{"gene":"EVC2","run_date":"2026-04-28T17:46:03","timeline":{"discoveries":[{"year":2012,"finding":"Hedgehog agonists promote association between Smoothened (Smo) and EVC2; this Smo-EVC2 complex formation is spatially restricted to a distinct ciliary compartment called the EvC zone. Mutant EVC2 proteins that localize in cilia but are displaced from the EvC zone act as dominant inhibitors of Hh signaling, and disabling EVC2 blocks Hh signaling between Smo and the downstream regulators PKA and Suppressor of Fused, preventing Gli transcription factor activation.","method":"Co-immunoprecipitation, dominant-negative localization mutants, epistasis analysis with PKA and Sufu, live-cell imaging of ciliary localization","journal":"Developmental cell","confidence":"High","confidence_rationale":"Tier 2 — reciprocal Co-IP, dominant-negative mutants, epistasis, spatial localization with functional consequence; replicated in same year by two independent labs","pmids":["22981989"],"is_preprint":false},{"year":2012,"finding":"EVC and EVC2 are mutually required for localizing to primary cilia and for maintaining normal protein levels of each other. Smo co-precipitates with the EVC/EVC2 complex. In EVC2-deficient chondrocytes, Gli3 recruitment to cilia tips is reduced and Sufu/Gli3 dissociation is impaired after Hh activation, while Smo translocation to the cilium remains normal.","method":"Mouse knockout models, co-immunoprecipitation, immunofluorescence of cilia, Western blot of protein levels, SAG-stimulated Gli3 trafficking assay","journal":"Human molecular genetics","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods (KO mice, Co-IP, ciliary imaging, epistasis), replicated across labs","pmids":["23026747"],"is_preprint":false},{"year":2012,"finding":"EVC and EVC2 act downstream of Smo but upstream of Sufu to transduce the Hh signal. Hh stimulates binding of EVC/EVC2 to Smo dependent on phosphorylation of the Smo C-terminal intracellular tail. This binding is abolished in Kif3a-/- cilium-deficient cells, demonstrating that the interaction requires primary cilia. Loss of EVC/EVC2 does not affect Smo phosphorylation or ciliary localization but impedes Hh pathway activation by constitutively active Smo.","method":"Co-immunoprecipitation, epistasis with Sufu-/- cells and constitutively active Smo constructs, phosphorylation-dependent binding assays, Kif3a-/- cilium-deficient cell line","journal":"Cell research","confidence":"High","confidence_rationale":"Tier 1-2 — phosphorylation-dependent binding assay, epistasis with multiple genetic backgrounds, replicated by concurrent independent labs","pmids":["22986504"],"is_preprint":false},{"year":2011,"finding":"EVC2 is a positive regulator of the Hh signaling pathway essential for pathway activation in response to the Smo agonist purmorphamine. EVC2 and EVC interact directly (identified by yeast two-hybrid and confirmed by immunoprecipitation). Co-localization of EVC and EVC2 at the basal body and primary cilia is co-dependent: basal body/cilia localization requires co-transfection of both constructs. EVC2 is a transmembrane protein with extracellular N-terminus and intracellular C-terminus and is also found in the nucleus (full-length EVC2 but not EVC).","method":"Yeast two-hybrid, co-immunoprecipitation, immunofluorescence of primary cilia and basal body, subcellular fractionation/Western blot, topology analysis","journal":"BMC biology","confidence":"High","confidence_rationale":"Tier 2 — yeast two-hybrid confirmed by Co-IP, multiple localization methods, functional assay with Smo agonist","pmids":["21356043"],"is_preprint":false},{"year":2014,"finding":"The EFCAB7-IQCE complex anchors EVC-EVC2 in a signaling microdomain at the base of cilia (EvC zone). EFCAB7 directly binds a C-terminal disordered region of EVC2 that is deleted in Weyers acrofacial dysostosis patients. EFCAB7 depletion causes mislocalization of EVC-EVC2 within cilia and impairs activation of the transcription factor GLI2, phenocopying the Weyers cellular defect.","method":"Co-immunoprecipitation, shRNA-mediated depletion, immunofluorescence of ciliary localization, GLI2 activation assay, evolutionary/bioinformatic analysis","journal":"Developmental cell","confidence":"High","confidence_rationale":"Tier 2 — direct binding mapping, functional knockdown with defined cellular phenotype, disease-variant validation; strong mechanistic evidence from single lab with multiple methods","pmids":["24582806"],"is_preprint":false},{"year":2009,"finding":"Expression of Weyers acrofacial dysostosis EVC2 exon 22 missense variants (dominant mutations) in NIH 3T3 cells disrupts Hedgehog signal transduction, whereas expression of a truncated EVC2 protein mimicking an Ellis-van Creveld syndrome loss-of-function mutation does not impair Hh signaling in the same assay, consistent with the dominant-negative mechanism of Weyers mutations.","method":"Ectopic expression of murine EVC2 exon 22 variants in NIH 3T3 cells, Hh reporter assay","journal":"Human mutation","confidence":"Medium","confidence_rationale":"Tier 2 — functional cell-based assay distinguishing dominant-negative from loss-of-function alleles, single lab","pmids":["19810119"],"is_preprint":false},{"year":2015,"finding":"Homozygous Evc2 knockout mice show no ciliary localization of EVC2 or EVC and display reduced Hedgehog signaling activity with associated skeletal and oral defects. Cartilage-specific disruption of Evc2 causes skeletal defects; neural crest-specific disruption causes defective incisor growth; osteoblast-specific disruption does not cause overt skeletal changes, defining tissue-specific requirements for EVC2 in mineralized tissue formation.","method":"Conditional and conventional knockout mouse generation (IRES-LacZ knock-in), immunofluorescence for ciliary localization, Hh signaling assays, tissue-specific Cre lines","journal":"Genesis (New York, N.Y. : 2000)","confidence":"High","confidence_rationale":"Tier 2 — multiple tissue-specific KO lines with defined phenotypes, direct ciliary localization assay, Hh pathway readout; rigorous genetic dissection","pmids":["26219237"],"is_preprint":false},{"year":2016,"finding":"Elevated FGF signaling, mainly due to increased Fgf18 expression upon inactivation of Evc2 in the perichondrium, critically contributes to limb dwarfism pathogenesis. Partial rescue of dwarfism is achieved by inactivation of one Fgf18 allele in Evc2 mutant mice, establishing FGF pathway upregulation as a downstream pathogenic mechanism in EVC2-deficient bone.","method":"Evc2 mutant mouse analysis, in vivo and in vitro growth plate cultures, FGF signaling assays, genetic rescue (Evc2 mut x Fgf18 het intercross)","journal":"PLoS genetics","confidence":"High","confidence_rationale":"Tier 2 — genetic epistasis with Fgf18 rescue, in vivo and in vitro confirmation, mechanistic pathway placement","pmids":["28027321"],"is_preprint":false},{"year":2023,"finding":"EVC-EVC2 complex stability and ciliary targeting are regulated by ubiquitin and SUMO modification. Monoubiquitination of EVC-EVC2 cytosolic tails reduces protein levels. SUMOylation with SUMO3 enhances EVC-EVC2 accumulation at the EvC zone, possibly via increased binding to the EFCAB7-IQCE complex. EvC zone targeting requires two separate EFCAB7-binding motifs within EVC2's Weyers-deleted peptide. An endogenous EVC interactome screen confirmed EVC2, IQCE, and EFCAB7 as main interactors and identified USP7 as a new interactor.","method":"Endogenous protein interactome (mass spectrometry), ubiquitination assays, SUMOylation assays, immunofluorescence of EvC zone localization, Evc-null cell lines, domain mapping","journal":"Frontiers in cell and developmental biology","confidence":"High","confidence_rationale":"Tier 1-2 — mass spectrometry interactome, biochemical PTM assays, domain mapping with functional localization readout; multiple orthogonal methods in one study","pmids":["37576597"],"is_preprint":false},{"year":2017,"finding":"Dental mesenchymal-specific deletion of Evc2 phenocopies the hypomorphic enamel and tooth abnormalities of global Evc2 knockout mice, demonstrating that EVC2 function in dental mesenchymal cells (regulating dental mesenchymal stem cell homeostasis and odontoblast differentiation) is required for normal ameloblast maturation and enamel formation.","method":"Conditional knockout mice (dental mesenchyme-specific Cre), histology, ameloblast marker immunohistochemistry, preameloblast differentiation assays","journal":"Journal of dental research","confidence":"Medium","confidence_rationale":"Tier 2 — tissue-specific KO with defined cellular phenotype, histological markers; single lab","pmids":["28081373"],"is_preprint":false},{"year":2025,"finding":"EVC and EVC2 are aberrantly overexpressed in a subset of AML and are essential for leukemogenic properties of AML cells. Loss of EVC/EVC2 impairs leukemia cell proliferation, promotes differentiation, and blocks AML progression in vivo. Mechanistically, the leukemogenic role of EVC/EVC2 is mediated through MYC pathway activation, independent of their canonical Hedgehog signaling function. Elevated EVC/EVC2 expression is associated with gained AML1-ETO occupancy or enhanced chromatin interactions at EVC/EVC2 promoter regions in t(8;21) or ASXL1-mutant AML.","method":"shRNA/CRISPR loss-of-function in AML cell lines, in vivo AML progression assay, MYC pathway gene expression analysis, chromatin immunoprecipitation/chromatin interaction assays, differentiation assays","journal":"Leukemia","confidence":"Medium","confidence_rationale":"Tier 2 — functional KO with in vivo phenotype and pathway analysis; single lab with multiple methods but novel finding not yet replicated","pmids":["41249566"],"is_preprint":false}],"current_model":"EVC2 is a ciliary transmembrane protein that forms a heterodimeric complex with EVC, anchored to the EvC zone at the base of primary cilia by the EFCAB7-IQCE module; upon Hedgehog stimulation, phosphorylated Smoothened recruits the EVC-EVC2 complex, which acts between Smo and Sufu to promote Gli transcription factor activation by facilitating Sufu/Gli dissociation and Gli trafficking to the ciliary tip, with complex stability and EvC zone targeting regulated by monoubiquitination (destabilizing) and SUMO3 modification (enhancing), while in AML cells EVC2 can also drive MYC pathway activation independently of its canonical Hh signaling role."},"narrative":{"teleology":[{"year":2009,"claim":"Distinguishing dominant-negative from loss-of-function EVC2 alleles established that the C-terminal region disrupted in Weyers acrofacial dysostosis exerts a distinct pathomechanism from Ellis–van Creveld truncation mutations, linking specific EVC2 domains to Hh signaling output.","evidence":"Ectopic expression of Weyers exon 22 missense variants versus EvC-syndrome truncation constructs in NIH 3T3 Hh reporter assay","pmids":["19810119"],"confidence":"Medium","gaps":["Only a single reporter cell line tested; no endogenous locus validation","Mechanism of dominant-negative action not resolved"]},{"year":2011,"claim":"Identification of EVC2 as a transmembrane protein that directly binds EVC and co-dependently localizes to the basal body/primary cilium established the EVC-EVC2 complex as a functional unit required for Hh pathway activation.","evidence":"Yeast two-hybrid confirmed by co-IP; topology analysis; co-dependent ciliary localization by immunofluorescence; Smo agonist functional assay","pmids":["21356043"],"confidence":"High","gaps":["Whether EVC2 nuclear localization has a signaling role remained unresolved","Stoichiometry and structural basis of the EVC-EVC2 complex unknown"]},{"year":2012,"claim":"Three concurrent studies placed EVC-EVC2 in the Hh pathway between phosphorylated Smoothened and Sufu/Gli, showing that Smo phosphorylation-dependent recruitment of EVC2 to a defined ciliary EvC zone is necessary for Gli3 trafficking to cilia tips and Sufu/Gli dissociation.","evidence":"Co-IP of Smo-EVC2, epistasis with Sufu−/− and Kif3a−/− cells, dominant-negative EvC zone displacement mutants, Gli3 ciliary tip assays in EVC2-deficient chondrocytes from KO mice","pmids":["22981989","23026747","22986504"],"confidence":"High","gaps":["Biochemical mechanism by which EVC-EVC2 promotes Sufu/Gli dissociation undefined","Whether EVC2 directly contacts Gli or Sufu unknown"]},{"year":2014,"claim":"Discovery that the EFCAB7-IQCE module anchors EVC-EVC2 to the EvC zone through a C-terminal disordered region of EVC2 explained how spatial restriction of the complex enables signaling and why Weyers deletions of that region act as dominant negatives.","evidence":"Co-IP mapping of EFCAB7-EVC2 binding domain, shRNA knockdown of EFCAB7 causing EVC-EVC2 mislocalization and impaired GLI2 activation","pmids":["24582806"],"confidence":"High","gaps":["Structural details of EFCAB7-EVC2 interaction absent","Role of IQCE beyond scaffolding not elucidated"]},{"year":2015,"claim":"Conditional knockout studies defined tissue-specific requirements: EVC2 is essential in cartilage for skeletal growth and in neural crest for incisor development, but dispensable in osteoblasts, revealing context-dependent Hh transduction needs.","evidence":"Conventional and conditional Evc2 KO mice with cartilage-, neural crest-, and osteoblast-specific Cre drivers; Hh pathway and localization assays","pmids":["26219237"],"confidence":"High","gaps":["Whether other signaling pathways compensate in osteoblasts is unknown","Heart and other organ phenotypes not thoroughly characterized"]},{"year":2016,"claim":"The finding that Evc2 loss in perichondrium upregulates Fgf18, and that heterozygous Fgf18 deletion partially rescues dwarfism, identified FGF pathway hyperactivation as a key downstream pathogenic effector of EVC2 deficiency in skeletal growth.","evidence":"Evc2 mutant mice crossed with Fgf18 heterozygotes; in vivo and in vitro growth plate cultures with FGF signaling readouts","pmids":["28027321"],"confidence":"High","gaps":["Whether Fgf18 upregulation is directly due to reduced Hh output or a parallel mechanism is unclear","Rescue was partial — additional pathogenic pathways likely contribute"]},{"year":2017,"claim":"Dental mesenchyme-specific deletion showed that EVC2's role in Hh-dependent stem cell homeostasis within the dental mesenchyme non-cell-autonomously drives ameloblast maturation, extending the pathway's tissue requirement beyond bone.","evidence":"Conditional KO mice with dental mesenchyme-specific Cre; histology and ameloblast marker analysis","pmids":["28081373"],"confidence":"Medium","gaps":["Single lab; independent replication pending","Molecular mediators of the non-cell-autonomous signal to ameloblasts unidentified"]},{"year":2023,"claim":"Post-translational regulation of the EVC-EVC2 complex was defined: monoubiquitination destabilizes the complex while SUMO3 modification enhances EvC zone accumulation, and two distinct EFCAB7-binding motifs within EVC2 are both required for EvC zone targeting; USP7 was identified as a novel interactor.","evidence":"Endogenous EVC interactome by mass spectrometry, ubiquitination and SUMOylation biochemical assays, domain mapping with ciliary localization readout in Evc-null cells","pmids":["37576597"],"confidence":"High","gaps":["Whether USP7 deubiquitinates EVC-EVC2 directly not shown","SUMO3 ligase and deubiquitinase identities remain unknown","Functional Hh signaling consequences of SUMO/ubiquitin perturbation not measured"]},{"year":2025,"claim":"EVC2 was found to be aberrantly overexpressed in AML subsets and to drive leukemogenesis through MYC pathway activation independently of Hh signaling, revealing a non-canonical oncogenic function.","evidence":"shRNA/CRISPR KO in AML cell lines, in vivo AML progression assays, MYC pathway expression analysis, ChIP and chromatin interaction assays for AML1-ETO and ASXL1-mutant contexts","pmids":["41249566"],"confidence":"Medium","gaps":["Novel finding not yet independently replicated","Mechanism linking EVC-EVC2 to MYC activation is undefined","Whether EVC2 transmembrane/ciliary topology is relevant in AML cells unclear"]},{"year":null,"claim":"The direct biochemical mechanism by which the EVC-EVC2 complex promotes Sufu/Gli dissociation remains unknown, and no structural model of the EVC-EVC2-EFCAB7-IQCE assembly or its interface with Smoothened exists.","evidence":"","pmids":[],"confidence":"High","gaps":["No reconstituted in vitro system demonstrating EVC-EVC2 sufficiency for Sufu-Gli dissociation","No high-resolution structure of EVC-EVC2 or its EvC zone complex","Signaling role of nuclear EVC2 unresolved"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[0,1,2,4]}],"localization":[{"term_id":"GO:0005929","term_label":"cilium","supporting_discovery_ids":[0,1,3,4,6,8]},{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[3]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0,1,2,3,5,6]},{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[6,7,9]}],"complexes":["EVC-EVC2 complex","EVC-EVC2-EFCAB7-IQCE complex"],"partners":["EVC","EFCAB7","IQCE","SMO","USP7"],"other_free_text":[]},"mechanistic_narrative":"EVC2 is a ciliary transmembrane protein that functions as a core positive transducer of Hedgehog (Hh) signaling, linking activated Smoothened to downstream Gli transcription factor processing at primary cilia. EVC2 forms an obligate heterodimeric complex with EVC — each partner required for the other's ciliary localization and protein stability — and upon Hh stimulation, phosphorylated Smoothened recruits this complex at a specialized ciliary microdomain (the EvC zone), where it promotes Sufu/Gli dissociation and Gli trafficking to the ciliary tip [PMID:22981989, PMID:23026747, PMID:22986504]. The EvC zone positioning of EVC-EVC2 is anchored by the EFCAB7-IQCE module binding two motifs in the EVC2 C-terminal disordered region, and complex turnover is regulated by destabilizing monoubiquitination and stabilizing SUMO3 modification [PMID:24582806, PMID:37576597]. Loss-of-function mutations in EVC2 cause Ellis–van Creveld syndrome, characterized by skeletal dwarfism partly driven by ectopic FGF18 upregulation, while dominant-negative C-terminal mutations underlie Weyers acrofacial dysostosis [PMID:19810119, PMID:28027321]."},"prefetch_data":{"uniprot":{"accession":"Q86UK5","full_name":"Limbin","aliases":["Ellis-van Creveld syndrome protein 2","EVC2"],"length_aa":1308,"mass_kda":147.9,"function":"Component of the EvC complex that positively regulates ciliary Hedgehog (Hh) signaling. Plays a critical role in bone formation and skeletal development. May be involved in early embryonic morphogenesis","subcellular_location":"Cell membrane; Cytoplasm, cytoskeleton, cilium basal body; Cell projection, cilium; Cell projection, cilium membrane; Nucleus","url":"https://www.uniprot.org/uniprotkb/Q86UK5/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/EVC2","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/EVC2","total_profiled":1310},"omim":[{"mim_id":"619142","title":"CARDIOACROFACIAL DYSPLASIA 1; CAFD1","url":"https://www.omim.org/entry/619142"},{"mim_id":"618123","title":"POLYDACTYLY, POSTAXIAL, TYPE A8; PAPA8","url":"https://www.omim.org/entry/618123"},{"mim_id":"617632","title":"EF-HAND CALCIUM-BINDING DOMAIN-CONTAINING PROTEIN 7; EFCAB7","url":"https://www.omim.org/entry/617632"},{"mim_id":"617631","title":"IQ DOMAIN-CONTAINING PROTEIN E; IQCE","url":"https://www.omim.org/entry/617631"},{"mim_id":"617088","title":"SHORT-RIB THORACIC DYSPLASIA 15 WITH POLYDACTYLY; SRTD15","url":"https://www.omim.org/entry/617088"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Plasma membrane","reliability":"Approved"},{"location":"Microtubules","reliability":"Approved"},{"location":"Cytokinetic bridge","reliability":"Approved"},{"location":"Primary cilium","reliability":"Approved"},{"location":"Nucleoplasm","reliability":"Additional"},{"location":"Primary cilium tip","reliability":"Additional"},{"location":"Basal body","reliability":"Additional"},{"location":"Cytosol","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in many","driving_tissues":[],"url":"https://www.proteinatlas.org/search/EVC2"},"hgnc":{"alias_symbol":["LBN"],"prev_symbol":[]},"alphafold":{"accession":"Q86UK5","domains":[{"cath_id":"2.60.40.1510","chopping":"159-286","consensus_level":"high","plddt":77.97,"start":159,"end":286}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q86UK5","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q86UK5-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q86UK5-F1-predicted_aligned_error_v6.png","plddt_mean":72.69},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=EVC2","jax_strain_url":"https://www.jax.org/strain/search?query=EVC2"},"sequence":{"accession":"Q86UK5","fasta_url":"https://rest.uniprot.org/uniprotkb/Q86UK5.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q86UK5/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q86UK5"}},"corpus_meta":[{"pmid":"22981989","id":"PMC_22981989","title":"A Smoothened-Evc2 complex transduces the Hedgehog signal at primary cilia.","date":"2012","source":"Developmental cell","url":"https://pubmed.ncbi.nlm.nih.gov/22981989","citation_count":149,"is_preprint":false},{"pmid":"23026747","id":"PMC_23026747","title":"The ciliary Evc/Evc2 complex interacts with Smo and controls Hedgehog pathway activity in chondrocytes by regulating Sufu/Gli3 dissociation and Gli3 trafficking in primary cilia.","date":"2012","source":"Human molecular genetics","url":"https://pubmed.ncbi.nlm.nih.gov/23026747","citation_count":93,"is_preprint":false},{"pmid":"22986504","id":"PMC_22986504","title":"Smoothened transduces Hedgehog signal by forming a complex with Evc/Evc2.","date":"2012","source":"Cell research","url":"https://pubmed.ncbi.nlm.nih.gov/22986504","citation_count":86,"is_preprint":false},{"pmid":"17024374","id":"PMC_17024374","title":"Sequencing EVC and EVC2 identifies mutations in two-thirds of Ellis-van Creveld syndrome patients.","date":"2006","source":"Human 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biology","url":"https://pubmed.ncbi.nlm.nih.gov/21356043","citation_count":75,"is_preprint":false},{"pmid":"23220543","id":"PMC_23220543","title":"Novel and recurrent EVC and EVC2 mutations in Ellis-van Creveld syndrome and Weyers acrofacial dyostosis.","date":"2012","source":"European journal of medical genetics","url":"https://pubmed.ncbi.nlm.nih.gov/23220543","citation_count":59,"is_preprint":false},{"pmid":"19810119","id":"PMC_19810119","title":"Widening the mutation spectrum of EVC and EVC2: ectopic expression of Weyer variants in NIH 3T3 fibroblasts disrupts Hedgehog signaling.","date":"2009","source":"Human mutation","url":"https://pubmed.ncbi.nlm.nih.gov/19810119","citation_count":52,"is_preprint":false},{"pmid":"16404586","id":"PMC_16404586","title":"A novel heterozygous deletion in the EVC2 gene causes Weyers acrofacial dysostosis.","date":"2006","source":"Human 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Part A","url":"https://pubmed.ncbi.nlm.nih.gov/21815252","citation_count":20,"is_preprint":false},{"pmid":"29321360","id":"PMC_29321360","title":"Ellis-van Creveld syndrome and profound deafness resulted by sequence variants in the EVC/EVC2 and TMC1 genes.","date":"2017","source":"Journal of genetics","url":"https://pubmed.ncbi.nlm.nih.gov/29321360","citation_count":19,"is_preprint":false},{"pmid":"28027321","id":"PMC_28027321","title":"Elevated Fibroblast Growth Factor Signaling Is Critical for the Pathogenesis of the Dwarfism in Evc2/Limbin Mutant Mice.","date":"2016","source":"PLoS genetics","url":"https://pubmed.ncbi.nlm.nih.gov/28027321","citation_count":18,"is_preprint":false},{"pmid":"28950429","id":"PMC_28950429","title":"The Role of Ellis-Van Creveld 2(EVC2) in Mice During Cranial Bone Development.","date":"2017","source":"Anatomical record (Hoboken, N.J. : 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complex formation is spatially restricted to a distinct ciliary compartment called the EvC zone. Mutant EVC2 proteins that localize in cilia but are displaced from the EvC zone act as dominant inhibitors of Hh signaling, and disabling EVC2 blocks Hh signaling between Smo and the downstream regulators PKA and Suppressor of Fused, preventing Gli transcription factor activation.\",\n      \"method\": \"Co-immunoprecipitation, dominant-negative localization mutants, epistasis analysis with PKA and Sufu, live-cell imaging of ciliary localization\",\n      \"journal\": \"Developmental cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal Co-IP, dominant-negative mutants, epistasis, spatial localization with functional consequence; replicated in same year by two independent labs\",\n      \"pmids\": [\"22981989\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"EVC and EVC2 are mutually required for localizing to primary cilia and for maintaining normal protein levels of each other. Smo co-precipitates with the EVC/EVC2 complex. In EVC2-deficient chondrocytes, Gli3 recruitment to cilia tips is reduced and Sufu/Gli3 dissociation is impaired after Hh activation, while Smo translocation to the cilium remains normal.\",\n      \"method\": \"Mouse knockout models, co-immunoprecipitation, immunofluorescence of cilia, Western blot of protein levels, SAG-stimulated Gli3 trafficking assay\",\n      \"journal\": \"Human molecular genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods (KO mice, Co-IP, ciliary imaging, epistasis), replicated across labs\",\n      \"pmids\": [\"23026747\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"EVC and EVC2 act downstream of Smo but upstream of Sufu to transduce the Hh signal. Hh stimulates binding of EVC/EVC2 to Smo dependent on phosphorylation of the Smo C-terminal intracellular tail. This binding is abolished in Kif3a-/- cilium-deficient cells, demonstrating that the interaction requires primary cilia. Loss of EVC/EVC2 does not affect Smo phosphorylation or ciliary localization but impedes Hh pathway activation by constitutively active Smo.\",\n      \"method\": \"Co-immunoprecipitation, epistasis with Sufu-/- cells and constitutively active Smo constructs, phosphorylation-dependent binding assays, Kif3a-/- cilium-deficient cell line\",\n      \"journal\": \"Cell research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — phosphorylation-dependent binding assay, epistasis with multiple genetic backgrounds, replicated by concurrent independent labs\",\n      \"pmids\": [\"22986504\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"EVC2 is a positive regulator of the Hh signaling pathway essential for pathway activation in response to the Smo agonist purmorphamine. EVC2 and EVC interact directly (identified by yeast two-hybrid and confirmed by immunoprecipitation). Co-localization of EVC and EVC2 at the basal body and primary cilia is co-dependent: basal body/cilia localization requires co-transfection of both constructs. EVC2 is a transmembrane protein with extracellular N-terminus and intracellular C-terminus and is also found in the nucleus (full-length EVC2 but not EVC).\",\n      \"method\": \"Yeast two-hybrid, co-immunoprecipitation, immunofluorescence of primary cilia and basal body, subcellular fractionation/Western blot, topology analysis\",\n      \"journal\": \"BMC biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — yeast two-hybrid confirmed by Co-IP, multiple localization methods, functional assay with Smo agonist\",\n      \"pmids\": [\"21356043\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"The EFCAB7-IQCE complex anchors EVC-EVC2 in a signaling microdomain at the base of cilia (EvC zone). EFCAB7 directly binds a C-terminal disordered region of EVC2 that is deleted in Weyers acrofacial dysostosis patients. EFCAB7 depletion causes mislocalization of EVC-EVC2 within cilia and impairs activation of the transcription factor GLI2, phenocopying the Weyers cellular defect.\",\n      \"method\": \"Co-immunoprecipitation, shRNA-mediated depletion, immunofluorescence of ciliary localization, GLI2 activation assay, evolutionary/bioinformatic analysis\",\n      \"journal\": \"Developmental cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — direct binding mapping, functional knockdown with defined cellular phenotype, disease-variant validation; strong mechanistic evidence from single lab with multiple methods\",\n      \"pmids\": [\"24582806\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Expression of Weyers acrofacial dysostosis EVC2 exon 22 missense variants (dominant mutations) in NIH 3T3 cells disrupts Hedgehog signal transduction, whereas expression of a truncated EVC2 protein mimicking an Ellis-van Creveld syndrome loss-of-function mutation does not impair Hh signaling in the same assay, consistent with the dominant-negative mechanism of Weyers mutations.\",\n      \"method\": \"Ectopic expression of murine EVC2 exon 22 variants in NIH 3T3 cells, Hh reporter assay\",\n      \"journal\": \"Human mutation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — functional cell-based assay distinguishing dominant-negative from loss-of-function alleles, single lab\",\n      \"pmids\": [\"19810119\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Homozygous Evc2 knockout mice show no ciliary localization of EVC2 or EVC and display reduced Hedgehog signaling activity with associated skeletal and oral defects. Cartilage-specific disruption of Evc2 causes skeletal defects; neural crest-specific disruption causes defective incisor growth; osteoblast-specific disruption does not cause overt skeletal changes, defining tissue-specific requirements for EVC2 in mineralized tissue formation.\",\n      \"method\": \"Conditional and conventional knockout mouse generation (IRES-LacZ knock-in), immunofluorescence for ciliary localization, Hh signaling assays, tissue-specific Cre lines\",\n      \"journal\": \"Genesis (New York, N.Y. : 2000)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple tissue-specific KO lines with defined phenotypes, direct ciliary localization assay, Hh pathway readout; rigorous genetic dissection\",\n      \"pmids\": [\"26219237\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Elevated FGF signaling, mainly due to increased Fgf18 expression upon inactivation of Evc2 in the perichondrium, critically contributes to limb dwarfism pathogenesis. Partial rescue of dwarfism is achieved by inactivation of one Fgf18 allele in Evc2 mutant mice, establishing FGF pathway upregulation as a downstream pathogenic mechanism in EVC2-deficient bone.\",\n      \"method\": \"Evc2 mutant mouse analysis, in vivo and in vitro growth plate cultures, FGF signaling assays, genetic rescue (Evc2 mut x Fgf18 het intercross)\",\n      \"journal\": \"PLoS genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic epistasis with Fgf18 rescue, in vivo and in vitro confirmation, mechanistic pathway placement\",\n      \"pmids\": [\"28027321\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"EVC-EVC2 complex stability and ciliary targeting are regulated by ubiquitin and SUMO modification. Monoubiquitination of EVC-EVC2 cytosolic tails reduces protein levels. SUMOylation with SUMO3 enhances EVC-EVC2 accumulation at the EvC zone, possibly via increased binding to the EFCAB7-IQCE complex. EvC zone targeting requires two separate EFCAB7-binding motifs within EVC2's Weyers-deleted peptide. An endogenous EVC interactome screen confirmed EVC2, IQCE, and EFCAB7 as main interactors and identified USP7 as a new interactor.\",\n      \"method\": \"Endogenous protein interactome (mass spectrometry), ubiquitination assays, SUMOylation assays, immunofluorescence of EvC zone localization, Evc-null cell lines, domain mapping\",\n      \"journal\": \"Frontiers in cell and developmental biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — mass spectrometry interactome, biochemical PTM assays, domain mapping with functional localization readout; multiple orthogonal methods in one study\",\n      \"pmids\": [\"37576597\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Dental mesenchymal-specific deletion of Evc2 phenocopies the hypomorphic enamel and tooth abnormalities of global Evc2 knockout mice, demonstrating that EVC2 function in dental mesenchymal cells (regulating dental mesenchymal stem cell homeostasis and odontoblast differentiation) is required for normal ameloblast maturation and enamel formation.\",\n      \"method\": \"Conditional knockout mice (dental mesenchyme-specific Cre), histology, ameloblast marker immunohistochemistry, preameloblast differentiation assays\",\n      \"journal\": \"Journal of dental research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — tissue-specific KO with defined cellular phenotype, histological markers; single lab\",\n      \"pmids\": [\"28081373\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"EVC and EVC2 are aberrantly overexpressed in a subset of AML and are essential for leukemogenic properties of AML cells. Loss of EVC/EVC2 impairs leukemia cell proliferation, promotes differentiation, and blocks AML progression in vivo. Mechanistically, the leukemogenic role of EVC/EVC2 is mediated through MYC pathway activation, independent of their canonical Hedgehog signaling function. Elevated EVC/EVC2 expression is associated with gained AML1-ETO occupancy or enhanced chromatin interactions at EVC/EVC2 promoter regions in t(8;21) or ASXL1-mutant AML.\",\n      \"method\": \"shRNA/CRISPR loss-of-function in AML cell lines, in vivo AML progression assay, MYC pathway gene expression analysis, chromatin immunoprecipitation/chromatin interaction assays, differentiation assays\",\n      \"journal\": \"Leukemia\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — functional KO with in vivo phenotype and pathway analysis; single lab with multiple methods but novel finding not yet replicated\",\n      \"pmids\": [\"41249566\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"EVC2 is a ciliary transmembrane protein that forms a heterodimeric complex with EVC, anchored to the EvC zone at the base of primary cilia by the EFCAB7-IQCE module; upon Hedgehog stimulation, phosphorylated Smoothened recruits the EVC-EVC2 complex, which acts between Smo and Sufu to promote Gli transcription factor activation by facilitating Sufu/Gli dissociation and Gli trafficking to the ciliary tip, with complex stability and EvC zone targeting regulated by monoubiquitination (destabilizing) and SUMO3 modification (enhancing), while in AML cells EVC2 can also drive MYC pathway activation independently of its canonical Hh signaling role.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"EVC2 is a ciliary transmembrane protein that functions as a core positive transducer of Hedgehog (Hh) signaling, linking activated Smoothened to downstream Gli transcription factor processing at primary cilia. EVC2 forms an obligate heterodimeric complex with EVC — each partner required for the other's ciliary localization and protein stability — and upon Hh stimulation, phosphorylated Smoothened recruits this complex at a specialized ciliary microdomain (the EvC zone), where it promotes Sufu/Gli dissociation and Gli trafficking to the ciliary tip [PMID:22981989, PMID:23026747, PMID:22986504]. The EvC zone positioning of EVC-EVC2 is anchored by the EFCAB7-IQCE module binding two motifs in the EVC2 C-terminal disordered region, and complex turnover is regulated by destabilizing monoubiquitination and stabilizing SUMO3 modification [PMID:24582806, PMID:37576597]. Loss-of-function mutations in EVC2 cause Ellis–van Creveld syndrome, characterized by skeletal dwarfism partly driven by ectopic FGF18 upregulation, while dominant-negative C-terminal mutations underlie Weyers acrofacial dysostosis [PMID:19810119, PMID:28027321].\",\n  \"teleology\": [\n    {\n      \"year\": 2009,\n      \"claim\": \"Distinguishing dominant-negative from loss-of-function EVC2 alleles established that the C-terminal region disrupted in Weyers acrofacial dysostosis exerts a distinct pathomechanism from Ellis–van Creveld truncation mutations, linking specific EVC2 domains to Hh signaling output.\",\n      \"evidence\": \"Ectopic expression of Weyers exon 22 missense variants versus EvC-syndrome truncation constructs in NIH 3T3 Hh reporter assay\",\n      \"pmids\": [\"19810119\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Only a single reporter cell line tested; no endogenous locus validation\", \"Mechanism of dominant-negative action not resolved\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Identification of EVC2 as a transmembrane protein that directly binds EVC and co-dependently localizes to the basal body/primary cilium established the EVC-EVC2 complex as a functional unit required for Hh pathway activation.\",\n      \"evidence\": \"Yeast two-hybrid confirmed by co-IP; topology analysis; co-dependent ciliary localization by immunofluorescence; Smo agonist functional assay\",\n      \"pmids\": [\"21356043\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether EVC2 nuclear localization has a signaling role remained unresolved\", \"Stoichiometry and structural basis of the EVC-EVC2 complex unknown\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Three concurrent studies placed EVC-EVC2 in the Hh pathway between phosphorylated Smoothened and Sufu/Gli, showing that Smo phosphorylation-dependent recruitment of EVC2 to a defined ciliary EvC zone is necessary for Gli3 trafficking to cilia tips and Sufu/Gli dissociation.\",\n      \"evidence\": \"Co-IP of Smo-EVC2, epistasis with Sufu−/− and Kif3a−/− cells, dominant-negative EvC zone displacement mutants, Gli3 ciliary tip assays in EVC2-deficient chondrocytes from KO mice\",\n      \"pmids\": [\"22981989\", \"23026747\", \"22986504\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Biochemical mechanism by which EVC-EVC2 promotes Sufu/Gli dissociation undefined\", \"Whether EVC2 directly contacts Gli or Sufu unknown\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Discovery that the EFCAB7-IQCE module anchors EVC-EVC2 to the EvC zone through a C-terminal disordered region of EVC2 explained how spatial restriction of the complex enables signaling and why Weyers deletions of that region act as dominant negatives.\",\n      \"evidence\": \"Co-IP mapping of EFCAB7-EVC2 binding domain, shRNA knockdown of EFCAB7 causing EVC-EVC2 mislocalization and impaired GLI2 activation\",\n      \"pmids\": [\"24582806\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural details of EFCAB7-EVC2 interaction absent\", \"Role of IQCE beyond scaffolding not elucidated\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Conditional knockout studies defined tissue-specific requirements: EVC2 is essential in cartilage for skeletal growth and in neural crest for incisor development, but dispensable in osteoblasts, revealing context-dependent Hh transduction needs.\",\n      \"evidence\": \"Conventional and conditional Evc2 KO mice with cartilage-, neural crest-, and osteoblast-specific Cre drivers; Hh pathway and localization assays\",\n      \"pmids\": [\"26219237\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether other signaling pathways compensate in osteoblasts is unknown\", \"Heart and other organ phenotypes not thoroughly characterized\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"The finding that Evc2 loss in perichondrium upregulates Fgf18, and that heterozygous Fgf18 deletion partially rescues dwarfism, identified FGF pathway hyperactivation as a key downstream pathogenic effector of EVC2 deficiency in skeletal growth.\",\n      \"evidence\": \"Evc2 mutant mice crossed with Fgf18 heterozygotes; in vivo and in vitro growth plate cultures with FGF signaling readouts\",\n      \"pmids\": [\"28027321\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether Fgf18 upregulation is directly due to reduced Hh output or a parallel mechanism is unclear\", \"Rescue was partial — additional pathogenic pathways likely contribute\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Dental mesenchyme-specific deletion showed that EVC2's role in Hh-dependent stem cell homeostasis within the dental mesenchyme non-cell-autonomously drives ameloblast maturation, extending the pathway's tissue requirement beyond bone.\",\n      \"evidence\": \"Conditional KO mice with dental mesenchyme-specific Cre; histology and ameloblast marker analysis\",\n      \"pmids\": [\"28081373\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab; independent replication pending\", \"Molecular mediators of the non-cell-autonomous signal to ameloblasts unidentified\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Post-translational regulation of the EVC-EVC2 complex was defined: monoubiquitination destabilizes the complex while SUMO3 modification enhances EvC zone accumulation, and two distinct EFCAB7-binding motifs within EVC2 are both required for EvC zone targeting; USP7 was identified as a novel interactor.\",\n      \"evidence\": \"Endogenous EVC interactome by mass spectrometry, ubiquitination and SUMOylation biochemical assays, domain mapping with ciliary localization readout in Evc-null cells\",\n      \"pmids\": [\"37576597\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether USP7 deubiquitinates EVC-EVC2 directly not shown\", \"SUMO3 ligase and deubiquitinase identities remain unknown\", \"Functional Hh signaling consequences of SUMO/ubiquitin perturbation not measured\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"EVC2 was found to be aberrantly overexpressed in AML subsets and to drive leukemogenesis through MYC pathway activation independently of Hh signaling, revealing a non-canonical oncogenic function.\",\n      \"evidence\": \"shRNA/CRISPR KO in AML cell lines, in vivo AML progression assays, MYC pathway expression analysis, ChIP and chromatin interaction assays for AML1-ETO and ASXL1-mutant contexts\",\n      \"pmids\": [\"41249566\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Novel finding not yet independently replicated\", \"Mechanism linking EVC-EVC2 to MYC activation is undefined\", \"Whether EVC2 transmembrane/ciliary topology is relevant in AML cells unclear\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The direct biochemical mechanism by which the EVC-EVC2 complex promotes Sufu/Gli dissociation remains unknown, and no structural model of the EVC-EVC2-EFCAB7-IQCE assembly or its interface with Smoothened exists.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No reconstituted in vitro system demonstrating EVC-EVC2 sufficiency for Sufu-Gli dissociation\", \"No high-resolution structure of EVC-EVC2 or its EvC zone complex\", \"Signaling role of nuclear EVC2 unresolved\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [0, 1, 2, 4]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005929\", \"supporting_discovery_ids\": [0, 1, 3, 4, 6, 8]},\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [3]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 1, 2, 3, 5, 6]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [6, 7, 9]}\n    ],\n    \"complexes\": [\n      \"EVC-EVC2 complex\",\n      \"EVC-EVC2-EFCAB7-IQCE complex\"\n    ],\n    \"partners\": [\n      \"EVC\",\n      \"EFCAB7\",\n      \"IQCE\",\n      \"SMO\",\n      \"USP7\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}