{"gene":"EYA3","run_date":"2026-04-28T17:46:03","timeline":{"discoveries":[{"year":2010,"finding":"EYA3 partners with SIX1 to synergistically activate TSHβ expression in the pars tuberalis, with further enhancement by TEF and HLF, positioning EYA3 as an upstream transcriptional activator in the photoperiodic signaling cascade.","method":"Genome-wide expression analysis, transcriptional reporter assays, acute light-stimulation experiments in CBA/N mice","journal":"Current biology : CB","confidence":"Medium","confidence_rationale":"Tier 2 — defined pathway position and direct transcriptional activation shown in vivo and in vitro, single lab","pmids":["21129973"],"is_preprint":false},{"year":2010,"finding":"EYA3 is the strongest long-photoperiod-activated gene in the sheep pars tuberalis, identifying it as a conserved molecular photoperiodic signal upstream of TSH-mediated reproductive regulation in mammals.","method":"Microarray, in situ hybridization under controlled photoperiod conditions in sheep","journal":"Current biology : CB","confidence":"Medium","confidence_rationale":"Tier 2 — direct in vivo expression mapping tied to functional photoperiodic pathway, single lab","pmids":["20434341"],"is_preprint":false},{"year":2008,"finding":"EYA3 forms a complex with SKI and SIX1 via the SKI Dachshund homology domain to activate transcription from the MEF3 site on the Myogenin (MYOG) regulatory region, promoting muscle terminal differentiation.","method":"Co-immunoprecipitation, chromatin immunoprecipitation (ChIP), transcriptional reporter assays, retroviral overexpression/knockdown in C2C12 myoblasts","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 — reciprocal Co-IP, ChIP, reporter assays, and KD phenotype with multiple orthogonal methods","pmids":["19008232"],"is_preprint":false},{"year":2008,"finding":"Loss of EYA3 in mice produces pleiotropic physiological defects including decreased bone mineral content, shorter body length, reduced tidal volume, cardiac conduction changes, and reduced muscle strength, with no apparent eye defect, demonstrating broad developmental roles distinct from the Drosophila eye phenotype.","method":"EYA3 knockout mouse phenotypic analysis within the German Mouse Clinic; in situ hybridization; beta-Gal staining; differential gene expression analysis","journal":"BMC developmental biology","confidence":"Medium","confidence_rationale":"Tier 2 — comprehensive KO mouse phenotyping with multiple physiological readouts, single lab","pmids":["19102749"],"is_preprint":false},{"year":2012,"finding":"EWS/FLI1 upregulates EYA3 in Ewing sarcoma by repressing miR-708, which targets the EYA3 3'-UTR; elevated EYA3 promotes cell survival and chemoresistance by enabling more effective DNA damage repair.","method":"miRNA target site analysis, EYA3 knockdown in Ewing sarcoma cell lines, clonogenic survival assays, DNA damage repair assays with chemotherapeutics","journal":"Molecular cancer research : MCR","confidence":"High","confidence_rationale":"Tier 2 — miR-708/EYA3 3'-UTR targeting validated, KD with defined survival/chemoresistance phenotype and DNA repair readout, corroborated in human tumor samples","pmids":["22723308"],"is_preprint":false},{"year":2018,"finding":"The N-terminal domain Ser/Thr phosphatase activity of EYA3 is not intrinsic but arises from direct interaction with PP2A-B55α holoenzyme; EYA3 redirects PP2A-B55α to dephosphorylate c-Myc at pT58 (stabilizing c-Myc), contrasting with PP2A-B56α-mediated dephosphorylation of pS62 (which destabilizes c-Myc).","method":"Co-immunoprecipitation, in vitro phosphatase assays, mutagenesis, xenograft breast cancer model","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1-2 — in vitro reconstitution of phosphatase activity, direct interaction demonstrated, site-specific mutagenesis, in vivo xenograft validation","pmids":["29535359"],"is_preprint":false},{"year":2018,"finding":"EYA3 utilizes its Thr phosphatase activity to dephosphorylate Myc at pT58, stabilizing Myc, which drives PD-L1 upregulation and tumor immune suppression; EYA3 knockdown reduces tumor growth in immune-competent mice via increased CD8+ T cell infiltration.","method":"EYA3 knockdown/rescue experiments, phosphatase-dead mutants, CD8+ T cell depletion, PD-L1 rescue assays, syngeneic breast tumor models","journal":"The Journal of clinical investigation","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods including phosphatase-dead mutant, immune cell depletion rescue, and in vivo tumor models","pmids":["29757193"],"is_preprint":false},{"year":2018,"finding":"WDR1 is an EYA3-specific substrate: Src kinase phosphorylates WDR1 on tyrosine residues, and EYA3 (but not EYA1) efficiently dephosphorylates these sites; loss of WDR1 tyrosine phosphorylation causes major reorganization of the actin cytoskeleton. Additionally, Src phosphorylates EYA3 itself, controlling its nuclear and cytoskeletal localization, and EYA3 can autodephosphorylate these sites.","method":"Phosphotyrosine peptide microarray, in vitro phosphatase assays, Src kinase assays, subcellular fractionation/localization, actin cytoskeleton imaging","journal":"Scientific reports","confidence":"High","confidence_rationale":"Tier 1-2 — peptide microarray substrate identification, in vitro phosphatase reconstitution, localization experiments with functional consequence","pmids":["29440662"],"is_preprint":false},{"year":2019,"finding":"EYA3 tyrosine phosphatase activity promotes survival of pulmonary arterial smooth muscle cells under DNA-damaging conditions; transgenic mice with an inactivating EYA3 tyrosine phosphatase domain mutation are protected from vascular remodeling, and pharmacological inhibition of EYA3 tyrosine phosphatase reverses remodeling in a rat pulmonary hypertension model.","method":"Transgenic knock-in mice with catalytic-dead EYA3 mutation, pharmacological inhibition, rat angio-obliterative PAH model, cell survival assays","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1-2 — catalytic-dead knock-in mouse, pharmacological inhibition in two animal models, defined cellular survival phenotype","pmids":["31515519"],"is_preprint":false},{"year":2019,"finding":"EYA3 is phosphorylated by Src kinase at 13 tyrosine residues (including Y77, Y96, Y237, Y508); EYA3 autodephosphorylates these sites; specific residues Y77, Y96, and Y237 control HEK293T cell proliferation, as their mutation abolishes the pro-proliferative effect of EYA3 overexpression.","method":"Native and bottom-up mass spectrometry phosphosite mapping, site-directed mutagenesis, cell cycle analysis, Src kinase assays","journal":"International journal of molecular sciences","confidence":"High","confidence_rationale":"Tier 1 — MS-based phosphosite mapping, mutagenesis with functional proliferation readout","pmids":["31847183"],"is_preprint":false},{"year":2021,"finding":"EYA3 tyrosine phosphatase activity promotes Ewing sarcoma tumor growth and angiogenesis by regulating VEGFA levels and promoting DNA damage repair; pharmacological inhibition of EYA3 tyrosine phosphatase elevates its substrate H2AX-pY142 in tumor tissue and inhibits tumor growth in cell line and patient-derived xenografts.","method":"Genetic knockdown, pharmacological inhibition (Benzarone), xenograft and patient-derived xenograft models, H2AX-pY142 substrate engagement assay","journal":"Molecular cancer therapeutics","confidence":"High","confidence_rationale":"Tier 2 — in vivo target engagement demonstrated via substrate phosphorylation, multiple models including PDX","pmids":["33649104"],"is_preprint":false},{"year":2021,"finding":"A missense variant (p.Asn358Ser) in EYA3 associated with oculo-auriculo-vertebral spectrum increases the half-life of the mutated protein without impairing its ability to dephosphorylate H2AFX following DNA damage; eya3 knockdown in zebrafish embryos causes specific craniofacial abnormalities.","method":"Exome sequencing, cellular protein stability assays, H2AFX dephosphorylation assay, zebrafish morpholino knockdown, proteomics","journal":"Human genetics","confidence":"Medium","confidence_rationale":"Tier 2 — direct phosphatase activity assay on patient variant, zebrafish KD with craniofacial phenotype, proteomics, single lab","pmids":["33475861"],"is_preprint":false},{"year":2022,"finding":"EYA3 assembles a complex with SIX5 and histone acetyltransferase p300 in hypoxic colorectal cancer cells; this EYA3-SIX5-p300 complex binds the promoters of EGFR, VEGFD, and multiple MMPs to activate their transcription and drive tumor progression.","method":"Co-immunoprecipitation, mass spectrometry, chromatin immunoprecipitation (ChIP), pharmacological inhibition with benzarone in xenograft model","journal":"Annals of translational medicine","confidence":"Medium","confidence_rationale":"Tier 2 — reciprocal Co-IP/MS for complex identification, ChIP for promoter binding, in vivo xenograft validation, single lab","pmids":["35957720"],"is_preprint":false},{"year":2023,"finding":"EYA3 expression is required for myoblast proliferation and differentiation; alternatively spliced EYA3 isoforms (regulated by RBFOX2) differentially interact with SIX4 or ZBTB1 to control distinct transcriptional programs during myogenesis; the exon 7 splicing event is tissue-specific and developmentally regulated.","method":"Mass spectrometry proteomics, genome-wide transcriptomics, alternative splicing analysis, EYA3 knockdown in myoblasts with proliferation/differentiation readouts","journal":"iScience","confidence":"Medium","confidence_rationale":"Tier 2 — MS-validated protein interactions, transcriptomics, KD phenotype, multiple orthogonal methods; single lab","pmids":["38026174"],"is_preprint":false},{"year":2025,"finding":"Cryo-EM and NMR structures of the PP2A:B55α holoenzyme with EYA3 reveal that EYA3 binds B55α through an extended peptide in its N-terminal domain using a core B55 recruitment motif conserved across the EYA family; this binding overlaps with substrate recruitment sites but differs mechanistically from substrate binding; B55 recruitment by EYA3 directs selective dephosphorylation of specific phosphosites.","method":"Cryo-electron microscopy, NMR spectroscopy, NMR-based dephosphorylation assays, structural comparison","journal":"Nature structural & molecular biology","confidence":"High","confidence_rationale":"Tier 1 — cryo-EM structure with NMR functional validation and dephosphorylation assays; defines molecular mechanism of substrate recruitment","pmids":["40247147"],"is_preprint":false},{"year":2025,"finding":"Cryo-EM structures of PP2A-B55α bound with EYA3 show EYA3 binds B55α via an extended peptide in its N-terminal domain at a similar surface as substrates and peptide inhibitors; a designed inhibitory peptide (B55i) disrupts the EYA3-B55α interaction in vitro and increases Myc pT58 while reducing Myc protein levels in TNBC cells.","method":"Cryo-EM structure determination, in vitro binding/competition assays, B55i peptide expression in TNBC cells, Myc pT58 phosphorylation assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 — cryo-EM structures, in vitro inhibition assay, cellular functional validation with specific phosphorylation readout","pmids":["40414499"],"is_preprint":false},{"year":2025,"finding":"EYA3 upregulates NF-κB signaling to enhance CCL2 expression in triple-negative breast cancer cells; secreted CCL2 suppresses cytotoxic NK cell activation in vitro and reduces NK cell infiltration into the pre-metastatic niche in vivo, promoting metastasis independently of primary tumor effects.","method":"EYA3 knockdown, NF-κB signaling rescue, CCL2 re-expression rescue, NK cell depletion/activation assays in vitro, syngeneic pre-metastatic niche models in vivo","journal":"Science advances","confidence":"High","confidence_rationale":"Tier 2 — epistatic rescue experiments (NF-κB and CCL2 re-expression), in vivo NK cell phenotyping, multiple orthogonal approaches in single study","pmids":["40333987"],"is_preprint":false}],"current_model":"EYA3 is a multifunctional protein that acts as (1) a transcriptional co-activator partnering with SIX1/SIX4/SIX5, SKI, and p300 to regulate developmental and oncogenic gene expression programs; (2) a tyrosine phosphatase (HAD-family) that dephosphorylates substrates including H2AX-pY142 to promote DNA damage repair and cell survival, and WDR1-pY to remodel the actin cytoskeleton; and (3) a regulator of PP2A specificity, whereby its N-terminal domain—through a conserved recruitment motif that binds B55α at a structurally defined interface—redirects PP2A-B55α to dephosphorylate c-Myc pT58, stabilizing Myc to drive tumor progression and PD-L1-mediated immune suppression, while also engaging NF-κB/CCL2 signaling to suppress cytotoxic NK cells in the pre-metastatic niche."},"narrative":{"teleology":[{"year":2008,"claim":"Establishing EYA3 as a transcriptional co-activator in muscle differentiation resolved how EYA3 engages chromatin: it forms a SKI–SIX1–EYA3 complex on the MEF3 site of the Myogenin promoter to activate terminal differentiation genes.","evidence":"Reciprocal Co-IP, ChIP, reporter assays, and knockdown in C2C12 myoblasts","pmids":["19008232"],"confidence":"High","gaps":["Whether EYA3 phosphatase activity is required for its co-activator function in myogenesis was not tested","Other genomic targets of the SKI–SIX1–EYA3 complex were not mapped"]},{"year":2008,"claim":"EYA3 knockout mice revealed broad developmental functions—reduced bone mineral content, shorter body length, cardiac conduction defects, reduced muscle strength—without eye defects, establishing that mammalian EYA3 has pleiotropic roles distinct from the Drosophila eye specification paradigm.","evidence":"Comprehensive phenotyping of Eya3-null mice at the German Mouse Clinic","pmids":["19102749"],"confidence":"Medium","gaps":["Molecular targets responsible for each phenotypic defect were not identified","Redundancy with other EYA family members was not formally assessed"]},{"year":2010,"claim":"Identification of EYA3 as the strongest photoperiod-activated gene in the pars tuberalis, synergistically activating TSHβ with SIX1, placed EYA3 atop a conserved mammalian photoperiodic signaling cascade regulating seasonal reproduction.","evidence":"Microarray and in situ hybridization in sheep under controlled photoperiod; transcriptional reporter assays and acute light stimulation in mice","pmids":["20434341","21129973"],"confidence":"Medium","gaps":["Upstream signals that induce EYA3 expression in response to light were not fully defined","Whether EYA3 phosphatase activity contributes to photoperiodic signaling is unknown"]},{"year":2012,"claim":"Discovery that EWS/FLI1 upregulates EYA3 by repressing miR-708 revealed a mechanism by which Ewing sarcoma exploits EYA3 for DNA damage repair and chemoresistance, establishing EYA3's phosphatase as a survival factor in cancer.","evidence":"miR-708/EYA3 3′-UTR targeting validation, EYA3 knockdown with clonogenic survival and DNA repair assays in Ewing sarcoma cell lines","pmids":["22723308"],"confidence":"High","gaps":["The specific EYA3 phosphatase substrates mediating chemoresistance were not identified at this stage","In vivo validation of miR-708-EYA3 axis was limited"]},{"year":2018,"claim":"Three concurrent studies redefined EYA3 enzymology: its reported N-terminal Ser/Thr phosphatase activity was shown to arise from recruited PP2A-B55α, which EYA3 redirects to dephosphorylate c-Myc pT58, stabilizing Myc to drive PD-L1 upregulation and tumor immune evasion; separately, WDR1 was identified as an EYA3-specific tyrosine phosphatase substrate controlling actin cytoskeleton remodeling, and Src-dependent phosphorylation of EYA3 itself was shown to regulate its subcellular localization.","evidence":"Co-IP and in vitro phosphatase reconstitution with PP2A-B55α; phosphatase-dead mutant rescue and CD8+ T cell depletion in syngeneic tumor models; phosphotyrosine peptide microarray for WDR1 substrate identification; Src kinase assays with subcellular fractionation","pmids":["29535359","29757193","29440662"],"confidence":"High","gaps":["The structural basis for EYA3–B55α interaction was unknown","How EYA3 selectively recruits PP2A-B55α versus B56α was unresolved","Whether WDR1 dephosphorylation by EYA3 is relevant in vivo was not shown"]},{"year":2019,"claim":"Catalytic-dead EYA3 knock-in mice and pharmacological inhibition demonstrated that EYA3 tyrosine phosphatase activity is required for pathological vascular remodeling in pulmonary hypertension, validating EYA3 as a druggable target in vivo.","evidence":"Transgenic mice with inactivating mutation in EYA3 tyrosine phosphatase domain; pharmacological inhibition in rat pulmonary arterial hypertension model","pmids":["31515519"],"confidence":"High","gaps":["The direct vascular substrate(s) of EYA3 tyrosine phosphatase in smooth muscle cells were not identified","Long-term therapeutic window and off-target effects of pharmacological inhibition were not assessed"]},{"year":2019,"claim":"Mass spectrometry mapping of 13 Src-phosphorylated tyrosine residues on EYA3, with mutagenesis showing Y77, Y96, and Y237 are required for its pro-proliferative activity, defined the regulatory phosphorylation landscape of EYA3 itself.","evidence":"Native and bottom-up MS phosphosite mapping, site-directed mutagenesis with cell cycle analysis in HEK293T cells","pmids":["31847183"],"confidence":"High","gaps":["Which downstream signaling pathways are affected by each specific phosphosite remains unknown","Physiological contexts in which Src-EYA3 phosphorylation occurs in vivo were not established"]},{"year":2021,"claim":"In vivo target engagement was demonstrated: pharmacological EYA3 tyrosine phosphatase inhibition elevated H2AX-pY142 in Ewing sarcoma tumors and suppressed growth in cell line and patient-derived xenografts, confirming H2AX-pY142 as a bona fide in vivo substrate and linking EYA3 phosphatase to tumor angiogenesis via VEGFA.","evidence":"Benzarone treatment of xenograft and PDX models with H2AX-pY142 substrate engagement assay","pmids":["33649104"],"confidence":"High","gaps":["Whether VEGFA regulation requires EYA3 phosphatase activity or its transcriptional co-activator function was not fully dissected","Selectivity of benzarone for EYA3 versus other targets is a concern"]},{"year":2021,"claim":"A missense variant (p.Asn358Ser) in EYA3 was linked to oculo-auriculo-vertebral spectrum, with the mutant protein showing increased stability without loss of H2AX dephosphorylation; zebrafish eya3 knockdown produced craniofacial defects, establishing EYA3 as a craniofacial development gene.","evidence":"Exome sequencing, protein stability assays, H2AFX dephosphorylation assay, zebrafish morpholino knockdown","pmids":["33475861"],"confidence":"Medium","gaps":["The variant was identified in a single family; broader genetic evidence is needed","How increased protein stability causes disease pathology is unclear","Whether gain-of-function or neomorphic activity underlies the phenotype was not determined"]},{"year":2022,"claim":"Identification of the EYA3–SIX5–p300 complex on EGFR, VEGFD, and MMP promoters in hypoxic colorectal cancer cells extended EYA3's transcriptional co-activator role to hypoxia-driven tumor progression programs.","evidence":"Co-IP/mass spectrometry, ChIP on target promoters, benzarone inhibition in xenograft model","pmids":["35957720"],"confidence":"Medium","gaps":["Whether the phosphatase activity of EYA3 is required for p300 recruitment and histone acetylation at these loci is unknown","The hypoxia-responsive element driving EYA3 expression/activity was not identified"]},{"year":2023,"claim":"RBFOX2-regulated alternative splicing of EYA3 exon 7 was shown to generate isoforms with differential binding to SIX4 versus ZBTB1, controlling distinct transcriptional programs during myoblast proliferation and differentiation, revealing a splicing-based mechanism for EYA3 functional diversification.","evidence":"Mass spectrometry proteomics, transcriptomics, splicing analysis, EYA3 knockdown with proliferation/differentiation readouts in myoblasts","pmids":["38026174"],"confidence":"Medium","gaps":["The structural basis for isoform-specific partner selection is unknown","In vivo relevance of the exon 7 splicing switch in muscle development has not been tested"]},{"year":2025,"claim":"Cryo-EM and NMR structures of PP2A-B55α bound to EYA3 revealed that a conserved N-terminal peptide of EYA3 engages the B55α substrate-recruitment groove, providing the structural basis for how EYA3 redirects PP2A specificity; a designed inhibitory peptide (B55i) disrupts this interaction and destabilizes Myc in triple-negative breast cancer cells.","evidence":"Cryo-EM structure determination, NMR spectroscopy and dephosphorylation assays, B55i peptide competition in vitro and in TNBC cells with Myc pT58 readout","pmids":["40247147","40414499"],"confidence":"High","gaps":["In vivo efficacy and pharmacokinetic properties of B55i or derivatives are untested","Whether the EYA3–B55α interaction has substrates beyond c-Myc in cells is not known","Structural data for full-length EYA3 bound to PP2A holoenzyme is lacking"]},{"year":2025,"claim":"EYA3 was shown to activate NF-κB/CCL2 signaling in TNBC cells, with secreted CCL2 suppressing NK cell cytotoxicity and reducing NK infiltration in the pre-metastatic niche to promote metastasis, adding innate immune evasion to EYA3's oncogenic repertoire.","evidence":"EYA3 knockdown with NF-κB and CCL2 epistatic rescue, NK cell activation/depletion assays in vitro, syngeneic pre-metastatic niche models","pmids":["40333987"],"confidence":"High","gaps":["Whether EYA3's phosphatase or co-activator function drives NF-κB activation is unresolved","The direct molecular link between EYA3 and NF-κB pathway activation has not been identified"]},{"year":null,"claim":"Key unresolved questions include: how EYA3's dual phosphatase and transcriptional activities are coordinated in physiological contexts; the full spectrum of PP2A-B55α substrates redirected by EYA3 beyond c-Myc; the structural basis for isoform-specific partner selection; and whether therapeutic disruption of the EYA3–B55α interface is effective in vivo.","evidence":"","pmids":[],"confidence":"Low","gaps":["No integrated model connects EYA3 phosphatase and transcriptional activities in the same cellular context","Full-length EYA3 structure remains undetermined","In vivo therapeutic targeting of the EYA3–B55α interface is untested"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[7,8,9,10,11]},{"term_id":"GO:0140110","term_label":"transcription regulator activity","supporting_discovery_ids":[0,2,12,13]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[5,6,14,15]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[2,7,12]},{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[7]}],"pathway":[{"term_id":"R-HSA-74160","term_label":"Gene expression (Transcription)","supporting_discovery_ids":[0,2,12,13]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[4,6,10,16]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[6,16]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[5,6,14,15,16]},{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[2,3,13]}],"complexes":["SKI-SIX1-EYA3","EYA3-SIX5-p300","PP2A-B55α-EYA3"],"partners":["SIX1","SIX4","SIX5","SKI","PPP2R2A","WDR1","EP300","ZBTB1"],"other_free_text":[]},"mechanistic_narrative":"EYA3 is a multifunctional transcriptional co-activator and phosphatase that integrates developmental gene regulation, DNA damage repair, cytoskeletal remodeling, and immune evasion. As a transcriptional co-activator, EYA3 partners with SIX-family homeoproteins (SIX1, SIX4, SIX5), SKI, and p300 to drive expression of genes controlling myogenesis, photoperiodic signaling, and tumor-promoting programs such as VEGFA and EGFR; alternatively spliced isoforms regulated by RBFOX2 confer partner selectivity and distinct transcriptional outputs during myogenic differentiation [PMID:19008232, PMID:21129973, PMID:35957720, PMID:38026174]. Its C-terminal HAD-family tyrosine phosphatase domain dephosphorylates H2AX-pY142 to promote DNA damage repair and cell survival—a function exploited in Ewing sarcoma chemoresistance and pulmonary vascular remodeling—and dephosphorylates WDR1-pY to reorganize the actin cytoskeleton [PMID:22723308, PMID:29440662, PMID:31515519, PMID:33649104]. The N-terminal domain lacks intrinsic Ser/Thr phosphatase activity but instead recruits PP2A-B55α holoenzyme through a conserved peptide motif that binds the B55α substrate-recruitment surface, redirecting PP2A to dephosphorylate c-Myc at pT58, thereby stabilizing Myc to drive PD-L1-mediated immune suppression and NF-κB/CCL2-dependent NK cell exclusion in the pre-metastatic niche [PMID:29535359, PMID:29757193, PMID:40247147, PMID:40333987]."},"prefetch_data":{"uniprot":{"accession":"Q99504","full_name":"Protein phosphatase EYA3","aliases":["Eyes absent homolog 3"],"length_aa":573,"mass_kda":62.7,"function":"Tyrosine phosphatase that specifically dephosphorylates 'Tyr-142' of histone H2AX (H2AXY142ph). 'Tyr-142' phosphorylation of histone H2AX plays a central role in DNA repair and acts as a mark that distinguishes between apoptotic and repair responses to genotoxic stress. Promotes efficient DNA repair by dephosphorylating H2AX, promoting the recruitment of DNA repair complexes containing MDC1 (PubMed:19234442, PubMed:19351884). Its function as histone phosphatase probably explains its role in transcription regulation during organogenesis. Coactivates SIX1, and seems to coactivate SIX2, SIX4 and SIX5. The repression of precursor cell proliferation in myoblasts by SIX1 is switched to activation through recruitment of EYA3 to the SIX1-DACH1 complex and seems to be dependent on EYA3 phosphatase activity (By similarity). May be involved in development of the eye","subcellular_location":"Cytoplasm; Nucleus","url":"https://www.uniprot.org/uniprotkb/Q99504/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/EYA3","classification":"Not Classified","n_dependent_lines":10,"n_total_lines":1208,"dependency_fraction":0.008278145695364239},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/EYA3","total_profiled":1310},"omim":[{"mim_id":"608389","title":"BRANCHIOOTIC SYNDROME 3; BOS3","url":"https://www.omim.org/entry/608389"},{"mim_id":"603550","title":"EYA TRANSCRIPTIONAL COACTIVATOR AND PHOSPHATASE 4; EYA4","url":"https://www.omim.org/entry/603550"},{"mim_id":"601655","title":"EYA TRANSCRIPTIONAL COACTIVATOR AND PHOSPHATASE 3; EYA3","url":"https://www.omim.org/entry/601655"},{"mim_id":"601654","title":"EYA TRANSCRIPTIONAL COACTIVATOR AND PHOSPHATASE 2; EYA2","url":"https://www.omim.org/entry/601654"},{"mim_id":"601653","title":"EYA TRANSCRIPTIONAL COACTIVATOR AND PHOSPHATASE 1; EYA1","url":"https://www.omim.org/entry/601653"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Enhanced","locations":[{"location":"Nucleoplasm","reliability":"Enhanced"},{"location":"Centrosome","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/EYA3"},"hgnc":{"alias_symbol":["DKFZp686C132"],"prev_symbol":[]},"alphafold":{"accession":"Q99504","domains":[{"cath_id":"3.40.50.12350","chopping":"303-386_405-569","consensus_level":"medium","plddt":97.1333,"start":303,"end":569}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q99504","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q99504-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q99504-F1-predicted_aligned_error_v6.png","plddt_mean":65.19},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=EYA3","jax_strain_url":"https://www.jax.org/strain/search?query=EYA3"},"sequence":{"accession":"Q99504","fasta_url":"https://rest.uniprot.org/uniprotkb/Q99504.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q99504/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q99504"}},"corpus_meta":[{"pmid":"21129973","id":"PMC_21129973","title":"Acute induction of Eya3 by late-night light stimulation triggers TSHβ expression in photoperiodism.","date":"2010","source":"Current biology : CB","url":"https://pubmed.ncbi.nlm.nih.gov/21129973","citation_count":96,"is_preprint":false},{"pmid":"22723308","id":"PMC_22723308","title":"EWS/FLI1 regulates EYA3 in Ewing sarcoma via modulation of miRNA-708, resulting in increased cell survival and chemoresistance.","date":"2012","source":"Molecular cancer research : MCR","url":"https://pubmed.ncbi.nlm.nih.gov/22723308","citation_count":78,"is_preprint":false},{"pmid":"29535359","id":"PMC_29535359","title":"Eya3 partners with PP2A to induce c-Myc stabilization and tumor progression.","date":"2018","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/29535359","citation_count":73,"is_preprint":false},{"pmid":"20434341","id":"PMC_20434341","title":"Identification of Eya3 and TAC1 as long-day signals in the sheep pituitary.","date":"2010","source":"Current biology : CB","url":"https://pubmed.ncbi.nlm.nih.gov/20434341","citation_count":69,"is_preprint":false},{"pmid":"29757193","id":"PMC_29757193","title":"Eya3 promotes breast tumor-associated immune suppression via threonine phosphatase-mediated PD-L1 upregulation.","date":"2018","source":"The Journal of clinical investigation","url":"https://pubmed.ncbi.nlm.nih.gov/29757193","citation_count":40,"is_preprint":false},{"pmid":"19008232","id":"PMC_19008232","title":"Ski regulates muscle terminal differentiation by transcriptional activation of Myog in a complex with Six1 and Eya3.","date":"2008","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/19008232","citation_count":36,"is_preprint":false},{"pmid":"19102749","id":"PMC_19102749","title":"Pleiotropic effects in Eya3 knockout mice.","date":"2008","source":"BMC developmental biology","url":"https://pubmed.ncbi.nlm.nih.gov/19102749","citation_count":33,"is_preprint":false},{"pmid":"31515519","id":"PMC_31515519","title":"The EYA3 tyrosine phosphatase activity promotes pulmonary vascular remodeling in pulmonary arterial hypertension.","date":"2019","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/31515519","citation_count":27,"is_preprint":false},{"pmid":"29440662","id":"PMC_29440662","title":"WDR1 is a novel EYA3 substrate and its dephosphorylation induces modifications of the cellular actin cytoskeleton.","date":"2018","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/29440662","citation_count":20,"is_preprint":false},{"pmid":"33475861","id":"PMC_33475861","title":"A recurrent missense variant in EYA3 gene is associated with oculo-auriculo-vertebral spectrum.","date":"2021","source":"Human genetics","url":"https://pubmed.ncbi.nlm.nih.gov/33475861","citation_count":20,"is_preprint":false},{"pmid":"34395419","id":"PMC_34395419","title":"CircGNG4 Promotes the Progression of Prostate Cancer by Sponging miR-223 to Enhance EYA3/c-myc Expression.","date":"2021","source":"Frontiers in cell and developmental biology","url":"https://pubmed.ncbi.nlm.nih.gov/34395419","citation_count":19,"is_preprint":false},{"pmid":"33649104","id":"PMC_33649104","title":"Targeting EYA3 in Ewing Sarcoma Retards Tumor Growth and Angiogenesis.","date":"2021","source":"Molecular cancer therapeutics","url":"https://pubmed.ncbi.nlm.nih.gov/33649104","citation_count":15,"is_preprint":false},{"pmid":"35957720","id":"PMC_35957720","title":"Both a hypoxia-inducible EYA3 and a histone acetyltransferase p300 function as coactivators of SIX5 to mediate tumorigenesis and cancer progression.","date":"2022","source":"Annals of translational medicine","url":"https://pubmed.ncbi.nlm.nih.gov/35957720","citation_count":11,"is_preprint":false},{"pmid":"40247147","id":"PMC_40247147","title":"Cryo-EM structures of PP2A:B55 with p107 and Eya3 define substrate recruitment.","date":"2025","source":"Nature structural & molecular biology","url":"https://pubmed.ncbi.nlm.nih.gov/40247147","citation_count":7,"is_preprint":false},{"pmid":"38026174","id":"PMC_38026174","title":"RBFOX2 regulated EYA3 isoforms partner with SIX4 or ZBTB1 to control transcription during myogenesis.","date":"2023","source":"iScience","url":"https://pubmed.ncbi.nlm.nih.gov/38026174","citation_count":7,"is_preprint":false},{"pmid":"31847183","id":"PMC_31847183","title":"Analysis of EYA3 Phosphorylation by Src Kinase Identifies Residues Involved in Cell Proliferation.","date":"2019","source":"International journal of molecular sciences","url":"https://pubmed.ncbi.nlm.nih.gov/31847183","citation_count":7,"is_preprint":false},{"pmid":"34089890","id":"PMC_34089890","title":"Neuroendocrine regulation of reproduction in Atlantic cod (Gadus morhua): Evidence of Eya3 as an integrator of photoperiodic cues and nutritional regulation to initiate sexual maturation.","date":"2021","source":"Comparative biochemistry and physiology. Part A, Molecular & integrative physiology","url":"https://pubmed.ncbi.nlm.nih.gov/34089890","citation_count":6,"is_preprint":false},{"pmid":"35633536","id":"PMC_35633536","title":"Inhibitors of EYA3 Protein in Ewing Sarcoma.","date":"2022","source":"Asian Pacific journal of cancer prevention : APJCP","url":"https://pubmed.ncbi.nlm.nih.gov/35633536","citation_count":5,"is_preprint":false},{"pmid":"40333987","id":"PMC_40333987","title":"EYA3 regulation of NF-κB and CCL2 suppresses cytotoxic NK cells in the premetastatic niche to promote TNBC metastasis.","date":"2025","source":"Science advances","url":"https://pubmed.ncbi.nlm.nih.gov/40333987","citation_count":4,"is_preprint":false},{"pmid":"36588754","id":"PMC_36588754","title":"Cytogenomic Characterization of a Novel de novo Balanced Reciprocal Translocation t(1;12) by Genome Sequencing Leading to Fusion Gene Formation of EYA3/EFCAB4b.","date":"2022","source":"Molecular syndromology","url":"https://pubmed.ncbi.nlm.nih.gov/36588754","citation_count":3,"is_preprint":false},{"pmid":"29884726","id":"PMC_29884726","title":"An Immunosuppressive Role for Eya3 in TNBC.","date":"2018","source":"Cancer discovery","url":"https://pubmed.ncbi.nlm.nih.gov/29884726","citation_count":2,"is_preprint":false},{"pmid":"33473370","id":"PMC_33473370","title":"Expression analysis of DIO2, EYA3, KISS1 and GPR54 genes in year-round estrous and seasonally estrous rams.","date":"2020","source":"Archives animal breeding","url":"https://pubmed.ncbi.nlm.nih.gov/33473370","citation_count":2,"is_preprint":false},{"pmid":"39211066","id":"PMC_39211066","title":"An EYA3/NF-κB/CCL2 signaling axis suppresses cytotoxic NK cells in the pre-metastatic niche to promote triple negative breast cancer metastasis.","date":"2024","source":"bioRxiv : the preprint server for biology","url":"https://pubmed.ncbi.nlm.nih.gov/39211066","citation_count":2,"is_preprint":false},{"pmid":"39550476","id":"PMC_39550476","title":"EYA3 promotes the tumorigenesis of gastric cancer through activation of the mTORC1 signaling pathway and inhibition of autophagy.","date":"2024","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/39550476","citation_count":1,"is_preprint":false},{"pmid":"40743761","id":"PMC_40743761","title":"HOTAIR Promotes Spiral Ganglion Neuron Proliferation via miR-211-5p/EYA3 Regulation.","date":"2025","source":"Critical reviews in immunology","url":"https://pubmed.ncbi.nlm.nih.gov/40743761","citation_count":1,"is_preprint":false},{"pmid":"40435734","id":"PMC_40435734","title":"Loss of circular EYA3 attenuates formaldehyde-induced cardiomyocyte pyroptosis and congenital heart defects by regulating Smad5 stability.","date":"2025","source":"The Science of the total environment","url":"https://pubmed.ncbi.nlm.nih.gov/40435734","citation_count":1,"is_preprint":false},{"pmid":"40414499","id":"PMC_40414499","title":"Cryo-EM structures reveal the PP2A-B55α and Eya3 interaction that can be disrupted by a peptide inhibitor.","date":"2025","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/40414499","citation_count":0,"is_preprint":false},{"pmid":"39975004","id":"PMC_39975004","title":"Cryo-EM structures reveal the PP2A-B55α and Eya3 interaction that can be disrupted by a peptide inhibitor.","date":"2025","source":"bioRxiv : the preprint server for biology","url":"https://pubmed.ncbi.nlm.nih.gov/39975004","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":14402,"output_tokens":4223,"usd":0.053276},"stage2":{"model":"claude-opus-4-6","input_tokens":7727,"output_tokens":4160,"usd":0.213952},"total_usd":0.267228,"stage1_batch_id":"msgbatch_0118RScg3WGaGkaZRtWZbtce","stage2_batch_id":"msgbatch_01Cpfzvtbj42aqp5oYZ5Te2s","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2010,\n      \"finding\": \"EYA3 partners with SIX1 to synergistically activate TSHβ expression in the pars tuberalis, with further enhancement by TEF and HLF, positioning EYA3 as an upstream transcriptional activator in the photoperiodic signaling cascade.\",\n      \"method\": \"Genome-wide expression analysis, transcriptional reporter assays, acute light-stimulation experiments in CBA/N mice\",\n      \"journal\": \"Current biology : CB\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — defined pathway position and direct transcriptional activation shown in vivo and in vitro, single lab\",\n      \"pmids\": [\"21129973\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"EYA3 is the strongest long-photoperiod-activated gene in the sheep pars tuberalis, identifying it as a conserved molecular photoperiodic signal upstream of TSH-mediated reproductive regulation in mammals.\",\n      \"method\": \"Microarray, in situ hybridization under controlled photoperiod conditions in sheep\",\n      \"journal\": \"Current biology : CB\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — direct in vivo expression mapping tied to functional photoperiodic pathway, single lab\",\n      \"pmids\": [\"20434341\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"EYA3 forms a complex with SKI and SIX1 via the SKI Dachshund homology domain to activate transcription from the MEF3 site on the Myogenin (MYOG) regulatory region, promoting muscle terminal differentiation.\",\n      \"method\": \"Co-immunoprecipitation, chromatin immunoprecipitation (ChIP), transcriptional reporter assays, retroviral overexpression/knockdown in C2C12 myoblasts\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal Co-IP, ChIP, reporter assays, and KD phenotype with multiple orthogonal methods\",\n      \"pmids\": [\"19008232\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Loss of EYA3 in mice produces pleiotropic physiological defects including decreased bone mineral content, shorter body length, reduced tidal volume, cardiac conduction changes, and reduced muscle strength, with no apparent eye defect, demonstrating broad developmental roles distinct from the Drosophila eye phenotype.\",\n      \"method\": \"EYA3 knockout mouse phenotypic analysis within the German Mouse Clinic; in situ hybridization; beta-Gal staining; differential gene expression analysis\",\n      \"journal\": \"BMC developmental biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — comprehensive KO mouse phenotyping with multiple physiological readouts, single lab\",\n      \"pmids\": [\"19102749\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"EWS/FLI1 upregulates EYA3 in Ewing sarcoma by repressing miR-708, which targets the EYA3 3'-UTR; elevated EYA3 promotes cell survival and chemoresistance by enabling more effective DNA damage repair.\",\n      \"method\": \"miRNA target site analysis, EYA3 knockdown in Ewing sarcoma cell lines, clonogenic survival assays, DNA damage repair assays with chemotherapeutics\",\n      \"journal\": \"Molecular cancer research : MCR\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — miR-708/EYA3 3'-UTR targeting validated, KD with defined survival/chemoresistance phenotype and DNA repair readout, corroborated in human tumor samples\",\n      \"pmids\": [\"22723308\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"The N-terminal domain Ser/Thr phosphatase activity of EYA3 is not intrinsic but arises from direct interaction with PP2A-B55α holoenzyme; EYA3 redirects PP2A-B55α to dephosphorylate c-Myc at pT58 (stabilizing c-Myc), contrasting with PP2A-B56α-mediated dephosphorylation of pS62 (which destabilizes c-Myc).\",\n      \"method\": \"Co-immunoprecipitation, in vitro phosphatase assays, mutagenesis, xenograft breast cancer model\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — in vitro reconstitution of phosphatase activity, direct interaction demonstrated, site-specific mutagenesis, in vivo xenograft validation\",\n      \"pmids\": [\"29535359\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"EYA3 utilizes its Thr phosphatase activity to dephosphorylate Myc at pT58, stabilizing Myc, which drives PD-L1 upregulation and tumor immune suppression; EYA3 knockdown reduces tumor growth in immune-competent mice via increased CD8+ T cell infiltration.\",\n      \"method\": \"EYA3 knockdown/rescue experiments, phosphatase-dead mutants, CD8+ T cell depletion, PD-L1 rescue assays, syngeneic breast tumor models\",\n      \"journal\": \"The Journal of clinical investigation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods including phosphatase-dead mutant, immune cell depletion rescue, and in vivo tumor models\",\n      \"pmids\": [\"29757193\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"WDR1 is an EYA3-specific substrate: Src kinase phosphorylates WDR1 on tyrosine residues, and EYA3 (but not EYA1) efficiently dephosphorylates these sites; loss of WDR1 tyrosine phosphorylation causes major reorganization of the actin cytoskeleton. Additionally, Src phosphorylates EYA3 itself, controlling its nuclear and cytoskeletal localization, and EYA3 can autodephosphorylate these sites.\",\n      \"method\": \"Phosphotyrosine peptide microarray, in vitro phosphatase assays, Src kinase assays, subcellular fractionation/localization, actin cytoskeleton imaging\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — peptide microarray substrate identification, in vitro phosphatase reconstitution, localization experiments with functional consequence\",\n      \"pmids\": [\"29440662\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"EYA3 tyrosine phosphatase activity promotes survival of pulmonary arterial smooth muscle cells under DNA-damaging conditions; transgenic mice with an inactivating EYA3 tyrosine phosphatase domain mutation are protected from vascular remodeling, and pharmacological inhibition of EYA3 tyrosine phosphatase reverses remodeling in a rat pulmonary hypertension model.\",\n      \"method\": \"Transgenic knock-in mice with catalytic-dead EYA3 mutation, pharmacological inhibition, rat angio-obliterative PAH model, cell survival assays\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — catalytic-dead knock-in mouse, pharmacological inhibition in two animal models, defined cellular survival phenotype\",\n      \"pmids\": [\"31515519\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"EYA3 is phosphorylated by Src kinase at 13 tyrosine residues (including Y77, Y96, Y237, Y508); EYA3 autodephosphorylates these sites; specific residues Y77, Y96, and Y237 control HEK293T cell proliferation, as their mutation abolishes the pro-proliferative effect of EYA3 overexpression.\",\n      \"method\": \"Native and bottom-up mass spectrometry phosphosite mapping, site-directed mutagenesis, cell cycle analysis, Src kinase assays\",\n      \"journal\": \"International journal of molecular sciences\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — MS-based phosphosite mapping, mutagenesis with functional proliferation readout\",\n      \"pmids\": [\"31847183\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"EYA3 tyrosine phosphatase activity promotes Ewing sarcoma tumor growth and angiogenesis by regulating VEGFA levels and promoting DNA damage repair; pharmacological inhibition of EYA3 tyrosine phosphatase elevates its substrate H2AX-pY142 in tumor tissue and inhibits tumor growth in cell line and patient-derived xenografts.\",\n      \"method\": \"Genetic knockdown, pharmacological inhibition (Benzarone), xenograft and patient-derived xenograft models, H2AX-pY142 substrate engagement assay\",\n      \"journal\": \"Molecular cancer therapeutics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — in vivo target engagement demonstrated via substrate phosphorylation, multiple models including PDX\",\n      \"pmids\": [\"33649104\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"A missense variant (p.Asn358Ser) in EYA3 associated with oculo-auriculo-vertebral spectrum increases the half-life of the mutated protein without impairing its ability to dephosphorylate H2AFX following DNA damage; eya3 knockdown in zebrafish embryos causes specific craniofacial abnormalities.\",\n      \"method\": \"Exome sequencing, cellular protein stability assays, H2AFX dephosphorylation assay, zebrafish morpholino knockdown, proteomics\",\n      \"journal\": \"Human genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — direct phosphatase activity assay on patient variant, zebrafish KD with craniofacial phenotype, proteomics, single lab\",\n      \"pmids\": [\"33475861\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"EYA3 assembles a complex with SIX5 and histone acetyltransferase p300 in hypoxic colorectal cancer cells; this EYA3-SIX5-p300 complex binds the promoters of EGFR, VEGFD, and multiple MMPs to activate their transcription and drive tumor progression.\",\n      \"method\": \"Co-immunoprecipitation, mass spectrometry, chromatin immunoprecipitation (ChIP), pharmacological inhibition with benzarone in xenograft model\",\n      \"journal\": \"Annals of translational medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal Co-IP/MS for complex identification, ChIP for promoter binding, in vivo xenograft validation, single lab\",\n      \"pmids\": [\"35957720\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"EYA3 expression is required for myoblast proliferation and differentiation; alternatively spliced EYA3 isoforms (regulated by RBFOX2) differentially interact with SIX4 or ZBTB1 to control distinct transcriptional programs during myogenesis; the exon 7 splicing event is tissue-specific and developmentally regulated.\",\n      \"method\": \"Mass spectrometry proteomics, genome-wide transcriptomics, alternative splicing analysis, EYA3 knockdown in myoblasts with proliferation/differentiation readouts\",\n      \"journal\": \"iScience\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — MS-validated protein interactions, transcriptomics, KD phenotype, multiple orthogonal methods; single lab\",\n      \"pmids\": [\"38026174\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Cryo-EM and NMR structures of the PP2A:B55α holoenzyme with EYA3 reveal that EYA3 binds B55α through an extended peptide in its N-terminal domain using a core B55 recruitment motif conserved across the EYA family; this binding overlaps with substrate recruitment sites but differs mechanistically from substrate binding; B55 recruitment by EYA3 directs selective dephosphorylation of specific phosphosites.\",\n      \"method\": \"Cryo-electron microscopy, NMR spectroscopy, NMR-based dephosphorylation assays, structural comparison\",\n      \"journal\": \"Nature structural & molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — cryo-EM structure with NMR functional validation and dephosphorylation assays; defines molecular mechanism of substrate recruitment\",\n      \"pmids\": [\"40247147\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Cryo-EM structures of PP2A-B55α bound with EYA3 show EYA3 binds B55α via an extended peptide in its N-terminal domain at a similar surface as substrates and peptide inhibitors; a designed inhibitory peptide (B55i) disrupts the EYA3-B55α interaction in vitro and increases Myc pT58 while reducing Myc protein levels in TNBC cells.\",\n      \"method\": \"Cryo-EM structure determination, in vitro binding/competition assays, B55i peptide expression in TNBC cells, Myc pT58 phosphorylation assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — cryo-EM structures, in vitro inhibition assay, cellular functional validation with specific phosphorylation readout\",\n      \"pmids\": [\"40414499\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"EYA3 upregulates NF-κB signaling to enhance CCL2 expression in triple-negative breast cancer cells; secreted CCL2 suppresses cytotoxic NK cell activation in vitro and reduces NK cell infiltration into the pre-metastatic niche in vivo, promoting metastasis independently of primary tumor effects.\",\n      \"method\": \"EYA3 knockdown, NF-κB signaling rescue, CCL2 re-expression rescue, NK cell depletion/activation assays in vitro, syngeneic pre-metastatic niche models in vivo\",\n      \"journal\": \"Science advances\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — epistatic rescue experiments (NF-κB and CCL2 re-expression), in vivo NK cell phenotyping, multiple orthogonal approaches in single study\",\n      \"pmids\": [\"40333987\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"EYA3 is a multifunctional protein that acts as (1) a transcriptional co-activator partnering with SIX1/SIX4/SIX5, SKI, and p300 to regulate developmental and oncogenic gene expression programs; (2) a tyrosine phosphatase (HAD-family) that dephosphorylates substrates including H2AX-pY142 to promote DNA damage repair and cell survival, and WDR1-pY to remodel the actin cytoskeleton; and (3) a regulator of PP2A specificity, whereby its N-terminal domain—through a conserved recruitment motif that binds B55α at a structurally defined interface—redirects PP2A-B55α to dephosphorylate c-Myc pT58, stabilizing Myc to drive tumor progression and PD-L1-mediated immune suppression, while also engaging NF-κB/CCL2 signaling to suppress cytotoxic NK cells in the pre-metastatic niche.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"EYA3 is a multifunctional transcriptional co-activator and phosphatase that integrates developmental gene regulation, DNA damage repair, cytoskeletal remodeling, and immune evasion. As a transcriptional co-activator, EYA3 partners with SIX-family homeoproteins (SIX1, SIX4, SIX5), SKI, and p300 to drive expression of genes controlling myogenesis, photoperiodic signaling, and tumor-promoting programs such as VEGFA and EGFR; alternatively spliced isoforms regulated by RBFOX2 confer partner selectivity and distinct transcriptional outputs during myogenic differentiation [PMID:19008232, PMID:21129973, PMID:35957720, PMID:38026174]. Its C-terminal HAD-family tyrosine phosphatase domain dephosphorylates H2AX-pY142 to promote DNA damage repair and cell survival—a function exploited in Ewing sarcoma chemoresistance and pulmonary vascular remodeling—and dephosphorylates WDR1-pY to reorganize the actin cytoskeleton [PMID:22723308, PMID:29440662, PMID:31515519, PMID:33649104]. The N-terminal domain lacks intrinsic Ser/Thr phosphatase activity but instead recruits PP2A-B55α holoenzyme through a conserved peptide motif that binds the B55α substrate-recruitment surface, redirecting PP2A to dephosphorylate c-Myc at pT58, thereby stabilizing Myc to drive PD-L1-mediated immune suppression and NF-κB/CCL2-dependent NK cell exclusion in the pre-metastatic niche [PMID:29535359, PMID:29757193, PMID:40247147, PMID:40333987].\",\n  \"teleology\": [\n    {\n      \"year\": 2008,\n      \"claim\": \"Establishing EYA3 as a transcriptional co-activator in muscle differentiation resolved how EYA3 engages chromatin: it forms a SKI–SIX1–EYA3 complex on the MEF3 site of the Myogenin promoter to activate terminal differentiation genes.\",\n      \"evidence\": \"Reciprocal Co-IP, ChIP, reporter assays, and knockdown in C2C12 myoblasts\",\n      \"pmids\": [\"19008232\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether EYA3 phosphatase activity is required for its co-activator function in myogenesis was not tested\", \"Other genomic targets of the SKI–SIX1–EYA3 complex were not mapped\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"EYA3 knockout mice revealed broad developmental functions—reduced bone mineral content, shorter body length, cardiac conduction defects, reduced muscle strength—without eye defects, establishing that mammalian EYA3 has pleiotropic roles distinct from the Drosophila eye specification paradigm.\",\n      \"evidence\": \"Comprehensive phenotyping of Eya3-null mice at the German Mouse Clinic\",\n      \"pmids\": [\"19102749\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular targets responsible for each phenotypic defect were not identified\", \"Redundancy with other EYA family members was not formally assessed\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Identification of EYA3 as the strongest photoperiod-activated gene in the pars tuberalis, synergistically activating TSHβ with SIX1, placed EYA3 atop a conserved mammalian photoperiodic signaling cascade regulating seasonal reproduction.\",\n      \"evidence\": \"Microarray and in situ hybridization in sheep under controlled photoperiod; transcriptional reporter assays and acute light stimulation in mice\",\n      \"pmids\": [\"20434341\", \"21129973\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Upstream signals that induce EYA3 expression in response to light were not fully defined\", \"Whether EYA3 phosphatase activity contributes to photoperiodic signaling is unknown\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Discovery that EWS/FLI1 upregulates EYA3 by repressing miR-708 revealed a mechanism by which Ewing sarcoma exploits EYA3 for DNA damage repair and chemoresistance, establishing EYA3's phosphatase as a survival factor in cancer.\",\n      \"evidence\": \"miR-708/EYA3 3′-UTR targeting validation, EYA3 knockdown with clonogenic survival and DNA repair assays in Ewing sarcoma cell lines\",\n      \"pmids\": [\"22723308\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"The specific EYA3 phosphatase substrates mediating chemoresistance were not identified at this stage\", \"In vivo validation of miR-708-EYA3 axis was limited\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Three concurrent studies redefined EYA3 enzymology: its reported N-terminal Ser/Thr phosphatase activity was shown to arise from recruited PP2A-B55α, which EYA3 redirects to dephosphorylate c-Myc pT58, stabilizing Myc to drive PD-L1 upregulation and tumor immune evasion; separately, WDR1 was identified as an EYA3-specific tyrosine phosphatase substrate controlling actin cytoskeleton remodeling, and Src-dependent phosphorylation of EYA3 itself was shown to regulate its subcellular localization.\",\n      \"evidence\": \"Co-IP and in vitro phosphatase reconstitution with PP2A-B55α; phosphatase-dead mutant rescue and CD8+ T cell depletion in syngeneic tumor models; phosphotyrosine peptide microarray for WDR1 substrate identification; Src kinase assays with subcellular fractionation\",\n      \"pmids\": [\"29535359\", \"29757193\", \"29440662\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"The structural basis for EYA3–B55α interaction was unknown\", \"How EYA3 selectively recruits PP2A-B55α versus B56α was unresolved\", \"Whether WDR1 dephosphorylation by EYA3 is relevant in vivo was not shown\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Catalytic-dead EYA3 knock-in mice and pharmacological inhibition demonstrated that EYA3 tyrosine phosphatase activity is required for pathological vascular remodeling in pulmonary hypertension, validating EYA3 as a druggable target in vivo.\",\n      \"evidence\": \"Transgenic mice with inactivating mutation in EYA3 tyrosine phosphatase domain; pharmacological inhibition in rat pulmonary arterial hypertension model\",\n      \"pmids\": [\"31515519\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"The direct vascular substrate(s) of EYA3 tyrosine phosphatase in smooth muscle cells were not identified\", \"Long-term therapeutic window and off-target effects of pharmacological inhibition were not assessed\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Mass spectrometry mapping of 13 Src-phosphorylated tyrosine residues on EYA3, with mutagenesis showing Y77, Y96, and Y237 are required for its pro-proliferative activity, defined the regulatory phosphorylation landscape of EYA3 itself.\",\n      \"evidence\": \"Native and bottom-up MS phosphosite mapping, site-directed mutagenesis with cell cycle analysis in HEK293T cells\",\n      \"pmids\": [\"31847183\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Which downstream signaling pathways are affected by each specific phosphosite remains unknown\", \"Physiological contexts in which Src-EYA3 phosphorylation occurs in vivo were not established\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"In vivo target engagement was demonstrated: pharmacological EYA3 tyrosine phosphatase inhibition elevated H2AX-pY142 in Ewing sarcoma tumors and suppressed growth in cell line and patient-derived xenografts, confirming H2AX-pY142 as a bona fide in vivo substrate and linking EYA3 phosphatase to tumor angiogenesis via VEGFA.\",\n      \"evidence\": \"Benzarone treatment of xenograft and PDX models with H2AX-pY142 substrate engagement assay\",\n      \"pmids\": [\"33649104\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether VEGFA regulation requires EYA3 phosphatase activity or its transcriptional co-activator function was not fully dissected\", \"Selectivity of benzarone for EYA3 versus other targets is a concern\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"A missense variant (p.Asn358Ser) in EYA3 was linked to oculo-auriculo-vertebral spectrum, with the mutant protein showing increased stability without loss of H2AX dephosphorylation; zebrafish eya3 knockdown produced craniofacial defects, establishing EYA3 as a craniofacial development gene.\",\n      \"evidence\": \"Exome sequencing, protein stability assays, H2AFX dephosphorylation assay, zebrafish morpholino knockdown\",\n      \"pmids\": [\"33475861\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"The variant was identified in a single family; broader genetic evidence is needed\", \"How increased protein stability causes disease pathology is unclear\", \"Whether gain-of-function or neomorphic activity underlies the phenotype was not determined\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Identification of the EYA3–SIX5–p300 complex on EGFR, VEGFD, and MMP promoters in hypoxic colorectal cancer cells extended EYA3's transcriptional co-activator role to hypoxia-driven tumor progression programs.\",\n      \"evidence\": \"Co-IP/mass spectrometry, ChIP on target promoters, benzarone inhibition in xenograft model\",\n      \"pmids\": [\"35957720\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether the phosphatase activity of EYA3 is required for p300 recruitment and histone acetylation at these loci is unknown\", \"The hypoxia-responsive element driving EYA3 expression/activity was not identified\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"RBFOX2-regulated alternative splicing of EYA3 exon 7 was shown to generate isoforms with differential binding to SIX4 versus ZBTB1, controlling distinct transcriptional programs during myoblast proliferation and differentiation, revealing a splicing-based mechanism for EYA3 functional diversification.\",\n      \"evidence\": \"Mass spectrometry proteomics, transcriptomics, splicing analysis, EYA3 knockdown with proliferation/differentiation readouts in myoblasts\",\n      \"pmids\": [\"38026174\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"The structural basis for isoform-specific partner selection is unknown\", \"In vivo relevance of the exon 7 splicing switch in muscle development has not been tested\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Cryo-EM and NMR structures of PP2A-B55α bound to EYA3 revealed that a conserved N-terminal peptide of EYA3 engages the B55α substrate-recruitment groove, providing the structural basis for how EYA3 redirects PP2A specificity; a designed inhibitory peptide (B55i) disrupts this interaction and destabilizes Myc in triple-negative breast cancer cells.\",\n      \"evidence\": \"Cryo-EM structure determination, NMR spectroscopy and dephosphorylation assays, B55i peptide competition in vitro and in TNBC cells with Myc pT58 readout\",\n      \"pmids\": [\"40247147\", \"40414499\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo efficacy and pharmacokinetic properties of B55i or derivatives are untested\", \"Whether the EYA3–B55α interaction has substrates beyond c-Myc in cells is not known\", \"Structural data for full-length EYA3 bound to PP2A holoenzyme is lacking\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"EYA3 was shown to activate NF-κB/CCL2 signaling in TNBC cells, with secreted CCL2 suppressing NK cell cytotoxicity and reducing NK infiltration in the pre-metastatic niche to promote metastasis, adding innate immune evasion to EYA3's oncogenic repertoire.\",\n      \"evidence\": \"EYA3 knockdown with NF-κB and CCL2 epistatic rescue, NK cell activation/depletion assays in vitro, syngeneic pre-metastatic niche models\",\n      \"pmids\": [\"40333987\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether EYA3's phosphatase or co-activator function drives NF-κB activation is unresolved\", \"The direct molecular link between EYA3 and NF-κB pathway activation has not been identified\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Key unresolved questions include: how EYA3's dual phosphatase and transcriptional activities are coordinated in physiological contexts; the full spectrum of PP2A-B55α substrates redirected by EYA3 beyond c-Myc; the structural basis for isoform-specific partner selection; and whether therapeutic disruption of the EYA3–B55α interface is effective in vivo.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No integrated model connects EYA3 phosphatase and transcriptional activities in the same cellular context\", \"Full-length EYA3 structure remains undetermined\", \"In vivo therapeutic targeting of the EYA3–B55α interface is untested\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [7, 8, 9, 10, 11]},\n      {\"term_id\": \"GO:0140110\", \"supporting_discovery_ids\": [0, 2, 12, 13]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [5, 6, 14, 15]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [2, 7, 12]},\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [7]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"GO:0073894\", \"supporting_discovery_ids\": [4, 8, 10]},\n      {\"term_id\": \"R-HSA-74160\", \"supporting_discovery_ids\": [0, 2, 12, 13]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [4, 6, 10, 16]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [6, 16]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [5, 6, 14, 15, 16]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [2, 3, 13]}\n    ],\n    \"complexes\": [\n      \"SKI-SIX1-EYA3\",\n      \"EYA3-SIX5-p300\",\n      \"PP2A-B55α-EYA3\"\n    ],\n    \"partners\": [\n      \"SIX1\",\n      \"SIX4\",\n      \"SIX5\",\n      \"SKI\",\n      \"PPP2R2A\",\n      \"WDR1\",\n      \"EP300\",\n      \"ZBTB1\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}