Nucleus

Cajal bodies (CBs) are nuclear condensates known for their role in sn- and snoRNA maturation in animal, but their function in plant stress responses remains poorly understood. We compared wild-type (WT) Arabidopsis thaliana with the CB-lacking ncb-1 mutant during hypoxia and reoxygenation, focusing on nuclear retention of transcripts. In both genotypes, hypoxia caused nuclear accumulation of poly(A+) RNA and extensive reprogramming of gene expression. However, the transcriptional response, including induction of hypoxia-responsive genes, was considerably weaker in ncb-1, both during stress and recovery. In nuclear RNA, introns with low (intron retention index, IRX < 10%) and high (IRX > 50%) retention showed opposite, genotype-dependent dynamics. During hypoxia, efficiently spliced introns (IRX < 10%) accounted for over 70% of introns detected in nuclear RNA. Relative to normoxia, this proportion increased two- to threefold in WT but only moderately in the mutant. Together with poly(A+) RNA accumulation, this suggests that largely spliced transcripts, including those of genes involved in protein transport and ribosome biogenesis, are held in the nucleus during stress. Conversely, strongly retained introns, often terminal and located in genes related to hypoxia and other abiotic stresses, were most abundant in WT nuclear RNA under normoxia. Their proportion dropped sharply during hypoxia, and much less so in ncb-1. This indicate that WT plants maintain a nuclear reserve of incompletely spliced stress-related transcripts that are processed upon stress. The mutant also showed reduced interaction between U2 snRNA and U2B'', which leads to splicing defects. Our results indicate that CBs regulate which transcripts are held in the nucleus and how they are processed, contributing to plant tolerance of oxygen deprivation and recovery.

Chromosome was "self-assembled" into a complex three-dimensional topology in the nucleus, which provided the structural basis for important biological processes such as gene expression regulation and cell differentiation. The current understanding of the three-dimensional organization of the genome is based on high-throughput sequencing and imaging technologies to visualize the conformation of chromatin in its native environment. This study proposes a new research perspective and technical means to use fusion genes to determine the interaction information and three-dimensional structure of chromosomes. This three-dimensional information provided by the fusion gene spectrum not only shows the universality of the spatial layout of chromosomes, but also increases the interpretability of the occurrence of fusion genes. The chromatin conformation capture data of multiple tissues and periods is significantly correlated with the strength of fusion genes. For example, the frequency of trans-interaction between chromosomes 11 and 17 was the highest, and the frequency of fusion gene occurrence corresponding to KMT2A on chromosome 11 was the highest. Therefore, we infer that this structural universality is a common feature of the human genome, which provides a theoretical basis and data for predicting chromosome fusion events.

Background: X-chromosome inactivation (XCI) evolved independently in eutherian and marsupial mammals, where it is orchestrated by the unrelated long non-coding RNAs Xist and RSX, respectively. Xist organizes a repressive nuclear compartment through multivalent RNA-protein interactions, but whether RSX exploits similar biophysical principles remains unknown. A recent paper has identified bona fide RSX interacting proteins. Results: We integrated proteome-scale RNA-protein interaction prediction, experimental validation, phase-separation propensity analysis, functional annotation and comparative RNA-structure modelling to characterize the RSX interaction landscape. Using catRAPID, we ranked 1,168 RNA-binding proteins from the native Monodelphis domestica proteome. Predictions were significantly enriched for experimentally identified RSX interactors, with 4.85-fold enrichment among the top 50 candidates (P approximately 1.6 x 10-5), increasing to approximately eightfold for proteins shared by the experimental RSX and Xist interactomes (P approximately 2 x 10-6). Among 30 high-confidence RSX interactors, 13 were experimentally supported, 17 were previously unrecognized candidates and 17 exhibited high phase-separation propensity. The network was enriched in ribonucleoprotein granules and nuclear bodies and converged on m6A regulators and SR-family splicing factors. Comparative modelling detected no conserved secondary or tertiary architecture between RSX and Xist. Conclusions: RSX and Xist appear to have converged not through RNA sequence or global structure, but through recruitment of related, condensation-prone protein networks. These findings identify interaction-network and biophysical convergence as a potential principle of lncRNA-mediated chromosome regulation and provide testable candidates for determining whether RSX establishes a condensate-like compartment on the marsupial inactive X.

Samp1 is an inner nuclear membrane protein required for myogenic differentiation and involved in chromatin organization at the nuclear periphery. Here, we investigated whether these functions are connected by studying the effects of Samp1 depletion during C2C12 myogenic differentiation using immunofluorescence microscopy, RNA sequencing, FRIC, and chromosome-positioning analysis. Samp1-depleted cells showed strongly reduced MyHC expression and virtually abrogated multinucleated fiber formation. Although cell-cycle withdrawal was not prevented, the transcriptional program driving differentiation was drastically perturbed, with reduced muscle-associated transcripts and incomplete repression of genes normally downregulated during myogenesis. Samp1 depletion also disrupted peripheral chromatin organization and prevented the accumulation of peripheral heterochromatin typically seen during differentiation. In addition, radial chromosome distribution was disrupted, evidenced by the failure of chromosome 8 to reposition to the nuclear periphery during differentiation. Together, these findings link the requirement for Samp1 in myogenic differentiation to its role in genome organization at the nuclear periphery.

Calcium (Ca2+) is a universal second messenger that orchestrates plant growth, development, and stress responses. Plant calcineurin B-like (CBL) proteins sense and relay Ca2+; signals in response to pathogens to activate downstream defense signalling pathways. Early blight disease, caused by the necrotrophic fungal pathogen Alternaria solani, affects tomato crops and severely limits their productivity. In the present study, we found that the tolerant tomato cultivar (Arka Rakshak) restricts A. solani infection and disease progression compared to the susceptible cultivar (PKM-1). Comparative transcriptome and qRT-PCR analysis of A. solani-infected tomato leaves showed that SlCBL1-2 (Solyc08g007160.3), a Ca2+; sensor, is up-regulated in the tolerant cultivar, both relative to its own uninfected control and the infected susceptible cultivar, whereas it was not significantly induced in the uninfected susceptible cultivar. Protein sequence analysis indicates that SlCBL1-2 contains four EF-hand motifs, an N-terminal N-myristoylation/S-acylation module and a C-terminal FPSF motif, and it is predicted to interact with several CBL-interacting serine/threonine protein kinases (CIPKs). However, the evolutionary analysis, conformational transitions upon binding to Ca2+, and the Ca2+-binding affinity of SlCBL1-2 have not been previously characterized. Molecular Dynamics (MD) simulation of the AlphaFold structure of SlCBL1-2 indicates a well-folded structure and binding of two Ca2+ ions at EF-hands 3 and 4. Further, far-UV circular dichroism and tryptophan fluorescence spectroscopy of recombinant SlCBL1-2 indicate a natively folded structure in the absence of Ca2+ and undergo minimal secondary and tertiary structural changes upon Ca2+-binding, in contrast to several Ca2+-binding proteins. Of note, SlCBL1-2 binds Ca2+ with apparent KD values of 526 {+/-} 113 {micro}M and 28.7 {+/-} 9.47 {micro}M, as evidenced by isothermal titration calorimetry. Together, our study provides an experimental description of the Ca2+-binding properties of tomato CBL protein and defines the molecular basis by which pathogen-induced SlCBL1-2 may act as a Ca2+ sensor in defense signalling.

Nucleolar alterations are characteristic features of cancer cells; however, how changes in the overall morphology relate to internal subcompartment organization and ribosomal RNA (rRNA) dynamics remains unclear. In this study, the nucleolar organization of non-transformed MCF-10A mammary epithelial cells and MDA-MB-231 breast cancer cells was quantitatively compared. MDA-MB-231 cells exhibited differences in nucleolar morphology, accompanied by altered spatial organization of the dense fibrillar component and granular component, and accelerated intranucleolar trafficking of nascent rRNA. Pharmacological perturbation with SU056 shifted multiple structural and dynamic parameters toward an MCF-10A-like profile. Conversely, transient physicochemical perturbation with 1,6-hexanediol induced several MDA-MB-231-like nucleolar features in MCF-10A cells, which returned to their untreated profile after washout. These findings support the view that cancer-associated nucleolar alterations are an integrated reorganization across morphological, spatial, and dynamic dimensions. The observed reversibility further indicates that cancer-associated nucleolar organization can be remodeled in response to perturbations. By linking the morphology, subcompartment organization, and nascent rRNA dynamics within a common framework, this study provides a basis for exploring the establishment, maintenance, and remodeling of distinct nucleolar profiles in cancer.

The onset of mitosis in animal cells is marked by nuclear envelope breakdown (NEBD), which requires coordinated disassembly of nuclear pore complexes (NPCs) and the nuclear lamina. Polo-like kinase 1 (Plk1/PLK-1) initiates NEBD through phosphorylation of NPC components, but mechanisms that spatially regulate PLK-1 between NPCs and the nuclear interior are unclear. Here, we identify the Caenorhabditis elegans TMEM209 homolog NPP-28 as a transmembrane nucleoporin that concentrates PLK-1 at the nuclear envelope while limiting its nuclear accumulation to promote NEBD. We identify docking sites within NPP-28 that mediate PLK-1 nuclear envelope-association. NPP-28 is phosphorylated by PLK-1 in vitro, and mutational analysis suggests that NPP-28-mediated docking facilitates NEBD, whereas phosphorylation prevents premature NEBD. Simultaneous loss of npp-28 and the transmembrane nucleoporin npp-12/gp210 exacerbates mitotic NEBD defects in early embryos and causes severe defects in germline development. Together, our findings identify NPP-28 as a spatial regulator of PLK-1 during mitosis with functional relationships with other conserved transmembrane nucleoporins depending on context.

Infertility affects one in six couples worldwide and has a significant genetic component. Genome sequencing efforts have identified increasing numbers of potentially pathogenic variants, including in conserved meiotic genes. However, the lack of functional validation remains a major barrier to establishing variant pathogenicity and clinical diagnoses. Here, we establish an experimental pipeline integrating C. elegans and mouse in vivo models with in silico protein structure predictions to functionally validate potentially pathogenic infertility-associated variants. Using CRISPR-Cas9 we generated worm and mouse models of a homozygous missense variant (P638L) in MSH4, a mismatch repair protein essential for meiotic recombination, identified in a patient with non-obstructive azoospermia (NOA). Homozygous msh-4P546L worms exhibited severe meiotic defects including an absence of chiasmata, accumulation of DNA damage, and loss of crossover markers. Comparable defects were observed in spermatocytes of homozygous Msh4P660L/P660L mice, while Msh4P660L/P660L ovaries lacked oocyte-containing follicles, demonstrating that this variant causes infertility in both sexes. Together, these findings support a model in which the MSH4 P638L substitution disrupts the conversion of early recombination events into crossovers, leading to NOA in men and primary ovarian insufficiency in women. Further worm modelling of human MSH4 genetic variants identified exclusively in heterozygosity (G649R) or also in homozygosity (Y589C) demonstrates the potential of worm models to predict the pathogenicity of missense variants in meiotic proteins found in the human population. We propose that this integrated worm-mouse pipeline provides a powerful framework for accelerating the genetic diagnosis of infertility and informing the clinical management of affected individuals.

The LINC (linker of nucleoskeleton and cytoskeleton) complex is a central mechanotransductive apparatus that physically couples cytoskeletal forces to the nuclear interior, thereby regulating nuclear architecture, chromatin organization, and downstream signaling. Using atomistic molecular dynamics simulations, we identify and characterize force gates within the LINC complex, structural interactions that maintain mechanical connectivity under load. By introducing targeted mutations into the KASH peptide and SUN protein, we provide computational evidence that a key disulfide bond (SUN2 Cys563 to KASH2 Cys6862) and specific residues within the KASH {beta} strand interaction motif (KASH6875, KASH6877) act as distinct mechanical elements during force transmission across the SUN and KASH interface. Leveraging this framework, we show that the disulfide bond and the {beta} sheet network contribute unequally: removing the disulfide reduces both sustained and peak force transmission approximately twofold, whereas disrupting the {beta} sheet network leaves peak force largely intact while altering interfacial coupling and the duration over which load is borne. Localized molecular changes therefore reshape mechanical coupling to the nucleus through distinct routes rather than through a single graded loss of force. Such altered LINC mechanics may be relevant to laminopathies, including Hutchinson Gilford Progeria Syndrome (HGPS), in which defective nuclear lamina compromises force transmission; however, implications for specific diseases remain speculative and require experimental validation. By resolving how distinct molecular contacts contribute to force transmission in space and sequence, this work establishes a framework for dissecting mechanical coupling at protein interfaces under load. These findings provide a foundation for future studies aimed at experimentally testing how individual interface interactions govern nuclear force transmission, a prerequisite for therapeutically targeting LINC mechanics. More broadly, the force gate framework may guide strategies to modulate interfaces that bear mechanical loads in other mechanically regulated proteins. Together, these findings establish a computational framework for investigating force transmission at mechanically loaded protein interfaces and provide experimentally testable predictions for future studies.

Naive T cells are poised for activation and contain a pool of translationally repressed ribosomal protein (RP) mRNA prepared to induce ribosome biogenesis to support protein synthesis, cell growth and proliferation. RP mRNA are the prototypical members of a class of transcripts initiating at cytosine followed by a CU rich element called terminal oligo pyrimidine (TOP) RNAs. TOP RNAs are regulated by an RNA binding protein LARP1, which promotes transcript stabilisation and translational repression. We investigated LARP1 function in T cell activation by generating cross-linking immunoprecipitation (CLIP) datasets detailing the LARP1-RNA interactions in naive and activated CD4+ T cells and identifying novel TOP RNAs. TOP RNAs identified by this analysis were functionally diverse. RP mRNAs were typified by high stability, and translational repression in naive T cells followed by MTORC1-dependent translation increases following T cell activation. However, other TOP RNAs varied in these aspects of their regulation. Notably, TOP RNAs with longer 3'UTRs had a relaxed dependency on LARP1 for stability and a reduced dependency on MTORC1 for their translation. Transcription start site heterogeneity also impacted TOP RNA regulation by generating a mixture of transcript isoforms with different TOP motif lengths. Longer terminal oligo pyrimidine stretches were associated with a greater dependency on MTORC1 for translation. Differential regulation of TOP RNAs may allow tuneable translational responses to MTORC1 and indicates potential roles for LARP1 beyond translation regulation and stability.

Uterine leiomyomas, commonly known as fibroids, are benign tumors of the female reproductive tract. They are the most common tumor of the female reproductive system and the leading cause of hysterectomy in the United States. Progesterone, estrogen, and their receptors are important drivers for the development and growth of leiomyomas. How estrogen drives leiomyoma growth remains unclear. We have previously shown that simvastatin can inhibit leiomyoma growth by modulating intracellular calcium. This suggests that calcium signaling is important for leiomyoma growth. We therefore hypothesized that estrogen may drive leiomyoma growth by stimulating calcium release through the membrane estrogen receptor GPR30 (also known as GPER). We found that estrogen dose-dependently induced calcium release in leiomyoma cells. The GPR30-specific agonist G-1 also induced calcium release, indicating that GPR30 mediates estrogen-mediated calcium release. The GPR30 antagonist G-15 blocked estrogen-induced calcium release and significantly reduced leiomyoma proliferation. Our results demonstrate that GPR30 is a major driver of estrogen-dependent growth in leiomyoma cells by modulating intracellular calcium. These findings may have high therapeutic relevance to leiomyomas and other estrogen-dependent tumors.

Functionally linking enhancers to gene promoters at high resolution often entails analysis of 3C datasets, including viewpoint-based methods such as Capture-(Hi)C. Quantitative comparison across samples and experiments, together with visualization and integration of promoter interactions with other datasets are of critical importance. While existing statistical tools identify significant interactions and quantify underlying signal, systematically comparing the shape and extent of this signal across large sets of intervals remains challenging. Moreover, experimental artefacts, such as inefficient crosslinking, can affect downstream interpretation, but are difficult to detect systematically. Here we introduce PostChicago, an R/Bioconductor package that fills this gap. Building upon the widely used interaction-calling package Chicago, PostChicago provides a complete workflow for quality control, visualization, integration and quantitative analysis of promoter interactions between multiple samples, including annotation and quantification of genomic regions based on external datasets. PostChicago extends visual quantitative comparisons beyond individual viewpoints by computing normalized metaprofiles around large sets of intervals that visualize the distribution of interaction signal, enabling comparison between narrow and broad interaction patterns. We further demonstrate how PostChicago can identify experimental crosslinking artefacts. Together, PostChicago enables integrative, shape-sensitive analysis and quality control of promoter Capture-(Hi)C datasets at a scale that would otherwise require extensive custom scripting.

Tumors in men often lose the Y chromosome. In some tumor types, this is predictive of poor outcome. Given the male predominance and high LOY frequency in clear cell renal cell carcinoma (ccRCC), we asked whether LOY might impact the transcriptome of ccRCC. We determined somatic Y chromosome status and performed differential expression analysis on tumor RNA-sequencing samples of two human ccRCC cohorts (TCGA-KIRC, CPTAC-ccRCC) and in an autochthonous murine ccRCC model with renal epithelium-specific deletion of Vhl, Trp53 and Rb1. We additionally derived five isogenic Y-deficient and five Y chromosome-retaining clones of the human ccRCC cell line 786-O. Across all three settings, transcriptional changes were largely restricted to the expected gene dosage effects of male-specific Y (MSY) and pseudoautosomal region (PAR) genes, without large effects on autosomal genes. At the pathway level, we identified a modest but reproducible response in the human cohorts, converging on cell cycle, ribosomal biogenesis and DNA repair genes. A pan-cancer meta-analysis across TCGA entities recovered the same pattern, with no autosomal gene showing a large consistent effect. In some entities, but not in ccRCC, cancer/testis antigen expression was higher in LOY tumors. We conclude that, apart from direct gene dosage, modest pathway-level shifts and altered silencing of cancer/testis antigens, LOY is transcriptionally silent in adult humans and mice. Y-chromosomal genes are therefore unlikely to act as transcriptional drivers of ccRCC and other cancer entities.

Palmitate-associated lipotoxic stress activates the endoplasmic-reticulum stress sensor protein IRE1, but how IRE1 signaling influences genotoxic-stress responses in triple-negative breast cancer remains unclear. Here, we investigated IRE1 and XBP1 signaling in MDA-MB-231 cells exposed to palmitate and etoposide. Under combined lipotoxic and genotoxic stress, XBP1 depletion increased DNA-damage accumulation in IRE1 wild-type cells but not in IRE1-deficient cells, whereas IRE1 re-expression reduced DNA damage. Pharmacologic IRE1 RNase inhibition also increased etoposide-associated DNA damage, supporting a protective role for IRE1 RNase-dependent signaling. IRE1 status and XBP1 manipulation differentially affected H2A.X and {gamma}H2A.X abundance, indicating context-dependent regulation of chromatin-associated DNA-damage signaling. At the RNA level, H2A.X was enriched in IRE1 immunoprecipitates, and the H2A.X-linked DNA-damage-response transcripts HUWE1 and BRCA2 also associated with IRE1 under combined stress. These findings link palmitate-activated IRE1-XBP1 signaling to genome-protective responses during genotoxic stress and identify a network of IRE1-associated DNA-damage-response transcripts in triple-negative breast cancer cells. Direct IRE1-mediated cleavage of these transcripts remains to be established.

Dysfunctions in transcription cause severe human diseases. In eukaryotes, transcription initiation by RNA polymerase (Pol) II requires the formation of the pre-initiation complex (PIC) composed of general transcription factors (GTFs), Pol II, and an essential multiprotein coactivator, Mediator. In vitro experiments with GTFs and Pol II have depicted a linear sequence of PIC assembly. However, PIC assembly paths in vivo at the genomic scale and Mediator's role in these paths remain poorly understood. One strategy characterizes ChIP-seq occupancy alterations in Mediator mutants, but analysis of these data still lacks a formalized interpretation framework. In this study, we address this need by developing a quantitative framework that scores candidate assembly paths according to their ability to explain these alterations. This framework combines a model of PIC assembly based on a system of differential equations with parameter optimization based on a penalized criterion. We applied this new framework to datasets from two yeast Mediator mutants, in the Med17 and Med10 subunits. Our results show that the best-supported PIC assembly paths differ from those inferred in vitro. In particular, we show that it is important to distinguish the core TFIIH module from the TFIIK kinase module, and that similar paths starting with Mediator or TBP receive different levels of support. Furthermore, although multiple paths fit the data equally well, they differ markedly for specific gene groups. Taken together, the results show that the path can depend on the gene, and are also compatible with more than one path being operational in vivo for a single gene. This work improves our understanding of PIC assembly paths and transcription initiation, and opens perspectives for future modeling and experimental studies.

Salivary gland adenoid cystic carcinoma (SACC) is a rare, poorly understood malignancy. Here, using spatial transcriptomics, we show that disturbances in epigenetic regulation including chromatin remodeling, epitranscriptomic control by m6A RNA methylation, and RNA splicing may contribute to SACC. We demonstrated that machineries responsible for these processes-SWI/SNF chromatin remodeling complex (CRC), NineTeen Complex (NTC), and m6A RNA methylation complex interact directly. Immunohistochemistry on 48 SACC tumor samples indicated altered abundance of METTL3, METTL14, WTAP (m6A RNA methylation) and PRP19, CDC5L, PLRG1 (NTC-splicing) proteins. Chromatin immunoprecipitation indicated direct regulation of genes encoding subunits of SWI/SNF, NTC and RNA methylation complexes, and some EMT (epithelial-to-mesenchymal) marker genes by BRM (BRAHMA). Transcriptomic re-analysis revealed broad alterations in transcript usage, and splicing events in SACC. Collectively, our findings provide the first evidence that coordinated disruption of chromatin remodeling, RNA methylation, and splicing contributes to SACC development and progression, highlighting new avenues for therapeutic exploration.

In our past study on human cilia from primary ciliary dyskinesia (PCD) patients using cryo-electron tomography and mass spectrometry, defects in the DNAH5 gene were proven to cause the loss of the entire outer dynein arm along with other proteins. This phenomenon was not observed in the unicellular green alga Chlamydomonas. In this study, we examined the loss of ciliary components caused by defects in various genes, based on proteomic and structural analyses of motile cilia from multiple PCD patients. The absence of single outer arm dynein genes causes a loss or significant decrease in other inner and outer arm dyneins. Interestingly, some patients with outer dynein defects show decreased intraflagellar transport (IFT) proteins, suggesting an influence of cargo components or assembly on transport. These phenomena were not observed in Chlamydomonas, suggesting a more complex mechanism of ciliogenesis in humans despite the high similarity in the final 3D architecture of the 9+2 axoneme. Defects in certain central pair proteins unexpectedly reduced components on the doublets as well. Our results demonstrate protein-protein interactions during stages of human motile ciliogenesis that are more complex than those in unicellular organisms.

A cell's ability to maintain its volume is a prerequisite for its essential physiological functions. Changes in extracellular osmolarity induce the flux of water and solutes across the cell and nuclear membrane and, therefore, alters cell and nuclear volumes. Here, we investigated the equilibrium and dynamic volumetric responses of cells and nuclei in adherent human hepatoma (HepG2) cell monolayers using live three-dimensional imaging. Through image segmentation, 3D reconstruction, and volume rendering, precise cell and nuclear volumes were quantified. We found that during hypoosmotic stress, cells swell and lose osmolytes through active volume regulation, and nuclei undergo greater relative expansion than cells. During hyperosmotic stress, we found that both cells and nuclei shrink rapidly, develop irregular morphologies and undergo passive volume regulation. Furthermore, we explored the osmotic responses in these cells by restoring isosmotic conditions following osmotic stress and found that nuclei returned to volumes greater than their initial isosmotic volume, whereas cytoplasmic volume returned to a value below its initial isosmotic volume following swelling. By fitting coupled transport models water and solute permeability estimates for both cells and nuclei were made from their dynamic responses to different anisosmotic media with and without the presence of permeating solutes dimethyl sulfoxide and ethylene glycol. Together, these quantitative measurements of cell, cytoplasm, and nuclear volumetric responses and associated transport parameters have important implications for understanding cell physiology and the mechanism of osmoregulation.

The association of cytoplasmic localization of VDR in TNBC tumors and disease aggressivity prompted investigation of its regulation by XPO1. In TNBC cell lines, the XPO1 inhibitors selinexor and eltanexor increased nuclear VDR localization. Their effects were particularly strong when combined with vitamin D treatment. Similarly, XPO1 knockdown increased nuclear VDR levels, both alone and in combination with vitamin D. These results identify XPO1 as an important regulator of VDR subcellular distribution. XPO1 inhibition also enhanced vitamin D-dependent VDR transcriptional activity. Indeed, selinexor, eltanexor, and XPO1 silencing increased vitamin D-dependent CYP24A1 reporter activity and the expression of genes such as CYP24A1 or CDH1. RNA sequencing showed that XPO1 modulation markedly reshaped the vitamin D transcriptional response. The affected genes were mainly involved in metabolism, cell-cycle regulation, protein localization, and cellular organization. Functional analyses demonstrated that cytoplasmic VDR promoted TNBC cell proliferation. Conversely, nuclear VDR was required for the antiproliferative effects of vitamin D. Accordingly, XPO1 inhibition or depletion potentiated vitamin D-mediated growth suppression. This finding suggested functional cooperation between XPO1-dependent export and VDR activity. Finally, high VDR expression was associated with poor disease-free or overall survival only in tumors with high XPO1 expression. Overall, XPO1 negatively regulates VDR nuclear localization and vitamin D responsiveness in TNBC. Targeting XPO1 may therefore enhance the therapeutic potential of vitamin D-based strategies in TNBC.

DNA double strand breaks (DSBs) generated by genotoxic stress frequently contain chemically diverse termini that require end processing before ligation by non-homologous end joining (NHEJ). How end chemistry influences assembly and persistence of NHEJ synaptic complexes remains unclear. Using single-molecule imaging approaches in Xenopus egg extract, we measured formation and stability of the long-range (LRC) and short-range (SRC) complexes on nine DNA substrates spanning kinase-, polymerase- and nuclease-dependent processing requirements. LRC formation and lifetime were comparable across end types, and SRC formation and stability were largely insensitive to end chemistry. DNA hairpins, physiological intermediates of V(D)J recombination, were a notable exception that formed LRCs but inefficiently transitioned to SRCs. Depletion of cognate processing factors had modest effects on SRC formation and persistence, indicating that core NHEJ factors provide the principal structural support for synapsis across diverse termini. Although XLF was required for SRC formation on all substrates, reducing an XLF dimer from two intact tail/Ku-binding motifs to one selectively reduced productive SRC formation on ends requiring processing. Together, these results support a model in which partially redundant protein-protein interactions between core NHEJ factors maintain synapsis while end-specific processing reactions shape repair outcome.