PLOS Genetics: New Articles

The regulation of Xrp1 expression by uORFs and main ORF sequences and its function in Drosophila disease models

2026-06-10T14:00:00Z

by Hidetaka Katow, Thao Nguyen, Sarah Hyunsoh Park, Hyung Don Ryoo

The Integrated Stress Response (ISR) mediates cellular adaptation to endoplasmic reticulum (ER) stress, amino acid deprivation, and mitochondrial dysfunction. The ISR regulates gene expression in part by preferentially translating the transcription factor ATF4, a process regulated by upstream open reading frames (uORFs) in its 5’ leader. In Drosophila, Xrp1 is another transcription factor induced during the ISR, but the precise underlying mechanism remains unclear. Here, we report that Xrp1 induction in response to ER stress is regulated by both its uORFs and the main ORF sequence. Xrp1 has seven splice isoforms, and the two predominant transcripts expressed in eye imaginal discs contain uORFs. Expressing the ER stress-imposing ninaE^G69D transgene in this tissue induced Xrp1 expression without significantly changing the Xrp1 splice isoform composition. The uORF-containing 5’ leaders, particularly the AUG codon of the second uORF, inhibited DsRed expression when placed upstream of the reporter. Unlike ATF4, the uORF-containing 5’ leader alone was insufficient to mediate the main ORF induction, but Xrp1 induction occurred in ninaE^G69D-expressing discs when Xrp1’s 5’ leader and the main ORF sequence were both present. Functionally, Xrp1 was required to maintain the integrity of Drosophila photoreceptors exposed to constant light. In a different disease model, parkin mutants activated Xrp1 target gene expression in specific tissues and Xrp1 loss enhanced the viability of parkin mutant flies during adult eclosion. These results provide molecular and pathological insights into Xrp1 regulation and function in disease models.

The importance of nonsense errors: Estimating the rates and implications of ribosome drop-off during protein synthesis

2026-06-09T14:00:00Z

by Alexander L. Cope, Denizhan Pak, Michael A. Gilchrist

The process of translation is both energetically costly and relatively error-prone compared to transcription and replication. Nonsense errors during translation occur when a ribosome drops off a transcript before reaching a stop codon, resulting in energetic investment in an incomplete and likely non-functional protein. Nonsense errors impose a potentially significant energy burden on the cell, making it critical to quantify their frequency and energetic cost. Here, we present a model of ribosome movement for estimating protein production, elongation, and nonsense error rates from high-throughput ribosome profiling data. Applying this model to an exemplary ribosome profiling dataset in S. cerevisiae, we find that nonsense error rates vary substantially between codons and that these types of errors place an energetic burden on cells comparable to ribosome pausing. Overall, we present multiple lines of evidence that selection against nonsense errors is a prominent force shaping protein-coding sequence evolution and codon usage bias, in particular.

Rv3839-Rv3840 links the endogenous heme biosynthesis pathway with Mycobacterium tuberculosis adaptation to nitric oxide and iron limitation stress

2026-06-08T14:00:00Z

by Natalia F. Quirk, Kate N. Gregory, Yasu S. Morita, Shumin Tan

During infection, Mycobacterium tuberculosis (Mtb) encounters multiple environmental stressors, including nitric oxide (NO) and iron limitation, and an ability to mount an integrated response is essential for the bacterium’s adaptation and continued survival. Iron-containing prosthetic groups in key enzymes are critical for Mtb sensing and detoxification of NO, and there is significant overlap between NO- and low iron-responsive genes. However, how Mtb adapts to these two stressors concurrently is largely unknown. Here, we find that exposure to NO globally augments expression of low iron-responsive genes and vice versa, with a two gene operon, rv3839-rv3840, among the most highly upregulated. Deletion of rv3839-rv3840 resulted in increased growth under prolonged iron limitation and early exit of Mtb from an adaptive state of growth arrest induced upon exposure to NO/low iron. ∆rv3839-rv3840 Mtb exhibited an elongated cell morphology compared to wild type Mtb in NO/low iron conditions, indicating effects of this operon on cell growth and division under stress conditions, with Rv3839 as the key driver of this phenotype. Coproporphyrin III tetramethyl ester (TMC), a modified precursor molecule in the endogenous Mtb heme biosynthesis pathway, was found to accumulate in ∆rv3839-rv3840 Mtb under iron limiting conditions. Further, intrabacterial heme levels were increased in ∆rv3839-rv3840 Mtb under NO stress and iron limitation. Together, these findings reveal Rv3839-Rv3840 as proteins involved in the downregulation of heme biosynthesis under NO stress and iron limitation, and highlight the link between Mtb growth control in response to NO/low iron and endogenous heme biosynthesis.

Tbx16 and mesogenin 1 promote presomitic mesoderm differentiation by repressing the mesodermal progenitor cell state

2026-06-08T14:00:00Z

by Guoyu Zhu, Miriam A. Genuth, Yanrong Xiao, Abigail A. Kindberg, Kayleigh Hackett, Scott A. Holley

During zebrafish embryonic body elongation, differentiation of mesodermal progenitors into presomitic mesoderm requires the transcription factors tbx16 and mesogenin 1. Here, by using temporally controlled tbx16 and mesogenin 1 overexpression and RNAseq to identify immediate downstream changes in gene expression, we elucidate how these genes promote presomitic mesoderm differentiation. Using machine learning and game theory, we integrated differentially expressed genes with wild-type scRNAseq data and identified genes downstream of tbx16 and mesogenin 1 during mesoderm differentiation. This data-driven analysis indicates that mesogenin 1 and tbx16 primarily repress expression of genes as mesodermal progenitors differentiate. Strikingly, the genes that are most important for defining transcriptional cell states during mesoderm differentiation are most strongly repressed by tbx16 and mesogenin 1. Moreover, these downstream effectors are enriched for genes with known roles in mesoderm development and body elongation such as Fgf, Wnt and Bmp pathways and the transcription factors tbxta, eve1, hoxd12a, hoxd13b, lef1, cdx4, tbx16l, ved, vent and vox. Gradients of Fgf and Wnt specify the mesodermal progenitor state in the posterior tailbud and activate many of these transcription factors indicating that tbx16 and mesogenin 1 promote mesoderm differentiation by repressing this progenitor state.

RNA Polymerase III subunit Polr3a is required for craniofacial cartilage and bone development in zebrafish

2026-06-08T14:00:00Z

by Bailey T. Lubash, Roxana Gutierrez, Nicole A. Hansen, Kade Fink, Colette A. Hopkins, Lauren B. Sands, Jessica C. Nelson, Kristin E. N. Watt

Transcription by RNA Polymerase III (Pol III) is essential for ribosome biogenesis and translation in all cells, but pathogenic variants in genes encoding subunits of Pol III lead to tissue-specific phenotypes including craniofacial differences. To understand the function of Pol III in craniofacial development, we examined polr3a mutant zebrafish. These mutants display hypoplasia of the neural crest cell-derived craniofacial cartilage and bone but, surprisingly, no significant changes were observed in neural crest cell proliferation or survival during embryogenesis. At larval stages, increased cell death was observed throughout the head, including in the craniofacial cartilage. These changes coincide with reduced transcription of transfer RNAs and reduced ribosome biogenesis in polr3a mutant zebrafish. To determine tissue-specific transcriptional changes, we performed single-cell RNA-sequencing. Analysis revealed both global and cartilage-specific changes, including upregulation of tp53. However, Tp53 inhibition alone was not sufficient to rescue craniofacial cartilage and bone, indicating that additional factors are important to support cartilage and bone growth in polr3a mutants. Altogether, our study provides new mechanistic insights into the functions of Pol III in craniofacial development.

Promotion of shade avoidance by BBX5 involves activation of PIF4 along with auxin biosynthetic and signaling genes

2026-06-05T14:00:00Z

by Fengyue Zhao, Rongbo Yang, Zhaoqing Song, Yeting Bian, Yuntao Xiao, Dongqing Xu

Neighbor proximity triggers changes in light quality that regulate various developmental and physiological processes in plants. phytochrome B (phyB)-PHYTOCHROMEINTERACTING FACTOR 4 (PIF4) module serves as a central regulatory hub enabling plants to accurately perceive and respond to shade cues. Here, we identify B-box PROTEIN 5 (BBX5) as a positive regulator of shade avoidance. phyB interacts with BBX5 and promotes its protein stability. Conversely, the E3 ubiquitin ligase CONSTITUTIVELY PHOTOMORPHOGENICLY 1 (COP1) associates with and destabilizes BBX5 via the 26S proteasome system in shade. BBX5 binds to the PIF4 promoter to upregulate its expression during the early phase of shade exposure, and directly associates with the promoters of auxin biosynthetic and signaling genes YUCCA8 (YUC8) and INDOLE-3-ACETIC ACID INDUCIBLE 19 (IAA19) to activate their expression in shade. Our study reveals that BBX5 acts as a transcriptional activator of PIF4, YUC8 and IAA19 to promote plant growth and development in response to shade signals.

Chromatin insulators homie and nhomie can interact with distant copies either together or separately, with distinct outcomes for enhancer-promoter interactions

2026-06-05T14:00:00Z

by Miki Fujioka, Wenfan Ke, Paul Schedl, James B. Jaynes

Chromatin insulators, a.k.a. boundary elements, separate regions of the chromosome with distinct chromatin characteristics, including distinct histone modifications. This activity affects gene expression by allowing chromatin domains to be stably regulated and maintained. Insulators also block enhancer-promoter interactions and, somewhat paradoxically, facilitate other interactions, particularly when they stitch together distant regions of the chromosome by pairing with specific partners. Here we explore how long-range interactions facilitated by insulator pairing are affected by the presence of two potentially competing partners. Our results show that when two partners are present, they can reduce each other’s effects on distant gene expression, suggesting that enhancer-promoter interactions are best facilitated by pairwise insulator interactions. When a distant copy of an eve insulator (homie or nhomie) is present, it can interact with either or both endogenous insulators. But when one endogenous insulator is removed, the remaining one interacts more strongly with the transgenic copy, biasing the induced enhancer-promoter interactions toward those nearest the remaining endogenous insulator. On the other hand, physical interaction data suggest that strictly pairwise interactions are not the rule, suggesting a more complex model involving tripartite interactions. We further show that removing one or both endogenous eve insulators significantly reduces endogenous eve function at a critical early stage of development, and that the eve Polycomb domain expands in both directions when its insulator boundaries are removed, showing that insulators in their native context are required for each of the main functions that have been ascribed to them based on transgene assays.

The stoichiometry of minor-to-major pilins regulates the dynamic activity of the type IVa competence pilus in Vibrio cholerae

2026-06-04T14:00:00Z

by Nicholas D. Christman, Triana N. Dalia, Jennifer L. Chlebek, Ankur B. Dalia

Type IVa pili (T4aP) are bacterial surface appendages that perform various functions including twitching motility, reversible surface attachment, microcolony formation, surface sensing, and DNA uptake for natural transformation. Pivotal to each of these functions is the ability of T4aP to be dynamically extended and retracted from the cell surface. However, the factors that regulate this dynamic activity remain poorly understood in most systems. To address this question, we employ the competence T4aP from Vibrio cholerae as a model system. T4aP are composed of major and minor pilin subunits, named based on their relative abundance in the pilus filament. Prior work has established that minor pilins form a complex that initiates T4aP assembly. This allows for the subsequent addition of major pilins to the filament, which promotes T4aP extension. Here, we uncover that the stoichiometry of minor-to-major pilins is a crucial determinant of T4aP dynamic activity. Specifically, we show that either 1) overexpressing minor pilins or 2) underexpressing the major pilin results in a dramatic increase in the frequency of T4aP dynamics. These results indicate that the stoichiometry of minor-to-major pilins, not their absolute abundance, is one mechanism that regulates T4aP dynamic activity.

Barrier-to-autointegration factor protects against the cGAS-STING response to chromatin bridges

2026-06-03T14:00:00Z

by Laura Chastant, Karine Normandin, Firas El-Mortada, Marc J. Servant, Vincent Archambault

Cellular damage or stress can lead to disorganization, mislocalization or damage to self-DNA that can activate intracellular innate immune response mechanisms. Micronuclei, such as can occur following mitotic defects, have been proposed as a source of DNA capable of activating the cGAS-STING pathway, resulting in IRF3-dependent proinflammatory transcription. However, to what extent micronuclei per se or other concurrent defects contribute to the cGAS-STING response remains unclear. To better understand the ability of post-mitotic defects to induce this response, we compared the effects resulting from inhibition of the Spindle-Assembly Checkpoint (through MPS1 inhibition) or interference with nuclear reassembly (through inactivation of BAF). We found that combining both perturbations synergistically enhances the cGAS-STING response. This effect is not due to an increase in post-mitotic nuclear deformations including micronucleation and lobulation but instead correlates with an increase in destabilized chromatin bridges resulting in structures that potently recruit cGAS. Our results suggest that by stabilizing chromatin bridges, BAF contributes to preventing their degeneration into cGAS-activating chromatin structures. This work helps better understand how the innate immune system detects mitotic defects.

Epistasis among clustered lineage-specific amino acid substitutions in the Drosophila Trio protein

2026-06-03T14:00:00Z

by Flora Borne, Andrew M. Taverner, Peter Andolfatto

Intramolecular epistasis is increasingly recognized as a key factor shaping patterns of evolutionary rate variation among protein sites and constraining adaptive evolution. While genome-wide analyses have revealed that intramolecular epistatic interactions can drive the spatial clustering of amino acid substitutions, direct empirical evidence for such interactions and their evolutionary consequences remains limited. Using a population genetic screen for spatially-clustered and lineage-specific adaptive amino acid substitutions in Drosophila proteins, we systematically identify experimentally tractable candidates for functional analysis. As proof of concept, we focus on the Trio protein, a Rho guanine nucleotide exchange factor that exhibits three spatially-clustered putatively adaptive amino acid substitutions in the D. melanogaster lineage. By systematically reconstructing evolutionary intermediates in vivo using genome editing, we find that all possible intermediate states exhibit reduced viability and/or locomotor defects, providing strong evidence for epistatic constraints on evolutionary trajectories. Notably, these deleterious effects are recessive, suggesting that intermediate combinations of epistatically interacting amino acid substitutions can accumulate in heterozygotes prior to fixation, thereby circumventing apparent constraints imposed by maladaptive intermediate states. Together, these findings provide a rare empirical view of the fitness landscape shaped by intramolecular epistasis and establish a framework for investigating the constraints on adaptive protein evolution in diploid multicellular organisms.

Dual-function enzyme acts as a global c-di-GMP sink and local anti sigma factor antagonist to drive cellular differentiation

2026-06-03T14:00:00Z

by Buse Cınar Cakmak, Johanna D. Saric, Katrin Wrede, Neil A. Holmes, Julian Haist, Maria A. Schumacher, Mark J. Buttner, Natalia Tschowri

Soil bacteria of the genus Streptomyces are natural producers of over two-thirds of clinically used antibiotics. Their ability to generate these valuable metabolites is tightly linked to a developmental program involving the transition from vegetative hyphae to spores. The second messenger cyclic di-GMP (c-di-GMP) stabilizes effector complexes that block sporulation, including the RsiG-σ^WhiG complex leading to sequestration of the developmental sigma factor by its anti sigma factor. How signal termination and disruption of effector complexes is achieved to allow sporulation, remains poorly understood. Here, we identify the phosphodiesterase RmdB as a dual-function regulator that terminates c-di-GMP signaling both globally and locally. We show that deletion of the rmdB gene leads to increase of the global c-di-GMP pool and delayed development. Using genetic complementation, we demonstrate that both the EAL motif and the GGDEF domain are essential for the physiological function of RmdB. Our co-immunoprecipitation and co-elution assays revealed that RmdB interacts directly with the sigma factor σ^WhiG via its GGDEF domain, thus preventing binding of the anti sigma factor RsiG to σ^WhiG and promoting sporulation. Our bacterial two-hybrid analyses identify RmdB as an interaction hub connecting to multiple diguanylate cyclases (DGCs), including CdgE, which also interacts with σ^WhiG. These findings establish a novel principle of bacterial signaling in which a phosphodiesterase serves as an antagonist of an anti sigma factor, integrating global second messenger degradation with local effector complex formation to control cell fate decisions.

Canalization of neural dynamics by δ-protocadherins in the developing zebrafish optic tectum

2026-06-01T14:00:00Z

by Sayantanee Biswas, Michelle R. Emond, Grace S. Philip, James D. Jontes

Brain dynamics are constrained by the underlying topology of neuronal networks. How genes collaborate to organize these neural networks during development remains an enduring mystery. In humans, large numbers of genes have been implicated in neurodevelopmental disorders that are characterized by variable and overlapping phenotypes. The complexity of the brain and the heterogeneity of the disorders makes understanding the relationships between genes, development and neural function challenging. Beginning in the 1940s, Waddington suggested the concept of canalization to describe the role of genes as buffering developmental trajectories against genetic and environmental variation, leading to precise outcomes. Here, we show that members of the δ-protocadherin family of homophilic cell adhesion molecules, Protocadherin-19 and Protocadherin-17, contribute to developmental canalization of neural dynamics in the visual system of larval zebrafish. We provided oriented visual stimuli to zebrafish larvae and performed in vivo 2-photon calcium imaging in the optic tectum. The latent dynamics resulting from the population activity were remarkably conserved among different wild type larvae, allowing quantitative comparisons within and among genotypes. In both Protocadherin-19 and Protocadherin-17 mutants, the latent dynamics diverged stochastically from wild type, suggesting that the loss of these adhesion molecules leads to stochastic phenotypic variability and introduced disruptions of circuit organization that varied among individual mutants. These results are consistent with the developmental canalization of a vertebrate neural circuit, and suggest a framework for understanding the observed variability in complex brain disorders.

Sequence context and methylation interact to shape germline mutation rate variation at CpG sites

2026-06-01T14:00:00Z

by Sheel Chandra, Ziyue Gao

A prominent example of sequence context-dependent mutation rate variation is the elevated transition rate at CpG sites, which is largely attributed to cytosine methylation. CpGs with different flanking sequences also exhibit mutation rate variation, but this variation is only partially correlated with context-specific methylation level. Here, we quantify the CpG mutation rate and mutagenic effect of methylation across sequence contexts. Using a regression framework that accounts for recurrent mutations, we analyze human polymorphisms from the gnomAD dataset to estimate mutation rates of unmethylated and methylated CpGs separately in each unique 4-mer or 6-mer context. We find that CpG mutation rate variation in the human genome is shaped by methylation at the focal cytosine, the flanking nucleotides, and interactions between them, suggesting distinct context-dependent mutation patterns for unmethylated and methylated cytosines. Our analysis further reveals that the context effects are driven by largely independent effects of upstream and downstream sequences. Notably, an upstream adenine markedly increases CpG mutation rates regardless of methylation status or downstream sequences. Furthermore, upstream and downstream sequences have similar effects in chimpanzee and rhesus macaque, indicating that some conserved, intrinsic sequence features shape CpG mutability. On the other hand, some inter-species differences, which are especially pronounced at methylated sites on the chimpanzee lineage, point to recent evolutionary changes, possibly in context-specificity of proteins governing DNA demethylation and repair processes.

Yap1 regulates motility and vertebral development and prevents kyphoscoliosis in zebrafish

2026-05-28T14:00:00Z

by Victoria C. Williams-Ward, Kees Wanders, Simon M. Hughes

Scoliosis affects 2–3% of people, often developing during and after adolescence, and currently has a lifetime chance of surgical intervention of ~0.1% in high income countries. Understanding of causal genetic and environmental factors is improving, with mechanical feedback interactions between the neuromuscular and skeletal systems thought to be important. While examining mechanosignalling in the zebrafish musculoskeletal system, we observed transient expression of yap1 mRNA in precursor cells of muscle and notochord and wwtr1 mRNA accumulation in differentiated muscle. Yap1 and Wwtr1/Taz are transcriptional coactivators that mediate Hippo pathway signalling, often in response to mechanosignals. Loss of function mutation of either gene alone transiently altered early larval motility and reduced survival to adulthood, but mutation of yap1 specifically diminished overall growth without an obvious histological muscle defect. Yap1 mutants had a temperature-sensitive phenotype of oedema in cardiac and other tissues, which could be rescued by rearing at low temperature. Rescued yap1 mutants showed focal defects in hypochordal col8a1a mRNA expression at 1–2 days post-fertilisation (dpf), an early motility defect at 5 dpf and subsequently developed a fully penetrant vertebral dysmorphology, reflected by a decrease in posterior vertebral height. Thereafter, frank kyphoscoliosis accompanied by additional vertebral defects developed in around a third of the surviving yap1 mutants and was first detected at 11 dpf. Thus, the mild initial vertebral defect can, in a predisposing genetic or environmental background, gradually develop into full kyphoscoliosis through a positive feedback mechanism, analogous to the Hueter-Volkmann ‘Law’. Although the cell type/s of cell autonomous yap1 action remain unclear, we hypothesise that Yap1 mechanosensation mediates feedback between bone, muscle and tendon to restrain vertebral overgrowth and protect against the development of kyphoscoliosis.

Species-specific coevolution of RecA–RecN interfaces governs DNA double-strand break repair in Escherichia coli

2026-05-28T14:00:00Z

by Mizuki Inoue, Genki Akanuma, Masafumi Hayashi, Takashi Hishida

DNA double-strand breaks (DSBs) threaten genome stability and cell survival but can be faithfully repaired through homologous recombination (HR). RecN, a bacterial protein closely related to the structural maintenance of chromosomes family, cooperates with RecA in HR-dependent DSB repair, yet the molecular basis and physiological relevance of their interaction remain unclear. Here, we investigated the functional interplay between RecA and RecN during DSB repair by heterologously expressing Pseudomonas aeruginosa RecA (paRecA) and RecN (paRecN) in Escherichia coli. We found that in E. coli ∆recA ∆recN cells, co-expression of paRecA and paRecN fully restored MMC resistance, whereas co-expression of E. coli RecA (ecRecA) with paRecN conferred partial resistance to mitomycin C (MMC), demonstrating species-specific compatibility. Expression analysis revealed that paRecN was poorly expressed in E. coli, but codon optimization significantly enhanced its abundance and repair activity. We further identified gain-of-function paRecN mutants (I73T and R453H) that restored repair without increased expression. These mutants displayed species-specific adaptation, which improved compatibility with ecRecA but reduced functionality with paRecA. Fluorescence microscopy revealed that MMC-induced nucleoid localization was increased in paRecN^I73T and paRecN^R453H compared with paRecN. Collectively, these findings demonstrate that coevolution optimizes the RecA–RecN interface to ensure efficient DSB repair.

FabF and FadM cooperate to recycle fatty acids and rescue ∆plsX lethality in Staphylococcus aureus

2026-05-27T14:00:00Z

by Paprapach Wongdontree, Milya Palmier, Clara Louche, Vincent Leguillier, Carine Machado Rodrigues, Karine Gloux, David Halpern, Céline Henry, Jamila Anba-Mondoloni, Alexandra Gruss

Phospholipids are essential components of most cell membranes. In Staphylococcus aureus, PlsX acyltransferase is considered indispensable for initiating phospholipid synthesis, unless exogenous fatty acids (FAs) are available to bypass this requirement. We report that S. aureus can capture internal FA sources to overcome PlsX essentiality in a ∆plsX mutant via point mutations in either of two genes: fabF, which encodes the FA synthesis enzyme 3-oxoacyl-(acyl-carrier-protein) synthase II, or fadM, which encodes an understudied bifunctional acyl-CoA thioesterase and ACP binding protein. Despite growth rescue, both ∆plsX suppressors differ from the parental strain by producing phospholipids with shortened FA lengths, suggesting that both suppressors lead to premature FA release during synthesis. Additionally, both suppressors display increased sensitivity to β-lactam antibiotics. The similar behavior of both suppressors led us to show that fabF suppressors require the presence of fadM, indicative of FabF-FadM cooperation. We propose that reduced processivity of FabF suppressor variants, or greater availability of FadM for ACP binding in FadM variants, facilitates FA release from FabF-acyl-ACP intermediates. A FabF-FadM relay leading to FA release may contribute to homeostasis between FASII and phospholipid synthesis pathways.

Predicting antifolate resistance in the unculturable fungal pathogen Pneumocystis jirovecii

2026-05-27T14:00:00Z

by Francois D. Rouleau, Alexandre K. Dubé, Alicia Pageau, Lyne Désautels, Philippe J. Dufresne, Christian R. Landry

Pneumocystis jirovecii is an opportunistic fungal pathogen responsible for Pneumocystis pneumonia (PCP) in immunocompromised patients. Antifolate drugs targeting the dihydrofolate reductase (DHFR), including trimethoprim (TMP), remain central to treatment, but studying the effects of mutations in DHFR on resistance to treatment is limited by our inability to culture this organism in vitro or in animal models. We expressed P. jirovecii DHFR (PjDHFR) in Saccharomyces cerevisiae and performed deep mutational scanning (DMS) on this protein to measure the effects of all single amino-acid substitutions on enzyme function and resistance to methotrexate (MTX), a model antifolate which shares structural features with TMP. We integrated experimental results with structural and evolutionary features from multiple biophysical modeling approaches, and by using an interpretable machine-learning framework, we trained a random forest model to classify MTX resistance-conferring mutations in PjDHFR. We then leveraged this framework as a prediction tool to model the effects of mutations on resistance to TMP, which cannot be directly assayed experimentally. Functional measurements from DMS were the strongest contributors to resistance prediction and generally outperformed purely computational features. Resistance-conferring mutations were constrained by function, revealing a functional–resistance trade-off within this essential protein. Feature contribution analyses highlighted key predictors such as distance to ligand, flexibility, stability, and functional trade-off as determinants of resistance. When extrapolated to TMP, the model identified candidate resistance mutations consistent with known biochemical constraints of DHFR. We demonstrate how experimentally measured functional landscapes can be combined with biophysical modeling to help understand and predict antifolate resistance in an unculturable fungal pathogen. Our results provide biological insight into the constraints affecting the evolution of resistance in PjDHFR, and support that resistance arises from mutations altering drug interactions while preserving function. We illustrate how DMS data can enable generalizable, mechanistically interpretable models of drug resistance across structurally related antifolates.

mFABIO: An integrative multi-tissue TWAS fine-mapping approach to prioritize potentially causal genes and tissues underlying binary traits

2026-05-27T14:00:00Z

by Haihan Zhang, Kevin He, Lam C. Tsoi, Xiang Zhou

Recent advances in transcriptome-wide association study (TWAS) fine-mapping have enabled the joint modeling of multiple genes to improve causal gene prioritization. However, existing methods have been developed primarily for quantitative traits and most of them rely on gene expression data from a single tissue. Here, we present mFABIO, a multi-tissue TWAS fine-mapping method specifically designed for binary traits. mFABIO employs a probit model to directly link genetically regulated expression (GReX) of genes within a locus across multiple tissues to a binary outcome, while accounting for correlations in GReX across genes and tissues. As a result, mFABIO offers substantial power gains for binary traits, while maintaining robust control of false discovery rates (FDR). We evaluated mFABIO through extensive simulations and applied it to an in-depth analysis of six binary disease traits (asthma, breast cancer, gout, hypertension, prostate cancer, and rheumatoid arthritis) in the UK Biobank, using expression data spanning 38 Genotype-Tissue Expression (GTEx) tissues. mFABIO identified an average of 42 likely causal genes and 65 tissue-gene pairs per disease (FDR < 0.05). Notably, 60.9% of the genes and 77.2% of the gene-tissue pairs were supported by existing TWAS or GWAS evidence. This represented at least a 14.9% increase in evidence-supported genes and a 14.8% increase in evidence-supported gene-tissue pairs, compared to existing approaches. Additionally, mFABIO was also able to narrow down the list of potentially causal candidates by at least 51.3% for genes, and 50.8% for gene-tissue pairs, compared to single-tissue approaches. Leveraging its improved power, mFABIO successfully prioritized multiple potentially causal gene-tissue pairs associated with these diseases, with biological support. Notable examples include D2HGDH in lung tissue for asthma, CYBRD1 in breast mammary tissue for breast cancer, and CCR6 in spleen tissue for rheumatoid arthritis. Overall, mFABIO serves as an effective tool for multi-tissue TWAS fine-mapping of binary traits.

Pre-cuticle DPY-6 acts as a blueprint for aECM periodic organization in C. elegans

2026-05-26T14:00:00Z

by Sophie Mazzoli, Thomas Sonntag, Emma Cadena, Claire Valotteau, Susanna K. Birnbaum, Meera V. Sundaram, Nathalie Pujol

Apical extracellular matrices (aECMs) are essential for tissue integrity and function in multicellular organisms, but there is limited understanding of how such matrices are assembled and organized in the extracellular environment. The Caenorhabditis elegans cuticle, a model aECM that undergoes morphogenesis during each of the worm’s four larval molts, requires periodic circumferential furrows for structural integrity and immune regulation. Here, we show that furrow collagens must be cleaved from their N-terminal transmembrane domain for secretion and depend on the mucin-like pre-cuticle protein DPY-6 for their periodic assembly. While DPY-6 is dispensable for initial embryonic furrow formation, it acts as a mold during subsequent molts, ensuring pattern replication via its C-terminal cysteine cradle domain. These results reveal a central role for a transient matrix factor in organizing a complex periodically structured aECM.

MR2G: A novel framework for causal network inference using GWAS summary data

2026-05-26T14:00:00Z

by Zhaotong Lin, Wei Pan, Haoran Xue

Inferring a causal network among multiple traits is essential for unraveling complex biological relationships and informing interventions. Mendelian randomization (MR) has emerged as a powerful tool for causal inference, utilizing genetic variants as instrumental variables (IVs) to estimate causal effects. However, when the directions of causal relationships among traits are unknown, reconstructing the underlying causal network becomes challenging. In particular, the presence of cycles or feedback loops, which are common in biological systems, poses additional challenges for causal network inference, and remains largely under-studied with standard MR approaches and existing IV-based network inference methods. To address these issues, we introduce MR2G, a new statistical framework that enables robust inference of causal networks, including those with cycles, directly from GWAS summary statistics. MR2G is built on a formally defined recursive causal graph model that rigorously links direct causal effects to (univariable) MR estimands. It recovers a biologically interpretable causal network from pairwise MR effect estimates, while incorporating a network-informed IV screening strategy to reduce pleiotropic bias and improve robustness. Through realistic simulations, MR2G demonstrates superior accuracy and robustness in recovering complex causal structures, including those involving feedback loops. We apply MR2G to GWAS summary statistics for six complex diseases and nine cardiometabolic risk factors. MR2G not only recovers well-established causal pathways but also uncovers multiple feedback relationships, highlighting its utility in disentangling complex and biologically plausible causal networks from large-scale genetic data.

ZBP1 Drives CD8⁺ T cell-mediated anti-tumor immunity in head and neck squamous cell carcinoma

2026-05-26T14:00:00Z

by Yu Min, Ge Song, Lianlian Yang, Ling He, Shihong Xu, Kun Gao, Zheran Liu, Xingchen Peng, Lei Dai

Head and neck squamous cell carcinoma (HNSCC) frequently resists PD-1 blockade due to an immunologically “cold” tumor microenvironment (TME). Here, we identify Z-DNA binding protein 1 (ZBP1) as a key immunoregulator that reprograms immune-suppressive TMEs. Integrated TCGA/SangerBox analyses revealed ZBP1 as a hub gene strongly correlated with cytotoxic CD8⁺ T cells (r = 0.48, p < 0.0001) and M1 macrophages (r = 0.39, p < 0.0001). Multi-model validation in 92 HNSCC specimens revealed elevated ZBP1 expression versus normal tissues (p < 0.01), co-localized with infiltrating CD8⁺/CD4⁺ T cells and CD68⁺ macrophages through multiplex immunofluorescence. Clinically, high ZBP1 predicted improved survival (HR = 0.61 for overall survival; HR = 0.45 for disease specific survival; p < 0.0001) and early-stage presentation (p = 0.004). Mechanistically, ZBP1 overexpression in SCC-7/MOC2 models suppressed tumor growth while enhancing IFN-γ⁺ CD8⁺ T cell activation and reducing M2 polarization (CD206⁺: 16.91% vs 38.19% in ZBP1-high vs control, p < 0.001). Single-cell transcriptomics uncovered ZBP1-driven TME remodeling through chemokine signaling networks and expanded effector T cell compartments, validated by 1.49-fold increased CD8⁺ T cell infiltration via flow cytometry. Spatial analysis revealed ZBP1 overexpression amplified immune cell crosstalk (1.65-fold interaction increase, p < 0.001), upregulating CD8⁺ T cell chemotaxis (CXCR3/CCR5-CCL5 axis) and effector functions (p < 0.0001). Concurrently, it suppressed immunosuppressive pathways through metabolic reprogramming, establishing ZBP1 as a dual regulator synchronizing lymphocyte recruitment and myeloid suppression. Our integrative approach bridges computational biology with functional validation, demonstrating ZBP1’s capacity to convert “cold” tumors into immunologically active niches. This work positions ZBP1 as both a stratification biomarker for checkpoint inhibitor response and a therapeutic target for TME reprogramming in HNSCC.

An intronic variant in Ferredoxin Reductase (FDXR) creates a cryptic exon in Quarter Horses with Equine Juvenile Spinocerebellar Ataxia

2026-05-20T14:00:00Z

by Briana N. Brown, Anna R. Dahlgren, Sharmila Ghosh, Blythe Durbin-Johnson, Andrew Willis, Cassandra Olivas, Daniel York, Robert Grahn, Rebecca R. Bellone, Gino A. Cortopassi, Andrew D. Miller, C. Titus Brown, Kevin Woolard, Carrie J. Finno

Equine Juvenile Spinocerebellar Ataxia (EJSCA) is a novel autosomal recessive neurologic disease in Quarter Horses. Affected foals display a progressive proprioceptive ataxia by 1–5 weeks of age, leading to recumbency and necessitating euthanasia. Whole genome sequencing was performed on 7 EJSCA cases and unaffected horses that included 4 obligate carriers, 4 unaffected half or full-siblings, and 28 unrelated, unaffected control Quarter Horses. An 82 kb region of association was identified (EquCab3.0, chr11: 6963986–7045999), containing 9 candidate SNPs across four genes (FADS6, FDXR, GRIN2C and TMEM104). Decreased FDXR mRNA expression and a cryptic exon was identified in spinal cord tissue from EJSCA cases via RNA-sequencing. One of the 9 associated SNPs (FDXR-203 c.177 + 1778G > C) was the eighth base pair of this cryptic exon. Affected foals were all homozygous for the variant. Protein concentrations of FDXR were lower in EJSCA cases in spinal cord and liver compared to unaffected controls. The FDXR-203 c.177 + 1778G > C mutation represents the first non-coding neurological genetic variant in horses. Additionally, this is the first genetic cause of a degenerative axonopathy in the horse and a spontaneous disease model to study FDXR pathology in humans.

Comparative whole-genome analyses of articular chondrocytes and skin fibroblasts reveal distinct genome instability landscapes in mesenchymal cell types

2026-05-20T14:00:00Z

by Safia Mahabub Sauty, Jacqueline Shine, Hamed Bostan, Jian-Liang Li, Piotr A. Mieczkowski, Richard F. Loeser, Brian O. Diekman, Dmitry A. Gordenin

DNA damage lesions can result in mutations and genome rearrangements that are associated with cellular aging and diseases. The landscape of somatic mutations in individual tissue and cell types are dictated by their unique physiological states, cellular functions, mutagenic exposures, and efficiency of DNA repair. Articular chondrocytes and skin fibroblasts are two cell types of mesodermal origin with distinct exposure to internal and external sources of DNA damage. While somatic genome instability features of skin fibroblasts have been well detailed, knowledge about mechanisms underlying genome changes in chondrocytes is scarce. Here, we took a whole-genome sequencing approach to evaluate the load, sources, and patterns of genome changes in 18 primary human chondrocyte clones from donors with and without osteoarthritis (OA). Findings in chondrocyte clones largely agreed with a recent study of 100 single-cell sequenced chondrocytes. We compared genome changes in chondrocytes with clonally-expanded human skin fibroblasts sequenced in our previous studies. We demonstrated that skin fibroblasts show a higher burden of somatic mutations, with an increased rate of mutation accumulation per cell division. Motif-centered analyses of mutation catalogues identified only endogenous sources of mutations in chondrocytes, as opposed to skin fibroblasts which also showed a heavy burden of UV-induced mutations. Spontaneous deamination of meCpG and mutagenesis by exposure to small epoxides and S_N2 electrophiles showed higher mutagenic activities in chondrocytes compared to skin fibroblasts. Chondrocytes showed ubiquitous prevalence of indels in homonucleotide runs of ≥5 bases, while skin fibroblasts showed high contributions of UV-associated deletions of ≥5 bp not in repeats. Structural variants in rearrangement hotspots colocalized with human common fragile sites in skin fibroblasts, but not in chondrocytes. Together, our study comprehensively recorded genome instability features in chondrocytes and highlighted the unique mutagenesis landscapes of two mesenchymal cell types.

Correction: Alanine-scanning mutagenesis library of MreB reveals distinct roles for regulating cell shape and viability

2026-05-20T14:00:00Z

by Suman Maharjan, Ryan Sloan, Jada Lusk, Rose Bevienguevarr, Jacob Surber, Randy M. Morgenstein

Correction: Eliciting priors and relaxing the single causal variant assumption in colocalisation analyses

2026-05-19T14:00:00Z

by Chris Wallace

Why recombination hotspots?

2026-05-19T14:00:00Z

by Julien Joseph, Thomas Brazier, Marie Raynaud, Sylvain Glémin, Frédéric Baudat, Bernard de Massy, Nicolas Lartillot, Laurent Duret

Meiotic recombination is the process by which DNA is exchanged between parental chromosomes during the production of gametes in eukaryotes. This phenomenon has important implications for fertility, genetic diversity and genome stability. Intriguingly, not all regions of the genome are equally susceptible to recombine during meiosis. Instead, in many eukaryotes, recombination events are concentrated in short genomic segments called recombination hotspots. Since the first discovery of recombination hotspots, several theories have emerged to explain their existence. In this review, we discuss the relevance of these theories in regards of recent advances in characterizing the diversity and determinants of fine-scale recombination landscapes. Finally, we outline new research avenues for elucidating the evolutionary origins of recombination hotspots.

Cas9-expressing HC-04 hepatocytes facilitate CRISPR-based analysis of Plasmodium falciparum sporozoite-host interactions

2026-05-18T14:00:00Z

by Lisa H. Verzier, Eva Hesping, Marcel Doerflinger, Marco J. Herold, Justin A. Boddey

Sporozoites of Plasmodium falciparum, the deadliest malaria parasite, are injected into the skin by infected mosquitoes and must reach the liver to initiate infection. There, they invade hepatocytes and develop into exoerythrocytic merozoites that eventually enter the bloodstream and invade erythrocytes, causing malaria. The sporozoite’s journey requires cell traversal, where sporozoites transiently enter and exit host cells, lysing membranes to move deeper into tissue and evade immune cell destruction. After reaching the liver and traversing several hepatocytes, sporozoites productively invade a final hepatocyte to establish an exoerythrocytic form. The molecular mechanisms underlying traversal, invasion, and intracellular development remain incompletely understood, particularly with respect to host factors. To address this, we engineered human HC-04 hepatocytes, the only known cell line supporting P. falciparum liver-stage development, to express Cas9-mCherry, enabling CRISPR-based functional genomics studies. We validated Cas9 activity of HC-04.2B3 and demonstrated successful guide-RNA-directed gene disruption via non-homologous end joining. Optimized traversal and invasion assays led to a robust cytometric readout suitable for screening human genes involved in P. falciparum infection. Disruption of 10 human genes previously implicated in infection by bacterial and viral pathogens confirmed utility of this platform. This study provides the basis for genome-wide CRISPR screens to uncover hepatocyte biology and host determinants of infection.

Spatiotemporal characterization of single-stranded DNA intermediates after UV irradiation: II. Rapid growth and effects of recA and recJ

2026-05-14T14:00:00Z

by Remy A. A. Ripandelli, Elizabeth A. Wood, Andrew Robinson, Antoine M. van Oijen, Michael M. Cox

Irradiation of E. coli with UV light results in the formation of post-replication gaps and induction of the SOS response. Here, we investigate the dynamics of single strand gap formation and resolution in cells growing with a 50 min doubling time within specially designed microfluidic chips, making observations on fluorescent gap markers in individual cells over the course of 8 hours. In cells proficient for gap repair, irradiation with UV at 5 J/m² triggers an immediate increase in the number and intensity of gap markers. Cells lacking recF, recO, or recA cannot repair gaps via the canonical gap repair pathway and exhibit elevated gap numbers and intensities over many hours. Major conclusions include: (1) Post-replication gaps are a major feature of DNA metabolism after UV irradiation. (2) The long-lived, high-intensity foci observed in recF, recO, or recA mutants are completely dependent on the RecJ nuclease. In the absence of other RecFOR pathway functions, RecJ-mediated enlargement of many gaps continues unabated for extended periods. (3) In the absence of RecA or other RecFOR pathway functions, cells accumulate repair intermediates that are bound by large numbers of SSB molecules. (4) In the absence of RecJ, other pathways, perhaps involving TLS, displace SSB in gaps. Our results also confirm, using a different experimental setup and protocol, that: (a) UV-related repair activities, including nucleotide excision repair of at least some lesions, may continue for multiple cell generations after exposure; and (b) we again see no evidence that RecF facilitates lesion skipping. Overall patterns of gap formation and resolution under rapid growth conditions are consistent with a burst of post-replication gap formation following UV irradiation, postulated in the accompanying report to rapidly trigger the SOS response.

Spatiotemporal characterization of single-stranded DNA Intermediates after UV Irradiation: I: Post-replication gaps formed during slow growth

2026-05-14T14:00:00Z

by Nischal Sharma, Megan E. Cherry, Camille Henry, Elizabeth A. Wood, Andrew Robinson, Antoine van Oijen, Harshad Ghodke, Michael M. Cox

When E. coli cells are UV irradiated, replisome encounters with some DNA lesions lead to lesion skipping and formation of ssDNA gaps. These gaps are protected by SSB and repaired through the RecFOR recombinational DNA repair pathway. However, many questions about this pathway remain unanswered. Here, we used a fluorescent SSB fusion that supports normal growth in the absence of WT SSB under most conditions to directly visualize the real-time formation and resolution of ssDNA intermediates in cells lacking factors (RecB, RecJ, RecF, and RecO), that facilitate recombinational DNA repair pathways under slow growth conditions. Upon DNA damage, SSB-bound features of various sizes increased within these cells. In WT cells, ssDNA gaps appeared and were resolved at a steady state level that persisted for hours. Formation of most ssDNA gaps was not dependent on RecB function. Large increases in ssDNA gaps were observed in cells lacking RecFORJ functions, particularly in the absence of RecO. These findings indicate that: (a) When hundreds of UV lesions are introduced into the genome, at least some lesions remain unaddressed by nucleotide excision repair (NER) for several hours under slow growth conditions. (b) Replisome encounters with DNA lesions rapidly generate ssDNA gaps. (c) A relatively small portion of the ssDNA foci appearing in WT cells may reflect breaks processed by the RecBCD system. (d) Most prominent SSB features reflect post-replication gaps repaired by RecFORJ. Lack of RecFORJ functions leads to accumulation of unresolved gaps over time. (e) RecF is not required for post-replication gap formation. Overall, the results provide direct visualization of complex UV-induced changes in DNA metabolism caused by replisome encounters with UV-generated pyrimidine dimers. Combined with a decades-long literature of related results and proposals, a unified view of how E. coli responds to UV irradiation can be put forward.

Design and interpretation of eQTL-GWAS colocalisation studies: Lessons from a large-scale evaluation

2026-05-08T14:00:00Z

by Guillermo Reales, Jeffrey M. Pullin, Ichcha Manipur, Elena Vigorito, Chris Wallace

Colocalisation analysis integrates GWAS and molecular QTL datasets to identify candidate effector genes. Even with a wide range of molecular QTLs, 40% or more of GWAS loci remain unexplained, leaving a “colocalisation gap”. We systematically characterised two large-scale eQTL colocalisation studies, to describe the determinants of this gap and ultimately inform the selection and design of eQTL studies to close the gap. We analyse over 1.3 million colocalisation tests from Open Targets Genetics (OTG) and perform and analyse colocalisations from 14 immune-mediated disease (IMD) GWAS and 12 diverse immune cell eQTL studies, selected to cover a range of cellular granularities and sample sizes. We find that 50% of GWAS peaks in OTG and 34% in IMDs colocalised and were more likely to colocalise if they were located nearer to genes and had a more common lead variant. Colocalisation was also more likely to occur in disease relevant tissues. The lowest granularity immune cell eQTL studies had the largest sample sizes, the greatest eQTL discovery and produced the largest number of colocalisations, particularly for lower-frequency variants. However, while higher resolution eQTL studies detected fewer eQTLs, each of those eQTLs was more likely to colocalise with a GWAS peak, emphasising the importance of cell specific eQTLs. Indeed, over 50% of colocalisations were found in only one cell type. Overall, our results suggest that a diverse set of cells in different contexts, and large, high granularity studies will be needed to identify remaining colocalisations. In addition, we observed that 47% of GWAS peaks colocalised with multiple genes in OTG and 37% in IMDs. Through simulations, sensitivity analyses, and integration of enhancer-promoter capture data we find that multiple colocalisations likely represent coregulation. While disentangling causality from horizontal pleiotropy will ultimately require experimental perturbation, triangulation using different sources of observational data is likely to be necessary for gene prioritisation.

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An intronic variant in Ferredoxin Reductase (FDXR) creates a cryptic exon in Quarter Horses with Equine Juvenile Spinocerebellar Ataxia

Comparative whole-genome analyses of articular chondrocytes and skin fibroblasts reveal distinct genome instability landscapes in mesenchymal cell types

Correction: Alanine-scanning mutagenesis library of MreB reveals distinct roles for regulating cell shape and viability

Correction: Eliciting priors and relaxing the single causal variant assumption in colocalisation analyses

Why recombination hotspots?

Cas9-expressing HC-04 hepatocytes facilitate CRISPR-based analysis of Plasmodium falciparum sporozoite-host interactions

Spatiotemporal characterization of single-stranded DNA intermediates after UV irradiation: II. Rapid growth and effects of recA and recJ

Spatiotemporal characterization of single-stranded DNA Intermediates after UV Irradiation: I: Post-replication gaps formed during slow growth

Design and interpretation of eQTL-GWAS colocalisation studies: Lessons from a large-scale evaluation

ZBP1 Drives CD8⁺ T cell-mediated anti-tumor immunity in head and neck squamous cell carcinoma