiological and pathological conditions.

PGC-1α activation to enhance macrophage immune function in mycobacterial infections

Nontuberculous Mycobacteria (NTM) are a heterogeneous group of environmental microorganisms with distinct human pathogenesis. Their incidence and prevalence are rising worldwide, due in part to elevated antimicrobial resistance which complicates treatment and potential successful outcomes. Although information exists on the clinical significance of NTMs, little is known about host immune response to infection. NTM infections alter macrophage mitochondrial capacity and decrease ATP production, efficient immune response, and bacterial clearance. Transcription factor peroxisome proliferator activated receptor (PPAR) γ coactivator-1α (PGC-1α) is a master regulator of mitochondrial biogenesis, influencing metabolism, mitochondrial pathways, and antioxidant response. Mitochondrial transcription factor A (TFAM) is a protein essential for mitochondrial DNA (mtDNA) genome stability, integrity, and metabolism. Both PGC-1α and TFAM regulate mitochondrial biogenesis and activity, and their disruption is linked to inflammatory signaling and altered macrophage function. We show that NTM causes macrophage mitochondrial damage and disrupted bioenergetics. Mechanistically we show that this is related to attenuation of expression of PGC-1α and TFAM in infected macrophages. Importantly, rescuing expression of PGC-1α and TFAM using pharmacologic approaches restored macrophage immune function. Our results suggest that pharmacologic approaches to enhance mitochondrial function provide a novel approach to target macrophage immune function and means to combat NTM infections.

Retrograde mitochondrial signaling governs the identity and maturity of metabolic tissues

Mitochondrial damage is a hallmark of metabolic diseases, including diabetes, yet the consequences of compromised mitochondria in metabolic tissues are often unclear. Here, we report that dysfunctional mitochondrial quality control engages a retrograde (mitonuclear) signaling program that impairs cellular identity and maturity in β-cells, hepatocytes, and brown adipocytes. Targeted deficiency throughout the mitochondrial quality control pathway, including genome integrity, dynamics, or turnover, impaired the oxidative phosphorylation machinery, activating the mitochondrial integrated stress response, eliciting chromatin remodeling, and promoting cellular immaturity rather than apoptosis to yield metabolic dysfunction. Indeed, pharmacologic blockade of the integrated stress response in vivo restored β-cell identity following loss of mitochondrial quality control. Targeting mitochondrial retrograde signaling may therefore be promising in the treatment or prevention of metabolic disorders.

Integrating genome-wide association studies and transcriptomics prioritizes drug targets for meningioma

Abstract Meningioma, a prevalent central nervous system tumor, presents a significant challenge in neuro-oncology. This study harnesses genome-wide association studies (GWAS) and transcriptomic analysis to illuminate the pathological underpinnings of meningioma and spearhead the discovery of novel drug targets. By employing Summary-data-based Mendelian Randomization (SMR), colocalization analyses, and Mendelian randomization, we pinpointed four genes as pivotal therapeutic targets. The integration of bulk and single-cell RNA sequencing confirmed the upregulated expression of three of the genes (XBP1, TTC28, and TRPC6) in meningioma tissues, unraveling their cellular distribution and hinting at the tumor's intrinsic heterogeneity. Molecular docking further identified dexamethasone and levonorgestrel as potential modulators of these targets, paving the way for personalized meningioma treatment strategies. This research advances our understanding of meningioma's molecular landscape and illustrates the power of genomic and transcriptomic integration in the realm of precision oncology.

Impact of trainability on telomere dynamics of pet dogs (Canis lupus familiaris): An explorative study in aging dogs.

This research studied the impact of various factors (including social and physiological parameters) on telomere dynamics in pet dogs. Telomeres, essential for maintaining genomic integrity, undergo shortening with each cell division, leading to cellular senescence. Previous studies in humans have linked cognitive and social factors with telomere dynamics but in animals, such associations remain understudied. This study is based on a previous study, where behavioral and cognitive changes in aging pet dogs were investigated. Together with standard variables (sex, age, body weight, diet), behavioral predictors that were assessed in the "Modified Vienna Canine Cognitive Battery" were used. This study aimed to investigate the influence of these factors on telomere dynamics in aging pet dogs. The relative telomere length of 63 dogs was measured, using a qPCR method and a model selection approach was applied to assess which variables can explain the found telomere patterns. Results revealed a strong association of the behavioral factor called trainability and telomere change. Trainability was the best predictor for telomere change over time and was the only predictor having a relative variable importance (RVI) above 0.7. This finding suggests that higher trainability positively affects telomere dynamics in aging dogs and factors like age, sex, diet, and other cognitive parameters are less important. The study sheds light on the potential role of cognitive factors in canine aging and offers insights into improving the quality of life for aging dogs, but further research is needed to comprehensively understand the interplay between behavior, cognition, and telomere dynamics in dogs.

Integrated In-silico and In-vivo Assessments of Betaine's Effect on the Hypothalamic-Pituitary-Testicular (HPT) Axis in Fluoride-Treated Rats.

Toxicity is associated with undue sodium fluoride (NaF) exposure, and Betaine (BET) is recognised for its nutraceutical benefits. Although it is necessary to reduce toxic level exposure to fluoride, the literature lacks information on the role of BET in mitigating fluoride-induced reproductive toxicity. Therefore, this study assesses the impact of BET on NaF-induced reproductive perturbation in male rats. Wistar rats were treated with NaF (9mg/kg) alone or co-treated with BET (50 or 100mg/kg) for 28 d. Our findings indicate that BET significantly mitigated alterations in sperm functionality indices caused by NaF treatment. BET substantially increased reproductive hormone levels and averted NaF-induced increases in oxidative stress biomarkers and testicular enzymes. NaF-induced increases in inflammatory markers in the testis, epididymis, and hypothalamus were effectively reversed upon BET co-treatment. Also, co-treatment with BET protected genome integrity, as evidenced by p53 and apoptotic markers Bax and Bcl-2 levels, abating damages in the testes, epididymis, and hypothalamus of NaF-treated rats. Also, our findings from in-silico studies revealed that BET moderately inhibits the molecular activation of the inhibitor of nuclear factor-κB kinase, hypoxia-inducible factor -1 alpha, and proviral integration for the Moloney murine leukaemia virus-1 kinase. While it is preferable to reduce fluoride exposure, the relevant findings here indicate that BET exhibits anti-inflammatory, antioxidant, and anti-apoptotic properties that ameliorate inadvertent NaF-mediated toxicities in experimental rats exposed to NaF.

UV-induced reorganization of 3D genome mediates DNA damage response

While it is well-established that UV radiation threatens genomic integrity, the precise mechanisms by which cells orchestrate DNA damage response and repair within the context of 3D genome architecture remain unclear. Here, we address this gap by investigating the UV-induced reorganization of the 3D genome and its critical role in mediating damage response. Employing temporal maps of contact matrices and transcriptional profiles, we illustrate the immediate and holistic changes in genome architecture post-irradiation, emphasizing the significance of this reconfiguration for effective DNA repair processes. We demonstrate that UV radiation triggers a comprehensive restructuring of the 3D genome organization at all levels, including loops, topologically associating domains and compartments. Through the analysis of DNA damage and excision repair maps, we uncover a correlation between genome folding, gene regulation, damage formation probability, and repair efficacy. We show that adaptive reorganization of the 3D genome is a key mediator of the damage response, providing new insights into the complex interplay of genomic structure and cellular defense mechanisms against UV-induced damage, thereby advancing our understanding of cellular resilience.

RECQ4-MUS81 interaction contributes to telomere maintenance with implications to Rothmund-Thomson syndrome

Replication stress, particularly in hard-to-replicate regions such as telomeres and centromeres, leads to the accumulation of replication intermediates that must be processed to ensure proper chromosome segregation. In this study, we identify a critical role for the interaction between RECQ4 and MUS81 in managing such stress. We show that RECQ4 physically interacts with MUS81, targeting it to specific DNA substrates and enhancing its endonuclease activity. Loss of this interaction, results in significant chromosomal segregation defects, including the accumulation of micronuclei, anaphase bridges, and ultrafine bridges (UFBs). Our data further demonstrate that the RECQ4-MUS81 interaction plays an important role in ALT-positive cells, where MUS81 foci primarily colocalise with telomeres, highlighting its role in telomere maintenance. We also observe that a mutation associated with Rothmund-Thomson syndrome, which produces a truncated RECQ4 unable to interact with MUS81, recapitulates these chromosome instability phenotypes. This underscores the importance of RECQ4-MUS81 in safeguarding genome integrity and suggests potential implications for human disease. Our findings demonstrate the RECQ4-MUS81 interaction as a key mechanism in alleviating replication stress at hard-to-replicate regions and highlight its relevance in pathological conditions such as RTS.

Perspectives and opportunities in forensic human, animal, and plant integrative genomics in the Pangenome era.

The Human Pangenome Reference Consortium, the Chinese Pangenome Consortium, and other plant and animal pangenome projects have announced the completion of pilot work aimed at constructing high-quality, haplotype-resolved reference graph genomes representative of global ethno-linguistically different populations or different plant and animal species. These graph-based, gapless pangenome references, which are enriched in terms of genomic diversity, completeness, and contiguity, have the potential for enhancing long-read sequencing (LRS)-based genomic research, as well as improving mappability and variant genotyping on traditional short-read sequencing platforms. We comprehensively discuss the advancements in pangenome-based genomic integrative genomic discoveries across forensic-related species (humans, animals, and plants) and summarize their applications in variant identification and forensic genomics, epigenetics, transcriptomics, and microbiome research. Recent developments in multiplexed array sequencing have introduced a highly efficient and programmable technique to overcome the limitations of short forensic marker lengths in LRS platforms. This technique enables the concatenation of short RNA transcripts and DNA fragments into LRS-optimal molecules for sequencing, assembly, and genotyping. The integration of new pangenome reference coordinates and corresponding computational algorithms will benefit forensic integrative genomics by facilitating new marker identification, accurate genotyping, high-resolution panel development, and the updating of statistical algorithms. This review highlights the necessity of integrating LRS-based platforms, pangenome-based study designs, and graph-based pangenome references in short-read mapping and LRS-based innovations to achieve precision forensic science.

The Urogenital System Microbiota: Is It a New Gamechanger in Urogenital Cancers?

The human microbiome, which encompasses microbial communities and their genetic material, significantly influences health and disease, including cancer. The urogenital microbiota, naturally present in the urinary and genital tracts, interact with factors such as age, lifestyle, and health conditions to affect homeostasis and carcinogenesis. Studies suggest that alterations in this microbiota contribute to the development and progression of genitourinary cancers, emphasizing the concept of oncobiome, which refers to microbial genetic contributions to cancer. Similarly, gut microbiota can influence hormone levels and systemic inflammation, impacting cancers such as cervical and prostate cancer. Advanced studies indicate that microbial communities in genitourinary cancers have distinct profiles that may serve as diagnostic biomarkers or therapeutic targets. Dysbiosis of the urinary microbiota correlates with bladder and kidney cancer. Additionally, gut microbiota influence the effectiveness of cancer treatments. However, further research is necessary to clarify causality, the role of microbial metabolites, and hormonal regulation. The aim of this review is to understand that these dynamics present opportunities for innovative cancer diagnostics and therapies, highlighting the need for integration of microbiology, oncology, and genomics to explore the role of microbiota in genitourinary cancers. For this, a comprehensive search of relevant databases was conducted, applying specific inclusion and exclusion criteria to identify studies examining the association between microbiota and urogenital cancers. Research into the mechanisms by which microbiota influence urogenital cancers may pave the way for new diagnostic and therapeutic approaches, ultimately improving patient outcomes.

Inhibitory Effects of Garlic Extract on Hop Stunt Viroid in Micropropagated Grapevine Plantlets

Hop stunt viroid (HSVd) is a major pathogen that affects grapevine health and causes substantial economic losses in grape cultivation. Many studies have been conducted to control grapevine diseases, but effective control methods after plant infections remain lacking. This study aimed to assess the antiviral potential of garlic extract, a natural substance that inhibits HSVds in grapevine plants after micropropagation. Garlic extract was diluted 1,000-fold and applied to grapevine plants, and its effect on HSVd accumulation was evaluated using reverse transcription polymerase chain reaction and digital PCR. The results showed that HSVd accumulation was significantly reduced, with an inhibition rate of 74.45%; meanwhile, higher garlic extract concentrations resulted in contamination and plant damage. Nanopore sequencing confirmed that the integrity of the HSVd genome was compromised after treatment. Furthermore, garlic extract inhibited the HSVd and promoted plant growth by enhancing shoot and root development. Additionally, inhibition of the HSVd was sustained in regenerated grapevine plants. Moreover, the garlic extract showed inhibitory effects against HSVds in natural host cucumber plants. These results suggest that garlic extract could be a cost-effective and sustainable alternative for viroid control in grapevine cultivation, providing long-term protection and broader antiviral activity across plant species.

La revista Science considera al fármaco lenacapavir el avance científico más importante del año 2024 por su papel en la profilaxis preexposición (PrEP) frente al VIH

Since the appearance of the first cases of infection in 1981 in New York and San Francisco by a virus unknown at the time and later identified in 1983 as the human immunodeficiency virus (HIV), which causes AIDS and is responsible for some 42 million deaths and 88 million infections since the beginning of the pandemic, fundamental discoveries have been made that have led to great advances in HIV control. These discoveries range from methods of rapid detection of the virus, basic knowledge of the structure and function of viral proteins, mechanisms of cell penetration, receptors, interaction with the host, transport and genomic integration, stability, pathology, to the development of control procedures, especially antivirals. For years, HIV infection was synonymous with death, until the scientific community and the pharmaceutical sector developed antiretrovirals. There are currently multiple specific compounds directed against different stages in viral replication, such as reverse transcriptase inhibitors, nucleoside analogues and non-analogues, protease inhibitors, inhibitors of fusion and penetration of the virus, antagonists of co-receptors on the surface of lymphocytes, integrase blockers, inhibitors of the fusion of the virus to the cell membrane, capsid inhibitors, pharmacokinetic enhancers. These drugs are combined, using at least two drugs in a single pharmaceutical form with two different classes of action for oral treatment and also in long-acting intravenous administration. In this way, it has been possible to turn a fatal infection into a chronic one, with a very high long-term survival rate.

Exonuclease action of replicative polymerase gamma drives damage-induced mitochondrial DNA clearance.

Mitochondrial DNA (mtDNA) replication is essential for mitochondrial function. This is carried out by a dedicated DNA polymerase gamma, with 5'-3' polymerase and 3'-5' proofreading/ exonuclease activity. Perturbations to either property can have pathological consequences. Predominant sources for replication stress are DNA lesions, such as those induced by oxidative damage. How mtDNA lesions affect the polymerase activity and mtDNA stability in vivo is not fully understood. To address this, we induce mtDNA-specific damage in S. cerevisiae. We observe that mtDNA damage results in significant mtDNA loss. This loss occurs independent of cell cycle progression or cell division, suggesting an active mechanism for damaged mtDNA clearance. We implicate the 3'-5' exonuclease activity of the mtDNA polymerase in this clearance, with rates of loss being affected by cellular dNTP levels. Overall, our findings reveal context-dependent, selective regulation of two critical but opposing functions of polymerase gamma to ensure mitochondrial genome integrity.

Identification of DNA damage repair-related genes in sepsis using bioinformatics and machine learning: An observational study.

Sepsis is a life-threatening disease with a high mortality rate, for which the pathogenetic mechanism still unclear. DNA damage repair (DDR) is essential for maintaining genome integrity. This study aimed to explore the role of DDR-related genes in the development of sepsis and further investigated their molecular subtypes to enrich potential diagnostic biomarkers. Two Gene Expression Omnibus datasets (GSE65682 and GSE95233) were implemented to investigate the underlying role of DDR-related genes in sepsis. Three machine learning algorithms were utilized to identify the optimal feature genes. The diagnostic value of the selected genes was evaluated using the receiver operating characteristic curves. A nomogram was built to assess the diagnostic ability of the selected genes via "rms" package. Consensus clustering was subsequently performed to identify the molecular subtypes for sepsis. Furthermore, CIBERSORT was used to evaluate the immune cell infiltration of samples. Three different expressed DDR-related genes (GADD45A, HMGB2, and RPS27L) were identified as sepsis biomarkers. Receiver operating characteristic curves revealed that all 3 genes showed good diagnostic value. The nomogram including these 3 genes also exhibited good diagnostic efficiency. A notable difference in the immune microenvironment landscape was discovered between sepsis patients and healthy controls. Furthermore, all 3 genes were significantly associated with various immune cells. Our findings identify potential new diagnostic markers for sepsis that shed light on novel pathogenetic mechanism of sepsis and, therefore, may offer opportunities for potential intervention and treatment strategies.

Construction of a Multiple Cyclic Ligation-Promoted Exponential Recombinase Polymerase Amplification Platform for Sensitive and Simultaneous Monitoring of Cancer Biomarkers Fpg and FEN1.

Formamidopyrimidine DNA glycosylase (Fpg) and flap endonuclease 1 (FEN1) are essential to sustaining genomic stability and integrity, while the abnormal activities of Fpg and FEN1 may lead to various diseases and cancers. The development of simple methods for simultaneously monitoring Fpg and FEN1 is highly desirable. Herein, we construct a multiple cyclic ligation-promoted exponential recombinase polymerase amplification (RPA) platform for sensitive and simultaneous monitoring of Fpg and FEN1 in cells and clinical tissues. We designed two programmable substrate probes with 8-oxo-7,8-dihydroguanine (8-oxoG) damage sites and 5' flaps that can be identified/cleaved by Fpg and FEN1 to produce nicking sites. The juxtaposition of the cleavage sites is ligated by DNA ligase to form intact double-stranded DNA (dsDNA) templates that can be amplified via RPA to produce abundant dsDNA products labeled with Cy5 and Cy3 fluorophores and biotin, respectively. The resultant dsDNA can be captured by magnetic beads and subsequently disassembled into dispersed Cy3 and Cy5 molecules upon heat treatment, generating significant fluorescence signals. This assay exhibits a limit of detection of 1.12 × 10-10 U μL-1 for Fpg and 1.66 × 10-9 U μL-1 for FEN1, and it can be used for the analysis of enzymatic kinetic parameters, screening of inhibitors, and simultaneous monitoring of Fpg and FEN1 in a single cell and in clinic tissue samples. Moreover, the proposed strategy can be applied to monitor other DNA repair proteins by merely changing the recognition sites of dsDNA substrate probes, providing a promising platform for clinical diagnosis, biomedical research, and drug discovery.

RECQL4 requires PARP1 for recruitment to DNA damage, and PARG dePARylation facilitates its associated role in end joining

RecQ helicases, highly conserved proteins with pivotal roles in DNA replication, DNA repair and homologous recombination, are crucial for maintaining genomic integrity. Mutations in RECQL4 have been associated with various human diseases, including Rothmund–Thomson syndrome. RECQL4 is involved in regulating major DNA repair pathways, such as homologous recombination and nonhomologous end joining (NHEJ). RECQL4 has more prominent single-strand DNA annealing activity than helicase activity. Its ability to promote DNA damage repair and the precise role of its DNA annealing activity in DNA repair are unclear. Here we demonstrate that PARP1 interacts with RECQL4, increasing its single-stranded DNA strand annealing activity. PARP1 specifically promoted RECQL4 PARylation at both its N- and C-terminal regions, promoting RECQL4 recruitment to DNA double-strand breaks (DSBs). Inhibition or depletion of PARP1 significantly diminished RECQL4 recruitment and occupancy at specific DSB sites on chromosomes. After DNA damage, PARG dePARylated RECQL4 and stimulated its end-joining activity. RECQL4 actively displaced replication protein A from single-stranded DNA, promoting microhomology annealing in vitro. Furthermore, depletion of PARP1 or RECQL4 substantially impacted classical-NHEJ- and alternative-NHEJ-mediated DSB repair. Consequently, the combined activities of PARP1, PARG and RECQL4 modulate DNA repair.

The BLM-TOP3A-RMI1-RMI2 proximity map reveals that RAD54L2 suppresses sister chromatid exchanges.

Homologous recombination is a largely error-free DNA repair mechanism conserved across all domains of life and is essential for the maintenance of genome integrity. Not only are the mutations in homologous recombination repair genes probable cancer drivers, some also cause genetic disorders. In particular, mutations in the Bloom (BLM) helicase cause Bloom Syndrome, a rare autosomal recessive disorder characterized by increased sister chromatid exchanges and predisposition to a variety of cancers. The pathology of Bloom Syndrome stems from the impaired activity of the BLM-TOP3A-RMI1-RMI2 (BTRR) complex which suppresses crossover recombination to prevent potentially deleterious genome rearrangements. We provide a comprehensive BTRR proximal proteome, revealing proteins that suppress crossover recombination. We find that RAD54L2, a SNF2-family protein, physically interacts with BLM and suppresses sister chromatid exchanges. RAD54L2 is important for recruitment of BLM to chromatin and requires an intact ATPase domain to promote non-crossover recombination. Thus, the BTRR proximity map identifies a regulator of recombination.

How human papillomavirus (HPV) targets DNA repair pathways for viral replication: from guardian to accomplice.

SUMMARYHuman papillomaviruses (HPVs) are small DNA viruses that are responsible for significant disease burdens worldwide, including cancers of the cervix, anogenital tract, and oropharynx. HPVs infect stratified epithelia at a variety of body locations and link their productive life cycles to the differentiation of the host cell. These viruses have evolved sophisticated mechanisms to exploit cellular pathways, such as DNA damage repair (DDR), to regulate their life cycles. HPVs activate key DDR pathways such as ATM, ATR, and FA, which are critical for maintaining genomic integrity but are often dysregulated in cancers. Importantly, these DDR pathways are essential for HPV replication in undifferentiated cells and amplification upon differentiation. The ability to modulate these DDR pathways not only enables HPV persistence but also contributes to cellular transformation. In this review, we discuss the recent advances in understanding the mechanisms by which HPV manipulates the host DDR pathways and how these depend upon enhanced topoisomerase activity and R-loop formation. Furthermore, the strategies to manipulate DDR pathways utilized by high-risk HPVs are compared with those used by other DNA viruses that exhibit similarities and distinct differences.

On the origin of an insular hybrid butterfly lineage.

A new species can form through hybridization between species. Hybrid speciation in animals has been intensely debated, partly because hard evidence for the process has been difficult to obtain. Here we report the discovery of a European hybrid butterfly lineage, a finding that can be considered surprising given the intense and long-term study of European butterflies. The lineage we describe is mainly inhabiting an island in the Baltic Sea and was previously designated as a subspecies (horkei) of one of the parental species (Aricia artaxerxes). By analysing whole-genome resequencing data and developing a novel cluster analysis based on historical recombination events (Fisher junctions), we determine that horkei originated by hybridization between the non-sister species A. artaxerxes and A. agestis. This hybridization event occurred approximately 54,000 years ago, predating the last glaciation of the current distribution range. Horkei must therefore have persisted long enough to be able to colonize its current range, despite that this area lies between the current distributions of the parental species. The hybrid origin, the maintenance of genomic integrity across times of dramatic climate change and the expression of a combination of parental traits suggest that horkei could be in the process of hybrid speciation.