技术资料

Retinal neurogenesis is mediated by the coordinated activities of a complex gene regulatory network (GRN) of transcription factors (TFs) in multipotent retinal progenitor cells (RPCs). How this GRN mechanistically guides neural competence remains poorly understood. In this study,we present integrated transcriptional,genetic and genomic analyses to uncover the regulatory mechanisms of SOX2,a key factor in establishing neural identity in RPCs. We show that SOX2 is preferentially enriched in the RPC-specific enhancer landscape associated with essential regulators of retinogenesis. Disruption of SOX2 expression impairs retinogenesis,marked by a selective loss of enhancer activity near genes essential for RPC proliferation and lineage specification. We identified the RPC transcription factor VSX2 as a binding partner for SOX2 and,together,SOX2 and VSX2 co-target a core,retina-specific chromatin repertoire characterized by enhanced TF binding and robust chromatin accessibility. This cooperative binding establishes a shared SOX2-VSX2 transcriptional code that promotes the expression of crucial regulators of neurogenesis while repressing the acquisition of alternative lineage cell fate. Our data illuminate fundamental biological insights on how transcription factors act in concert to drive chromatin-based genetic programs underlying retinal neural identity. View Publication

Background: Recurrent/metastatic head and neck squamous cell carcinoma ((R/M) HNSCC) represents one of the most aggressive and immunosuppressive cancers. Despite the introduction of immune checkpoint inhibitors (ICIs),only a limited number of patients obtain long-term benefits. In (R/M) HNSCC patients,the antitumor immune response is defective,conferring resistance and promoting tumor progression. Therefore,the identification of novel biomarkers for superior clinical outcomes and easily accessible in standard clinical settings is still an unmet clinical need. Methods: Blood liquid biopsies obtained from (R/M) HNSCC patients undergoing pembrolizumab therapy (monotherapy or in combination with chemotherapy) were analyzed by flow cytometry to evaluate the levels of circulating immunosuppressive regulatory T cells (Tregs) and myeloid derived suppressor cells (MDSCs),at baseline and during therapy. Correlations between these immunosuppressive immune cell subsets and clinical parameters (clinical response rate,progression-free survival (PFS),overall survival (OS) and performance status (PS)) were performed. Results: Univariate analysis showed that before therapy,higher circulating levels of both CD137⁺Tregs and LOX-1⁺PMN-MDSCs,identified patients with significantly worse survival. Furthermore,CD137⁺Tregs resulted also positively correlated with worse PS,while high levels of LOX-1⁺PMN-MDSCs negatively affected response to pembrolizumab,with a significant increase in non-responsive patients during therapy. Interestingly,both CD137⁺Tregs as well as LOX-1⁺PMN-MDSCs exerted a higher immunosuppression on T cell proliferation than CD137−Tregs and LOX-1⁻PMN-MDSCs,respectively. Multivariate analysis revealed that the circulating LOX-1⁺PMN-MDSC subset resulted as an independent prognostic factor for survival by multivariate analysis,as confirmed in an independent validation cohort. Conclusions: The levels of blood circulating LOX-1⁺PMN-MDSCs may be proposed as non-invasive biomarkers to predict clinical outcomes of (R/M) HNSCC patients developing resistance to immunotherapy,improving patient selection and suggesting novel personalized therapies. View Publication

The selection of cells that have acquired a desired gene edit is often done by the introduction of additional genes that confer drug resistance or encode fluorophores. However,such marker genes can have unintended physiological effects and are not compatible with editing of single nucleotides. Here,we present SNIPE,a method that allows the marker-free selection of edited cells based on single nucleotide differences to unedited cells. SNIPE drastically enriches for cells,which have been precisely edited (median 7-fold). We validate the approach for 42 different edits using Cas9 or Cas12a in different cell types and species. We use it to enrich for combinations of substitutions that change missense mutations carried by all people today back to the ancestral state seen in Neandertals and Denisovans. We also show that it can be used to kill cultured tumor cells with aberrant genotypes and to repair heterozygous tumorigenic mutations. Genome editing often requires marker genes for selection of edited cells. Here,the authors present SNIPE,a marker-free method that selects cells based on DNA sequence,enabling precise enrichment of edited cells and applications from evolutionary research to the elimination of cancer cells. View Publication

Induced pluripotent stem cells (iPSCs) are commonly used to model human genetic diseases. Two main strategies are used. The first involves making iPSC lines from individual cases with a disease,and the second involves making disease-relevant gene edits in established iPSC lines. Because generating gene-edited lines is time consuming and expensive,most studies begin with one starting iPSC stock line and evaluate several gene-edited sublines. The current studies focus on gene-editing to model Lesch–Nyhan disease (LND),which is caused by mutations in the HPRT1 gene. The same pathogenic c.508C>T edit was made in four well-established stock lines,and three gene-edited lines were isolated from each. RNA sequencing (RNAseq) was,then,used to evaluate the impact of the gene edit. Gene-edited lines were compared to their corresponding stock lines,as well as to each other. An aggregate analysis of all lines combined was also conducted to determine the most robust findings across all lines. Results from gene editing were further compared with iPSC lines derived from individual cases with LND,to determine how closely findings from gene editing match results obtained with case-derived lines. There were two main findings. First,the same gene edit has a different impact on gene expression when starting with different starting stock lines. Second,the gene editing strategy does not produce the same results as the case-derived strategy. Potential explanations for these differences are addressed,along with the relevance of these two different strategies for disease modeling. View Publication

The T-cell receptor (TCR) initiates T-lymphocyte activation,but the mechanism of TCR activation remains uncertain. Here,we present cryogenic electron microscopy structures for the unliganded and human leukocyte antigen (HLA)-bound human TCR–CD3 complex in nanodiscs that provide a native-like lipid environment. Distinct from the open and extended conformation seen in detergent,the unliganded TCR–CD3 in nanodiscs adopts two related closed and compacted conformations that represent its physiologic resting state in vivo. By contrast,the HLA-bound complex adopts the open and extended conformation,and conformation-locking disulfide mutants show that ectodomain opening is necessary for maximal ligand-dependent T-cell activation. These structures also reveal conformation-dependent protein–lipid and glycan–glycan interactions within the TCR. Together,these results establish allosteric conformational change during TCR activation,reveal avenues for immunotherapeutic engineering,and highlight the importance of native-like lipid environments for membrane protein structure determination. The T-cell receptor (TCR) activation mechanism has remained uncertain. Here,the authors present molecular structures for the apo and ligand-bound human TCR–CD3 complex in lipid nanodiscs,revealing large conformational changes during activation. View Publication

Staphylococcal enterotoxins (SE) crosslink the MHC‐II on antigen‐presenting cells (APC) with the T‐cell receptor,inducing a polyclonal T‐cell response. Although APCs are the initial targets of SE and are critical in shaping subsequent T‐cell activation,the effects of SE on APC function remain poorly understood. This study investigates the immunomodulatory effects of staphylococcal enterotoxin A (SEA) on monocytes and their differentiation into monocyte‐derived dendritic cells (moDC) or macrophages (MDM). Transcriptomic analyses of human monocytes via RNA sequencing revealed SEA‐induced enrichment of gene pathways associated with inflammation,infection,and dermatitis,effects that were amplified in the presence of T cells. Phenotypic and functional characterization showed that SEA‐primed monocytes differentiated into MDM with an altered polarization,deviating from classical M1/M2 pathways. SEA‐primed MDM exhibited downregulation of key markers,including HLA‐DR,CD80,CD86,and PD‐L1. Functional assays demonstrated that SEA‐primed MDM pushed hyperinflammatory T‐cell responses,with significantly enhanced proliferation and IFN‐γ secretion. In contrast,following SEA‐priming,moDC retained robust antigen‐presenting capabilities and displayed enhanced expression of molecules involved in T‐cell interactions. These findings provide mechanistic insights into SEA‐mediated immune modulation,illustrating how SEA reprograms MDM functions and amplifies proinflammatory T‐cell responses. This advances our understanding of superantigen‐driven immune interactions,offering a foundation for developing therapeutic strategies to mitigate superantigen‐mediated immune conditions. Staphylococcal enterotoxin A (SEA) alters monocyte differentiation and function,while preserving T cell stimulatory capacity. SEA‐primed macrophages downregulate antigen‐presenting markers yet drive heightened T‐cell proliferation and IFN‐γ secretion. These findings reveal mechanisms of SEA‐mediated immune modulation and superantigen‐driven inflammation. View Publication

Hypertrophic cardiomyopathy (HCM) is a common and deadly cardiac disease characterized by enlarged myocytes,increased myocardial wall thickening,and fibrosis. A majority of HCM cases are associated with mutations in the β-myosin heavy chain (MYH7) converter domain locus,which leads to varied pathophysiological and clinical manifestations. Using base-editing technology,we generated mutant human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) harboring HCM-causing myosin converter domain mutations (MYH7 c.2167C>T [R723C]; MYH6 c.2173C>T [R725C]) to define HCM pathogenesis in vitro. In this study,we integrated transcriptomic analysis with phenotypic and molecular analyses to dissect the HCM disease mechanisms using MYH6/7 myosin mutants. Our KEGG analysis of bulk RNA-sequencing data revealed significant upregulation of transcripts associated with HCM in the mutant hiPSC-CMs. Further,in-depth transcriptomic analysis using Gene-Ontology (GO-term) analysis for biological process showed upregulation of several transcripts associated with heart development and disease. Notably,our analysis showed robust upregulation of cytoskeletal transcripts,including actin-cytoskeleton networks,sarcomere components,and other structural proteins in the mutant CMs. Furthermore,cellular and nuclear morphological analysis showed that the MYH6/7 mutation induced cellular hypertrophy and increased aspect ratio compared to the isogenic control. Immunostaining experiments showed marked sarcomere disorganization with lower sarcomeric order and higher dispersion in the mutant hiPSC-CMs,highlighting the remodeling of the myofibril arrangement. Notably,the MYH6/7 mutant showed reduced cortical F-actin expression and increased central F-actin expression compared to the isogenic control,confirming the cytoskeletal remodeling and sarcomeric organization during HCM pathogenesis. These pathological changes accumulated progressively over time,underscoring the chronic and evolving nature of HCM driven by the MYH6/7 mutations. Together,our findings provide critical insights into the cellular and molecular underpinnings of MYH6/7-mutation-associated disease. These findings offer valuable insights into HCM pathogenesis,aiding in future therapies. View Publication

Multiple myeloma (MM) is a plasma cell malignancy that disrupts bone homeostasis by suppressing osteogenesis and promoting osteoclast activity. While most therapeutic interventions to date have focused on targeting tumor cells and reducing osteolysis,we investigate whether osteoinductive strategies can restore bone formation and counteract disease progression. Using a human bone marrow-like scaffold model that enables direct in vivo evaluation of tumor–stroma interactions and human bone formation,we demonstrate that MM-derived mesenchymal stromal cells (MSCs) retain osteogenic potential but are functionally suppressed by MM cells. Transcriptomic profiling of MM-primed MSCs revealed the downregulation of small leucine-rich proteoglycans (SLRPs),ASPN,OGN,and OMD,key mediators of bone morphogenetic protein (BMP) signaling,which governs osteoblast differentiation. Among the BMPs analyzed,BMP6 emerged as a potent inducer of osteogenesis and regulator of the expression of these SLRPs. Notably,BMP6 selectively promoted bone formation without enhancing osteoclastogenesis and attenuated inflammatory and tumor-supportive MSC phenotypes. BMP6 also directly inhibited MM cell proliferation and suppressed IL6-induced growth. These findings highlight BMP6 as a distinct multifunctional regulator warranting further investigation as a potential therapeutic approach,while establishing the humanized model as a valuable platform for dissecting tumor–bone interactions in MM. View Publication

The development of new alternative models is essential to overcome the limitations of traditional two-dimensional (2D) cell cultures and animal models. Three-dimensional (3D) models,such as organoids,better mimic the structural and functional complexity of mammalian organs,thereby reducing the ethical and economic issues related to animal experimentation. These systems provide more physiologically relevant environments,improving the accuracy of disease modeling and drug response prediction. In this context,we have developed mouse hepatic organoids from livers of adult BALB/c mice and characterized them by microscopy and transcriptional analysis. This model was applied to a robust and reproducible high-throughput screening (HTS) platform for testing cytotoxicity at the preclinical stage of drug discovery. In addition,mouse hepatic organoids were co-cultured with amastigotes of Leishmania donovani parasites to establish a model of host–parasite interaction,which was characterized by RNA-seq linked to differential expression analysis and cytokine production by the hepatic organoids. The findings provided in this work establish mouse hepatic organoids as an alternative model for drug discovery and pathogenesis studies. View Publication

Aberrant deposition of β‐amyloid (Aβ) and hyperphosphorylated tau,along with neuroinflammation,are key drivers of Alzheimer's disease (AD) pathology. Here,we identify ramalin,a natural antioxidant,as a promising therapeutic agent that alleviates AD pathology by modulating β‐site APP cleaving enzyme 1 (BACE1),histone deacetylase 6 (HDAC6),and the mitogen‐activated protein kinases (MAPK) pathway. Ramalin reduced BACE1 protein levels,independently of its transcription,translation,or enzymatic activity,an effect mediated by inhibition of HDAC6. Consistently,HDAC6 knockout similarly decreased BACE1 levels,highlighting HDAC6 as a key regulator of BACE1. Ramalin further suppressed neuroinflammatory responses by downregulating inducible nitric oxide synthase (iNOS) and the NLR family pyrin domain containing 3 (NLRP3) inflammasome. In AD mouse models,ramalin treatment significantly attenuated neuroinflammation,Aβ plaque burden,and tau hyperphosphorylation,while improving cognitive performance. Notably,ramalin reversed Aβ oligomer‐induced synaptic transmission impairment and restored synaptic vesicle recycling in hippocampal neurons. Transcriptomic analysis identified modulation of the MAPK pathway,with reduced phosphorylation of c‐Jun N‐terminal kinase (JNK) and extracellular signal‐regulated kinase (ERK) implicated in tau pathology. These findings establish ramalin as a disease‐modifying intervention that provides neuroprotection through concurrent regulation of BACE1,HDAC6,and MAPK signaling pathway. Collectively,our findings highlight ramalin as a compelling disease‐modifying candidate with the potential to drive a breakthrough approach targeting AD pathology. Ramalin alleviates Alzheimer's disease pathology by selectively inhibiting HDAC6,reducing BACE1 levels,and suppressing neuroinflammation through downregulation of the NLRP3 inflammasome and iNOS. It restores synaptic function impaired by Aβ toxicity and improves cognitive performance in AD mouse models,APP/PS1 and 3xTg‐AD. Additionally,ramalin modulates the MAPK signaling pathway,reducing tau phosphorylation by inhibiting JNK and ERK activation. View Publication

TAR DNA-binding protein 43 (TDP-43) dysfunction is a hallmark of several neurodegenerative diseases,including frontotemporal dementia,amyotrophic lateral sclerosis,and Alzheimer’s disease. Although cryptic exon inclusion is a well-characterized consequence of TDP-43 loss of function,emerging evidence reveals broader roles in RNA metabolism,notably in the regulation of alternative polyadenylation (APA) of disease-relevant transcripts. In the present study,we examined 3′ untranslated region lengthening events in the brains of individuals with frontotemporal lobar degeneration with TDP-43 pathology (FTLD-TDP),focusing on the functional impact of APA dysregulation. To investigate whether TDP-43-mediated APA events occur in the postmortem brain,we measured the 3′ untranslated region length of the retromer component vacuolar protein sorting 35 (VPS35) and the ETS transcription factor (ELK1) in the frontal cortex of a large cohort of FTLD-TDP patients and of healthy controls,and evaluated if these APA events are associated with FTLD-TDP clinical characteristic,markers of TDP-43 pathology [e.g.,hyperphosphorylated TDP-43 and cryptic stathmin-2 RNA],or the expression of VPS35 and VPS29 proteins,the latter being essential to the retromer complex. We identified robust 3′ untranslated region lengthening of VPS35 and ELK1 in FTLD-TDP,which strongly associated with markers of TDP-43 pathology,and ELK1 APA also associated with an earlier age of disease onset. Functionally,VPS35 APA was associated with reduced VPS35 and VPS29 protein expression,and lower VPS35 levels were associated with increased hyperphosphorylated TDP-43 and cryptic stathmin-2 RNA. Together,these data implicate APA dysregulation as a critical downstream consequence of TDP-43 dysfunction and suggest that TDP-43 loss may contribute to retromer impairment through APA-mediated repression of retromer subunits. Recent work has shown that TDP-43 loss in frontotemporal dementia (FTD) induces changes in alternative polyadenylation,but the functional consequences of this are unclear. This study reports that 3′UTR lengthening of VPS35 in FTD patient brain samples correlates with reduced VPS35 and VPS29 protein levels,suggesting that TDP-43 loss induces retromer dysfunction. View Publication

Super enhancers (SEs),characterized by clusters of enhancers,are instrumental in shaping cellular identity and function. Given this crucial involvement of SEs in cell lineage commitment,and considering the pivotal position of surface ectoderm in differentiating into a wide array of cell types,the study of these SEs holds immense promise for advancing cell-based therapeutic applications. In this study,we profiled the SE landscape in surface ectoderm cells derived from pluripotent stem cell differentiation. By leveraging 3D genomic data,we discerned active histone modifications and frequent chromatin interactions of SEs with target genes. Notably,perturbing specific SE using a CRISPR-dCas9-mediated approach resulted in decreased expression of the connected gene. Subsequently,we constructed a regulatory network of core transcription factors (TFs) operating on SEs and uncovered their control over the differentiation process by forming regulatory network with key TFs,such as TEAD1. Knocking down TEADs attenuated the differentiation process and target gene activation,whereas YAP-TEAD activation expedited the differentiation process by promoting the early establishment of SEs. Collectively,our findings shed light on the crucial role of SEs and identify YAP-TEAD as vital regulators controlling surface ectoderm commitment,thereby providing a novel insight into lineage commitment and stem cell-based epithelial regeneration. View Publication