{"gene":"CGN","run_date":"2026-04-28T17:28:52","timeline":{"discoveries":[{"year":1999,"finding":"Cingulin (CGN) is a ~140–160 kDa phosphoprotein at the cytoplasmic surface of epithelial tight junctions containing an N-terminal globular head domain (residues 1–439), a central α-helical rod domain (440–1,325), and a C-terminal tail (1,326–1,368). The rod domain mediates formation of coiled-coil parallel dimers via electron microscopy and pull-down assays. The N-terminal head fragment (residues 1–378) directly interacts with ZO-1 (Kd ~5 nM), ZO-2, ZO-3, myosin, and AF-6 in pull-down assays from epithelial, insect, and reticulocyte lysates, while ZO-1 and ZO-2 immunoprecipitates contain cingulin in vivo. Full-length cingulin but not individual fragments correctly localizes to tight junctions in transfected MDCK cells, indicating both head and rod domains are required for TJ targeting.","method":"Full-length Xenopus cDNA cloning, electron microscopy, in vitro pull-down assays (Kd measurement), co-immunoprecipitation from epithelial cells, transfection/localization in MDCK cells","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 1–2 — multiple orthogonal methods (in vitro binding with Kd, EM, Co-IP, cell transfection) in a single rigorous study","pmids":["10613913"],"is_preprint":false},{"year":2000,"finding":"Junctional adhesion molecule (JAM) physically associates with cingulin (CGN) at tight junctions. Co-precipitation experiments in Caco-2 epithelial cells showed JAM in a complex with cingulin, and this interaction required the amino-terminal globular head domain of cingulin. JAM transfection into CHO cells enhanced junctional localization of endogenous ZO-1, and JAM–ZO-1 interaction required the C-terminal PDZ-binding motif of JAM.","method":"Co-immunoprecipitation from detergent-insoluble fractions of Caco-2 cells, in vitro binding assays, transfection of CHO cells","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 — reciprocal Co-IP plus in vitro domain-mapping binding assays","pmids":["10877843"],"is_preprint":false},{"year":2005,"finding":"Cingulin (CGN) directly binds to GEF-H1/Lfc, a RhoA-specific guanine nucleotide exchange factor, via its junctional adaptor function. Cingulin binding to GEF-H1 inhibits RhoA activation and downstream signaling. In confluent MDCK epithelial cells, increased cingulin expression sequesters GEF-H1, downregulating RhoA. RNAi depletion of cingulin causes irregular monolayers and RhoA activation, while RNAi of GEF-H1 or transfection of GEF-H1-binding cingulin mutants inhibits G1/S cell-cycle progression.","method":"Direct interaction assays (pull-down), RhoA activation assays (GTPase pull-down), regulated RNAi in MDCK cells, G1/S phase analysis, dominant-negative mutant transfection","journal":"Developmental cell","confidence":"High","confidence_rationale":"Tier 2 — direct interaction demonstrated, multiple orthogonal functional readouts (RhoA activity, cell cycle, RNAi phenotype) replicated across constructs","pmids":["15866167"],"is_preprint":false},{"year":2010,"finding":"CGN promoter undergoes hypermethylation during TGFβ-driven epithelial-mesenchymal transition (EMT) in breast cancer cells, leading to transcriptional silencing. Disrupting Smad signaling in mesenchymal breast cancer cells reversed CGN promoter methylation and restored CGN expression, accompanied by reacquisition of epithelial morphology and suppressed invasion. This silencing was linked to reduced DNA-binding activity of DNMT1.","method":"Breast cancer cell-based EMT model, bisulfite sequencing/methylation analysis, Smad pathway disruption, gene re-expression assay, invasion assay, DNMT1 DNA-binding activity assay","journal":"Cancer research","confidence":"Medium","confidence_rationale":"Tier 2 — functional reversal of epigenetic silencing with mechanistic link to DNMT1, single lab study","pmids":["20086175"],"is_preprint":false},{"year":2021,"finding":"miR-125b directly targets CGN (cingulin) mRNA in colorectal cancer cells, as validated by dual-luciferase reporter assays with the CGN 3′-UTR. Knockdown of CGN phenocopies miR-125b overexpression by enhancing cell migration and invasion. Mechanistically, CGN suppression by miR-125b activates the RhoA/ROCK signaling pathway, promoting CRC metastasis. CGN re-expression rescues the pro-migratory phenotype in vitro and in vivo.","method":"Dual-luciferase reporter assay, RNA-seq, siRNA knockdown, overexpression rescue, in vitro migration/invasion assays, in vivo xenograft model, RhoA/ROCK pathway activity measurement","journal":"Cancers","confidence":"Medium","confidence_rationale":"Tier 2 — direct 3′-UTR reporter validation plus functional rescue experiments in vitro and in vivo, single lab","pmids":["34830864"],"is_preprint":false},{"year":2023,"finding":"Knockout of CGN (cingulin) alone, CGNL1 (paracingulin) alone, or both in MDCK kidney epithelial cells causes a modest but significant increase in transepithelial resistance (TER) and decreased sodium permeability, without affecting 3 kDa dextran permeability. This phenotype is associated with reduced protein expression and junctional accumulation of the sodium pore-forming claudin-2. Overexpression of CGN or CGNL1 (but not ZO-1) rescues claudin-2 junctional localization, indicating a CGN-specific role in claudin-2 regulation independent of ZO-1.","method":"CRISPR/Cas9 knockout in MDCK cells, transepithelial resistance measurements, paracellular permeability assays (dextran, sodium), calcium-switch assay, immunofluorescence, western blot, qRT-PCR, rescue by overexpression","journal":"Cells","confidence":"Medium","confidence_rationale":"Tier 2 — clean KO with defined cellular phenotype and domain-specific rescue, single lab","pmids":["37566083"],"is_preprint":false},{"year":2025,"finding":"In ovarian cancer cells, polyphyllin H upregulates CGN (cingulin) expression, which in turn blocks the RhoA/Rock2 signaling pathway to inhibit cancer cell proliferation, invasion, and migration. This CGN/RhoA/Rock2 axis was identified through protein sequencing of drug-treated cells and functionally validated in cell line-derived and patient-derived xenograft mouse models.","method":"CCK-8 proliferation assay, invasion/migration assays, protein sequencing (proteomics), CDX and PDX mouse models, in vivo tumor growth measurement, pathway activity assays","journal":"International journal of surgery (London, England)","confidence":"Low","confidence_rationale":"Tier 3 — mechanistic pathway placement via proteomics and in vivo models, but no direct mutagenesis or binding assay for CGN–RhoA interaction; single lab","pmids":["40773225"],"is_preprint":false}],"current_model":"Cingulin (CGN) is a dimeric coiled-coil scaffolding protein at the cytoplasmic face of epithelial tight junctions, where its N-terminal globular head domain directly binds ZO-1/ZO-2/ZO-3, myosin, JAM, and GEF-H1/Lfc; by sequestering GEF-H1 it suppresses RhoA/ROCK signaling and links tight junction formation to inhibition of cell proliferation (G1/S block) and restraint of epithelial migration, while also regulating claudin-2 junctional accumulation and paracellular sodium permeability."},"narrative":{"teleology":[{"year":1999,"claim":"Resolving cingulin's domain architecture and direct binding partners established it as a multi-valent cytoplasmic scaffold at tight junctions, rather than a simple structural component.","evidence":"cDNA cloning of Xenopus cingulin, electron microscopy of coiled-coil dimers, in vitro pull-downs with Kd measurement for ZO-1, co-IP and transfection/localization in MDCK cells","pmids":["10613913"],"confidence":"High","gaps":["Binding affinities for ZO-2, ZO-3, myosin, and AF-6 not quantified","Post-translational regulation of cingulin junctional recruitment not addressed","No functional assay for junctional barrier or signaling consequences"]},{"year":2000,"claim":"Identification of JAM as a transmembrane binding partner of cingulin showed that cingulin physically links integral membrane components to the tight-junction plaque, expanding its adaptor role beyond cytoplasmic scaffolds.","evidence":"Co-immunoprecipitation from Caco-2 detergent-insoluble fractions, domain-mapping in vitro binding assays, CHO cell transfection","pmids":["10877843"],"confidence":"High","gaps":["Whether cingulin–JAM interaction is direct or bridged by ZO-1 was not fully resolved","Functional consequence of disrupting cingulin–JAM binding on barrier integrity unknown"]},{"year":2005,"claim":"Discovery that cingulin sequesters GEF-H1 to inhibit RhoA activation revealed a direct signaling function for a tight-junction scaffold, answering how junction formation feeds back on cell proliferation and morphology.","evidence":"Direct pull-down of GEF-H1, RhoA-GTP assays, cingulin/GEF-H1 RNAi in MDCK cells, G1/S cell-cycle analysis, dominant-negative mutant expression","pmids":["15866167"],"confidence":"High","gaps":["Structural basis of cingulin–GEF-H1 interaction undefined","Whether cingulin regulates additional GEFs or GAPs not tested","Relative contributions of RhoA inhibition versus other cingulin interactions to barrier phenotype unresolved"]},{"year":2010,"claim":"Demonstration that TGFβ/Smad-driven EMT epigenetically silences CGN via promoter hypermethylation provided a mechanism for loss of cingulin's anti-migratory function during cancer progression.","evidence":"Bisulfite sequencing in breast cancer EMT models, Smad pathway disruption, DNMT1 activity assays, invasion assays","pmids":["20086175"],"confidence":"Medium","gaps":["Whether CGN silencing is a cause or consequence of EMT progression not fully distinguished","In vivo relevance of CGN epigenetic silencing in patient tumors not assessed","Specific Smad complex binding to CGN promoter not mapped"]},{"year":2021,"claim":"Validation of miR-125b as a direct post-transcriptional repressor of CGN in colorectal cancer confirmed that CGN loss activates RhoA/ROCK to promote metastasis, and that CGN re-expression rescues the phenotype in vivo.","evidence":"Dual-luciferase 3′-UTR reporter, siRNA knockdown, overexpression rescue in vitro and in xenograft models, RhoA/ROCK activity measurement","pmids":["34830864"],"confidence":"Medium","gaps":["Clinical correlation of miR-125b/CGN axis in patient cohorts limited","Whether additional miRNAs converge on CGN regulation unknown","Downstream RhoA effectors beyond ROCK not delineated"]},{"year":2023,"claim":"CRISPR knockout of CGN revealed a specific role in promoting claudin-2 junctional accumulation and paracellular sodium permeability, mechanistically separable from ZO-1-dependent functions.","evidence":"CRISPR/Cas9 KO of CGN and CGNL1 in MDCK cells, TER and permeability assays, rescue by CGN overexpression, immunofluorescence and western blot","pmids":["37566083"],"confidence":"Medium","gaps":["Whether cingulin directly binds claudin-2 or acts via an intermediate is unknown","Impact on other claudin family members not systematically tested","In vivo kidney or intestinal barrier phenotype of CGN knockout not reported"]},{"year":null,"claim":"The structural basis of cingulin's multi-valent head-domain interactions, its regulation by phosphorylation, and its in vivo roles in tissue barrier function and tumor suppression remain unresolved.","evidence":"","pmids":[],"confidence":"Low","gaps":["No high-resolution structure of cingulin head domain or its complexes with GEF-H1, ZO proteins, or claudins","Phosphorylation sites are documented but functional consequences in signaling are not mapped","No conditional in vivo knockout phenotype in mammalian epithelial tissues reported in the timeline"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[0,1,2]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[2,4]},{"term_id":"GO:0008092","term_label":"cytoskeletal protein binding","supporting_discovery_ids":[0]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[0,1,5]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[2,4,6]},{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[2]}],"complexes":["tight junction plaque"],"partners":["TJP1","TJP2","TJP3","F11R","ARHGEF2","CGNL1","CLDN2"],"other_free_text":[]},"mechanistic_narrative":"Cingulin (CGN) is a dimeric coiled-coil scaffolding protein localized to the cytoplasmic face of epithelial tight junctions, where it integrates junctional assembly with Rho GTPase signaling and paracellular permeability control. Its N-terminal globular head domain directly binds ZO-1, ZO-2, ZO-3, myosin, AF-6, JAM, and the RhoA-specific exchange factor GEF-H1, while the coiled-coil rod domain mediates parallel dimerization and is required together with the head for correct tight-junction targeting [PMID:10613913, PMID:10877843]. Sequestration of GEF-H1 by cingulin inhibits RhoA/ROCK activation, linking tight-junction formation to suppression of cell proliferation (G1/S block) and restraint of epithelial migration; loss of CGN derepresses RhoA signaling and promotes invasion in both normal and cancer epithelial contexts [PMID:15866167, PMID:34830864]. CGN also promotes junctional accumulation of the pore-forming claudin-2, thereby regulating paracellular sodium permeability independently of ZO-1 [PMID:37566083]."},"prefetch_data":{"uniprot":{"accession":"Q9P2M7","full_name":"Cingulin","aliases":[],"length_aa":1203,"mass_kda":137.1,"function":"Probably plays a role in the formation and regulation of the tight junction (TJ) paracellular permeability barrier","subcellular_location":"Cell junction, tight junction","url":"https://www.uniprot.org/uniprotkb/Q9P2M7/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/CGN","classification":"Not Classified","n_dependent_lines":6,"n_total_lines":1208,"dependency_fraction":0.004966887417218543},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"CALD1","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/CGN","total_profiled":1310},"omim":[{"mim_id":"619990","title":"TRANSMEMBRANE p24 TRAFFICKING PROTEIN 7; TMED7","url":"https://www.omim.org/entry/619990"},{"mim_id":"616432","title":"RHO GUANINE NUCLEOTIDE EXCHANGE FACTOR 18; ARHGEF18","url":"https://www.omim.org/entry/616432"},{"mim_id":"614398","title":"JUNCTIONAL CADHERIN 5-ASSOCIATED PROTEIN; JCAD","url":"https://www.omim.org/entry/614398"},{"mim_id":"613735","title":"BRAIN MALFORMATIONS WITH OR WITHOUT URINARY TRACT DEFECTS; BRMUTD","url":"https://www.omim.org/entry/613735"},{"mim_id":"609473","title":"CINGULIN; CGN","url":"https://www.omim.org/entry/609473"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Plasma membrane","reliability":"Supported"},{"location":"Cell Junctions","reliability":"Supported"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in many","driving_tissues":[{"tissue":"intestine","ntpm":53.1}],"url":"https://www.proteinatlas.org/search/CGN"},"hgnc":{"alias_symbol":["KIAA1319"],"prev_symbol":[]},"alphafold":{"accession":"Q9P2M7","domains":[{"cath_id":"1.20.5","chopping":"1113-1140","consensus_level":"medium","plddt":87.4186,"start":1113,"end":1140}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9P2M7","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9P2M7-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9P2M7-F1-predicted_aligned_error_v6.png","plddt_mean":69.56},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=CGN","jax_strain_url":"https://www.jax.org/strain/search?query=CGN"},"sequence":{"accession":"Q9P2M7","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9P2M7.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9P2M7/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9P2M7"}},"corpus_meta":[{"pmid":"34830864","id":"PMC_34830864","title":"miR-125b Promotes Colorectal Cancer Migration and Invasion by Dual-Targeting CFTR and CGN.","date":"2021","source":"Cancers","url":"https://pubmed.ncbi.nlm.nih.gov/34830864","citation_count":30,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"23658230","id":"PMC_23658230","title":"Life without tRNAArg-adenosine deaminase TadA: evolutionary consequences of decoding the four CGN codons as arginine in Mycoplasmas and other Mollicutes.","date":"2013","source":"Nucleic acids research","url":"https://pubmed.ncbi.nlm.nih.gov/23658230","citation_count":28,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"35484716","id":"PMC_35484716","title":"Single-cell RNA Sequencing Identified Novel Nr4a1+ Ear2+ Anti-Inflammatory Macrophage Phenotype under Myeloid-TLR4 Dependent Regulation in Anti-Glomerular Basement Membrane (GBM) Crescentic Glomerulonephritis (cGN).","date":"2022","source":"Advanced science (Weinheim, Baden-Wurttemberg, Germany)","url":"https://pubmed.ncbi.nlm.nih.gov/35484716","citation_count":22,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"25747092","id":"PMC_25747092","title":"Transformation pathways in high-pressure solid nitrogen: from molecular N2 to polymeric cg-N.","date":"2015","source":"The Journal of chemical physics","url":"https://pubmed.ncbi.nlm.nih.gov/25747092","citation_count":15,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"35809677","id":"PMC_35809677","title":"Biophysical interaction between lanthanum chloride and (CG)n or (GC)n repeats: A reversible B-to-Z DNA transition.","date":"2022","source":"International journal of biological macromolecules","url":"https://pubmed.ncbi.nlm.nih.gov/35809677","citation_count":11,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"16717448","id":"PMC_16717448","title":"Isolation, mapping and identification of SNPs for four genes (ACP6, CGN, ANXA9, SLC27A3) from a bovine QTL region on BTA3.","date":"2006","source":"Cytogenetic and genome research","url":"https://pubmed.ncbi.nlm.nih.gov/16717448","citation_count":7,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"37566083","id":"PMC_37566083","title":"Knock Out of CGN and CGNL1 in MDCK Cells Affects Claudin-2 but Has a Minor Impact on Tight Junction Barrier Function.","date":"2023","source":"Cells","url":"https://pubmed.ncbi.nlm.nih.gov/37566083","citation_count":6,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"27029281","id":"PMC_27029281","title":"Where have all the inosines gone? Conflicting evidence for A-to-I editing of the anticodon of higher eukaryotic tRNAACGArg questions the dogma of a universal wobble-mediated decoding of CGN codons.","date":"2016","source":"IUBMB life","url":"https://pubmed.ncbi.nlm.nih.gov/27029281","citation_count":5,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"34562582","id":"PMC_34562582","title":"Hepatic antioxidant activity, immunomodulation, and pro-anti-inflammatory cytokines manipulation of κ-carrageenan (κ-CGN) in cobia, Rachycentron canadum against Lactococcus garvieae.","date":"2021","source":"Fish & shellfish immunology","url":"https://pubmed.ncbi.nlm.nih.gov/34562582","citation_count":3,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"8754409","id":"PMC_8754409","title":"[Evaluation of factors influencing platelet aggregation in patients with chronic glomerulonephritis (CGN)].","date":"1996","source":"Przeglad lekarski","url":"https://pubmed.ncbi.nlm.nih.gov/8754409","citation_count":2,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"38778164","id":"PMC_38778164","title":"Arginines of the CGN codon family are Achilles' heels of cancer genes.","date":"2024","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/38778164","citation_count":1,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"39628587","id":"PMC_39628587","title":"TLX3 regulates CGN progenitor proliferation during cerebellum development and its dysfunction can lead to autism.","date":"2024","source":"iScience","url":"https://pubmed.ncbi.nlm.nih.gov/39628587","citation_count":1,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"8754408","id":"PMC_8754408","title":"[Evaluation of platelet membrane structure in patients with chronic glomerulonephritis (CGN)].","date":"1996","source":"Przeglad lekarski","url":"https://pubmed.ncbi.nlm.nih.gov/8754408","citation_count":1,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"40773225","id":"PMC_40773225","title":"Polyphyllin H inhibits malignant progression of ovarian cancer in patient-derived xenograft mouse models by regulating CGN/RhoA/Rock2 axis: an experimental research.","date":"2025","source":"International journal of surgery (London, England)","url":"https://pubmed.ncbi.nlm.nih.gov/40773225","citation_count":0,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":null,"id":"bio_10.1101_2025.06.04.657904","title":"Tetraploid turnips ( <i>Brassica rapa</i> ssp. <i>rapa</i> ) are highly tolerant of tetravalent formation and aneuploidy","date":"2025-06-06","source":"bioRxiv","url":"https://doi.org/10.1101/2025.06.04.657904","citation_count":0,"is_preprint":true,"source_track":"pubmed_title"},{"pmid":null,"id":"bio_10.1101_2024.05.23.595506","title":"Genomic factors shaping codon usage across the Saccharomycotina subphylum","date":"2024-05-24","source":"bioRxiv","url":"https://doi.org/10.1101/2024.05.23.595506","citation_count":0,"is_preprint":true,"source_track":"pubmed_title"},{"pmid":"12477932","id":"PMC_12477932","title":"Generation and initial analysis of more than 15,000 full-length human and mouse cDNA sequences.","date":"2002","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/12477932","citation_count":1479,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"28514442","id":"PMC_28514442","title":"Architecture of the human interactome defines protein communities and disease networks.","date":"2017","source":"Nature","url":"https://pubmed.ncbi.nlm.nih.gov/28514442","citation_count":1085,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"26496610","id":"PMC_26496610","title":"A human interactome in three quantitative dimensions organized by stoichiometries and abundances.","date":"2015","source":"Cell","url":"https://pubmed.ncbi.nlm.nih.gov/26496610","citation_count":1015,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"12529927","id":"PMC_12529927","title":"Tight junctions of the blood-brain barrier: development, composition and regulation.","date":"2002","source":"Vascular pharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/12529927","citation_count":869,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"32296183","id":"PMC_32296183","title":"A reference map of the human binary protein interactome.","date":"2020","source":"Nature","url":"https://pubmed.ncbi.nlm.nih.gov/32296183","citation_count":849,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"14702039","id":"PMC_14702039","title":"Complete sequencing and characterization of 21,243 full-length human cDNAs.","date":"2003","source":"Nature genetics","url":"https://pubmed.ncbi.nlm.nih.gov/14702039","citation_count":754,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"17353931","id":"PMC_17353931","title":"Large-scale mapping of human protein-protein interactions by mass spectrometry.","date":"2007","source":"Molecular systems biology","url":"https://pubmed.ncbi.nlm.nih.gov/17353931","citation_count":733,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"33961781","id":"PMC_33961781","title":"Dual proteome-scale networks reveal cell-specific remodeling of the human interactome.","date":"2021","source":"Cell","url":"https://pubmed.ncbi.nlm.nih.gov/33961781","citation_count":705,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"21873635","id":"PMC_21873635","title":"Phylogenetic-based propagation of functional annotations within the Gene Ontology consortium.","date":"2011","source":"Briefings in bioinformatics","url":"https://pubmed.ncbi.nlm.nih.gov/21873635","citation_count":656,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"29395067","id":"PMC_29395067","title":"High-Density Proximity Mapping Reveals the Subcellular Organization of mRNA-Associated Granules and Bodies.","date":"2018","source":"Molecular cell","url":"https://pubmed.ncbi.nlm.nih.gov/29395067","citation_count":580,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"12665801","id":"PMC_12665801","title":"Exploring proteomes and analyzing protein processing by mass spectrometric identification of sorted N-terminal peptides.","date":"2003","source":"Nature biotechnology","url":"https://pubmed.ncbi.nlm.nih.gov/12665801","citation_count":485,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"35271311","id":"PMC_35271311","title":"OpenCell: Endogenous tagging for the cartography of human cellular organization.","date":"2022","source":"Science (New York, N.Y.)","url":"https://pubmed.ncbi.nlm.nih.gov/35271311","citation_count":432,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"15324660","id":"PMC_15324660","title":"Proteomic, functional, and domain-based analysis of in vivo 14-3-3 binding proteins involved in cytoskeletal regulation and cellular organization.","date":"2004","source":"Current biology : CB","url":"https://pubmed.ncbi.nlm.nih.gov/15324660","citation_count":386,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"24255178","id":"PMC_24255178","title":"Protein interaction network of the mammalian Hippo pathway reveals mechanisms of kinase-phosphatase interactions.","date":"2013","source":"Science signaling","url":"https://pubmed.ncbi.nlm.nih.gov/24255178","citation_count":383,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"10877843","id":"PMC_10877843","title":"Interaction of junctional adhesion molecule with the tight junction components ZO-1, cingulin, and occludin.","date":"2000","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/10877843","citation_count":361,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"34079125","id":"PMC_34079125","title":"A proximity-dependent biotinylation map of a human cell.","date":"2021","source":"Nature","url":"https://pubmed.ncbi.nlm.nih.gov/34079125","citation_count":339,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"15146197","id":"PMC_15146197","title":"Transcriptome characterization elucidates signaling networks that control human ES cell growth and differentiation.","date":"2004","source":"Nature biotechnology","url":"https://pubmed.ncbi.nlm.nih.gov/15146197","citation_count":266,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"10613913","id":"PMC_10613913","title":"Cingulin contains globular and coiled-coil domains and interacts with ZO-1, ZO-2, ZO-3, and myosin.","date":"1999","source":"The Journal of cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/10613913","citation_count":229,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"27173435","id":"PMC_27173435","title":"An organelle-specific protein landscape identifies novel diseases and molecular mechanisms.","date":"2016","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/27173435","citation_count":211,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"32203420","id":"PMC_32203420","title":"Systems analysis of RhoGEF and RhoGAP regulatory proteins reveals spatially organized RAC1 signalling from integrin adhesions.","date":"2020","source":"Nature cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/32203420","citation_count":194,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"15161933","id":"PMC_15161933","title":"Comprehensive proteomic analysis of interphase and mitotic 14-3-3-binding proteins.","date":"2004","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/15161933","citation_count":185,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"17979178","id":"PMC_17979178","title":"A novel tandem affinity purification strategy for the efficient isolation and characterisation of native protein complexes.","date":"2007","source":"Proteomics","url":"https://pubmed.ncbi.nlm.nih.gov/17979178","citation_count":183,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"15866167","id":"PMC_15866167","title":"Binding of GEF-H1 to the tight junction-associated adaptor cingulin results in inhibition of Rho signaling and G1/S phase transition.","date":"2005","source":"Developmental cell","url":"https://pubmed.ncbi.nlm.nih.gov/15866167","citation_count":177,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"25468996","id":"PMC_25468996","title":"E-cadherin interactome complexity and robustness resolved by quantitative proteomics.","date":"2014","source":"Science signaling","url":"https://pubmed.ncbi.nlm.nih.gov/25468996","citation_count":162,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"17567994","id":"PMC_17567994","title":"Prominent use of distal 5' transcription start sites and discovery of a large number of additional exons in ENCODE regions.","date":"2007","source":"Genome research","url":"https://pubmed.ncbi.nlm.nih.gov/17567994","citation_count":154,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"20086175","id":"PMC_20086175","title":"Smad signaling is required to maintain epigenetic silencing during breast cancer progression.","date":"2010","source":"Cancer research","url":"https://pubmed.ncbi.nlm.nih.gov/20086175","citation_count":154,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"15778465","id":"PMC_15778465","title":"Targeted proteomic analysis of 14-3-3 sigma, a p53 effector commonly silenced in cancer.","date":"2005","source":"Molecular & cellular proteomics : MCP","url":"https://pubmed.ncbi.nlm.nih.gov/15778465","citation_count":153,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"22586326","id":"PMC_22586326","title":"Functional proteomics establishes the interaction of SIRT7 with chromatin remodeling complexes and expands its role in regulation of RNA polymerase I transcription.","date":"2012","source":"Molecular & cellular proteomics : MCP","url":"https://pubmed.ncbi.nlm.nih.gov/22586326","citation_count":145,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"16710414","id":"PMC_16710414","title":"The DNA sequence and biological annotation of human chromosome 1.","date":"2006","source":"Nature","url":"https://pubmed.ncbi.nlm.nih.gov/16710414","citation_count":144,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"29117863","id":"PMC_29117863","title":"RNA-binding activity of TRIM25 is mediated by its PRY/SPRY domain and is required for ubiquitination.","date":"2017","source":"BMC biology","url":"https://pubmed.ncbi.nlm.nih.gov/29117863","citation_count":135,"is_preprint":false,"source_track":"gene2pubmed"}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":10008,"output_tokens":827,"usd":0.021215},"stage2":{"model":"claude-opus-4-6","input_tokens":4006,"output_tokens":1233,"usd":0.076283},"total_usd":0.281527,"stage1_batch_id":"msgbatch_011kfMfE81o6CTcyhVUqumuW","stage2_batch_id":"msgbatch_01DTputS6WNkH745x1nVSoBo","note":"batch pricing = 50% of standard","round2_stage1":{"model":"claude-sonnet-4-6","input_tokens":22626,"output_tokens":3595,"usd":0.060901},"round2_rules_fired":"R3","round2_stage2":{"model":"claude-opus-4-6","input_tokens":5837,"output_tokens":2116,"usd":0.123128}},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2023,\n      \"finding\": \"Knockout of CGN (cingulin) and CGNL1 in MDCK kidney epithelial cells reduces junctional accumulation and protein expression of claudin-2, leading to a modest increase in transepithelial resistance and decreased sodium permeability; rescue experiments showed CGN or CGNL1, but not ZO-1, overexpression restored claudin-2 localization, establishing CGN's role in regulating claudin-2 at tight junctions.\",\n      \"method\": \"CRISPR/Cas9 knockout, calcium switch assay, transepithelial resistance measurement, dextran permeability assay, rescue overexpression experiments, mRNA and protein quantification\",\n      \"journal\": \"Cells\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — clean KO with defined cellular phenotype, multiple orthogonal methods, rescue experiment in single rigorous study\",\n      \"pmids\": [\"37566083\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"miR-125b directly targets the 3'UTR of CGN (cingulin) in colorectal cancer cells, suppressing its expression and activating the RhoA/ROCK signaling pathway, thereby promoting migration and invasion; loss of CGN phenocopies miR-125b overexpression in promoting RhoA/ROCK activity.\",\n      \"method\": \"Dual-luciferase reporter assay, RNA-seq, miR-125b overexpression/knockdown, in vitro migration/invasion assays, in vivo xenograft experiments\",\n      \"journal\": \"Cancers\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — dual-luciferase validates direct targeting, in vitro and in vivo phenotype; single lab study\",\n      \"pmids\": [\"34830864\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Polyphyllin H upregulates CGN (cingulin) expression in ovarian cancer cells, which blocks the RhoA/Rock2 signaling pathway and inhibits proliferation, invasion, and migration of ovarian cancer cells in vitro and in vivo.\",\n      \"method\": \"Protein sequencing, CDX and PDX mouse models, cell proliferation/invasion/migration assays, western blot for pathway components\",\n      \"journal\": \"International journal of surgery (London, England)\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — single lab, mechanistic link inferred from protein sequencing and pathway inhibition without direct CGN manipulation\",\n      \"pmids\": [\"40773225\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"CGN (cingulin) is a cytoplasmic tight junction adaptor that tethers ZO-1 to the actomyosin and microtubule cytoskeletons, regulates junctional localization and expression of claudin-2 (thereby modulating paracellular sodium permeability), and suppresses the RhoA/ROCK signaling pathway to inhibit epithelial-mesenchymal transition, cell migration, and invasion; its expression is post-transcriptionally repressed by miR-125b.\"\n}\n```\n\n--- ROUND 2 (R2/R3, augmented corpus) ---\n\nI'll classify each paper first, then extract discoveries only from KEEP papers.\n\n**Classification:**\n\nRetrieved papers:\n1. PMID:34830864 — About CGN (cingulin) as miR-125b target in CRC → KEEP\n2. PMID:23658230 — CGN codons (arginine codons), not the gene CGN → EXCLUDE (alias collision)\n3. PMID:35484716 — cGN = crescentic glomerulonephritis abbreviation → EXCLUDE (alias collision)\n4. PMID:25747092 — cg-N = cubic gauche nitrogen phase → EXCLUDE (alias collision)\n5. PMID:35809677 — (CG)n DNA repeats → EXCLUDE (alias collision)\n6. PMID:16717448 — CGN = cingulin gene in bovine → KEEP\n7. PMID:37566083 — CGN/CGNL1 KO in MDCK cells → KEEP\n8. PMID:27029281 — CGN codons for arginine → EXCLUDE (alias collision)\n9. PMID:34562582 — κ-CGN = κ-carrageenan in fish → EXCLUDE (alias collision)\n10. PMID:8754409 — CGN = chronic glomerulonephritis → EXCLUDE (alias collision)\n11. PMID:38778164 — CGN codon family (arginine codons) → EXCLUDE (alias collision)\n12. PMID:39628587 — CGN = cerebellar granule neuron progenitors → EXCLUDE (alias collision)\n13. PMID:8754408 — CGN = chronic glomerulonephritis → EXCLUDE (alias collision)\n14. PMID:40773225 — CGN = cingulin in ovarian cancer → KEEP\n15. bio_10.1101_2025.06.04.657904 — CGN = germplasm bank abbreviation → EXCLUDE (alias collision)\n16. bio_10.1101_2024.05.23.595506 — CGN codons in yeast → EXCLUDE (alias collision)\n\nGene2pubmed curated papers:\n1. PMID:12477932 — cDNA sequences, no CGN-specific mechanism → EXCLUDE\n2. PMID:28514442 — BioPlex interactome, CGN appears as interactor → EXCLUDE (no specific mechanism)\n3. PMID:26496610 — HeLa interactome → EXCLUDE (no specific mechanism)\n4. PMID:32296183 — HuRI interactome → EXCLUDE (no specific mechanism)\n5. PMID:14702039 — FLJ cDNA collection → EXCLUDE\n6. PMID:17353931 — Large-scale MS interactions → EXCLUDE (no specific mechanism)\n7. PMID:33961781 — BioPlex 3.0 → EXCLUDE (no specific mechanism)\n8. PMID:21873635 — GO consortium → EXCLUDE\n9. PMID:29395067 — mRNA granule BioID, CGN not specifically studied → EXCLUDE\n10. PMID:12665801 — N-terminal proteomics → EXCLUDE\n11. PMID:35271311 — OpenCell proteomics → EXCLUDE (no specific mechanism)\n12. PMID:15324660 — 14-3-3 binding proteins, CGN listed → EXCLUDE (no specific CGN mechanism)\n13. PMID:24255178 — Hippo pathway network → EXCLUDE\n14. PMID:10877843 — JAM interaction with cingulin → KEEP\n15. PMID:34079125 — BioID map → EXCLUDE (no specific mechanism)\n16. PMID:15146197 — hES transcriptome → EXCLUDE\n17. PMID:10613913 — Cingulin domains and interactions → KEEP\n18. PMID:27173435 — Ciliary proteomics → EXCLUDE\n19. PMID:32203420 — RhoGEF/RhoGAP systems → EXCLUDE (no specific CGN mechanism)\n20. PMID:15161933 — 14-3-3 binding proteins → EXCLUDE\n21. PMID:17979178 — SF-TAP purification → EXCLUDE\n22. PMID:15866167 — GEF-H1/cingulin interaction → KEEP\n23. PMID:25468996 — E-cadherin interactome → EXCLUDE\n24. PMID:17567994 — ENCODE transcription → EXCLUDE\n25. PMID:20086175 — TGFbeta/Smad and CGN silencing → KEEP (epigenetic mechanism involving CGN)\n26. PMID:15778465 — 14-3-3 sigma proteomics → EXCLUDE\n27. PMID:22586326 — SIRT7 proteomics → EXCLUDE\n28. PMID:16710414 — Chromosome 1 sequence → EXCLUDE\n29. PMID:29117863 — TRIM25 → EXCLUDE\n30. PMID:12529927 — Blood-brain barrier TJ review, mentions cingulin → EXCLUDE (review, no direct experiment)\n\n```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1999,\n      \"finding\": \"Cingulin (CGN) is a ~140–160 kDa phosphoprotein at the cytoplasmic surface of epithelial tight junctions containing an N-terminal globular head domain (residues 1–439), a central α-helical rod domain (440–1,325), and a C-terminal tail (1,326–1,368). The rod domain mediates formation of coiled-coil parallel dimers via electron microscopy and pull-down assays. The N-terminal head fragment (residues 1–378) directly interacts with ZO-1 (Kd ~5 nM), ZO-2, ZO-3, myosin, and AF-6 in pull-down assays from epithelial, insect, and reticulocyte lysates, while ZO-1 and ZO-2 immunoprecipitates contain cingulin in vivo. Full-length cingulin but not individual fragments correctly localizes to tight junctions in transfected MDCK cells, indicating both head and rod domains are required for TJ targeting.\",\n      \"method\": \"Full-length Xenopus cDNA cloning, electron microscopy, in vitro pull-down assays (Kd measurement), co-immunoprecipitation from epithelial cells, transfection/localization in MDCK cells\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — multiple orthogonal methods (in vitro binding with Kd, EM, Co-IP, cell transfection) in a single rigorous study\",\n      \"pmids\": [\"10613913\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"Junctional adhesion molecule (JAM) physically associates with cingulin (CGN) at tight junctions. Co-precipitation experiments in Caco-2 epithelial cells showed JAM in a complex with cingulin, and this interaction required the amino-terminal globular head domain of cingulin. JAM transfection into CHO cells enhanced junctional localization of endogenous ZO-1, and JAM–ZO-1 interaction required the C-terminal PDZ-binding motif of JAM.\",\n      \"method\": \"Co-immunoprecipitation from detergent-insoluble fractions of Caco-2 cells, in vitro binding assays, transfection of CHO cells\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal Co-IP plus in vitro domain-mapping binding assays\",\n      \"pmids\": [\"10877843\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Cingulin (CGN) directly binds to GEF-H1/Lfc, a RhoA-specific guanine nucleotide exchange factor, via its junctional adaptor function. Cingulin binding to GEF-H1 inhibits RhoA activation and downstream signaling. In confluent MDCK epithelial cells, increased cingulin expression sequesters GEF-H1, downregulating RhoA. RNAi depletion of cingulin causes irregular monolayers and RhoA activation, while RNAi of GEF-H1 or transfection of GEF-H1-binding cingulin mutants inhibits G1/S cell-cycle progression.\",\n      \"method\": \"Direct interaction assays (pull-down), RhoA activation assays (GTPase pull-down), regulated RNAi in MDCK cells, G1/S phase analysis, dominant-negative mutant transfection\",\n      \"journal\": \"Developmental cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — direct interaction demonstrated, multiple orthogonal functional readouts (RhoA activity, cell cycle, RNAi phenotype) replicated across constructs\",\n      \"pmids\": [\"15866167\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"CGN promoter undergoes hypermethylation during TGFβ-driven epithelial-mesenchymal transition (EMT) in breast cancer cells, leading to transcriptional silencing. Disrupting Smad signaling in mesenchymal breast cancer cells reversed CGN promoter methylation and restored CGN expression, accompanied by reacquisition of epithelial morphology and suppressed invasion. This silencing was linked to reduced DNA-binding activity of DNMT1.\",\n      \"method\": \"Breast cancer cell-based EMT model, bisulfite sequencing/methylation analysis, Smad pathway disruption, gene re-expression assay, invasion assay, DNMT1 DNA-binding activity assay\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — functional reversal of epigenetic silencing with mechanistic link to DNMT1, single lab study\",\n      \"pmids\": [\"20086175\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"miR-125b directly targets CGN (cingulin) mRNA in colorectal cancer cells, as validated by dual-luciferase reporter assays with the CGN 3′-UTR. Knockdown of CGN phenocopies miR-125b overexpression by enhancing cell migration and invasion. Mechanistically, CGN suppression by miR-125b activates the RhoA/ROCK signaling pathway, promoting CRC metastasis. CGN re-expression rescues the pro-migratory phenotype in vitro and in vivo.\",\n      \"method\": \"Dual-luciferase reporter assay, RNA-seq, siRNA knockdown, overexpression rescue, in vitro migration/invasion assays, in vivo xenograft model, RhoA/ROCK pathway activity measurement\",\n      \"journal\": \"Cancers\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — direct 3′-UTR reporter validation plus functional rescue experiments in vitro and in vivo, single lab\",\n      \"pmids\": [\"34830864\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Knockout of CGN (cingulin) alone, CGNL1 (paracingulin) alone, or both in MDCK kidney epithelial cells causes a modest but significant increase in transepithelial resistance (TER) and decreased sodium permeability, without affecting 3 kDa dextran permeability. This phenotype is associated with reduced protein expression and junctional accumulation of the sodium pore-forming claudin-2. Overexpression of CGN or CGNL1 (but not ZO-1) rescues claudin-2 junctional localization, indicating a CGN-specific role in claudin-2 regulation independent of ZO-1.\",\n      \"method\": \"CRISPR/Cas9 knockout in MDCK cells, transepithelial resistance measurements, paracellular permeability assays (dextran, sodium), calcium-switch assay, immunofluorescence, western blot, qRT-PCR, rescue by overexpression\",\n      \"journal\": \"Cells\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — clean KO with defined cellular phenotype and domain-specific rescue, single lab\",\n      \"pmids\": [\"37566083\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"In ovarian cancer cells, polyphyllin H upregulates CGN (cingulin) expression, which in turn blocks the RhoA/Rock2 signaling pathway to inhibit cancer cell proliferation, invasion, and migration. This CGN/RhoA/Rock2 axis was identified through protein sequencing of drug-treated cells and functionally validated in cell line-derived and patient-derived xenograft mouse models.\",\n      \"method\": \"CCK-8 proliferation assay, invasion/migration assays, protein sequencing (proteomics), CDX and PDX mouse models, in vivo tumor growth measurement, pathway activity assays\",\n      \"journal\": \"International journal of surgery (London, England)\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — mechanistic pathway placement via proteomics and in vivo models, but no direct mutagenesis or binding assay for CGN–RhoA interaction; single lab\",\n      \"pmids\": [\"40773225\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"Cingulin (CGN) is a dimeric coiled-coil scaffolding protein at the cytoplasmic face of epithelial tight junctions, where its N-terminal globular head domain directly binds ZO-1/ZO-2/ZO-3, myosin, JAM, and GEF-H1/Lfc; by sequestering GEF-H1 it suppresses RhoA/ROCK signaling and links tight junction formation to inhibition of cell proliferation (G1/S block) and restraint of epithelial migration, while also regulating claudin-2 junctional accumulation and paracellular sodium permeability.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"CGN (cingulin) is a cytoplasmic tight junction adaptor protein that regulates paracellular permeability and suppresses RhoA/ROCK signaling in epithelial cells. Knockout of CGN in MDCK kidney epithelial cells reduces junctional accumulation and protein expression of claudin-2, leading to increased transepithelial resistance and decreased sodium permeability, with rescue experiments confirming that CGN overexpression restores claudin-2 localization independently of ZO-1 [PMID:37566083]. CGN also functions as an endogenous suppressor of the RhoA/ROCK pathway; miR-125b directly targets the CGN 3′UTR in colorectal cancer cells to repress its expression, thereby activating RhoA/ROCK signaling and promoting cell migration and invasion [PMID:34830864].\",\n  \"teleology\": [\n    {\n      \"year\": 2021,\n      \"claim\": \"Establishing that CGN suppresses RhoA/ROCK signaling and that its loss promotes epithelial cell migration and invasion answered how CGN participates in signaling beyond its structural role at tight junctions.\",\n      \"evidence\": \"Dual-luciferase reporter assay confirming miR-125b direct targeting of CGN 3′UTR, combined with migration/invasion assays and in vivo xenografts in colorectal cancer cells\",\n      \"pmids\": [\"34830864\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"The mechanism by which CGN inhibits RhoA activation (direct interaction vs. scaffolding of a GAP) is unknown\",\n        \"Findings from a single lab in one cancer cell type; independent replication in other systems is lacking\"\n      ]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Demonstrating that CGN knockout reduces claudin-2 junctional accumulation and protein expression, thereby altering paracellular sodium permeability, established CGN as a regulator of tight junction composition and ion selectivity.\",\n      \"evidence\": \"CRISPR/Cas9 knockout in MDCK cells with transepithelial resistance, dextran permeability, calcium-switch assays, and rescue by CGN overexpression\",\n      \"pmids\": [\"37566083\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Whether CGN regulates claudin-2 through protein stability, trafficking, or transcriptional mechanisms is not resolved\",\n        \"Findings restricted to MDCK cells; relevance in vivo or in other epithelial tissues is untested\"\n      ]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Pharmacological upregulation of CGN by Polyphyllin H in ovarian cancer cells inhibited proliferation, invasion, and migration through RhoA/Rock2 pathway suppression, providing additional context for CGN as a tumor-suppressive effector.\",\n      \"evidence\": \"Protein sequencing, CDX and PDX mouse models, and western blot analysis of pathway components in ovarian cancer cells\",\n      \"pmids\": [\"40773225\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\n        \"CGN was not directly manipulated (knocked out or overexpressed) in this study, so the causal link between CGN upregulation and phenotype is indirect\",\n        \"Polyphyllin H likely has multiple targets; specificity of the effect to CGN is unconfirmed\",\n        \"No structural or biochemical data on how CGN engages RhoA or Rock2\"\n      ]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The direct biochemical mechanism by which CGN suppresses RhoA/ROCK signaling — whether through scaffolding a RhoGAP, sequestering GEFs, or another mechanism — and how CGN controls claudin-2 protein levels remain unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"No structural model of CGN in complex with RhoA pathway components exists\",\n        \"The post-transcriptional vs. trafficking basis for CGN-dependent claudin-2 regulation is unknown\",\n        \"In vivo phenotypes of CGN loss in mammalian tissues are not reported in this literature\"\n      ]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [0]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [0]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [1]},\n      {\"term_id\": \"R-HSA-1500931\", \"supporting_discovery_ids\": [0]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\n      \"CLDN2\",\n      \"CGNL1\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```\n\n--- ROUND 2 (R2/R3, augmented corpus) ---\n\n```json\n{\n  \"mechanistic_narrative\": \"Cingulin (CGN) is a dimeric coiled-coil scaffolding protein localized to the cytoplasmic face of epithelial tight junctions, where it integrates junctional assembly with Rho GTPase signaling and paracellular permeability control. Its N-terminal globular head domain directly binds ZO-1, ZO-2, ZO-3, myosin, AF-6, JAM, and the RhoA-specific exchange factor GEF-H1, while the coiled-coil rod domain mediates parallel dimerization and is required together with the head for correct tight-junction targeting [PMID:10613913, PMID:10877843]. Sequestration of GEF-H1 by cingulin inhibits RhoA/ROCK activation, linking tight-junction formation to suppression of cell proliferation (G1/S block) and restraint of epithelial migration; loss of CGN derepresses RhoA signaling and promotes invasion in both normal and cancer epithelial contexts [PMID:15866167, PMID:34830864]. CGN also promotes junctional accumulation of the pore-forming claudin-2, thereby regulating paracellular sodium permeability independently of ZO-1 [PMID:37566083].\",\n  \"teleology\": [\n    {\n      \"year\": 1999,\n      \"claim\": \"Resolving cingulin's domain architecture and direct binding partners established it as a multi-valent cytoplasmic scaffold at tight junctions, rather than a simple structural component.\",\n      \"evidence\": \"cDNA cloning of Xenopus cingulin, electron microscopy of coiled-coil dimers, in vitro pull-downs with Kd measurement for ZO-1, co-IP and transfection/localization in MDCK cells\",\n      \"pmids\": [\"10613913\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Binding affinities for ZO-2, ZO-3, myosin, and AF-6 not quantified\",\n        \"Post-translational regulation of cingulin junctional recruitment not addressed\",\n        \"No functional assay for junctional barrier or signaling consequences\"\n      ]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"Identification of JAM as a transmembrane binding partner of cingulin showed that cingulin physically links integral membrane components to the tight-junction plaque, expanding its adaptor role beyond cytoplasmic scaffolds.\",\n      \"evidence\": \"Co-immunoprecipitation from Caco-2 detergent-insoluble fractions, domain-mapping in vitro binding assays, CHO cell transfection\",\n      \"pmids\": [\"10877843\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Whether cingulin–JAM interaction is direct or bridged by ZO-1 was not fully resolved\",\n        \"Functional consequence of disrupting cingulin–JAM binding on barrier integrity unknown\"\n      ]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Discovery that cingulin sequesters GEF-H1 to inhibit RhoA activation revealed a direct signaling function for a tight-junction scaffold, answering how junction formation feeds back on cell proliferation and morphology.\",\n      \"evidence\": \"Direct pull-down of GEF-H1, RhoA-GTP assays, cingulin/GEF-H1 RNAi in MDCK cells, G1/S cell-cycle analysis, dominant-negative mutant expression\",\n      \"pmids\": [\"15866167\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Structural basis of cingulin–GEF-H1 interaction undefined\",\n        \"Whether cingulin regulates additional GEFs or GAPs not tested\",\n        \"Relative contributions of RhoA inhibition versus other cingulin interactions to barrier phenotype unresolved\"\n      ]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Demonstration that TGFβ/Smad-driven EMT epigenetically silences CGN via promoter hypermethylation provided a mechanism for loss of cingulin's anti-migratory function during cancer progression.\",\n      \"evidence\": \"Bisulfite sequencing in breast cancer EMT models, Smad pathway disruption, DNMT1 activity assays, invasion assays\",\n      \"pmids\": [\"20086175\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Whether CGN silencing is a cause or consequence of EMT progression not fully distinguished\",\n        \"In vivo relevance of CGN epigenetic silencing in patient tumors not assessed\",\n        \"Specific Smad complex binding to CGN promoter not mapped\"\n      ]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Validation of miR-125b as a direct post-transcriptional repressor of CGN in colorectal cancer confirmed that CGN loss activates RhoA/ROCK to promote metastasis, and that CGN re-expression rescues the phenotype in vivo.\",\n      \"evidence\": \"Dual-luciferase 3′-UTR reporter, siRNA knockdown, overexpression rescue in vitro and in xenograft models, RhoA/ROCK activity measurement\",\n      \"pmids\": [\"34830864\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Clinical correlation of miR-125b/CGN axis in patient cohorts limited\",\n        \"Whether additional miRNAs converge on CGN regulation unknown\",\n        \"Downstream RhoA effectors beyond ROCK not delineated\"\n      ]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"CRISPR knockout of CGN revealed a specific role in promoting claudin-2 junctional accumulation and paracellular sodium permeability, mechanistically separable from ZO-1-dependent functions.\",\n      \"evidence\": \"CRISPR/Cas9 KO of CGN and CGNL1 in MDCK cells, TER and permeability assays, rescue by CGN overexpression, immunofluorescence and western blot\",\n      \"pmids\": [\"37566083\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Whether cingulin directly binds claudin-2 or acts via an intermediate is unknown\",\n        \"Impact on other claudin family members not systematically tested\",\n        \"In vivo kidney or intestinal barrier phenotype of CGN knockout not reported\"\n      ]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The structural basis of cingulin's multi-valent head-domain interactions, its regulation by phosphorylation, and its in vivo roles in tissue barrier function and tumor suppression remain unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\n        \"No high-resolution structure of cingulin head domain or its complexes with GEF-H1, ZO proteins, or claudins\",\n        \"Phosphorylation sites are documented but functional consequences in signaling are not mapped\",\n        \"No conditional in vivo knockout phenotype in mammalian epithelial tissues reported in the timeline\"\n      ]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [0, 1, 2]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [2, 4]},\n      {\"term_id\": \"GO:0008092\", \"supporting_discovery_ids\": [0]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [0, 1, 5]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"GO:1500931\", \"supporting_discovery_ids\": [0, 1, 5]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [2, 4, 6]},\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [2]}\n    ],\n    \"complexes\": [\n      \"tight junction plaque\"\n    ],\n    \"partners\": [\n      \"TJP1\",\n      \"TJP2\",\n      \"TJP3\",\n      \"F11R\",\n      \"ARHGEF2\",\n      \"CGNL1\",\n      \"CLDN2\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}