{"gene":"CLDN3","run_date":"2026-04-28T17:28:52","timeline":{"discoveries":[{"year":1999,"finding":"Claudin-3 (identified as mouse RVP1 homolog) is a four-transmembrane domain protein that concentrates exclusively at tight junctions in liver and kidney, as demonstrated by immunofluorescence and immunoelectron microscopy, establishing it as a bona fide tight junction strand component.","method":"Immunofluorescence microscopy, immunoelectron microscopy, transfection into MDCK cells","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1-2 — direct localization by immunoelectron microscopy with multiple orthogonal methods, foundational paper replicated across labs","pmids":["9892664"],"is_preprint":false},{"year":1999,"finding":"ZO-1, ZO-2, and ZO-3 directly bind the COOH-terminal YV sequence of claudin-3 (and claudins 1-8) through their PDZ1 domains in vitro, and ZO-1 is recruited to claudin-based networks at cell-cell borders in fibroblasts transfected with claudins.","method":"In vitro binding assay, transfection into L fibroblasts, co-localization by immunofluorescence","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 1 — in vitro binding assay with mutagenesis-level specificity (PDZ1 domain) plus cellular reconstitution, replicated across claudin family members","pmids":["10601346"],"is_preprint":false},{"year":1999,"finding":"Claudin-3 can form heteromeric tight junction strands with claudin-1 and claudin-2, and claudin-3 strands can associate laterally with claudin-1 and claudin-2 strands in trans (heterophilic interactions), whereas claudin-1 and claudin-2 strands do not interact with each other.","method":"Co-transfection into L fibroblasts, co-culture experiments, immunoreplica electron microscopy","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal approaches (co-transfection, co-culture, immunoEM) establishing specific heteromeric assembly rules","pmids":["10562289"],"is_preprint":false},{"year":1997,"finding":"Claudin-3 (then called the human RVP1 homolog) functions as a receptor for Clostridium perfringens enterotoxin (CPE): L929 cells transfected with claudin-3 cDNA became sensitive to CPE, demonstrating it is sufficient to mediate CPE binding and cytolysis.","method":"Transfection of L929 cells, CPE binding assay, cytotoxicity assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 — functional gain-of-function in naive cells demonstrating receptor sufficiency, replicated by multiple subsequent studies","pmids":["9334247"],"is_preprint":false},{"year":2001,"finding":"Claudin-3 promotes activation of pro-MMP-2 mediated by membrane-type MMPs (MT-MMPs): claudin-3 expression in 293T cells stimulated MT-MMP-mediated pro-MMP-2 processing, and direct interaction of claudin family members with MT1-MMP and MMP-2 was demonstrated by immunoprecipitation.","method":"Expression cloning, co-immunoprecipitation, pro-MMP-2 activation assay in 293T cells","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2-3 — functional assay with co-IP, but claudin-3 was one of several claudins tested and was not the primary focus","pmids":["11382769"],"is_preprint":false},{"year":2003,"finding":"In human airways, claudin-3 contributes to tight junction barrier function: stable expression of claudin-3 in NIH/3T3 and IB3.1 airway cells decreased solute permeability, and co-immunoprecipitation revealed heterophilic interactions between claudin-3 and other claudin species in cell lines and in freshly excised human airway epithelium.","method":"Stable transfection, transepithelial resistance measurement, permeability coefficients, co-immunoprecipitation","journal":"American journal of physiology. Lung cellular and molecular physiology","confidence":"Medium","confidence_rationale":"Tier 2 — functional transepithelial resistance plus co-IP in both cell lines and primary tissue, single lab","pmids":["12909588"],"is_preprint":false},{"year":2004,"finding":"Disease-causing mutant WNK4 kinase binds and phosphorylates claudins 1-4 (including claudin-3) at tight junctions, with mutant WNK4 causing greater claudin phosphorylation than wild-type, and this is associated with increased paracellular chloride permeability in MDCK II cells.","method":"Stable expression in MDCK II cells, transepithelial ion permeability measurement, in vitro phosphorylation assay, co-immunoprecipitation","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1-2 — in vitro kinase assay demonstrating direct phosphorylation, functional permeability measurements, gain-of-function mutation approach","pmids":["15070779"],"is_preprint":false},{"year":2005,"finding":"Claudin-3 expression in human ovarian surface epithelial (HOSE) cells increases cell invasion, motility, and survival, and is associated with increased MMP-2 activity; siRNA-mediated knockdown of claudin-3 in ovarian cancer cell lines reduces invasion.","method":"Stable transfection of HOSE cells, Boyden chamber invasion assay, wound-healing assay, siRNA knockdown, MMP-2 zymography","journal":"Cancer research","confidence":"High","confidence_rationale":"Tier 2 — reciprocal gain-of-function and loss-of-function with defined molecular readout (MMP-2 activity), multiple orthogonal cellular assays","pmids":["16103090"],"is_preprint":false},{"year":2007,"finding":"The CLDN3 promoter contains a minimal Sp1-binding site critical for transcriptional activity; Sp1 binds preferentially to the unmethylated promoter, providing a mechanism for epigenetic silencing. siRNA knockdown of Sp1 significantly decreases CLDN3 mRNA and protein expression.","method":"Promoter deletion analysis, ChIP assay, in vitro DNA-binding assay, siRNA knockdown, DNA methylation analysis, histone acetylation ChIP","journal":"Cancer biology & therapy","confidence":"Medium","confidence_rationale":"Tier 2 — ChIP plus in vitro binding plus siRNA knockdown with multiple orthogonal methods in a single lab","pmids":["17986852"],"is_preprint":false},{"year":2010,"finding":"Claudin-3 acts as a general sealing component of the tight junction paracellular pathway: stable transfection of MDCK II cells with human claudin-3 elevated transepithelial resistance, decreased permeability to mono- and divalent cations, anions, and uncharged solutes (fluorescein, FD-4), and altered tight junction strand morphology toward uninterrupted meshwork loops, without affecting water permeability.","method":"Stable transfection of MDCK II cells, transepithelial resistance measurement, ion permeability assays, tracer flux assays, electron microscopy of tight junction strands","journal":"Biochimica et biophysica acta","confidence":"High","confidence_rationale":"Tier 1-2 — comprehensive functional characterization with multiple ion/solute permeability assays and structural analysis, rigorous controls with two clone systems","pmids":["20655293"],"is_preprint":false},{"year":2010,"finding":"Derepression of CLDN3 in ovarian tumorigenesis correlates with loss of repressive histone modifications (H3K27me3 and H4K20me3) from its promoter. CLDN3 repression in normal ovarian epithelial cells is maintained by bivalent histone marks (H3K4me3 + H3K27me3), and DNA methylation is not required for CLDN3 repression in immortalized ovarian epithelial cells.","method":"ChIP assay for histone modifications, bisulfite sequencing, DNA methyltransferase and HDAC inhibitor treatments, quantitative RT-PCR","journal":"Carcinogenesis","confidence":"Medium","confidence_rationale":"Tier 2 — ChIP with multiple histone marks plus pharmacological intervention, single lab","pmids":["20053926"],"is_preprint":false},{"year":2020,"finding":"CLDN3 overexpression in human trophoblast HTR8/SVneo cells promotes proliferation, invasion, and migration while reducing apoptosis; this is associated with increased MMP-2 and MMP-9 expression and increased ERK1/2 phosphorylation, placing CLDN3 upstream of ERK1/2 signaling and MMP activation in trophoblasts.","method":"Lentiviral overexpression, CCK-8 assay, flow cytometry, Transwell assay, Western blot for ERK1/2 phosphorylation and MMP expression","journal":"Experimental and therapeutic medicine","confidence":"Medium","confidence_rationale":"Tier 3 — gain-of-function with defined molecular readouts (ERK1/2, MMPs) but single lab, no rescue or loss-of-function complement","pmids":["32855729"],"is_preprint":false},{"year":2024,"finding":"TET1 promotes CLDN3 transcription by demethylating the CLDN3 promoter region (-16 to +512 bp). PPM1G phosphatase catalyzes TET1 dephosphorylation, leading to TET1 protein destabilization, which impairs TET1-mediated CLDN3 promoter demethylation and suppresses epithelial-mesenchymal transition in cholangiocarcinoma cells.","method":"ChIP assay, bisulfite sequencing, phosphatase activity assay, co-immunoprecipitation, protein stability assays, EMT functional assays, pharmacological inhibitors","journal":"Advanced science","confidence":"Medium","confidence_rationale":"Tier 2 — mechanistic chain from PPM1G → TET1 dephosphorylation → CLDN3 demethylation established with ChIP and biochemical assays, single lab","pmids":["39477806"],"is_preprint":false},{"year":2025,"finding":"HSF1 directly binds to the CLDN3 promoter and activates CLDN3 transcription in colorectal cancer cells, as demonstrated by ChIP assay; HSF1 knockdown reduces CLDN3 expression and inhibits CRC cell proliferation, migration, and invasion, while HSF1 overexpression promotes these behaviors.","method":"ChIP assay, Western blot, PCR, stable knockdown and overexpression, in vivo xenograft","journal":"Neoplasma","confidence":"Medium","confidence_rationale":"Tier 2 — direct promoter binding by ChIP plus reciprocal gain/loss-of-function, single lab","pmids":["40162508"],"is_preprint":false},{"year":2026,"finding":"CLDN3 directly interacts with TRIM28 as demonstrated by co-immunoprecipitation and immunofluorescence; TRIM28 mediates SUMOylation and degradation of CLDN3 protein, establishing TRIM28 as a writer controlling CLDN3 stability. CLDN3 knockdown in CRC cells decreases proliferation and migration, and CLDN3 overexpression reduces sensitivity to 5-FU.","method":"Co-immunoprecipitation, immunofluorescence, Western blot for SUMOylation, siRNA knockdown, overexpression, proliferation and migration assays","journal":"IET systems biology","confidence":"Medium","confidence_rationale":"Tier 2-3 — co-IP plus SUMOylation Western blot with functional readout, single lab","pmids":["41762617"],"is_preprint":false},{"year":2025,"finding":"CLDN3 acts as a host antiviral defense protein against rotavirus (RV): the CLDN3 extracellular loop 1 (EC1) directly interacts with the N-terminal domain of RV outer capsid protein VP7, reducing viral adsorption. Structural analysis identified glutamic acid at position 74 (E74) of VP7 as critical for the CLDN3-VP7 interaction. Knockdown or knockout of CLDN3 promotes RV binding and entry, and the VP8* peptide of RV induces CLDN3 mislocalization from the plasma membrane as a counterdefense. The E74K mutation in VP7 disrupts the CLDN3-VP7 interaction and enhances viral pathogenicity in vivo.","method":"CLDN3 knockout/knockdown (RNAi, CRISPR), viral binding and entry assays, co-immunoprecipitation, structural studies, in vivo mouse infection model, site-directed mutagenesis (E74K)","journal":"PLoS pathogens","confidence":"High","confidence_rationale":"Tier 1-2 — structural validation + mutagenesis + KO/KD + in vivo pathogenicity, multiple orthogonal methods in single study","pmids":["41860939"],"is_preprint":false},{"year":2025,"finding":"In chick embryos, CLDN3 (expressed in non-neural ectoderm) is required for neural fold fusion in the spinal region. CLDN3 depletion decreases apical cell area at neural fold edges, increases phospho-MLC staining in the apical domain, and increases tissue tension in the non-neural ectoderm as measured by laser ablation. Treatment with the myosin II inhibitor blebbistatin partially rescues neural fold fusion defects, placing CLDN3 upstream of actomyosin contractility and apical cytoskeletal regulation.","method":"Morpholino knockdown in chick embryos, live imaging, cell segmentation analysis, laser ablation biomechanics, pMLC immunostaining, blebbistatin pharmacological rescue","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 2 — multiple orthogonal methods (live imaging, laser ablation, pharmacological rescue) but preprint, single lab","pmids":[],"is_preprint":true},{"year":2009,"finding":"Claudin-3 contributes to tight junction strand formation and paracellular barrier regulation in intestinal epithelial context; downregulation of claudin-3 (along with claudins -4, -5, -7, -8, -12) is observed in intestinal inflammatory disorders, suggesting a role in maintaining epithelial barrier homeostasis.","method":"Expression analysis in intestinal inflammatory disease context (review/summary of experimental data)","journal":"Genome biology","confidence":"Low","confidence_rationale":"Tier 4 — review/expression-based inference, not a primary mechanistic experiment for CLDN3 specifically","pmids":["19706201"],"is_preprint":false},{"year":2025,"finding":"ASCL2 physically interacts with CLDN3 as demonstrated by co-immunoprecipitation assay in breast cancer cells. Rescue experiments show that overexpression of CLDN3 can partly reverse the inhibitory effects of ASCL2 deletion on migration and invasion, indicating ASCL2 and CLDN3 act synergistically to promote malignant capacity of breast cancer cells.","method":"Co-immunoprecipitation, overexpression rescue experiments, proliferation/migration/invasion assays, xenograft tumor model","journal":"Scientific reports","confidence":"Low","confidence_rationale":"Tier 3 — single co-IP plus rescue experiment, single lab, limited mechanistic resolution of the interaction","pmids":["41318809"],"is_preprint":false}],"current_model":"CLDN3 is a four-transmembrane tight junction protein that forms heteromeric strands with other claudins via homophilic and heterophilic interactions, seals the paracellular pathway against ions of either charge and uncharged solutes (acting as a barrier-forming claudin), binds ZO-1/ZO-2/ZO-3 via their PDZ1 domains through its C-terminal YV motif, serves as a functional receptor for Clostridium perfringens enterotoxin, is phosphorylated by WNK4 kinase to regulate paracellular ion permeability, promotes MMP-2 activation and cell invasion in epithelial cancers, is transcriptionally regulated by Sp1 (methylation-sensitive), HSF1, and TET1 (controlled by PPM1G-mediated dephosphorylation), undergoes TRIM28-mediated SUMOylation and degradation, acts as an antiviral decoy receptor that blocks rotavirus VP7-mediated attachment through its EC1 loop, and regulates apical actomyosin contractility and tissue tension during neural fold morphogenesis."},"narrative":{"teleology":[{"year":1997,"claim":"Establishing that CLDN3 is sufficient to confer sensitivity to Clostridium perfringens enterotoxin (CPE) identified it as a functional CPE receptor before its tight junction role was fully appreciated.","evidence":"Gain-of-function transfection of L929 cells with CLDN3 cDNA followed by CPE binding and cytolysis assays","pmids":["9334247"],"confidence":"High","gaps":["Binding interface between CPE and CLDN3 not mapped","Whether CPE binding requires CLDN3 in assembled strands or monomers was unknown"]},{"year":1999,"claim":"Three concurrent studies established that CLDN3 is a bona fide tight junction strand component that forms heteromeric/heterophilic complexes with claudin-1 and claudin-2 and recruits ZO-1/ZO-2/ZO-3 through its C-terminal YV motif, defining the molecular architecture of claudin-based tight junctions.","evidence":"ImmunoEM in liver/kidney (PMID:9892664); co-transfection/co-culture/immunoEM in L fibroblasts (PMID:10562289); in vitro PDZ1 binding assay with mutagenesis (PMID:10601346)","pmids":["9892664","10562289","10601346"],"confidence":"High","gaps":["Stoichiometry and structural basis of heteromeric strand assembly not resolved","Functional consequence of ZO-protein recruitment on barrier selectivity not tested"]},{"year":2001,"claim":"Discovery that CLDN3 stimulates MT-MMP-mediated pro-MMP-2 activation revealed an unexpected signaling role beyond passive barrier formation, linking tight junction proteins to extracellular matrix remodeling.","evidence":"Expression cloning in 293T cells, co-immunoprecipitation with MT1-MMP and MMP-2, pro-MMP-2 processing assay","pmids":["11382769"],"confidence":"Medium","gaps":["Direct binding interface between CLDN3 and MT1-MMP uncharacterized","Whether CLDN3-MMP interaction occurs at tight junctions or elsewhere not determined"]},{"year":2004,"claim":"Identification of WNK4 as a kinase that directly phosphorylates CLDN3 and increases paracellular chloride permeability provided the first post-translational regulatory mechanism controlling CLDN3 barrier function.","evidence":"In vitro kinase assay, co-immunoprecipitation, transepithelial ion permeability in MDCK II cells expressing wild-type or mutant WNK4","pmids":["15070779"],"confidence":"High","gaps":["Specific phosphorylation site(s) on CLDN3 not mapped","Whether phosphorylation alters strand assembly or ZO-protein interaction not determined"]},{"year":2005,"claim":"Reciprocal gain- and loss-of-function experiments established that CLDN3 promotes cell invasion and motility in ovarian cancer cells through MMP-2 activation, extending the MMP-2 connection into a disease-relevant cell biological phenotype.","evidence":"Stable transfection of HOSE cells, siRNA knockdown in ovarian cancer lines, Boyden chamber invasion, wound healing, MMP-2 zymography","pmids":["16103090"],"confidence":"High","gaps":["Whether CLDN3-driven invasion depends on tight junction localization or mislocalized protein unclear","Downstream signaling intermediates not defined"]},{"year":2007,"claim":"Mapping the CLDN3 promoter revealed Sp1 as a critical transcription factor whose binding is methylation-sensitive, providing a mechanism for epigenetic silencing and derepression of CLDN3 in cancer.","evidence":"Promoter deletion analysis, ChIP, in vitro DNA binding, Sp1 siRNA knockdown, DNA methylation analysis","pmids":["17986852"],"confidence":"Medium","gaps":["Whether Sp1-driven transcription is tissue-specific not addressed","Upstream signals controlling promoter methylation not identified"]},{"year":2010,"claim":"Comprehensive permeability profiling confirmed CLDN3 as a general sealing claudin that restricts both charged and uncharged solute flux without affecting water permeability, while reshaping strand morphology into continuous meshwork loops.","evidence":"Stable CLDN3 expression in MDCK II cells, multi-ion and tracer flux assays, freeze-fracture EM of strand architecture","pmids":["20655293"],"confidence":"High","gaps":["Structural basis for non-charge-selective sealing not resolved at atomic level","Contribution of CLDN3 heteromeric strands versus homomeric strands not dissected"]},{"year":2010,"claim":"Epigenetic profiling showed that CLDN3 repression in normal ovarian epithelium depends on bivalent histone marks (H3K4me3 + H3K27me3), and derepression in tumors correlates with loss of H3K27me3 and H4K20me3, refining the chromatin-level regulation beyond DNA methylation alone.","evidence":"ChIP for histone modifications, bisulfite sequencing, DNA methyltransferase and HDAC inhibitor treatments in ovarian epithelial and cancer cells","pmids":["20053926"],"confidence":"Medium","gaps":["Causal writers/erasers of H3K27me3 at CLDN3 locus not identified","Whether bivalent state exists in non-ovarian tissues unknown"]},{"year":2024,"claim":"Establishing the PPM1G→TET1→CLDN3 promoter demethylation axis showed how upstream phosphatase activity controls CLDN3 transcription through TET1 stability, linking CLDN3 expression to epithelial-mesenchymal transition in cholangiocarcinoma.","evidence":"ChIP, bisulfite sequencing, phosphatase activity assay, co-immunoprecipitation, protein stability assays in cholangiocarcinoma cells","pmids":["39477806"],"confidence":"Medium","gaps":["Whether the PPM1G-TET1-CLDN3 axis operates in normal epithelia not tested","TET1 phosphorylation site affecting CLDN3 regulation not mapped"]},{"year":2025,"claim":"Identification of CLDN3 EC1 as a decoy receptor for rotavirus VP7 — with E74 on VP7 as a critical contact residue — revealed an innate antiviral function for a tight junction protein, where viral VP8* counter-displaces CLDN3 from the membrane.","evidence":"CRISPR knockout, RNAi, viral binding/entry assays, co-IP, structural analysis, E74K mutagenesis, in vivo mouse infection","pmids":["41860939"],"confidence":"High","gaps":["Atomic-resolution structure of CLDN3-VP7 complex not available","Whether other claudin family members share antiviral activity against rotavirus not tested"]},{"year":2025,"claim":"HSF1 was identified as a direct transcriptional activator of CLDN3 in colorectal cancer, adding a stress-responsive transcription factor to the regulatory repertoire alongside Sp1 and TET1.","evidence":"ChIP assay for HSF1 at CLDN3 promoter, reciprocal knockdown/overexpression, in vivo xenograft","pmids":["40162508"],"confidence":"Medium","gaps":["Whether heat shock or proteotoxic stress acutely induces CLDN3 through HSF1 not tested","Relationship between HSF1 and Sp1/TET1 pathways at the CLDN3 promoter not addressed"]},{"year":2025,"claim":"Discovery that TRIM28 mediates CLDN3 SUMOylation and subsequent degradation established a post-translational mechanism controlling CLDN3 protein stability, complementing the transcriptional and phosphorylation-based regulatory layers.","evidence":"Co-immunoprecipitation, SUMOylation Western blot, siRNA/overexpression in CRC cells","pmids":["41762617"],"confidence":"Medium","gaps":["Specific SUMOylation site(s) on CLDN3 not mapped","Whether TRIM28-mediated degradation is proteasomal or lysosomal not determined","Single lab, awaits independent replication"]},{"year":null,"claim":"Key unresolved questions include the atomic structure of CLDN3 homo- and heteromeric strands, the identity of WNK4 phosphorylation sites that regulate barrier function, whether CLDN3's antiviral and MMP-activating roles are functionally separable from its tight junction barrier activity, and the in vivo relevance of CLDN3-mediated actomyosin regulation in mammalian neural tube closure.","evidence":"","pmids":[],"confidence":"Low","gaps":["No high-resolution structure of CLDN3 in strand context","Phosphorylation site mapping incomplete","In vivo mammalian loss-of-function phenotype not well characterized"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0098631","term_label":"cell adhesion mediator activity","supporting_discovery_ids":[0,2,5,9]},{"term_id":"GO:0005198","term_label":"structural molecule activity","supporting_discovery_ids":[0,2,9]},{"term_id":"GO:0001618","term_label":"virus receptor activity","supporting_discovery_ids":[3]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[0,9,15]}],"pathway":[{"term_id":"R-HSA-1500931","term_label":"Cell-Cell communication","supporting_discovery_ids":[0,1,2,9]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[3,15]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[7,11]}],"complexes":["Tight junction strand (claudin heteromeric complex)"],"partners":["CLDN1","CLDN2","TJP1","TJP2","TJP3","WNK4","TRIM28","MMP14"],"other_free_text":[]},"mechanistic_narrative":"CLDN3 is a barrier-forming tight junction protein that seals the paracellular pathway against ions of both charges and uncharged solutes by assembling into homo- and heteromeric strands with claudin-1 and claudin-2 [PMID:10562289, PMID:20655293]. Its C-terminal YV motif recruits ZO-1, ZO-2, and ZO-3 via their PDZ1 domains, anchoring strands to the submembranous scaffold [PMID:10601346], and WNK4 kinase phosphorylates CLDN3 to modulate paracellular chloride permeability [PMID:15070779]. Beyond its structural barrier role, CLDN3 serves as a functional receptor for Clostridium perfringens enterotoxin [PMID:9334247] and as an antiviral decoy whose EC1 loop binds rotavirus VP7 to block viral attachment [PMID:41860939], and it promotes MMP-2 activation and cell invasion when overexpressed in epithelial cancers [PMID:16103090, PMID:11382769]."},"prefetch_data":{"uniprot":{"accession":"O15551","full_name":"Claudin-3","aliases":["Clostridium perfringens enterotoxin receptor 2","CPE-R 2","CPE-receptor 2","Rat ventral prostate.1 protein homolog","hRVP1"],"length_aa":220,"mass_kda":23.3,"function":"Barrier-forming claudin. Plays a major role in tight junction-specific obliteration of the intercellular space, through calcium-independent cell-adhesion activity","subcellular_location":"Cell junction, tight junction; Cell membrane","url":"https://www.uniprot.org/uniprotkb/O15551/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/CLDN3","classification":"Not Classified","n_dependent_lines":0,"n_total_lines":1208,"dependency_fraction":0.0},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/CLDN3","total_profiled":1310},"omim":[{"mim_id":"617579","title":"CLAUDIN 10; CLDN10","url":"https://www.omim.org/entry/617579"},{"mim_id":"611231","title":"CLAUDIN 8; CLDN8","url":"https://www.omim.org/entry/611231"},{"mim_id":"609131","title":"CLAUDIN 7; CLDN7","url":"https://www.omim.org/entry/609131"},{"mim_id":"603718","title":"CLAUDIN 1; CLDN1","url":"https://www.omim.org/entry/603718"},{"mim_id":"602910","title":"CLAUDIN 3; CLDN3","url":"https://www.omim.org/entry/602910"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Cell Junctions","reliability":"Supported"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in many","driving_tissues":[{"tissue":"intestine","ntpm":248.6},{"tissue":"pancreas","ntpm":92.7},{"tissue":"thyroid gland","ntpm":110.9}],"url":"https://www.proteinatlas.org/search/CLDN3"},"hgnc":{"alias_symbol":["RVP1","CPE-R2","HRVP1"],"prev_symbol":["C7orf1","CPETR2"]},"alphafold":{"accession":"O15551","domains":[{"cath_id":"1.20.140.150","chopping":"2-22_70-181","consensus_level":"high","plddt":90.7356,"start":2,"end":181}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/O15551","model_url":"https://alphafold.ebi.ac.uk/files/AF-O15551-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-O15551-F1-predicted_aligned_error_v6.png","plddt_mean":81.56},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=CLDN3","jax_strain_url":"https://www.jax.org/strain/search?query=CLDN3"},"sequence":{"accession":"O15551","fasta_url":"https://rest.uniprot.org/uniprotkb/O15551.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/O15551/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/O15551"}},"corpus_meta":[{"pmid":"20053926","id":"PMC_20053926","title":"Derepression of CLDN3 and CLDN4 during ovarian tumorigenesis is associated with loss of repressive histone modifications.","date":"2010","source":"Carcinogenesis","url":"https://pubmed.ncbi.nlm.nih.gov/20053926","citation_count":65,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"17986852","id":"PMC_17986852","title":"Regulation of the CLDN3 gene in ovarian cancer cells.","date":"2007","source":"Cancer biology & therapy","url":"https://pubmed.ncbi.nlm.nih.gov/17986852","citation_count":55,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"16685400","id":"PMC_16685400","title":"p16, MGMT, RARbeta2, CLDN3, CRBP and MT1G gene methylation in esophageal squamous cell carcinoma and its precursor lesions.","date":"2006","source":"Oncology reports","url":"https://pubmed.ncbi.nlm.nih.gov/16685400","citation_count":45,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"23051912","id":"PMC_23051912","title":"Recombinant protein rVP1 upregulates BECN1-independent autophagy, MAPK1/3 phosphorylation and MMP9 activity via WIPI1/WIPI2 to promote macrophage migration.","date":"2012","source":"Autophagy","url":"https://pubmed.ncbi.nlm.nih.gov/23051912","citation_count":38,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"9878248","id":"PMC_9878248","title":"Genes for the CPE receptor (CPETR1) and the human homolog of RVP1 (CPETR2) are localized within the Williams-Beuren syndrome deletion.","date":"1998","source":"Genomics","url":"https://pubmed.ncbi.nlm.nih.gov/9878248","citation_count":37,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"40057807","id":"PMC_40057807","title":"Development of a bispecific antibody-drug conjugate targeting EpCAM and CLDN3 for the treatment of multiple solid tumors.","date":"2025","source":"Experimental hematology & oncology","url":"https://pubmed.ncbi.nlm.nih.gov/40057807","citation_count":7,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"39477806","id":"PMC_39477806","title":"PPM1G Inhibits Epithelial-Mesenchymal Transition in Cholangiocarcinoma by Catalyzing TET1 Dephosphorylation for Destabilization to Impair Its Targeted Demethylation of the CLDN3 Promoter.","date":"2024","source":"Advanced science (Weinheim, Baden-Wurttemberg, Germany)","url":"https://pubmed.ncbi.nlm.nih.gov/39477806","citation_count":5,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"32855729","id":"PMC_32855729","title":"CLDN3 expression and function in pregnancy-induced hypertension.","date":"2020","source":"Experimental and therapeutic medicine","url":"https://pubmed.ncbi.nlm.nih.gov/32855729","citation_count":5,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"37078946","id":"PMC_37078946","title":"DPP10-AS1-Mediated Downregulation of MicroRNA-324-3p Is Conducive to the Malignancy of Pancreatic Cancer by Enhancing CLDN3 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Sp1 binds the unmethylated promoter more efficiently than methylated promoter, and siRNA-mediated knockdown of Sp1 significantly decreases CLDN3 expression at both mRNA and protein levels.\",\n      \"method\": \"In vitro binding assays, ChIP assays, promoter deletion analysis, siRNA knockdown\",\n      \"journal\": \"Cancer biology & therapy\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods (in vitro binding, ChIP, siRNA KD) in a single study demonstrating Sp1-dependent transcriptional regulation\",\n      \"pmids\": [\"17986852\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"CLDN3 expression is epigenetically regulated: cells expressing high CLDN3 show low DNA methylation and high histone H3 acetylation at the CLDN3 promoter; treatment with DNA methyltransferase or HDAC inhibitors induces CLDN3 expression in CLDN3-negative cells.\",\n      \"method\": \"Bisulfite sequencing, ChIP, pharmacological inhibitors (5-aza-dC, TSA)\",\n      \"journal\": \"Cancer biology & therapy\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods (ChIP, bisulfite sequencing, pharmacological manipulation) with clear functional readout\",\n      \"pmids\": [\"17986852\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"CLDN3 expression is repressed in ovarian epithelial cells by bivalent promoter histone modifications (H3K4me3 + H3K27me3); derepression during tumorigenesis correlates with loss of H3K27me3 and H4K20me3, without requiring DNA methylation for CLDN3 specifically.\",\n      \"method\": \"ChIP assays for histone modifications, pharmacological treatment (EZH2 inhibitors, HDAC inhibitors), gene expression analysis\",\n      \"journal\": \"Carcinogenesis\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal ChIP analyses across cell lines with pharmacological validation\",\n      \"pmids\": [\"20053926\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"CLDN3 overexpression in human trophoblast HTR8/SVneo cells increases proliferation, invasion, and migration while reducing apoptosis; the mechanism involves upregulation of MMP-2, MMP-9, and increased ERK1/2 phosphorylation.\",\n      \"method\": \"Lentiviral overexpression, CCK-8 assay, flow cytometry, Transwell assay, western blot\",\n      \"journal\": \"Experimental and therapeutic medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — single lab, overexpression with multiple phenotypic readouts but pathway placement is correlative\",\n      \"pmids\": [\"32855729\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"miR-324-3p directly targets and downregulates CLDN3; the lncRNA DPP10-AS1 acts as a competing endogenous RNA by sequestering miR-324-3p, thereby releasing CLDN3 expression and promoting pancreatic cancer cell invasion and migration.\",\n      \"method\": \"Luciferase reporter assays, RNA pulldown, scratch/transwell assays, xenograft mouse model\",\n      \"journal\": \"Pancreas\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — ceRNA mechanism demonstrated with functional assays and in vivo validation, single lab\",\n      \"pmids\": [\"37078946\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"TET1 promotes CLDN3 transcription by targeting the CLDN3 promoter region (-16 to +512) for demethylation; PPM1G catalyzes the dephosphorylation of TET1, destabilizing it and impairing its ability to demethylate the CLDN3 promoter, thereby suppressing CLDN3-driven EMT in cholangiocarcinoma.\",\n      \"method\": \"ChIP assays, co-immunoprecipitation, phosphatase activity assays, western blot, EMT functional assays\",\n      \"journal\": \"Advanced science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods (ChIP, Co-IP, phosphatase assays) establishing mechanistic axis PPM1G→TET1 dephosphorylation→CLDN3 promoter demethylation\",\n      \"pmids\": [\"39477806\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"HSF1 directly binds to the transcription factor binding site of CLDN3 and activates its transcription; HSF1 knockdown reduces CLDN3 expression, and this axis promotes CRC cell proliferation, migration, and invasion.\",\n      \"method\": \"ChIP assays, luciferase reporter, stable knockdown/overexpression, in vivo xenograft\",\n      \"journal\": \"Neoplasma\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — ChIP and functional KD/OE with in vivo validation, single lab\",\n      \"pmids\": [\"40162508\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"ASCL2 physically interacts with CLDN3 (confirmed by Co-IP); overexpression of CLDN3 partially rescues the inhibitory effects of ASCL2 deletion on proliferation, migration and invasion of breast cancer cells, indicating a synergistic functional relationship.\",\n      \"method\": \"Co-immunoprecipitation, rescue overexpression experiments, xenograft model\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — single Co-IP confirmed interaction with rescue functional validation, single lab\",\n      \"pmids\": [\"41318809\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"CLDN3 directly interacts with TRIM28; TRIM28 mediates SUMOylation and subsequent degradation of CLDN3, thereby regulating CLDN3 protein stability in colorectal cancer cells.\",\n      \"method\": \"Co-immunoprecipitation, immunofluorescence, western blot for SUMOylation\",\n      \"journal\": \"IET systems biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — Co-IP and SUMOylation western blot in a single study, single lab\",\n      \"pmids\": [\"41762617\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"CLDN3 acts as a decoy receptor that inhibits rotavirus attachment: the CLDN3 extracellular loop 1 (EC1) interacts with the N-terminal domain of rotavirus VP7, reducing viral adsorption; the VP7 E74K mutation disrupts this interaction and enhances viral attachment and pathogenicity in vivo.\",\n      \"method\": \"Knockout/knockdown of CLDN3, binding assays, structural studies, site-directed mutagenesis (E74K), in vivo viral challenge\",\n      \"journal\": \"PLoS pathogens\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — mutagenesis of critical residue, structural studies, KO/KD with binding assay, and in vivo validation in a single study with multiple orthogonal methods\",\n      \"pmids\": [\"41860939\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"In chick embryos, CLDN3 expressed in the non-neural ectoderm regulates apical cell shape and tissue tension during neural fold fusion; CLDN3 depletion increases phospho-MLC (pMLC) staining and tissue tension (laser ablation), decreases apical cell area at neural fold edges, and impairs spinal neural fold fusion ('buttoning'); partial rescue by blebbistatin (myosin II inhibitor) demonstrates the mechanism involves actomyosin regulation.\",\n      \"method\": \"Live imaging, cell segmentation, laser ablation biomechanics, immunofluorescence for pMLC, pharmacological rescue with blebbistatin, morpholino knockdown\",\n      \"journal\": \"bioRxiv (preprint)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods (live imaging, laser ablation, pharmacological rescue) but preprint only\",\n      \"pmids\": [\"bio_10.1101_2025.10.14.682463\"],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Rat CLDN3 (but not human CLDN3) functions as an entry factor for rat hepacivirus NRHV1; the host-specific entry function is determined by two amino acid residues (Ile44 and Trp46) in extracellular loop 1; CLDN3 expression in otherwise non-susceptible murine cell lines confers NRHV1 entry.\",\n      \"method\": \"Gene expression profiling of susceptible vs. non-susceptible cell lines, exogenous expression of CLDN3 orthologs, site-directed mutagenesis of EC1 loop residues\",\n      \"journal\": \"bioRxiv (preprint)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — functional gain-of-entry assay with mutagenesis identifying critical residues, preprint only\",\n      \"pmids\": [\"bio_10.1101_2025.02.27.640490\"],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"CLDN3 is a tight junction transmembrane protein whose transcription is regulated by Sp1-dependent promoter activity modulated by DNA methylation, histone modifications (H3K27me3, H4K20me3), and transcription factors including HSF1 and TET1 (controlled by PPM1G-mediated dephosphorylation); its protein stability is regulated by TRIM28-mediated SUMOylation and degradation; its extracellular loop 1 mediates interactions with viral proteins (rotavirus VP7) to inhibit viral attachment and with intracellular partners such as ASCL2; and it regulates epithelial cell behaviors including actomyosin-dependent tissue tension, MMP expression, and ERK1/2 signaling to control cell migration, invasion, and morphogenesis.\"\n}\n```\n\n--- ROUND 2 (R2/R3, augmented corpus) ---\n\n```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1999,\n      \"finding\": \"Claudin-3 (identified as mouse RVP1 homolog) is a four-transmembrane domain protein that concentrates exclusively at tight junctions in liver and kidney, as demonstrated by immunofluorescence and immunoelectron microscopy, establishing it as a bona fide tight junction strand component.\",\n      \"method\": \"Immunofluorescence microscopy, immunoelectron microscopy, transfection into MDCK cells\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — direct localization by immunoelectron microscopy with multiple orthogonal methods, foundational paper replicated across labs\",\n      \"pmids\": [\"9892664\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"ZO-1, ZO-2, and ZO-3 directly bind the COOH-terminal YV sequence of claudin-3 (and claudins 1-8) through their PDZ1 domains in vitro, and ZO-1 is recruited to claudin-based networks at cell-cell borders in fibroblasts transfected with claudins.\",\n      \"method\": \"In vitro binding assay, transfection into L fibroblasts, co-localization by immunofluorescence\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro binding assay with mutagenesis-level specificity (PDZ1 domain) plus cellular reconstitution, replicated across claudin family members\",\n      \"pmids\": [\"10601346\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"Claudin-3 can form heteromeric tight junction strands with claudin-1 and claudin-2, and claudin-3 strands can associate laterally with claudin-1 and claudin-2 strands in trans (heterophilic interactions), whereas claudin-1 and claudin-2 strands do not interact with each other.\",\n      \"method\": \"Co-transfection into L fibroblasts, co-culture experiments, immunoreplica electron microscopy\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal approaches (co-transfection, co-culture, immunoEM) establishing specific heteromeric assembly rules\",\n      \"pmids\": [\"10562289\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1997,\n      \"finding\": \"Claudin-3 (then called the human RVP1 homolog) functions as a receptor for Clostridium perfringens enterotoxin (CPE): L929 cells transfected with claudin-3 cDNA became sensitive to CPE, demonstrating it is sufficient to mediate CPE binding and cytolysis.\",\n      \"method\": \"Transfection of L929 cells, CPE binding assay, cytotoxicity assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — functional gain-of-function in naive cells demonstrating receptor sufficiency, replicated by multiple subsequent studies\",\n      \"pmids\": [\"9334247\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"Claudin-3 promotes activation of pro-MMP-2 mediated by membrane-type MMPs (MT-MMPs): claudin-3 expression in 293T cells stimulated MT-MMP-mediated pro-MMP-2 processing, and direct interaction of claudin family members with MT1-MMP and MMP-2 was demonstrated by immunoprecipitation.\",\n      \"method\": \"Expression cloning, co-immunoprecipitation, pro-MMP-2 activation assay in 293T cells\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — functional assay with co-IP, but claudin-3 was one of several claudins tested and was not the primary focus\",\n      \"pmids\": [\"11382769\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"In human airways, claudin-3 contributes to tight junction barrier function: stable expression of claudin-3 in NIH/3T3 and IB3.1 airway cells decreased solute permeability, and co-immunoprecipitation revealed heterophilic interactions between claudin-3 and other claudin species in cell lines and in freshly excised human airway epithelium.\",\n      \"method\": \"Stable transfection, transepithelial resistance measurement, permeability coefficients, co-immunoprecipitation\",\n      \"journal\": \"American journal of physiology. Lung cellular and molecular physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — functional transepithelial resistance plus co-IP in both cell lines and primary tissue, single lab\",\n      \"pmids\": [\"12909588\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Disease-causing mutant WNK4 kinase binds and phosphorylates claudins 1-4 (including claudin-3) at tight junctions, with mutant WNK4 causing greater claudin phosphorylation than wild-type, and this is associated with increased paracellular chloride permeability in MDCK II cells.\",\n      \"method\": \"Stable expression in MDCK II cells, transepithelial ion permeability measurement, in vitro phosphorylation assay, co-immunoprecipitation\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — in vitro kinase assay demonstrating direct phosphorylation, functional permeability measurements, gain-of-function mutation approach\",\n      \"pmids\": [\"15070779\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Claudin-3 expression in human ovarian surface epithelial (HOSE) cells increases cell invasion, motility, and survival, and is associated with increased MMP-2 activity; siRNA-mediated knockdown of claudin-3 in ovarian cancer cell lines reduces invasion.\",\n      \"method\": \"Stable transfection of HOSE cells, Boyden chamber invasion assay, wound-healing assay, siRNA knockdown, MMP-2 zymography\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal gain-of-function and loss-of-function with defined molecular readout (MMP-2 activity), multiple orthogonal cellular assays\",\n      \"pmids\": [\"16103090\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"The CLDN3 promoter contains a minimal Sp1-binding site critical for transcriptional activity; Sp1 binds preferentially to the unmethylated promoter, providing a mechanism for epigenetic silencing. siRNA knockdown of Sp1 significantly decreases CLDN3 mRNA and protein expression.\",\n      \"method\": \"Promoter deletion analysis, ChIP assay, in vitro DNA-binding assay, siRNA knockdown, DNA methylation analysis, histone acetylation ChIP\",\n      \"journal\": \"Cancer biology & therapy\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — ChIP plus in vitro binding plus siRNA knockdown with multiple orthogonal methods in a single lab\",\n      \"pmids\": [\"17986852\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Claudin-3 acts as a general sealing component of the tight junction paracellular pathway: stable transfection of MDCK II cells with human claudin-3 elevated transepithelial resistance, decreased permeability to mono- and divalent cations, anions, and uncharged solutes (fluorescein, FD-4), and altered tight junction strand morphology toward uninterrupted meshwork loops, without affecting water permeability.\",\n      \"method\": \"Stable transfection of MDCK II cells, transepithelial resistance measurement, ion permeability assays, tracer flux assays, electron microscopy of tight junction strands\",\n      \"journal\": \"Biochimica et biophysica acta\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — comprehensive functional characterization with multiple ion/solute permeability assays and structural analysis, rigorous controls with two clone systems\",\n      \"pmids\": [\"20655293\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Derepression of CLDN3 in ovarian tumorigenesis correlates with loss of repressive histone modifications (H3K27me3 and H4K20me3) from its promoter. CLDN3 repression in normal ovarian epithelial cells is maintained by bivalent histone marks (H3K4me3 + H3K27me3), and DNA methylation is not required for CLDN3 repression in immortalized ovarian epithelial cells.\",\n      \"method\": \"ChIP assay for histone modifications, bisulfite sequencing, DNA methyltransferase and HDAC inhibitor treatments, quantitative RT-PCR\",\n      \"journal\": \"Carcinogenesis\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — ChIP with multiple histone marks plus pharmacological intervention, single lab\",\n      \"pmids\": [\"20053926\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"CLDN3 overexpression in human trophoblast HTR8/SVneo cells promotes proliferation, invasion, and migration while reducing apoptosis; this is associated with increased MMP-2 and MMP-9 expression and increased ERK1/2 phosphorylation, placing CLDN3 upstream of ERK1/2 signaling and MMP activation in trophoblasts.\",\n      \"method\": \"Lentiviral overexpression, CCK-8 assay, flow cytometry, Transwell assay, Western blot for ERK1/2 phosphorylation and MMP expression\",\n      \"journal\": \"Experimental and therapeutic medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — gain-of-function with defined molecular readouts (ERK1/2, MMPs) but single lab, no rescue or loss-of-function complement\",\n      \"pmids\": [\"32855729\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"TET1 promotes CLDN3 transcription by demethylating the CLDN3 promoter region (-16 to +512 bp). PPM1G phosphatase catalyzes TET1 dephosphorylation, leading to TET1 protein destabilization, which impairs TET1-mediated CLDN3 promoter demethylation and suppresses epithelial-mesenchymal transition in cholangiocarcinoma cells.\",\n      \"method\": \"ChIP assay, bisulfite sequencing, phosphatase activity assay, co-immunoprecipitation, protein stability assays, EMT functional assays, pharmacological inhibitors\",\n      \"journal\": \"Advanced science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — mechanistic chain from PPM1G → TET1 dephosphorylation → CLDN3 demethylation established with ChIP and biochemical assays, single lab\",\n      \"pmids\": [\"39477806\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"HSF1 directly binds to the CLDN3 promoter and activates CLDN3 transcription in colorectal cancer cells, as demonstrated by ChIP assay; HSF1 knockdown reduces CLDN3 expression and inhibits CRC cell proliferation, migration, and invasion, while HSF1 overexpression promotes these behaviors.\",\n      \"method\": \"ChIP assay, Western blot, PCR, stable knockdown and overexpression, in vivo xenograft\",\n      \"journal\": \"Neoplasma\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — direct promoter binding by ChIP plus reciprocal gain/loss-of-function, single lab\",\n      \"pmids\": [\"40162508\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"CLDN3 directly interacts with TRIM28 as demonstrated by co-immunoprecipitation and immunofluorescence; TRIM28 mediates SUMOylation and degradation of CLDN3 protein, establishing TRIM28 as a writer controlling CLDN3 stability. CLDN3 knockdown in CRC cells decreases proliferation and migration, and CLDN3 overexpression reduces sensitivity to 5-FU.\",\n      \"method\": \"Co-immunoprecipitation, immunofluorescence, Western blot for SUMOylation, siRNA knockdown, overexpression, proliferation and migration assays\",\n      \"journal\": \"IET systems biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — co-IP plus SUMOylation Western blot with functional readout, single lab\",\n      \"pmids\": [\"41762617\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"CLDN3 acts as a host antiviral defense protein against rotavirus (RV): the CLDN3 extracellular loop 1 (EC1) directly interacts with the N-terminal domain of RV outer capsid protein VP7, reducing viral adsorption. Structural analysis identified glutamic acid at position 74 (E74) of VP7 as critical for the CLDN3-VP7 interaction. Knockdown or knockout of CLDN3 promotes RV binding and entry, and the VP8* peptide of RV induces CLDN3 mislocalization from the plasma membrane as a counterdefense. The E74K mutation in VP7 disrupts the CLDN3-VP7 interaction and enhances viral pathogenicity in vivo.\",\n      \"method\": \"CLDN3 knockout/knockdown (RNAi, CRISPR), viral binding and entry assays, co-immunoprecipitation, structural studies, in vivo mouse infection model, site-directed mutagenesis (E74K)\",\n      \"journal\": \"PLoS pathogens\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — structural validation + mutagenesis + KO/KD + in vivo pathogenicity, multiple orthogonal methods in single study\",\n      \"pmids\": [\"41860939\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"In chick embryos, CLDN3 (expressed in non-neural ectoderm) is required for neural fold fusion in the spinal region. CLDN3 depletion decreases apical cell area at neural fold edges, increases phospho-MLC staining in the apical domain, and increases tissue tension in the non-neural ectoderm as measured by laser ablation. Treatment with the myosin II inhibitor blebbistatin partially rescues neural fold fusion defects, placing CLDN3 upstream of actomyosin contractility and apical cytoskeletal regulation.\",\n      \"method\": \"Morpholino knockdown in chick embryos, live imaging, cell segmentation analysis, laser ablation biomechanics, pMLC immunostaining, blebbistatin pharmacological rescue\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods (live imaging, laser ablation, pharmacological rescue) but preprint, single lab\",\n      \"pmids\": [],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Claudin-3 contributes to tight junction strand formation and paracellular barrier regulation in intestinal epithelial context; downregulation of claudin-3 (along with claudins -4, -5, -7, -8, -12) is observed in intestinal inflammatory disorders, suggesting a role in maintaining epithelial barrier homeostasis.\",\n      \"method\": \"Expression analysis in intestinal inflammatory disease context (review/summary of experimental data)\",\n      \"journal\": \"Genome biology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 4 — review/expression-based inference, not a primary mechanistic experiment for CLDN3 specifically\",\n      \"pmids\": [\"19706201\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"ASCL2 physically interacts with CLDN3 as demonstrated by co-immunoprecipitation assay in breast cancer cells. Rescue experiments show that overexpression of CLDN3 can partly reverse the inhibitory effects of ASCL2 deletion on migration and invasion, indicating ASCL2 and CLDN3 act synergistically to promote malignant capacity of breast cancer cells.\",\n      \"method\": \"Co-immunoprecipitation, overexpression rescue experiments, proliferation/migration/invasion assays, xenograft tumor model\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — single co-IP plus rescue experiment, single lab, limited mechanistic resolution of the interaction\",\n      \"pmids\": [\"41318809\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"CLDN3 is a four-transmembrane tight junction protein that forms heteromeric strands with other claudins via homophilic and heterophilic interactions, seals the paracellular pathway against ions of either charge and uncharged solutes (acting as a barrier-forming claudin), binds ZO-1/ZO-2/ZO-3 via their PDZ1 domains through its C-terminal YV motif, serves as a functional receptor for Clostridium perfringens enterotoxin, is phosphorylated by WNK4 kinase to regulate paracellular ion permeability, promotes MMP-2 activation and cell invasion in epithelial cancers, is transcriptionally regulated by Sp1 (methylation-sensitive), HSF1, and TET1 (controlled by PPM1G-mediated dephosphorylation), undergoes TRIM28-mediated SUMOylation and degradation, acts as an antiviral decoy receptor that blocks rotavirus VP7-mediated attachment through its EC1 loop, and regulates apical actomyosin contractility and tissue tension during neural fold morphogenesis.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"CLDN3 is a tight junction transmembrane protein that functions in epithelial barrier formation, tissue morphogenesis, and host defense against viral entry. Its transcription is controlled by Sp1 binding to an unmethylated promoter and is epigenetically regulated through DNA methylation, histone acetylation, and bivalent histone marks (H3K4me3/H3K27me3), with additional transcriptional input from HSF1 and TET1-mediated promoter demethylation that is itself regulated by PPM1G-dependent TET1 dephosphorylation [PMID:17986852, PMID:20053926, PMID:39477806, PMID:40162508]. At the protein level, CLDN3 stability is controlled by TRIM28-mediated SUMOylation and degradation, and its extracellular loop 1 mediates species-specific interactions with viral proteins, functioning as a decoy receptor that inhibits rotavirus attachment through direct binding to the VP7 N-terminal domain [PMID:41762617, PMID:41860939]. CLDN3 overexpression promotes cell proliferation, migration, and invasion through MMP-2/9 upregulation and ERK1/2 phosphorylation, and in developing epithelia it regulates actomyosin-dependent tissue tension and apical cell shape during neural fold fusion [PMID:32855729, PMID:37078946].\",\n  \"teleology\": [\n    {\n      \"year\": 2007,\n      \"claim\": \"Establishing that CLDN3 transcription is directly driven by Sp1 and gated by promoter DNA methylation and histone acetylation resolved how this tight junction gene is silenced or activated in different cell types.\",\n      \"evidence\": \"Promoter deletion analysis, ChIP, bisulfite sequencing, siRNA knockdown, and pharmacological demethylation/HDAC inhibition in cancer cell lines\",\n      \"pmids\": [\"17986852\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether Sp1-dependent regulation operates in normal (non-cancer) epithelial tissues\", \"Identity of the methyltransferases responsible for CLDN3 promoter silencing\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Demonstrating that CLDN3 is held in a bivalent chromatin state (H3K4me3 + H3K27me3) in normal ovarian epithelium, with derepression via loss of H3K27me3 during tumorigenesis, revealed a Polycomb-dependent mechanism for CLDN3 overexpression in cancer independent of DNA methylation.\",\n      \"evidence\": \"ChIP for histone marks across normal and cancer ovarian cell lines with EZH2 and HDAC inhibitor treatments\",\n      \"pmids\": [\"20053926\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether EZH2 is the sole H3K27 methyltransferase acting at the CLDN3 locus\", \"Signals that trigger H3K27me3 loss during transformation\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Showing that CLDN3 overexpression increases proliferation, invasion, and migration via MMP-2/9 and ERK1/2 signaling provided the first downstream effector pathway linking CLDN3 to pro-invasive cell behavior.\",\n      \"evidence\": \"Lentiviral overexpression in HTR8/SVneo trophoblast cells with Transwell, CCK-8, and western blot readouts\",\n      \"pmids\": [\"32855729\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether CLDN3 activates ERK1/2 directly or through an intermediate signaling complex\", \"Lack of loss-of-function validation in the same system\", \"Generalizability beyond trophoblast cells\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Identifying miR-324-3p as a direct post-transcriptional suppressor of CLDN3, sponged by the lncRNA DPP10-AS1, revealed a ceRNA regulatory axis that modulates CLDN3-driven invasion in pancreatic cancer.\",\n      \"evidence\": \"Luciferase reporter assays, RNA pulldown, Transwell assays, and xenograft model in pancreatic cancer cells\",\n      \"pmids\": [\"37078946\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether additional miRNAs converge on CLDN3 regulation\", \"Quantitative contribution of ceRNA axis versus transcriptional regulation in vivo\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Elucidating that TET1 demethylates the CLDN3 promoter and that PPM1G dephosphorylates and destabilizes TET1 established a phosphatase–epigenetic writer–CLDN3 signaling axis controlling EMT in cholangiocarcinoma.\",\n      \"evidence\": \"ChIP, Co-IP, phosphatase activity assays, and EMT functional assays in cholangiocarcinoma cells\",\n      \"pmids\": [\"39477806\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Which kinase phosphorylates TET1 to counteract PPM1G\", \"Whether the PPM1G–TET1–CLDN3 axis operates in non-biliary epithelia\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Identifying HSF1 as a direct transcriptional activator of CLDN3 linked heat shock signaling to CLDN3-dependent proliferation and invasion in colorectal cancer.\",\n      \"evidence\": \"ChIP, luciferase reporter, knockdown/overexpression, and xenograft in CRC cells\",\n      \"pmids\": [\"40162508\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether HSF1 activation of CLDN3 is stress-inducible or constitutive in tumors\", \"Relative contribution of HSF1 versus Sp1 to CLDN3 transcription in the same cells\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Demonstrating a physical interaction between ASCL2 and CLDN3, with CLDN3 overexpression partially rescuing ASCL2-deletion phenotypes, linked a Wnt-regulated transcription factor to tight junction-mediated pro-tumorigenic signaling in breast cancer.\",\n      \"evidence\": \"Co-IP, rescue overexpression experiments, and xenograft in breast cancer cells\",\n      \"pmids\": [\"41318809\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether ASCL2–CLDN3 interaction is direct or bridged by other tight junction components\", \"Reciprocal Co-IP not reported\", \"Structural basis of the interaction unknown\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Showing that CLDN3 in non-neural ectoderm regulates actomyosin contractility and tissue tension during neural fold fusion established a developmental morphogenetic role for CLDN3 beyond barrier function.\",\n      \"evidence\": \"Morpholino knockdown in chick embryos with live imaging, laser ablation, pMLC immunofluorescence, and blebbistatin rescue (preprint)\",\n      \"pmids\": [\"bio_10.1101_2025.10.14.682463\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Preprint; not yet peer-reviewed\", \"How CLDN3 mechanistically controls myosin light chain phosphorylation\", \"Whether this role is conserved in mammalian neural tube closure\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Identifying TRIM28-mediated SUMOylation as the mechanism for CLDN3 protein degradation revealed a post-translational control point for CLDN3 protein stability in colorectal cancer.\",\n      \"evidence\": \"Co-IP, immunofluorescence, and SUMOylation western blot in CRC cells\",\n      \"pmids\": [\"41762617\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Specific SUMO isoform(s) conjugated to CLDN3 not identified\", \"Whether ubiquitin–proteasome or autophagy mediates downstream degradation\", \"Single-lab finding\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Demonstrating that CLDN3 extracellular loop 1 interacts with rotavirus VP7 to block viral attachment, with the VP7 E74K mutation abolishing this interaction and enhancing pathogenicity, established CLDN3 as an innate antiviral decoy receptor.\",\n      \"evidence\": \"CLDN3 KO/KD, binding assays, structural studies, VP7 E74K mutagenesis, and in vivo viral challenge\",\n      \"pmids\": [\"41860939\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether CLDN3 similarly restricts other rotavirus genotypes\", \"Structural resolution of the CLDN3 EC1–VP7 interface at atomic level\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How CLDN3 mechanistically couples tight junction engagement to intracellular signaling cascades (ERK1/2, actomyosin) remains unresolved, as does the structural basis for its diverse extracellular loop 1 interactions with viral proteins and cellular partners.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No structural model of full-length CLDN3 in complex with signaling partners\", \"Direct versus indirect activation of ERK1/2 not established\", \"Relative physiological importance of transcriptional versus post-translational regulation of CLDN3 levels in normal tissues unknown\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0001618\", \"supporting_discovery_ids\": [9, 11]},\n      {\"term_id\": \"GO:0005198\", \"supporting_discovery_ids\": [10]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [9, 10, 11]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1500931\", \"supporting_discovery_ids\": [3, 10]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [3]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [9]},\n      {\"term_id\": \"R-HSA-4839726\", \"supporting_discovery_ids\": [1, 2]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"TRIM28\", \"ASCL2\", \"SP1\", \"TET1\", \"HSF1\"],\n    \"other_free_text\": []\n  }\n}\n```\n\n--- ROUND 2 (R2/R3, augmented corpus) ---\n\n```json\n{\n  \"mechanistic_narrative\": \"CLDN3 is a barrier-forming tight junction protein that seals the paracellular pathway against ions of both charges and uncharged solutes by assembling into homo- and heteromeric strands with claudin-1 and claudin-2 [PMID:10562289, PMID:20655293]. Its C-terminal YV motif recruits ZO-1, ZO-2, and ZO-3 via their PDZ1 domains, anchoring strands to the submembranous scaffold [PMID:10601346], and WNK4 kinase phosphorylates CLDN3 to modulate paracellular chloride permeability [PMID:15070779]. Beyond its structural barrier role, CLDN3 serves as a functional receptor for Clostridium perfringens enterotoxin [PMID:9334247] and as an antiviral decoy whose EC1 loop binds rotavirus VP7 to block viral attachment [PMID:41860939], and it promotes MMP-2 activation and cell invasion when overexpressed in epithelial cancers [PMID:16103090, PMID:11382769].\",\n  \"teleology\": [\n    {\n      \"year\": 1997,\n      \"claim\": \"Establishing that CLDN3 is sufficient to confer sensitivity to Clostridium perfringens enterotoxin (CPE) identified it as a functional CPE receptor before its tight junction role was fully appreciated.\",\n      \"evidence\": \"Gain-of-function transfection of L929 cells with CLDN3 cDNA followed by CPE binding and cytolysis assays\",\n      \"pmids\": [\"9334247\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Binding interface between CPE and CLDN3 not mapped\", \"Whether CPE binding requires CLDN3 in assembled strands or monomers was unknown\"]\n    },\n    {\n      \"year\": 1999,\n      \"claim\": \"Three concurrent studies established that CLDN3 is a bona fide tight junction strand component that forms heteromeric/heterophilic complexes with claudin-1 and claudin-2 and recruits ZO-1/ZO-2/ZO-3 through its C-terminal YV motif, defining the molecular architecture of claudin-based tight junctions.\",\n      \"evidence\": \"ImmunoEM in liver/kidney (PMID:9892664); co-transfection/co-culture/immunoEM in L fibroblasts (PMID:10562289); in vitro PDZ1 binding assay with mutagenesis (PMID:10601346)\",\n      \"pmids\": [\"9892664\", \"10562289\", \"10601346\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Stoichiometry and structural basis of heteromeric strand assembly not resolved\", \"Functional consequence of ZO-protein recruitment on barrier selectivity not tested\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Discovery that CLDN3 stimulates MT-MMP-mediated pro-MMP-2 activation revealed an unexpected signaling role beyond passive barrier formation, linking tight junction proteins to extracellular matrix remodeling.\",\n      \"evidence\": \"Expression cloning in 293T cells, co-immunoprecipitation with MT1-MMP and MMP-2, pro-MMP-2 processing assay\",\n      \"pmids\": [\"11382769\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct binding interface between CLDN3 and MT1-MMP uncharacterized\", \"Whether CLDN3-MMP interaction occurs at tight junctions or elsewhere not determined\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Identification of WNK4 as a kinase that directly phosphorylates CLDN3 and increases paracellular chloride permeability provided the first post-translational regulatory mechanism controlling CLDN3 barrier function.\",\n      \"evidence\": \"In vitro kinase assay, co-immunoprecipitation, transepithelial ion permeability in MDCK II cells expressing wild-type or mutant WNK4\",\n      \"pmids\": [\"15070779\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Specific phosphorylation site(s) on CLDN3 not mapped\", \"Whether phosphorylation alters strand assembly or ZO-protein interaction not determined\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Reciprocal gain- and loss-of-function experiments established that CLDN3 promotes cell invasion and motility in ovarian cancer cells through MMP-2 activation, extending the MMP-2 connection into a disease-relevant cell biological phenotype.\",\n      \"evidence\": \"Stable transfection of HOSE cells, siRNA knockdown in ovarian cancer lines, Boyden chamber invasion, wound healing, MMP-2 zymography\",\n      \"pmids\": [\"16103090\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether CLDN3-driven invasion depends on tight junction localization or mislocalized protein unclear\", \"Downstream signaling intermediates not defined\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Mapping the CLDN3 promoter revealed Sp1 as a critical transcription factor whose binding is methylation-sensitive, providing a mechanism for epigenetic silencing and derepression of CLDN3 in cancer.\",\n      \"evidence\": \"Promoter deletion analysis, ChIP, in vitro DNA binding, Sp1 siRNA knockdown, DNA methylation analysis\",\n      \"pmids\": [\"17986852\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether Sp1-driven transcription is tissue-specific not addressed\", \"Upstream signals controlling promoter methylation not identified\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Comprehensive permeability profiling confirmed CLDN3 as a general sealing claudin that restricts both charged and uncharged solute flux without affecting water permeability, while reshaping strand morphology into continuous meshwork loops.\",\n      \"evidence\": \"Stable CLDN3 expression in MDCK II cells, multi-ion and tracer flux assays, freeze-fracture EM of strand architecture\",\n      \"pmids\": [\"20655293\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis for non-charge-selective sealing not resolved at atomic level\", \"Contribution of CLDN3 heteromeric strands versus homomeric strands not dissected\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Epigenetic profiling showed that CLDN3 repression in normal ovarian epithelium depends on bivalent histone marks (H3K4me3 + H3K27me3), and derepression in tumors correlates with loss of H3K27me3 and H4K20me3, refining the chromatin-level regulation beyond DNA methylation alone.\",\n      \"evidence\": \"ChIP for histone modifications, bisulfite sequencing, DNA methyltransferase and HDAC inhibitor treatments in ovarian epithelial and cancer cells\",\n      \"pmids\": [\"20053926\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Causal writers/erasers of H3K27me3 at CLDN3 locus not identified\", \"Whether bivalent state exists in non-ovarian tissues unknown\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Establishing the PPM1G→TET1→CLDN3 promoter demethylation axis showed how upstream phosphatase activity controls CLDN3 transcription through TET1 stability, linking CLDN3 expression to epithelial-mesenchymal transition in cholangiocarcinoma.\",\n      \"evidence\": \"ChIP, bisulfite sequencing, phosphatase activity assay, co-immunoprecipitation, protein stability assays in cholangiocarcinoma cells\",\n      \"pmids\": [\"39477806\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether the PPM1G-TET1-CLDN3 axis operates in normal epithelia not tested\", \"TET1 phosphorylation site affecting CLDN3 regulation not mapped\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Identification of CLDN3 EC1 as a decoy receptor for rotavirus VP7 — with E74 on VP7 as a critical contact residue — revealed an innate antiviral function for a tight junction protein, where viral VP8* counter-displaces CLDN3 from the membrane.\",\n      \"evidence\": \"CRISPR knockout, RNAi, viral binding/entry assays, co-IP, structural analysis, E74K mutagenesis, in vivo mouse infection\",\n      \"pmids\": [\"41860939\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Atomic-resolution structure of CLDN3-VP7 complex not available\", \"Whether other claudin family members share antiviral activity against rotavirus not tested\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"HSF1 was identified as a direct transcriptional activator of CLDN3 in colorectal cancer, adding a stress-responsive transcription factor to the regulatory repertoire alongside Sp1 and TET1.\",\n      \"evidence\": \"ChIP assay for HSF1 at CLDN3 promoter, reciprocal knockdown/overexpression, in vivo xenograft\",\n      \"pmids\": [\"40162508\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether heat shock or proteotoxic stress acutely induces CLDN3 through HSF1 not tested\", \"Relationship between HSF1 and Sp1/TET1 pathways at the CLDN3 promoter not addressed\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Discovery that TRIM28 mediates CLDN3 SUMOylation and subsequent degradation established a post-translational mechanism controlling CLDN3 protein stability, complementing the transcriptional and phosphorylation-based regulatory layers.\",\n      \"evidence\": \"Co-immunoprecipitation, SUMOylation Western blot, siRNA/overexpression in CRC cells\",\n      \"pmids\": [\"41762617\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Specific SUMOylation site(s) on CLDN3 not mapped\", \"Whether TRIM28-mediated degradation is proteasomal or lysosomal not determined\", \"Single lab, awaits independent replication\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Key unresolved questions include the atomic structure of CLDN3 homo- and heteromeric strands, the identity of WNK4 phosphorylation sites that regulate barrier function, whether CLDN3's antiviral and MMP-activating roles are functionally separable from its tight junction barrier activity, and the in vivo relevance of CLDN3-mediated actomyosin regulation in mammalian neural tube closure.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No high-resolution structure of CLDN3 in strand context\", \"Phosphorylation site mapping incomplete\", \"In vivo mammalian loss-of-function phenotype not well characterized\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0098631\", \"supporting_discovery_ids\": [0, 2, 5, 9]},\n      {\"term_id\": \"GO:0005198\", \"supporting_discovery_ids\": [0, 2, 9]},\n      {\"term_id\": \"GO:0001618\", \"supporting_discovery_ids\": [3]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [0, 9, 15]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"GO:0005198\", \"supporting_discovery_ids\": []},\n      {\"term_id\": \"R-HSA-1500931\", \"supporting_discovery_ids\": [0, 1, 2, 9]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [3, 15]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [7, 11]}\n    ],\n    \"complexes\": [\n      \"Tight junction strand (claudin heteromeric complex)\"\n    ],\n    \"partners\": [\n      \"CLDN1\",\n      \"CLDN2\",\n      \"TJP1\",\n      \"TJP2\",\n      \"TJP3\",\n      \"WNK4\",\n      \"TRIM28\",\n      \"MMP14\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}