EMBO Reports

RNF43 is best known for removing the Wnt-receptor complex from the cell surface, thereby maintaining Wnt-signaling at minimal essential levels. Recent studies reported that RNF43-mutant colorectal cancers carrying the common BRAFV600E mutation, respond more effectively to combined BRAF/EGFR inhibition. To determine whether RNF43 directly regulates EGFR or BRAF protein abundance, multiple pancreatic and colorectal cancer cell line models were generated in which RNF43 was knocked out, repaired, or stably overexpressed. Total and cell surface EGFR levels, as well as endogenous BRAF expression, were quantified. Across all models, no consistent evidence emerges that RNF43 modulates endogenous EGFR or BRAF levels. R-spondins likewise fail to alter EGFR levels or internalization. Notably, elevated EGFR expression observed in a subset of RNF43 knockout clones is induced by unintended CRISPR/Cas9 vector integration rather than the absence of RNF43 itself, highlighting a previously underappreciated artefact that can confound interpretations of EGFR regulation in genome edited lines. Overall, the data argue against a direct and general role for RNF43 in controlling EGFR or BRAF protein abundance, contradicting recent reports that propose degradation of these targets. Further studies are required to resolve these discrepancies and clarify the mechanistic basis underlying these conflicting observations.

Mitochondrial dynamics are critical for T cell activation, differentiation, and survival. The inner mitochondrial membrane ATP-dependent metalloprotease YME1L1 regulates proteostasis and the processing of optic atrophy protein 1 (OPA1), thereby shaping mitochondrial cristae architecture and respiratory function in many cell types. Whether YME1L1 fulfils similar roles in lymphocytes remains unknown. Here, we examined YME1L1 function in T cells using conditional knockout mice lacking YME1L1 in lymphocytes (YME1L1{Delta}TB). YME1L1 expression increased upon T cell activation, yet its absence did not alter thymic development, peripheral T cell homeostasis, or the proportions of naive, memory, and regulatory subsets. T cell activation and proliferation in response to anti-CD3{varepsilon} stimulation were also unaffected. Mitochondrial parameters such as mass, membrane potential, and reactive oxygen species production, were largely preserved, with only modest, transient increases in oxidative stress detected in CD4 T cells lacking YME1L1. Electron microscopy revealed no major changes in mitochondrial size or roundness but showed increased cristae branching and reduced tortuosity, indicating subtle alterations in ultrastructure. Additionally, {gamma}{delta} T cells in YME1L1{Delta}TB mice exhibited a mild shift toward interferon-{gamma}-producing phenotypes at the expense of interleukin-17-producing subsets. Collectively, our data indicate that YME1L1, despite its requirement for OPA1 cleavage, is dispensable for T cell development and acute activation but may contribute to fine-tune mitochondrial architecture and {gamma}{delta} T cell effector programming. These findings highlight cell-type-specific redundancies in mitochondrial quality control and underscore the value of negative data in refining the understanding of mitochondrial regulation in immune cells.

Background/ObjectivesRNA polymerase II is a multifunctional complex that is critical for gene regulation and environmental responses. Its POLR2I subunit in human is associated with various pathologies, including cancer chemoresistance. However, much of our understanding of how POLR2I could function indirectly derives from studies of its homologs in yeasts called Rpb9. Here, we endogenously humanized the rpb9 gene of the fission yeast Schizosaccharomyces pombe to examine the functional capabilities of POLR2I. MethodsWe edited the genomic rpb9 locus in S. pombe so that it encodes the human POLR2I protein, and investigated functional and structural conservation. ResultsWith our humanized yeast system, we find widespread functional complementation by human POLR2I of S. pombe rpb9 roles in yeast growth, chronological aging, and stress responses. We also find that POLR2I complements novel roles for yeast rpb9 in facultative heterochromatin assembly, resistance against the chemotherapy 5-fluorouracil, and resistance against the fungicide thiabendazole. In contrast, we find that POLR2I cannot complement the role of rpb9 in resistance against the transcription elongation inhibitor 6-azauracil (6-AU) in our system. Interestingly, POLR2I could complement 6-AU resistance if ectopically expressed. Lastly, we observe extensive structural homology between Rpb9 and POLR2I proteins. ConclusionsOur study establishes an endogenous cross-species gene complementation strategy that uncovers both conserved and rewired functions of fission yeast rpb9 and its human homolog, POLR2I. In addition to validating conserved roles, we also identified conservation of previously unrecognized roles of rpb9 in heterochromatin formation and chemoresistance.

The ability to flexibly adapt behavior to changing environmental contingencies is a core component of brain function and relies on experience-dependent remodeling of neural circuits. While cognitive flexibility has been primarily attributed to prefrontal-striatal networks, the contribution of hippocampus and their underlying molecular substrates remains less understood. Here, we show that the dorsal hippocampus has a key role in cognitive flexibility. In particular, Ring Finger Protein 10 (RNF10)-mediated signaling, linking activation of synaptic NMDARs to specific transcriptional programs in the dorsal CA1, is necessary for cognitive flexibility. In fact, in vivo downregulation, through gene deletion and silencing of RNF10, resulting in impaired long-term synaptic plasticity, suppressed cognitive flexibility. This was reflected in the impaired ability to disengage from previously acquired contextual, visual, and spatial information and to adapt behavior to changed context. Overall, our results identified RNF10 as a key in vivo player necessary for the balance between cognitive stability and flexibility.

The master kinases of the DNA damage response (DDR), ATR, ATM and DNA-PK, become active in response to DNA damage and orchestrate a downstream wave of phosphorylations contributing to DNA damage repair and preservation of cellular homeostasis. Of them, we recently demonstrated that ATM binds the pool of the lipid phosphatidyl-inositol-4-phosphate (PI4P) situated at the Golgi membrane. Depending on PI4P availability at Golgi membranes, ATM is more or less titrated away from the nucleus, which translates into responses to nuclear DNA damage of matching intensity. Building on this knowledge, in this work we asked if, beyond the Golgi merely serving as a docking platform that retains ATM away from the nucleus, ATM does exert any role important for Golgi biology. We found that ATM maintains Golgi morphology by counteracting its excessive deployment. This occurs both by its mere presence (likely antagonizing the Golgi-stretching action of the protein GOLPH3) and by phosphorylating Golgi-resident substrates. Of relevance, we also report that the morphological alterations caused to the Golgi without ATM affect the biology of a model Golgi cargo. Our findings nourish the growing evidence that kinases of ATMs family display functional interactions with membranes and highlights an underappreciated crosstalk between the Golgi and the nucleus.

Ewing sarcoma (EwS) is an aggressive, human-exclusive tumor typically driven by the EWS::FLI1 fusion protein. To assess whether the neomorphic functions of EWS::FLI1 are fundamentally dependent on evolutionarily recent cofactors such as ETS transcription factors (ETS-TFs), Plycomb group (PcG) proteins, CBP/p300, or specific subunits of the BAF complex, we expressed EWS::FLI1 in the model organism Saccharomyces cerevisiae. This minimal system was chosen because several key EWS::FLI 's cofactors possess greatly reduced sequence homology (e.g., BAF) or are lacking altogether (e.g., ETS-TFs, PcG, or CBP/p300). We used co-IP/MS to map the yeast interactome, Chip-Seq to identify gDNA binding sequences, RNA-Seq for global gene expression, and engineered reporters to test conversion of (GGAA) tandem repeats (GGAASat) into neoenhancers. We found that the yeast EWS::FLI1 interactome was more limited and qualitatively distinct from its human counterpart, sharing core machinery (e.g. RNA Polymerase II, FACT) but lacking the BAF/SWI-SNF and spliceosome complexes, and showing strong enrichment for the SAGA chromatin remodeling complex. We also found that EWS::FLI1 binds to hundreds of sites in the yeast genome with a clear preference for putative ETS-TF consensus sequences and (CA) dinucleotide repeats. Yet, EWS::FLI1 expressing cells presented only minimal transcriptional dysregulation, a stark contrast to the extensive changes observed in humans and Drosophila cells. Finally, we found that EWS::FLI1 successfully converted silent GGAASat sequences into active enhancers in yeast. This remarkable result occurs despite the absence of homologs for key human activators, such as CBP/p300, strongly suggesting that EWS::FLI1 can mobilize functionally related, non-homologous pathways to establish neoenhancers at GGAASat sites. Altogether, our results indicate that EWS::FLI1's core ability to drive GGAASat-dependent gene expression is a conserved, ancient property, while GGAASat-independent extensive transcriptome reprogramming is dependent on co-factors and pathways specific to animal cells.

MAIT are a highly versatile population of innate-like T cells that have been implicated in promoting tissue repair-associated process in a variety of tissue and diseases settings in the last years. While certain specific effector molecules responsible for MAIT-cell mediated have been identified, the mechanisms by which MAIT cells exert repair functions remain incompletely understood. Here, we show that hepatic MAIT cells express VEGFA, VEGFB and vimentin, an alternative ligand for the VEGFA-receptor VEGFR2 in both, regenerating and heathy tissue. Expression and secretion of these factors were induced in vitro by combined T cell receptor and cytokine stimulation. Supernatants of activated MAIT cells were able to promote proliferation of different epithelial and endothelial cells, including a liver sinusoidal endothelial-derived cell line in an VEGFR2-dependent manner. Together, our findings expand our understanding of MAIT cell function, especially in the liver and open new opens avenues for exploring MAIT therapeutic potential in modulating tissue repair.

The PBAF is one of three biochemically distinct BAF chromatin remodelers in humans. We previously proposed the role of ARID2, a PBAF component, as a bonafide tumor suppressor in colorectal cancer (CRC). Here, we validated loss of tumor suppression under conditions of ARID2 deficiency emanating from a marked reduction in PBAF complex assembly resulting from destabilization of PBAF-specific components BRD7, PHF10, and PBRM1. Transcriptome profiling of ARID2 deficient CRC cells revealed perturbation of disease processes, including CRC and neurodegenerative disorders, as well as CRC relevant pathways including Wnt/{beta}-catenin signalling, but transcript levels of PBAF-specific components remained unchanged, confirmed by RT-qPCR and TCGA data analysis. Our study establishes ARID2 as a critical stabilizer of the PBAF complex of relevance to CRC.

Hypoxia-inducible factors (HIFs) are key regulators of cellular responses to low oxygen (hypoxia), controlling the expression of genes required for survival and adaptation. KDM2B, a chromatin-modifying enzyme, is a direct target of HIF-1, but its precise role in regulating HIF and the hypoxia response remains unclear. Here, we investigated the role of KDM2B in the response to hypoxia in a variety of cell lines. Our analysis reveals that KDM2B depletion regulates HIF activity in a cell type dependent manner, with KDM2B depletion decreasing HIF activity in U2OS and MDA-MB-231 cells and increasing HIF activity in HeLa cells. We show that KDM2B depletion also reduces HIF-1 protein and RNA expression and reduces HIF-1 binding at hypoxia-response elements of its target genes in U2OS and MDA-MB-231 cells. Conversely, overexpression of KDM2B enhances HIF activity and HIF-1 levels in both U2OS and HEK293 cells. Mechanistically, we find that KDM2B requires its JmjC demethylase and CxxC DNA-binding domains for HIF regulation. Furthermore, we demonstrate that KDM2B is required for RNA Pol II recruitment to the promoter of HIF-1. At the cellular level, KDM2B supports cell proliferation, with its depletion impairing proliferation and reducing cell numbers under hypoxic conditions. Our work highlights a new function of KDM2B, as a key regulator of HIF-1 expression, acting through its demethylase and DNA-binding functions. Our data indicate that KDM2B is essential for cellular adaptation to hypoxia, impacting both HIF-dependent gene expression and cell survival, and has important implications for our understanding of HIF regulation.

Environmental stressors rarely affect just one brain circuit. Most studies assess single cognitive endpoints, obscuring whether vulnerabilities are global or circuit-selective and how effects distribute across interconnected systems. To address this, we used galactic cosmic radiation (GCR), a Mars mission-relevant stressor that disrupts the hippocampal-nucleus accumbens-prefrontal circuit. C57BL/6J mice received 33-ion GCR simulation (33-GCR, 0.75 Gy) or sham radiation with the Nrf2-activating compound CDDO-EA or vehicle, followed by multi-domain behavioral testing in both sexes. Under very high memory load, male Veh/33-GCR mice showed enhanced pattern separation compared to Veh/Sham males, an effect normalized by CDDO-EA. Female mice showed no radiation-induced changes in pattern separation but weighed 9-18% more than Veh/Sham females and had reduced locomotor activity. Reward-based learning differed by sex: males showed no changes, while female Veh/33-GCR mice displayed enhanced reward anticipation that was further increased by CDDO-EA alone, with both treatments contributing to elevated goal-tracking. For behavioral flexibility, CDDO-EA impaired reversal learning in males regardless of radiation, while 33-GCR impaired reversal learning in females regardless of CDDO-EA. Principal component analysis revealed that treatments disrupted specific circuit relationships while leaving others intact, consistent with selective rather than global cognitive effects. Fiber photometry showed enhanced dentate gyrus encoding activity in irradiated males under high memory load. Combined CDDO-EA/33-GCR selectively reduced dentate gyrus progenitors in females. Males and females showed distinct, circuit-selective vulnerability patterns, demonstrating that multi-domain, both-sex assessment is necessary to capture how stressors and interventions affect integrated brain function. CDDO-EA proved to be a double-edged sword: protecting one cognitive domain while impairing another, a trade-off invisible to single-endpoint assessment. This framework has immediate relevance for astronaut risk assessment and extends to any context where neuroprotective interventions are evaluated against environmental stressors.

Inflammation is a key driver of hematopoietic dysfunction in myeloid malignancies, but its role in the context of hypomethylating therapy remains incompletely understood. Although 5-Azacytidine is used posttransplant in high-risk myelodysplastic syndrome (MDS), only 50% of patients show a clinical response. We provide evidence that inherent inflammatory properties of healthy donor CD34+ stem cells exist that are likely to contribute to the "response" seen in MDS patients. These are linked to epigenetic priming of the myeloid niche, resulting in S100A8/A9-driven inflammatory program that promotes functionality of immature NK cells. Using in vitro differentiation systems, multi-omic profiling, and a S100A9-/- mouse model, we find that 5-AzaC modulates inflammatory transcriptional programs through epigenetic rewiring of upstream regulatory elements. Loss of S100A9 disrupts myeloid differentiation, impairs NK cell maturation, and alters key developmental regulators including CEBPB, JUN, and NFIL3. In vivo, 5-AzaC restores these defects and primes NK cells in a time- and context-dependent manner. Re-analysis of the published Australian MDS/CMML cohort shows that "responders" display increased S100A8/A9 expression together with enhanced IFN-{gamma}, IL6-JAK-STAT3, and TNF signaling. These findings suggest that inflammatory myeloid programs may serve as predictive biomarkers and therapeutic targets to enhance NK cell-mediated graft-versus-leukemia activity posttransplant. SummaryO_LIWe provide compelling evidence that inherent properties of healthy donor CD34+ hematopoietic stem cells (SCs) exist that are likely to contribute to the "response" seen upon pre-emptive posttransplant 5-AzaC therapy of patients with high-risk myelodysplastic syndrome (MDS). C_LIO_LIThese properties are linked to a distinct form of epigenetic plasticity at upstream-located transcription factor (TF) binding sites. This may indirectly contribute to acute S100A8/A9-driven inflammation, which is demonstrable in distinct monocyte subsets and, importantly, also in NK cells thereby determining the characteristics of inflammatory monocyte-NK cell crosstalk. C_LIO_LIMice with a targeted deletion of S100A9 fail to upregulate CEBPB / JUN and NFIL3 which results in impaired myeloid priming and dysfunctional NK cell maturation, respectively. C_LIO_LIRe-analysis of the Australian MDS/CMML cohort confirms that MDS patients that "respond" to 5-AzaC exhibit activated IFN-{gamma}, IL6-JAK-STAT3, and TNF-signaling pathways in the context of upregulated S100A8/A9 after six months of treatment. C_LIO_LIOur study indicates that screening of healthy donors SCs for specific inflammatory markers in early developing monocytes could be used as a marker to predict which donor will have the potential of generating a S100A8/A9-driven inflammatory response. This may help identify patients with MDS as well as AML who are likely to benefit from low-dose, short-term 5-AzaC therapy as early as day 7 after transplantation, potentially resulting in increased graft-versus-leukemia (GvL) activity. C_LI

Despite lacking a robust interferon response, pluripotent stem cells remain highly resistant to viral infection, in part through the constitutive expression of immune genes traditionally classified as interferon-stimulated genes. While interferon signaling has been shown to be incompatible with the maintenance of pluripotency, the molecular mechanisms underlying this relationship remain poorly understood. Here, we investigate the transcriptional response of human embryonic stem cells (hESCs) to infection with a potent activator of the interferon response, an influenza A virus mutant lacking the viral NS1 protein. Single-cell RNA sequencing revealed that while most hESCs remain unresponsive to infection, a distinct subpopulation expresses type I and III interferons. Notably, only interferon-expressing cells mounted a robust antiviral response, characterized by strong induction of interferon-stimulated genes. In contrast to the bulk hESC population, interferon responding cells exhibited reduced expression of core pluripotency factors as well as negative regulators of interferon signaling, such as SOCS1 and SPRY4. Depletion of SOCS1 enabled hESCs to respond robustly to interferon stimulation, showing that this negative regulator is a key suppressor of interferon signaling in pluripotent stem cells. We further show that SOCS1 and additional negative regulators of IFN signaling are intrinsically expressed in hESCs and are transcriptionally controlled by pluripotency factors, such as NANOG, SOX2 and OCT4. Together, our findings support a model in which pluripotency factors regulate intrinsic immune gene expression, including negative regulators of interferon signaling, thereby suppressing canonical interferon signaling to preserve pluripotency while maintaining antiviral resistance. IMPORTANCEBy combining single-cell transcriptomics with functional studies, we demonstrate that the pluripotency transcriptional program active in pluripotent stem cells coordinately regulates pluripotency factors, antiviral genes, and negative regulators of interferon signaling. This integrated control enables pluripotent stem cells to achieve effective protection against viral infection while preserving their differentiation potential, providing new insights into how innate immunity is selectively constrained in pluripotent stem cells. These findings have important implications for stem cell-based therapies, where transient modulation of antiviral responses without disrupting pluripotency could improve therapeutic efficacy. More broadly, this work advances our understanding of interferon regulation that could inform the development of antiviral strategies that enhance protective immune responses while limiting harmful or unwanted inflammatory signaling.

BackgroundCellular senescence is accompanied by extensive epigenomic reprogramming leading to changes in enhancer RNA levels, yet how enhancer activity is translated into functional RNA-level regulation remains unclear. Here we investigate how enhancer reprogramming during senescence impacts functional RNA-level regulation by eRNAs. ResultsBy integrating time-resolved transcriptomic analyses across multiple primary human cell types, we identify a set of recurrently dysregulated senescence-associated enhancer RNAs (SAeRs). We focus on one of these transcripts, EN526, which is reproducibly repressed during senescence while its locus remains broadly stable across cell states. EN526 eRNA exhibits cytoplasmic localisation and extensive eRNA-mRNA interactions, and cytoplasmic depletion of EN526 recapitulates its senescence-associated loss and alters the stability and translation of the cell-cycle regulator CDKN2C. EN526 perturbation further mediates stress responses, cellular survival, and extracellular remodelling associated with the senescence phenotype. ConclusionTogether, these findings show that SAeRs changes accompanying enhancer reprogramming in senescence are not merely passive events but can act as functional intermediates linking enhancer dynamics to post-transcriptional regulatory networks that phenocopy key senescence-associated cellular features. Extending this model, genetic associations at the EN526 locus further connect this regulatory axis to age-related traits and circulating protein phenotypes, supporting its broader relevance to human ageing and disease.

Hypoxia is a defining feature of the solid tumour microenvironment and a major determinant of therapeutic response. Hypoxia-inducible factors (HIFs) are central regulators of transcriptional reprogramming under hypoxic stress. Hypoxia can paradoxically elicit both tumour-promoting and tumour-suppressive outcomes, suggesting regulatory mechanisms beyond canonical HIF-dependent pathways. Emerging evidence indicates that hypoxia-responsive RNAs (HRRs) may also be regulated independently of HIFs, with posttranscriptional stabilization playing a critical determinant of hypoxic adaptation. Cytoplasmic mRNA recapping mediated by the cytoplasmic capping enzyme (cCE) has recently emerged as an important post-transcriptional regulatory process, yet its role in hypoxia-driven RNA regulation remains poorly understood. Here, we aimed to identify novel HRRs that modulate cellular adaptability to hypoxia and to determine whether these transcripts are regulated by cCE. Using CoCl2-induced hypoxia, we observed a significant reduction in osteosarcoma cell aggressiveness, characterized by decreased proliferation, clonogenic survival, and migratory capacity. Transcriptomic profiling of hypoxic osteosarcoma cells identified RORA and KCTD16 as significantly upregulated and function as suppressors of tumour cell aggressiveness. Integrative in-silico CAGE tag analysis followed by cap-specific biochemical assays confirmed that both transcripts are post-transcriptionally stabilized by cCE. Mechanistically, hypoxia-induced stabilization of HIF1 transcriptionally elevated RORA and KCTD16 expression, while cCE further reinforced their stability post-transcriptionally. Stabilization of these cCE-targeted HRRs resulted in suppression of the oncogenic proliferation driver c-Myc, thereby attenuating the aggressive phenotype of hypoxic osteosarcoma cells. Collectively, our findings identify cCE as a previously unrecognized post-transcriptional regulator in hypoxia biology and reveal a RNA-centric mechanism by which hypoxia can restrain tumour aggressiveness.

Ribosomes are increasingly recognized as heterogeneous regulators of gene expression, yet how ribosomal protein paralogs interface with nutrient signaling remains poorly understood. In Saccharomyces cerevisiae, ribosomal protein gene expression is governed by duplicated gene pairs, many of which exhibit functional divergence despite high sequence identity. A central regulator of ribosome biogenesis and translational control is the Target of Rapamycin (TOR) pathway, which integrates nutrient signals to modulate growth, stress adaptation, and lifespan. Target of Rapamycin Complex 1 (TORC1) influences ribosome activity by phosphorylating ribosomal protein S6 (Rps6), a modification that links nutrient availability to translational output. Here, we investigated the functional divergence of the Rpl12 ribosomal stalk protein paralogs RPL12a and RPL12b and found that rpl12b{Delta} produces phenotypes consistent with reduced TOR activity, including decreased Rps6 phosphorylation, G2/M cell-cycle accumulation, and significant extension of chronological lifespan as compared to rpl12a{Delta} and wildtype strain. Multi-omics analyses further indicate translational and metabolic reprogramming consistent with activation of a stress-adaptive program associated with Gcn4. Importantly, loss of RPL12b also reduces levels of the ribosome preservation factor Stm1, a TORC1-regulated protein required for stabilization of 80S ribosomes under stress. This finding links ribosomal stalk composition to ribosome stability and nutrient-responsive signaling. Together, our results demonstrate that ribosomal paralog specialization provides an additional regulatory layer connecting translation, TOR signaling, and cellular longevity.

Germ cells are the only cells of an organism that pass onto the next generation and, hence perpetuate the species. To ensure this, germ cells need dedicated transcriptional repertoire, that ensure specification, proliferation, differentiation and fate maintenance. We previously characterized LSL-1, a conserved zinc-finger transcription factor that acts as a major direct transcriptional activator of genes involved in germ cell development, fate specification, meiosis and genome stability. Here, we show that LSL-1 interacts with the transcription factor HIM-17, the chromatin proteins BRA-2 and XND-1. These proteins are functionally related to LSL-1 and they colocalize at germline gene promoters, forming most likely a transcription-promoting complex. Furthermore, LSL-1 lies in close proximity to members of the COMPASS and the MOF complexes, corroborating the observation that HIM-17 and LSL-1 are required to maintain normal level of H3K4 methylation in the gonad. Finally, we show that LSL-1 interacting partners are necessary to maintain germ cell fate. Altogether, we propose that LSL-1 interacts with transcription regulators and chromatin modifiers to ensure the establishment of the transcriptional repertoire appropriate for germline function as well as for cell fate maintenance.

Naked mole-rats (NMRs, Heterocephalus glaber) display unusual longevity and resistance to age-related decline, and accumulating evidence suggests that their autophagy-lysosome pathway (ALP) is regulated differently from that of conventional mammalian models. However, most studies in NMR cells have relied on static biochemical or ultrastructural readouts, leaving the dynamic organisation of autophagy in living cells poorly defined. Here, we establish a stable tandem fluorescent autophagy reporter in NMR skin fibroblasts using an mCherry-EGFP-LC3NMR construct to enable live-cell, single-cell resolution analysis of ALP dynamics. Under basal conditions, NMR skin fibroblasts exhibit a greater abundance of LC3-positive structures than HeLa cells, together with a mixed population of autophagosomes and autolysosomes, indicating a distinct steady-state organisation of the ALP. Chloroquine (CQ)-induced lysosomal stress caused the expected accumulation of LC3-positive structures but also triggered the formation of large cytoplasmic vacuoles in NMR skin fibroblasts. Importantly, this vacuolation was not associated with acute cytotoxicity and progressively resolved following CQ removal, accompanied by reorganisation of LC3-positive compartments and recovery of lysosomal acidity. Electron microscopy showed that CQ-induced vacuoles are membrane-bound, containing internal material and co-existing with multiple ALP-related vesicular compartments. Primary NMR skin fibroblasts display a similar vacuolation phenotype, indicating that this response is not an artefact of immortalisation or reporter expression. Together, these findings establish a live-cell platform for analysing autophagy in NMR cells and identify a distinctive, reversible vacuolation response to lysosomal stress, consistent with dynamic remodelling of the lysosomal system within NMR skin fibroblasts.

Sleep deprivation (SD) impairs information processing through alterations of prefrontal cortex (PFC) function, yet the molecular underpinnings of this process remain poorly understood. We previously showed that SD disrupts sensorimotor gating by elevating prefrontal levels of the neurosteroid allopregnanolone (AP), a positive allosteric modulator of GABA-A receptors. Here we identify a complementary, mechanistically independent process whereby SD alters GABA-A currents in the PFC of mice and rats. SD reduced membrane expression of the chloride exporter KCC2, leading to intracellular chloride accumulation and a depolarizing shift in GABA-A receptor reversal potential that weakened GABAergic inhibition. Pharmacological normalization of chloride homeostasis with bumetanide fully rescued SD-induced deficits in sensorimotor gating and information encoding. SD also upregulated BDNF, and intra-PFC antagonism of its receptor TrkB restored KCC2 expression and normalized information processing, identifying BDNF-TrkB signaling as an upstream driver of chloride dysregulation. Notably, blocking AP synthesis rescued behavioral deficits without correcting chloride imbalance, confirming mechanistic independence. Finally, combined administration of AP and a KCC2 blocker produced information-processing deficits akin to those induced by SD. These findings identify TrkB-dependent disruption of prefrontal chloride homeostasis as a druggable mechanism underlying sleep loss-induced cognitive dysfunction.

Immune elimination of chronic infection or cancer requires cytotoxic CD8+ T cells that adopt and maintain an effector phenotype. Cytotoxic T cell function is a bioenergetically demanding process and T cells subjected to chronic antigen exposure have compromised effector function despite high rates of glycolysis. Here we report the ability of the short-chain -hydroxy acid, D--hydroxybutyrate, to act as a signaling molecule that increases mitochondrial ATP production and drives the conversion of proliferating T cells into cytotoxic effector cells. DAHB signaling switches ATP production from glycolysis to oxidative phosphorylation supported by fatty acid oxidation, even in glucose-replete media. This conversion suppresses both AMPK phosphorylation and the integrated stress response (ISR) in activated T cells while significantly elevating the level of the phosphagen, phosphocreatine (PCr). Both the PCr bioenergetic reserve and oxidative phosphorylation were required for T cell effector differentiation. DAHB-induction of CD8-effector gene transcription was coupled to bioenergetics by enhanced ATP-dependent remodeling of chromatin accessibility at effector gene loci. DAHB enhanced CD8+ T cell antitumor activity both in vitro and in vivo, and DAHB treatment of transferred T cells led to persistent in vivo antitumor effects. Together, these findings link cellular bioenergetics to the regulation of chromatin accessibility and gene expression required to support effector function.

ELF4 is an ETS family transcription factor involved in immune regulation, and germline loss-of-function mutations in ELF4 have been known as deficiency in ELF4, X-linked (DEX). To date, ELF4-related disease has been exclusively associated with germline mutations. Here, we report a pediatric patient with recurrent mucocutaneous inflammation and periodic fever caused by a somatic truncating mutation in ELF4. By directly comparing ELF4-mutant and wild-type immune cells within the same individual using full-length single-cell RNA sequencing, we identified mutation-associated transcriptional alterations across multiple immune cell types. Pathway analyses revealed cell type-specific immune alterations, characterized by reduced antiviral and interferon-related signaling in NK cells and enhanced inflammatory pathways related to Th17 differentiation and inflammatory bowel disease in CD16 monocytes. This study expands the disease spectrum of ELF4 deficiency by identifying somatic truncation of ELF4 as a genetic mechanism underlying autoinflammatory diseases and biased immune programs.