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Genomic imprinting controls parental allele-specific gene expression via epigenetic mechanisms. Abnormal imprinting at the GNAS gene causes multiple phenotypes,including pseudohypoparathyroidism type-1B (PHP1B),a disorder of multihormone resistance. Microdeletions affecting the neighboring STX16 gene ablate an imprinting control region (STX16-ICR) of GNAS and lead to PHP1B upon maternal but not paternal inheritance. Mechanisms behind this imprinted inheritance mode remain unknown. Here,we show that the STX16-ICR forms different chromatin conformations with each GNAS parental allele and enhances two GNAS promoters in human embryonic stem cells. When these cells differentiate toward proximal renal tubule cells,STX16-ICR loses its effect,accompanied by a transition to a somatic cell-specific GNAS imprinting status. The activity of STX16-ICR depends on an OCT4 motif,whose disruption impacts transcript levels differentially on each allele. Therefore,a biallelically active embryonic enhancer dictates GNAS imprinting via different chromatin conformations,underlying the allele-specific pathogenicity of STX16-ICR microdeletions. STX16 microdeletions cause pseudohypoparathyroidism type-1B only on the maternal allele. Here,the authors show that the allele-specific pathogenicity reflects differential conformations of a biallelically active enhancer dictating GNAS imprinting. View Publication

Brain tumors are commonly treated with radiotherapy,but the efficacy of the treatment is limited by its toxicity to the normal tissue including post-irradiation contrast enhanced lesions often linked to necrosis. The poorly understood mechanisms behind such brain lesions were studied using cerebral organoids. Here we show that irradiation of such organoids leads to dose-dependent growth retardation and formation of liquid-filled cavities but is not correlated with necrosis. Instead,the radiation-induced changes comprise of an enhancement of cortical hem markers,altered neuroepithelial stem cell differentiation,and an increase of ZO1+/AQP1+/CLDN3+-choroid plexus (CP)-like structures accompanied by an upregulation of IGF2 mRNA,known to be expressed in CP and cerebrospinal fluid. The altered differentiation is attributed to changes in the WNT/BMP signaling pathways. We conclude that aberrant CP formation can be involved in radiation-induced brain lesions providing additional strategies for possible countermeasures. Human cerebral organoids provide insights into mechanisms behind the formation of choroid plexus (CP)-like structures that may contribute to radiation-induced brain image changes. View Publication

Huntington disease (HD) is a neurodegenerative disease caused by the abnormal expansion of a polyglutamine tract resulting from a mutation in the HTT gene. Oxidative stress has been identified as a significant contributing factor to the development of HD and other neurodegenerative diseases,and targeting anti-oxidative stress has emerged as a potential therapeutic approach. CHCHD2 is a mitochondria-related protein involved in regulating cell migration,anti-oxidative stress,and anti-apoptosis. Although CHCHD2 is highly expressed in HD cells,its specific role in the pathogenesis of HD remains uncertain. We postulate that the up-regulation of CHCHD2 in HD models represents a compensatory protective response against mitochondrial dysfunction and oxidative stress associated with HD. To investigate this hypothesis,we employed HD mouse striatal cells and human induced pluripotent stem cells (hiPSCs) as models to examine the effects of CHCHD2 overexpression (CHCHD2-OE) or knockdown (CHCHD2-KD) on the HD phenotype. Our findings demonstrate that CHCHD2 is crucial for maintaining cell survival in both HD mouse striatal cells and hiPSCs-derived neurons. Our study demonstrates that CHCHD2 up-regulation in HD serves as a compensatory protective response against oxidative stress,suggesting a potential anti-oxidative strategy for the treatment of HD. View Publication

The cytoplasmic RIG-I-like receptors (RLRs) recognize viral RNA and initiate innate antiviral immunity. RLR signaling also triggers glycolytic reprogramming through glucose transporters (GLUTs),whose role in antiviral immunity is elusive. Here,we unveil that insulin-responsive GLUT4 inhibits RLR signaling independently of glucose uptake in adipose and muscle tissues. At steady state,GLUT4 is docked at the Golgi matrix by ubiquitin regulatory X domain 9 (UBXN9,TUG). Following RNA virus infection,GLUT4 is released and translocated to the cell surface where it spatially segregates a significant pool of cytosolic RLRs,preventing them from activating IFN-? responses. UBXN9 deletion prompts constitutive GLUT4 trafficking,sequestration of RLRs,and attenuation of antiviral immunity,whereas GLUT4 deletion heightens RLR signaling. Notably,reduced GLUT4 expression is uniquely associated with human inflammatory myopathies characterized by hyperactive interferon responses. Overall,our results demonstrate a noncanonical UBXN9-GLUT4 axis that controls antiviral immunity via plasma membrane tethering of cytosolic RLRs. View Publication

BackgroundThe expansion of GGC repeats (typically exceeding 60 repeats) in the 5’ untranslated region (UTR) of the NOTCH2NLC gene (N2C) is linked to N2C-related repeat expansion disorders (NREDs),such as neuronal intranuclear inclusion disease (NIID),frontotemporal dementia (FTD),essential tremor (ET),and Parkinson’s disease (PD). These disorders share common clinical manifestations,including parkinsonism,dementia,seizures,and muscle weakness. Intermediate repeat sizes ranging from 40 to 60 GGC repeats,particularly those with AGC-encoded serine insertions,have been reported to be associated with PD; however,the functional implications of these intermediate repeats with serine insertion remain unexplored.MethodsHere,we utilized cellular models harbouring different sizes of N2C variant 2 (N2C2) GGC repeat expansion and CRISPR-Cas9 engineered transgenic mouse models carrying N2C2 GGC intermediate repeats with and without serine insertion to elucidate the underlying pathophysiology associated with N2C intermediate repeat with serine insertion in NREDs.ResultsOur findings revealed that the N2C2 GGC intermediate repeat with serine insertion (32G13S) led to mitochondrial dysfunction and cell death in vitro. The neurotoxicity was influenced by the length of the repeat and was exacerbated by the presence of the serine insertion. In 12-month-old transgenic mice,32G13S intensified intranuclear aggregation and exhibited early PD-like characteristics,including the formation of ?-synuclein fibers in the midbrain and the loss of tyrosine hydroxylase (TH)-positive neurons in both the cortex and striatum. Additionally,32G13S induced neuronal hyperexcitability and caused locomotor behavioural impairments. Transcriptomic analysis of the mouse cortex indicated dysregulation in calcium signaling and MAPK signaling pathways,both of which are critical for mitochondrial function. Notably,genes associated with myelin sheath components,including MBP and MOG,were dysregulated in the 32G13S mouse. Further investigations using immunostaining and transmission electron microscopy revealed that the N2C intermediate repeat with serine induced mitochondrial dysfunction-related hypermyelination in the cortex.ConclusionsOur in vitro and in vivo investigations provide the first evidence that the N2C-GGC intermediate repeat with serine promotes intranuclear aggregation of N2C,leading to mitochondrial dysfunction-associated hypermyelination and neuronal hyperexcitability. These changes contribute to motor deficits in early PD-like neurodegeneration in NREDs.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13024-024-00780-2. View Publication

Rett syndrome (RTT) is a neurodevelopmental disorder that is caused by mutations in melty-CpG binding protein 2 (MeCP2). MeCP2 is a non-cell type-specific DNA binding protein,and its mutation influences not only neural cells but also non-neural cells in the brain,including vasculature associated with endothelial cells. Vascular integrity is crucial for maintaining brain homeostasis,and its alteration may be linked to the pathology of neurodegenerative disease,but a non-neurogenic effect,especially the relationship between vascular alternation and Rett syndrome pathogenesis,has not been shown. Here,we recapitulate a microvascular network using Rett syndrome patient-derived induced pluripotent stem (iPS) cells that carry MeCP2[R306C] mutation to investigate early developmental vascular impact. To expedite endothelial cell differentiation,doxycycline (DOX)-inducible ETV2 expression vectors were inserted into the AAVS1 locus of Rett syndrome patient-derived iPS cells and its isogenic control by CRISPR/Cas9. With these endothelial cells,we established a disease microvascular network (Rett-dMVNs) and observed higher permeability in the Rett-dMVNs compared to isogenic controls,indicating altered barrier function by MeCP2 mutation. Furthermore,we unveiled that hyperpermeability is involved in the upregulation of miR126–3p in Rett syndrome patient-derived endothelial cells by microRNA profiling and RNAseq,and rescue of miR126–3p level can recover their phenotype. We discover miR126–3p-mediated vascular impairment in Rett syndrome patients and suggest the potential application of these findings for translational medicine. View Publication

ETV6::RUNX1,the most common oncogenic fusion in pediatric B cell precursor acute lymphoblastic leukemia (BCP-ALL),induces a clinically silent preleukemic state that can persist in carriers for over a decade and may progress to overt leukemia upon acquisition of secondary lesions. The mechanisms contributing to quiescence of ETV6::RUNX1+ preleukemic cells still remain elusive. In this study,we identify linker histone H1-0 as a critical mediator of the ETV6::RUNX1+ preleukemic state by employing human -induced pluripotent stem cell (hiPSC) models engineered by using CRISPR/Cas9 gene editing. Global gene expression analysis revealed upregulation of H1-0 in ETV6::RUNX1+ hiPSCs that was preserved upon hematopoietic differentiation. Moreover,whole transcriptome data of 1,727 leukemia patient samples showed significantly elevated H1-0 levels in ETV6::RUNX1+ BCP-ALL compared to other leukemia entities. Using dual-luciferase promoter assays,we show that ETV6::RUNX1 induces H1-0 promoter activity. We further demonstrate that depletion of H1-0 specifically inhibits ETV6::RUNX1 signature genes,including RAG1 and EPOR. Single-cell sequencing showed that H1-0 is highly expressed in quiescent hematopoietic cells. Importantly,H1-0 protein levels correspond to susceptibility of BCP-ALL cells towards histone deacetylase inhibitors (HDACis) and combinatorial treatment using the H1-0-inducing HDACi Quisinostat showed promising synergism with established chemotherapeutic drugs. Taken together,our data identify H1-0 as a key regulator of the ETV6::RUNX1+ transcriptome and indicate that the addition of Quisinostat may be beneficial to target non-responsive or relapsing ETV6::RUNX1+ BCP-ALL. View Publication

BackgroundNatural killer (NK) cells are key effector cells of antitumor immunity. However,tumors can acquire resistance programs to escape NK cell-mediated immunosurveillance. Identifying mechanisms that mediate this resistance enables us to define approaches to improve immune-mediate antitumor activity. In previous studies from our group,a genome-wide CRISPR-Cas9 screen identified Charged Multivesicular Body Protein 2A (CHMP2A) as a novel mechanism that mediates tumor intrinsic resistance to NK cell activity.MethodsHere,we use an immunocompetent mouse model to demonstrate that CHMP2A serves as a targetable regulator of not only NK cell-mediated immunity but also other immune cell populations. Using the recently characterized murine 4MOSC model system,a syngeneic,tobacco-signature murine head and neck squamous cell carcinoma model,we deleted mCHMP2A using CRISPR/Cas9-mediated knock-out (KO),following orthotopic transplantation into immunocompetent hosts.ResultsWe found that mCHMP2A KO in 4MOSC1 cells leads to more potent NK-mediated tumor cell killing in vitro in these tumor cells. Moreover,following orthotopic transplantation,KO of mCHMP2A in 4MOSC1 cells,but not the more immune-resistant 4MOSC2 cells enables both T cells and NK cells to better mediate antitumor activity compared with wild type (WT) tumors. However,there was no difference in tumor development between WT and mCHMP2A KO 4MOSC1 or 4MOSC2 tumors when implanted in immunodeficient mice. Mechanistically,we find that mCHMP2A KO 4MOSC1 tumors transplanted into the immunocompetent mice had significantly increased CD4+T cells,CD8+T cells. NK cell,as well as fewer myeloid-derived suppressor cells (MDSC).ConclusionsTogether,these studies demonstrate that CHMP2A is a targetable inhibitor of cellular antitumor immunity. View Publication

In sub-Saharan Africa,multidrug-resistant non-typhoidal Salmonella serovars are a common cause of fatal bloodstream infection. Malnutrition is a predisposing factor,but the underlying mechanisms are unknown. Here we show that vitamin A deficiency,one of the most prevalent micronutrient deficits afflicting African children,increases susceptibility to disseminated non-typhoidal Salmonella disease in mice and impairs terminal neutrophil maturation. Immature neutrophils had reduced expression of Slc11a1,a gene that encodes a metal ion transporter generally thought to restrict pathogen growth in macrophages. Adoptive transfer of SLC11A1-proficient neutrophils,but not SLC11A1-deficient neutrophils,reduced systemic Salmonella burden in Slc11a1−/− mice or mice with vitamin A deficiency. Loss of terminal granulopoiesis regulator CCAAT/enhancer-binding protein ϵ (C/EBPϵ) also decreased neutrophil-mediated control of Salmonella,but not that mediated by peritoneal macrophages. Susceptibility to infection increased in Cebpe−/− Slc11a1+/+ mice compared with wild-type controls,in an Slc11a1-expression-dependent manner. These data suggest that SLC11A1 deficiency impairs Salmonella control in part by blunting neutrophil-mediated defence. Vitamin A deficiency exacerbates invasive non-typhoidal Salmonella infection in mice,revealing a restrictive role for SLC11A1 in neutrophils. View Publication

IntroductionCurrent antiretroviral therapy (ART) for HIV infection reduces plasma viral loads to undetectable levels and has increased the life expectancy of people with HIV (PWH). However,this increased lifespan is accompanied by signs of accelerated aging and a higher prevalence of age-related comorbidities. Tat (Trans-Activator of Transcription) is a key protein for viral replication and pathogenesis. Tat is encoded by 2 exons,with the full-length Tat ranging from 86 to 101 aa (Tat101). Introducing a stop codon in position 73 generates a 1 exon,synthetic 72aa Tat (Tat72). Intracellular,full-length Tat activates the NF-κB pro-inflammatory pathway and increases antiapoptotic signals and ROS generation. These effects may initiate a cellular senescence program,characterized by cell cycle arrest,altered cell metabolism,and increased senescence-associated secretory phenotype (SASP) mediator release However,the precise role of HIV-Tat in inducing a cellular senescence program in CD4+ T-cells is currently unknown.MethodsJurkat Tetoff cell lines stably transfected with Tat72,Tat101,or an empty vector were used. Flow cytometry and RT-qPCR were used to address senescence biomarkers,and 105 mediators were assessed in cell supernatants with an antibody-based membrane array. Key results obtained in Jurkat-Tat cells were addressed in primary,resting CD4+ T-cells by transient electroporation of HIV-Tat-FLAG plasmid DNA.ResultsIn the Jurkat cell model,expression of Tat101 increased the levels of the senescence biomarkers BCL-2,CD87,and p21,and increased the release of sCD30,PDGF-AA,and sCD31,among other factors. Tat101 upregulated CD30 and CD31 co-expression in the Jurkat cell surface,distinguishing these cells from Tat72 and Tetoff Jurkats. The percentage of p21+,p16+,and γ-H2AX+ cells were higher in Tat-expressing CD4+ T-cells,detected as a FLAG+ population compared to their FLAG- (Tat negative) counterparts. Increased levels of sCD31 and sCD26 were also detected in electroporated CD4+ T-cell supernatants.DiscussionIntracellular,full-length HIV-Tat expression increases several senescence biomarkers in Jurkat and CD4+ T-cells,and SASP/Aging mediators in cell supernatants. Intracellular HIV-Tat may initiate a cellular senescence program,contributing to the premature aging phenotype observed in PWH. Graphical Abstract View Publication

BackgroundCD24 plays a crucial role not only in promoting tumor progression and metastasis but also in modulating macrophage-mediated anti-tumor immunity. However,its impact on the immune landscape of the tumor microenvironment (TME) remains unexplored. Here,we investigated the role of CD24a,the murine CD24 gene,in tumor progression and TME immune dynamics in a murine triple-negative breast cancer (TNBC) model.MethodsClustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/Cas9 knockout technology was employed to generate CD24a knockout in the murine TNBC cell line 4T1. Flow cytometry was utilized to analyze the immune cell populations,including myeloid-derived suppressor cells (MDSCs),natural killer cells,T cells,and macrophages,within tumors,spleens,and bone marrow in the orthotopic mouse 4T1 breast cancer model. Immunofluorescence (IF) staining was used to detect the immune cells in tumor sections. High-speed confocal was used to perform three-dimensional (3D) mapping of immune cells in the 4T1 orthotopic tumors.ResultsKnocking out CD24a significantly reduced tumor growth kinetics and prolonged mouse survival in vivo. Flow cytometry and IF analysis of tumor samples revealed that CD24a loss significantly promoted the infiltration of M1 macrophages and cytotoxic CD8+ T cells into the TME while reducing the recruitment and expansion of granulocytic MDSCs (gMDSCs). In vitro coculture experiments showed that CD24a deficiency significantly enhanced macrophage‐mediated phagocytosis and CD8⁺ T cell-mediated cytotoxicity,effects that were partially reversed by re‐expression of CD24a. Moreover,in vivo depletion of macrophages and CD8+ T cells reverted the delayed tumor growth caused by CD24a knockout,underscoring their critical role in tumor growth suppression associated with CD24a knockout. 3D mapping of immune cells in the TME confirmed the anti-tumor immune landscape in the CD24a knockout 4T1 tumors. Furthermore,in vitro analysis showed that CD24a loss upregulated macrophage colony-stimulating factor expression while suppressed levels of CXCL1,CXCL5,and CXCL10,chemokines known to recruit gMDSCs,further providing a molecular basis for enhanced macrophage recruitment and diminished gMDSC accumulation.ConclusionsOur findings suggest that CD24a may regulate immune suppression within the TNBC TME. Targeting CD24a enhances macrophage- and CD8⁺ T cell-mediated anti-tumor immune responses and is associated with a shift in the TME toward a more immunogenic state,thereby suppressing tumor growth. These results may support CD24 as a promising immunotherapeutic target for TNBC.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12929-025-01165-3. View Publication

Understanding diseases as the result of continuous transitions from a healthy system is more realistic than understanding them as discrete states. Here,we designed the spectrum formation approach (SFA),a machine learning-based method that extracts key features contributing to disease state continuity. We applied the SFA to transcriptomic data from patients with progressive liver disease and neurodegenerative movement disorders to examine its effectiveness in identifying biologically relevant gene sets. The SFA identified transcription factors that potentially regulate liver inflammation and voluntary movement. In neurodegenerative disorders,the SFA also identified genes regulated by ETS-1,with unclear effects on movement. In functional assessment using human iPSC-derived neurons,ETS-1 overexpression disrupted dopamine receptor balance,reduced GABA-producing enzyme levels,and promoted cell death. These findings suggest that the SFA enables the discovery of regulatory factors capable of modifying disease states and provides a framework for the continuity-based interpretation of biological systems. Subject terms: Diseases,Pathogenesis,Signs and symptoms View Publication