Search Results (78)

DNMT1 variants are linked to complex neurodegenerative syndromes, yet their variant-specific functional and epigenomic consequences remain poorly defined. DNMT1 variants were identified in eight patients using gene-panel or whole-exome sequencing. Functional effects were assessed by site-directed mutagenesis and transient expression in HEK293T cells. Genome-wide methylation profiling of peripheral blood leukocyte DNA was performed using Nanopore sequencing, enabling direct quantification of 5-methylcytosine (5mC). CpG island-level differential methylation and gene set enrichment analysis (GSEA) were conducted. Variants in the replication foci targeting sequence (RFTS) domain (p.Y511H, p.Y540C, p.H569R) exhibited reduced DNMT1 protein expression, decreased enzymatic activity, and cytosolic aggregation. Variants in the C-terminal catalytic domain (p.A1334V and p.P1546S) showed reduced protein expression with relatively mild enzymatic impairment. Patients carrying the p.Y511H variant demonstrated a significant reduction in global 5mC levels compared with controls. Principal component analysis revealed distinct methylomic profiles separating most patients from controls, with marked intra- and inter-familial heterogeneity. CpG island-level analysis identified a single significantly hypomethylated region in p.Y511H carriers, and GSEA revealed differential enrichment of multiple Gene Ontology biological pathways. This study defines domain-dependent functional effects of DNMT1 variants and provides the first nanopore-based methylome analysis, revealing variant-specific and heterogeneous epigenomic alterations. Full article

The SNF2-related chromatin remodeler Srcap is the principal ATPase responsible for the deposition of the histone variant H2A.Z at promoters and regulatory chromatin regions. Although this activity is known to modulate transcription, its contribution to DNA replication remains unexplored. Here we show that Srcap is required for efficient replication fork progression and origin firing in mammalian cells. Using RNA interference in human PC3 cells, we found that Srcap depletion leads to a ~25% reduction in fork elongation rate, decreased replication fork density, accumulation of the replication-stress marker γH2AX, and reduced chromatin-bound H2A.Z. High-resolution expansion microscopy further revealed diminished intensity and increased spacing of replication foci, consistent with reduced origin activation. Transcriptomic analysis of published data identified broad downregulation of replication-associated genes. These data uncover a dual mechanism by which Srcap sustains replication efficiency—through both H2A.Z-dependent chromatin organization and transcriptional maintenance of the replication machinery. Our findings establish Srcap as an important coordinator of replication dynamics, with implications for genome stability. Full article

Cellular senescence is a heterogeneous and dynamic state characterised by stable proliferation arrest, macromolecular damage and metabolic remodelling. Although markers such as SA-β-galactosidase staining, yH2AX foci and p53 activation are widely used as de facto standards, they are imperfect and differ in terms of sensitivity, specificity and dependence on context. We present a multifactorial imaging platform integrating scanning electron, flow cytometry and high-resolution confocal microscopy. This allows us to identify senescence phenotypes in three in vitro models: replicative ageing via serial passaging; dose-graded genotoxic stress under serum deprivation; and primary fibroblasts from young and elderly donors. We present a multimodal imaging framework to characterise senescence-associated phenotypes by integrating LysoTracker and MitoTracker microscopy and SA-β-gal/FACS, p16INK4a immunostaining provides independent confirmation of proliferative arrest. Combined nutrient deprivation and genotoxic challenge elicited the most pronounced and concordant organelle alterations relative to single stressors, aligning with age-donor differences. Our approach integrates structural and functional readouts across modalities, reducing the impact of phenotypic heterogeneity and providing reproducible multiparametric endpoints. Although the framework focuses on a robustly validated panel of phenotypes, it is extensible by nature and sensitive to distributional shifts. This allows both drug-specific redistribution of established markers and the emergence of atypical or transient phenotypes to be detected. This flexibility renders the platform suitable for comparative studies and the screening of senolytics and geroprotectors, as well as for refining the evolving landscape of senescence-associated states. Full article

Bacillus subtilis DinG/XPD-like paralogues, DinG and YpvA, have been implicated in overcoming replication stress. DinG possesses a DEDD exonuclease and DNA helicase domains, whereas YpvA lacks the DEDD exonuclease domain. We report that DinG·Mg²⁺ (hereafter referred to as DinG) degrades linear single-stranded (lss) DNA with 3′→5′ polarity and binds lssDNA with higher affinity than its exonuclease-deficient mutant DinG D10A E12A. DinG’s ssDNA-dependent ATPase activity neither stimulates nor inhibits DNA degradation. When bound to the 3′-end of forked DNA, DinG destabilises and degrades the substrate; however, in the presence of ATP, DinG dissociates before reaching the duplex junction. DinG degrades the RNA strand within RNA–DNA hybrids but does not cleave lssRNA unless complexed with Mn²⁺. DinG removes genomic R-loops, as RnhC and PcrA do. DinG physically interacts with RecA and PolA and functions in the same pathway as translesion synthesis (TLS) DNA polymerases (DNAPs) to respond to both spontaneous and methyl methanesulphonate (MMS)-induced mutagenesis. DinG-mGold forms spontaneous foci at or near replication forks, which become enriched following MMS or rifampicin treatment. We propose that DinG contributes to mitigating replication stress by degrading R-loop barriers and facilitating TLS, potentially via RecA-linked mechanisms. Full article

Cohesin organizes the genome into spatially segregated loops and topologically associated domains by loop extrusion. In addition, it ensures cohesion of sister chromatids after replication. Thus, cohesin is expected to limit chromatin dynamics by ensuring cohesion and compacting chromatin in the interphase. Nonetheless, loop extrusion is an example of chromatin dynamics; thus, cohesin could promote the dynamics of genomic loci at the scale of individual loops and contact domains. Moreover, given that the extruding activity of cohesin after replication is supplemented by its cohesive activity, the impact of cohesin on chromatin dynamics in different phases of the cell cycle may vary. Of particular interest is the cohesin’s role in the regulation of the dynamics of damaged chromatin, which remains insufficiently studied. Here, we visualized a genomic locus using the CRISPR-Sirius system in human cells with auxin-induced depletion of the cohesin subunit RAD21. Cohesin depletion increased the local spatial dynamics of the visualized locus on a time scale of fractions of a second to one minute. This effect was observed in both replicated and unreplicated chromatin. However, the increase in the mobility of the visualized locus upon cohesin depletion was more pronounced in the former. In addition, we showed that cohesin depletion did not affect the local mobility of double-strand break repair foci visualized using a fluorescent fragment of the repair factor 53BP1. Cohesin depletion did not affect the local mobility of repair foci in either replicated or unreplicated chromatin. The results indicate that cohesin constrains local spatial dynamics of genomic loci. At the same time, cohesive activity of cohesin is not indispensable for restricting chromatin dynamics, although it enhances the confinement effect. On the other hand, repair foci are less mobile structures than point chromatin loci, and cohesin does not affect their dynamics on the studied time scales. Full article

Hepatitis E virus (HEV) exists in two distinct forms: a non-enveloped form (neHEV), which is present in feces and bile, and a quasi-enveloped form (eHEV), found in circulating blood and culture supernatants. This study aimed to elucidate the roles of Ras-associated binding 13 (Rab13) and protein kinase A (PKA) in the entry mechanisms of both eHEV and neHEV, utilizing small interfering RNA (siRNA) and chemical inhibitors. The results demonstrated that the entry of both viral forms is dependent on Rab13 and PKA. Further investigation into the involvement of tight junction (TJ) proteins revealed that the targeted knockdown of zonula occludens-1 (ZO-1) significantly impaired the entry of both eHEV and neHEV. In addition, in ZO-1 knockout (KO) cells inoculated with either viral form, HEV RNA levels in culture supernatants did not increase, even up to 16 days post-inoculation. Notably, the absence of ZO-1 did not affect the adsorption efficiency of eHEV or neHEV, nor did it influence HEV RNA replication. In cell-to-cell spread assays, ZO-1 KO cells inoculated with eHEV showed a lack of expression of HEV ORF2 and ORF3 proteins. In contrast, neHEV-infected ZO-1 KO cells showed markedly reduced ORF2 and ORF3 protein expression within virus-infected foci, compared to non-targeting knockout (NC KO) cells. These findings underscore the crucial role of ZO-1 in facilitating eHEV entry and mediating the cell-to-cell spread of neHEV in infected cells. Full article

Rat Sarcoma (RAS)-driven cancers have been one of the main foci in the field of cancer science for over four decades. Despite significant improvement in understanding the biology of RAS oncogene, the method to target RAS-mutated cancers is still unclear. In recent years, the role for RAS beyond its hyperproliferation has been extensively documented. In this review, we systematically address and dwell on the details of the mechanisms of RAS oncogene-mediated alteration in the DNA replication and DNA damage response (DDR) pathways, focusing on lung cancers. We further extend this molecular connection towards cytosolic DNA accumulation, innate immune activation and senescence in RAS-addicted cancers. At the end, we briefly speculate on the potential strategies for targeting RAS mutated lung cancers, considering various approaches targeting DNA replication, DNA repair and the cGAS-STING pro-inflammatory pathway. These new lines of therapy, especially when used in combinations, may enhance treatment efficacy and overcome the challenges associated with these mutations. Full article

Firearm shooting performance training rightfully focuses on shooting accuracy; however, additional foci should include decision processing speed and reaction time associated with decision making to avoid reaction-only based shooting responses. While advancements in realistic training environments attempt to mimic “real-world” situations, many remain largely anticipatory or subject to a speed–accuracy trade-off (SAT). The purpose of this brief field trial was to demonstrate the feasibility of a random-action target system (RATS) on participants’ shooting performance (i.e., accuracy, omission, and commission rates) among a convenience sample of six retired police officers and competitive shooters (age range: 45–58 years, mean age = 52.5 ± 5.89). Observational data were gathered from a single-day, three-round trial to test shooting accuracy and shooting errors when shooters were unable to anticipate target appearance location and target exposure speed. In Trial 1, the target exposure time was 0.5 s, which increased to 0.7 s in Trial 2, and decreased back to 0.5 s in Trial 3. Shooting accuracy generally increased, while omission and commission generally decreased, when shooters were presented with targets exposed for longer durations. From Trial 1 to Trial 3 (both trials with 0.5 s target exposures), shooters showed higher median accuracy rates, lower median omission rates, and lower median commission rates. Findings suggest that a non-anticipatory, RATS holds promise for improving shooting performance and offset SAT among shooters with firearm experience. However, additional trials are needed with the RATS to replicate these findings among a larger and more diverse set of participants, who train with the RATS consistently, over longer durations. Full article

Survivin is known for its dual biological role in apoptosis inhibition and mitotic progression. In addition to its being part of the chromosomal passenger complex (CPC), recent findings suggest additional roles for Survivin in the DNA damage response, further contributing to therapy resistance. In this study, we investigated the role of Survivin and the CPC proteins in the cellular response to irradiation with a focus on DNA replication processes. As is known, ionizing radiation leads to an increased expression of Survivin and its accumulation in nuclear foci, which we now know to be specifically localized to centromeric heterochromatin. The depletion of Survivin and Aurora B increases the DNA damage marker γH2AX, indicative of an impaired repair capacity. The presence of Survivin and the CPC in nuclear foci that we already identified during the S phase co-localize with the proliferating cell nuclear antigen (PCNA), further implying a potential role during replication. Indeed, Survivin knockdown reduced replication fork speed as assessed via DNA fiber assays. Mechanistically, we identified a PIP-box motif in INCENP mediating the interaction with PCNA to assist in managing damage-induced replication stress. Survivin depletion forces cells to undergo unphysiological genome replication via mitotic DNA synthesis (MiDAS), resulting in chromosome breaks. Finally, we revealed that Aurora B kinase liberates Pol η by phosphorylating polymerase delta-interacting protein 2 (POLDIP2) to resume the replication of damaged sites via translesion synthesis. In this study, we assigned a direct function to the CPC in the transition from stalled replication forks to translesion synthesis, further emphasizing the ubiquitous overexpression of Survivin particularly in tumors. This study, for the first time, assigns a direct function to the chromosomal passenger complex, CPC, including Survivin, Aurora B kinase, Borealin, and INCENP, in the transition from stalled replication forks (involving PCNA binding) to translesion synthesis (liberating Pol η by phosphorylating POLDIP2), and thus in maintaining genomic integrity. Full article

The two basic approaches to elucidating how life began both date from Darwin. The first, that of the experimentalists, stems from Darwin’s famous “warm little pond” letter to Joseph Hooker of 1871. This approach, an attempt to replicate the sequential events leading to life’s origin, is exemplified by the “primordial soup” hypothesis of A.I. Oparin (1924) and J.B.S. Haldane (1929); the Miller–Urey laboratory synthesis of amino acids under possible primitive Earth conditions (1953); and Joan Oró’s nonbiological synthesis of the nucleic acid adenine (1959). The second approach, that of the observationalists who search for relevant evidence in the geological record, dates from Darwin’s 1859 On the Origin of Species, in which he laments the “inexplicable” absence of a pre-Cambrian fossil record. Darwin’s concern spurred a century of search that was ultimately rewarded by Stanley Tyler’s 1953 discovery of diverse microscopic fossils in the ~1900 Ma Gunflint Chert of southern Canada. Tyler’s find was soon followed by a cascade of discoveries worldwide; the establishment of a new field of science, Precambrian paleobiology; and, more recently, the discovery of 3400 and ~3465 Ma Paleoarchean microfossils, establishing that primordial life evolved early, far, and fast. Though progress has been made, much remains to be learned in the foci of this Origin of Life 2024 volume, for which this essay is the history-reviewing “stage setter”. Full article

The integrated stress response, especially stress granules (SGs), contributes to host immunity. Typical G3BP1⁺ stress granules (tSGs) are usually formed after virus infection to restrain viral replication and stimulate innate immunity. Recently, several SG-like foci or atypical SGs (aSGs) with proviral function have been found during viral infection. We have shown that the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid (N) protein induces atypical N⁺/G3BP1⁺ foci (N⁺foci), leading to the inhibition of host immunity and facilitation of viral infection. However, the precise mechanism has not been well clarified yet. In this study, we showed that the SARS-CoV-2 N (SARS2-N) protein inhibits dsRNA-induced growth arrest and DNA damage-inducible 34 (GADD34) expression. Mechanistically, the SARS2-N protein promotes the interaction between GADD34 mRNA and G3BP1, sequestering GADD34 mRNA into the N⁺foci. Importantly, we found that GADD34 participates in IRF3 nuclear translocation through its KVRF motif and promotes the transcription of downstream interferon genes. The suppression of GADD34 expression by the SARS2-N protein impairs the nuclear localization of IRF3 and compromises the host’s innate immune response, which facilitates viral replication. Taking these findings together, our study revealed a novel mechanism by which the SARS2-N protein antagonized the GADD34-mediated innate immune pathway via induction of N⁺foci. We think this is a critical strategy for viral pathogenesis and has potential therapeutic implications. Full article

Background: Cellular senescence in response to ionizing radiation (IR) limits the replication of damaged cells by causing permanent cell cycle arrest. However, IR can induce pro-survival signaling pathways that reduce the extent of radiation-induced cytotoxicity and promote the development of radioresistance. The differential incorporation of histone variant H2A.J has profound effects on higher-order chromatin organization and on establishing the epigenetic state of radiation-induced senescence. However, the precise epigenetic mechanism and function of H2A.J overexpression in response to IR exposure still needs to be elucidated. Methods: Primary (no target, NT) and genetically modified fibroblasts overexpressing H2A.J (H2A.J-OE) were exposed to 20 Gy and analyzed 2 weeks post-IR for radiation-induced senescence by immunohistochemistry and immunofluorescence microscopy. Transcriptome signatures were analyzed in (non-)irradiated NT and H2A.J-OE fibroblasts by RNA sequencing. Since H2A.J plays an important role in the epidermal homeostasis of human skin, the oncogenic potential of H2A.J was investigated in cutaneous squamous cell carcinoma (cSCC). The tissue microarrays of cSCC were analyzed for H2A.J protein expression pattern by automated image analysis. Results: In response to radiation-induced DNA damage, the overexpression of H2A.J impairs the formation of senescence-associated heterochromatin foci (SAHF), thereby inhibiting the SAHF-mediated silencing of proliferation-promoting genes. The dysregulated activation of cyclins and cyclin-dependent kinases disturbs cell cycle arrest in irradiated H2A.J-OE fibroblasts, thereby overcoming radiation-induced senescence. Comparative transcriptome analysis revealed significantly increased WNT16 signaling in H2A.J OE fibroblasts after IR exposure, promoting the fundamental mechanisms of tumor development and progression, including the activation of the epithelial–mesenchymal transition. The quantitative analysis of cSCCs revealed that undifferentiated tumors are associated with high nuclear H2A.J expression, related with greater oncogenic potential. Conclusion: H2A.J overexpression induces radioresistance and promotes oncogenic transformation through the activation of WNT16 signaling pathway functions. H2A.J-associated signatures may improve risk stratification by identifying patients with more aggressive cSCC who may require radiotherapy with increased doses. Full article

Recurrent computed tomography (CT) examination has become a common diagnostic procedure for several diseases and injuries. Though each singular CT scan exposes individuals at low doses of low linear energy transfer (LET) radiation, the cumulative dose received from recurrent CT scans poses an increasing concern for potential health risks. Here, we evaluated the biological effects of recurrent CT scans on the DNA damage response (DDR) in human fibroblasts and retinal pigment epithelial cells maintained in culture for five months and subjected to four CT scans, one every four weeks. DDR kinetics and eventual accumulation of persistent-radiation-induced foci (P-RIF) were assessed by combined immunofluorescence for γH2AX and 53BP1, i.e., γH2AX/53BP1 foci. We found that CT scan repetitions significantly increased both the number and size of γH2AX/53BP1 foci. In particular, after the third CT scan, we observed the appearance of giant foci that might result from the overlapping of individual small foci and that do not associate with irreversible growth arrest, as shown by DNA replication in the foci-carrying cells. Whether these giant foci represent coalescence of unrepaired DNA damage as reported following single exposition to high doses of high LET radiation is still unclear. However, morphologically, these giant foci resemble the recently described compartmentalization of damaged DNA that should facilitate the repair of DNA double-strand breaks but also increase the risk of chromosomal translocations. Overall, these results indicate that for a correct evaluation of the damage following recurrent CT examinations, it is necessary to consider the size and composition of the foci in addition to their number. Full article

Negative-strand RNA viruses form cytoplasmic inclusion bodies (IBs) representing virus replication foci through phase separation or biomolecular condensation of viral and cellular proteins, as a hallmark of their infection. Alternatively, mammalian cells form stalled mRNA containing antiviral stress granules (SGs), as a consequence of phosphorylation of eukaryotic initiation factor 2α (eIF2α) through condensation of several RNA-binding proteins including TIA-1. Whether and how Chandipura virus (CHPV), an emerging human pathogen causing influenza-like illness, coma and death, forms IBs and evades antiviral SGs remain unknown. By confocal imaging on CHPV-infected Vero-E6 cells, we found that CHPV infection does not induce formation of distinct canonical SGs. Instead, CHPV proteins condense and co-localize together with SG proteins to form heterogeneous IBs, which ensued independent of the activation of eIF2α and eIF2α kinase, protein kinase R (PKR). Interestingly, siRNA-mediated depletion of PKR or TIA-1 significantly decreased viral transcription and virion production. Moreover, CHPV infection also caused condensation and recruitment of PKR to IBs. Compared to SGs, IBs exhibited significant rapidity in disassembly dynamics. Altogether, our study demonstrating that CHPV replication co-optimizes with SG proteins and revealing an unprecedented proviral role of TIA-1/PKR may have implications in understanding the mechanisms regulating CHPV-IB formation and designing antiviral therapeutics. Importance: CHPV is an emerging tropical pathogen reported to cause acute influenza-like illness and encephalitis in children with a very high mortality rate of ~70%. Lack of vaccines and an effective therapy against CHPV makes it a potent pathogen for causing an epidemic in tropical parts of globe. Given these forewarnings, it is of paramount importance that CHPV biology must be understood comprehensively. Targeting of host factors offers several advantages over targeting the viral components due to the generally higher mutation rate in the viral genome. In this study, we aimed at understanding the role of SGs forming cellular RNA-binding proteins in CHPV replication. Our study helps understand participation of cellular factors in CHPV replication and could help develop effective therapeutics against the virus. Full article

Cellular senescence is an irreversible growth arrest that acts as a barrier to cancer initiation and progression. Histone alteration is one of the major events during replicative senescence. However, little is known about the function of H3.3 in cellular senescence. Here we found that the downregulation of H3.3 induced growth suppression with senescence-like phenotypes such as senescence-associated heterochromatin foci (SAHF) and β-galactosidase (SA-β-gal) activity. Furthermore, H3.3 depletion induced senescence-like phenotypes with the p53/p21-depedent pathway. In addition, we identified miR-22-3p, tumor suppressive miRNA, as an upstream regulator of the H3F3B (H3 histone, family 3B) gene which is the histone variant H3.3 and replaces conventional H3 in active genes. Therefore, our results reveal for the first time the molecular mechanisms for cellular senescence which are regulated by H3.3 abundance. Taken together, our studies suggest that H3.3 exerts functional roles in regulating cellular senescence and is a promising target for cancer therapy. Full article