{"gene":"EYA4","run_date":"2026-06-09T23:54:43","timeline":{"discoveries":[{"year":1999,"finding":"EYA4 encodes a 640 amino acid protein containing a highly conserved C-terminal domain of 271 amino acids (Eya homologous region) that mediates developmentally important protein-protein interactions, as established by characterization of the human and mouse gene.","method":"cDNA cloning, sequence analysis, domain characterization","journal":"Human molecular genetics","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — domain characterization by sequence homology to Drosophila eya, functional inference from conserved domain, single study","pmids":["9887327"],"is_preprint":false},{"year":2001,"finding":"EYA4 is the causative gene for postlingual progressive autosomal dominant hearing loss at the DFNA10 locus; mutations creating premature stop codons in two unrelated families established haploinsufficiency as the disease mechanism.","method":"Candidate gene sequencing, mutation identification, co-segregation analysis in two families","journal":"Human molecular genetics","confidence":"High","confidence_rationale":"Tier 2 / Strong — replicated in two independent families with different mutations, co-segregation confirmed","pmids":["11159937"],"is_preprint":false},{"year":2004,"finding":"EYA4 Eya homologous region (Eya4HR) interacts with SIX1 (demonstrated by yeast two-hybrid), but not with DACH1. Eya4HR localizes to the cytoplasm and is translocated to the nucleus upon co-expression with SIX1. Mutant truncating alleles are not translated, consistent with haploinsufficiency as the mechanism for DFNA10.","method":"Yeast two-hybrid, alpha-galactosidase activity assay, immunofluorescence, dual Eya-containing constructs","journal":"Journal of the Association for Research in Otolaryngology : JARO","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal methods (Y2H, immunofluorescence, functional assay), single lab","pmids":["15492887"],"is_preprint":false},{"year":2005,"finding":"EYA4 deletion causes dilated cardiomyopathy in addition to sensorineural hearing loss; eya4 morpholino knockdown in zebrafish produced heart failure. EYA4 peptides associated with SNHL-only mutations bound wild-type EYA4 and SIX proteins, whereas the cardiomyopathy-associated short peptide (193 aa) did not, indicating that SIX protein interaction is required for cardiac function.","method":"Zebrafish morpholino knockdown, biochemical interaction assays (co-immunoprecipitation), truncated peptide binding studies","journal":"Nature genetics","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (morpholino in vivo, biochemical interaction assays), mechanistic link established between SIX binding and cardiac phenotype","pmids":["15735644"],"is_preprint":false},{"year":2008,"finding":"EYA4 regulates Na+/K+-ATPase (specifically the atp1b2b subunit) in zebrafish; eya4 morphant fish had reduced atp1b2b levels, reduced hair cells, and heart failure. Morpholino knockdown of atp1b2b recapitulated eya4 deficiency phenotypes, and atp1b2b overexpression rescued eya4 morphant phenotypes, placing atp1b2b downstream of eya4 in sensory and cardiac development.","method":"Zebrafish morpholino knockdown, genetic epistasis (rescue by overexpression), hair cell counts, sensory response measurement","journal":"Development (Cambridge, England)","confidence":"High","confidence_rationale":"Tier 2 / Strong — epistasis via morpholino knockdown and rescue overexpression, multiple phenotypic readouts, single lab but multiple orthogonal methods","pmids":["18799547"],"is_preprint":false},{"year":2009,"finding":"EYA4 physically interacts with SIX3 (co-immunoprecipitation) and is recruited to the nucleus by SIX3. EYA4 cooperates functionally with SIX3 as a transcriptional coactivator (reporter gene assay), suggesting a role in forebrain development.","method":"Co-immunoprecipitation, confocal microscopy localization, luciferase reporter assay","journal":"Human mutation","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal co-IP and reporter assay, single lab, two orthogonal methods","pmids":["19606496"],"is_preprint":false},{"year":2015,"finding":"EYA4 overexpression in cardiac tissue induces hypertrophy via regulation of p27kip1 expression; chromatin immunoprecipitation and luciferase assays confirmed that EYA4 binds the p27 promoter and suppresses p27 expression. The downstream signaling involves casein kinase-2α and histone deacetylase 2 (HDAC2) phosphorylation. The truncating mutation E193 acts in an opposing manner, promoting dilated cardiomyopathy.","method":"Transgenic mice (cardiac-specific overexpression), luciferase reporter assay, chromatin immunoprecipitation, MRI/hemodynamic analysis, in vitro expression studies","journal":"Circulation. Cardiovascular genetics","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — ChIP, luciferase, in vivo transgenic models, multiple orthogonal methods in single study","pmids":["26499333"],"is_preprint":false},{"year":2016,"finding":"miR-431 directly targets Eya4 mRNA; luciferase reporter assay and western blotting confirmed that miR-431 inhibits Eya4 translation. Overexpression of miR-431 in transgenic mice led to reduced EYA4 protein in cochleae, decreased spiral ganglion neuron density, and hearing loss.","method":"Luciferase reporter assay, western blotting, transgenic mice, ABR measurement, SGN counting","journal":"Biochimica et biophysica acta","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — luciferase and western blot validate direct targeting, in vivo transgenic model, single lab","pmids":["27545760"],"is_preprint":false},{"year":2018,"finding":"EYA4 suppresses HCC cell growth and invasion by antagonizing NF-κB activity: EYA4 serine/threonine phosphatase activity inhibits phosphorylation and ubiquitination of IκBα, blocks nuclear translocation and transactivation of p65, thereby suppressing RAP1 transcription driven by NF-κB. Calyculin A (phosphatase inhibitor) abolished EYA4's suppression of NF-κB, confirming phosphatase-dependence.","method":"Stable transfection, xenograft, chromatin immunoprecipitation, immunofluorescence, cellular ubiquitination assay, NF-κB reporter, pharmacological inhibition","journal":"Cancer communications (London, England)","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — multiple orthogonal methods (ChIP, ubiquitination assay, phosphatase inhibitor, immunofluorescence, in vivo xenograft), single lab but rigorous","pmids":["29764501"],"is_preprint":false},{"year":2018,"finding":"EYA4 knockdown in ESCC cells increases migration and invasion; EYA4 overexpression promotes epithelial phenotype and reduces TGF-β1-induced EMT by reducing phosphorylation of Akt and GSK-3β, leading to inactivation of slug. TGF-β1 decreases EYA4 expression through DNMT3A-mediated DNA methylation.","method":"shRNA knockdown, overexpression, migration/invasion assays, western blotting for pAkt/pGSK3β/slug, treatment with 5-aza-dC/TSA","journal":"Cancer science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — loss-of-function and gain-of-function with defined pathway readouts, single lab","pmids":["29660222"],"is_preprint":false},{"year":2019,"finding":"EYA4 dephosphorylates β-catenin at Ser552 (serine/threonine phosphatase activity), reducing nuclear translocation of β-catenin and thereby suppressing β-catenin/LEF1-driven transcription of MYCBP. EYA4 KO increased MYCBP; siRNA of MYCBP in EYA4-overexpressing cells rescued proliferation and G2/M arrest phenotypes.","method":"EYA4 overexpression/KO in HCC cell lines, siRNA rescue, western blotting for phospho-β-catenin, qRT-PCR, nuclear fractionation","journal":"Cancer science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — phosphorylation site identified, epistasis by siRNA rescue, single lab, multiple readouts","pmids":["31385398"],"is_preprint":false},{"year":2020,"finding":"EYA4 truncating mutations in the EYA-HR domain result in absence of protein expression (confirmed in mammalian COS7 cells), consistent with haploinsufficiency. Missense mutant p.Glu369Asp shows normal expression and nuclear translocation in presence of SIX1. Minigene assays confirmed that synonymous and splice-site variants cause exon skipping leading to frameshifts.","method":"Transient expression in COS7 cells, confocal microscopy, minigene splicing assays in NIH3T3 cells, in silico splicing analysis","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — cell expression studies and functional minigene assays, single lab, multiple variant analyses","pmids":["32277154"],"is_preprint":false},{"year":2021,"finding":"EYA4 promotes glioma cell proliferation by directly suppressing expression of p27Kip1, and SIX1 is required for EYA4 to suppress p27Kip1.","method":"EYA4 overexpression, cell viability/BrdU assay, flow cytometry, gene expression analysis","journal":"Cellular physiology and biochemistry","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, limited mechanistic depth for direct p27 suppression claim, no ChIP or direct promoter assay described in abstract","pmids":["30231237"],"is_preprint":false},{"year":2023,"finding":"EYA4 serine/threonine phosphatase domain prevents replication-associated DNA damage and is required for replication fork progression. EYA4 depletion causes spontaneous replication stress (ATR pathway activation, γH2AX accumulation, sensitivity to hydroxyurea, endoreplication/polyploidy). EYA4 overexpression promotes breast cancer cell proliferation, migration, and metastasis.","method":"EYA4 KD/OE in breast cancer cells and xenografts, γH2AX staining, ATR pathway markers, hydroxyurea sensitivity, DNA fiber assay (replication fork), phosphatase domain mutant","journal":"Molecular cancer","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal methods including phosphatase-domain mutant, in vitro and in vivo, single lab","pmids":["37777742"],"is_preprint":false},{"year":2024,"finding":"EYA4 (and EYA1) are tyrosine phosphatases that dephosphorylate PLK1 at pY445 during G2 phase of the cell cycle. This dephosphorylation is required for centrosome maturation, PLK1 localization to centrosomes, and polo-box domain (PBD)-dependent interactions between PLK1 and PLK1-activation complexes. Depletion or chemical inhibition of EYA phosphatase activity reduces PLK1 activation, causing mitotic defects and cell death.","method":"Co-immunoprecipitation, phosphatase substrate assay, molecular dynamics simulation, EYA4/EYA1 depletion, chemical inhibition, immunofluorescence of centrosome maturation, mitosis assays","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro phosphatase assay identifying specific substrate site (pY445), co-IP, molecular dynamics, cell-based loss-of-function with multiple orthogonal readouts","pmids":["38360978"],"is_preprint":false},{"year":2024,"finding":"EYA4 tyrosine phosphatase activity dephosphorylates RAD51 at Tyr315, which regulates RAD51 localization, presynaptic filament formation, foci formation, and homologous recombination (HR) at DNA double-strand breaks. DNA binding stimulates EYA4 phosphatase activity. EYA4 depletion decreases ssDNA accumulation after DNA damage and impairs HR; overexpression promotes RAD51 dephosphorylation and nucleoprotein filament formation.","method":"In vitro phosphatase assay, HR reporter assay, RAD51 foci formation, DNA fiber assay, ssDNA accumulation measurement, EYA4 KD/OE","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro phosphatase assay with specific substrate site identification, multiple functional readouts (HR assay, foci, ssDNA), single rigorous study with multiple orthogonal methods","pmids":["38084915"],"is_preprint":false},{"year":2023,"finding":"TRIM69 E3 ligase promotes EYA4 polyubiquitylation and proteasomal turnover. ERK2 directly binds EYA4 at Leu512/514 (MAPK docking groove D-site) and phosphorylates EYA4 at Ser37, which is required for TRIM69-mediated EYA4 polyubiquitylation. Loss of EYA4 via this axis enables β-catenin/ID2 cascade activation promoting PDAC proliferation.","method":"Co-immunoprecipitation, GST pulldown, in vitro protein kinase assay, cycloheximide pulse-chase, cellular ubiquitination assay, chromatin immunoprecipitation, luciferase reporter, immunofluorescence","journal":"Journal of Cancer","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro kinase assay identifying phosphorylation site, multiple orthogonal methods (GST pulldown, co-IP, ubiquitination, CHX chase), single lab but rigorous","pmids":["36741265"],"is_preprint":false},{"year":2024,"finding":"EYA4 interacts with histone H2AX directly and facilitates DNA double-strand break repair via DDR pathway activation, reducing osteosarcoma sensitivity to doxorubicin.","method":"Co-immunoprecipitation (EYA4-H2AX interaction), EYA4 KD/OE, in vitro and in vivo doxorubicin sensitivity assays, γH2AX measurement","journal":"Biochemical pharmacology","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single co-IP reported for H2AX interaction, single lab, limited mechanistic detail in abstract","pmids":["38876260"],"is_preprint":false},{"year":2026,"finding":"EYA4 interacts with transcription factor SIX2 to promote transcription of p21 (CDKN1A), thereby accelerating cellular senescence. This transcriptional activation function is independent of EYA4 phosphatase activity (shown using phosphatase-deficient mutants). EYA4-SIX2-mediated p21 upregulation is p53-dependent.","method":"EYA4 KD, co-immunoprecipitation (EYA4-SIX2), reporter/expression assays for p21, phosphatase-deficient mutant, p53 KO epistasis, replicative and stress-induced senescence assays","journal":"Advanced biotechnology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP, phosphatase mutant, p53 epistasis, multiple readouts, single lab","pmids":["41991886"],"is_preprint":false},{"year":2025,"finding":"EYA4 and EYA1 promote PLK1 activation and cell survival in neuroblastoma and glioblastoma; combined chemical inhibition of EYA phosphatase and PLK1 kinase produces synergistic cell death. Mechanism involves decreased PLK1 activity and RAD51 foci formation, mitotic arrest. NuRD complex and SOX9 expression levels correlate with combination sensitivity.","method":"Chemical inhibition (EYA inhibitors + PLK1 inhibitors), cancer cell line viability assays, PLK1 activity measurement, RAD51 foci, mitotic arrest quantification, multi-omic correlation","journal":"bioRxiv","confidence":"Low","confidence_rationale":"Tier 3 / Weak — preprint, pharmacological inhibition only (no direct EYA4 substrate assay), single study","pmids":[],"is_preprint":true}],"current_model":"EYA4 is a dual-function transcriptional coactivator and protein phosphatase (with both serine/threonine and tyrosine phosphatase activities) that operates in a conserved PAX-SIX-EYA-DACH transcriptional network: it resides in the cytoplasm and is translocated to the nucleus by SIX family proteins (SIX1, SIX3), where it activates target gene transcription; its tyrosine phosphatase activity dephosphorylates PLK1 at pY445 to promote centrosome maturation and mitotic fidelity, and dephosphorylates RAD51 at pY315 to promote homologous recombination; its serine/threonine phosphatase activity suppresses NF-κB signaling (via IκBα dephosphorylation) and dephosphorylates β-catenin at Ser552 to suppress MYCBP transcription, and is also required for replication fork progression; in the heart, EYA4 regulates the p27kip1/CK2α/HDAC2 cascade to maintain normal cardiac function, and in the cochlea it regulates Na+/K+-ATPase expression required for sensory hair cell development; haploinsufficiency of EYA4 due to truncating mutations is the primary mechanism underlying DFNA10 sensorineural hearing loss, while more N-terminal truncations additionally disrupt SIX protein binding and cause dilated cardiomyopathy."},"narrative":{"mechanistic_narrative":"EYA4 is a dual-function protein that acts both as a SIX-dependent transcriptional coactivator and as a phosphatase, operating in developmental gene-regulatory networks and in genome-maintenance pathways [PMID:15492887, PMID:38360978]. It is built around a conserved C-terminal Eya homologous region that mediates protein-protein interactions [PMID:9887327]; through this domain EYA4 binds SIX family transcription factors (SIX1, SIX3), which recruit cytoplasmic EYA4 to the nucleus where the complex activates target gene transcription [PMID:15492887, PMID:19606496]. As a tyrosine phosphatase, EYA4 dephosphorylates PLK1 at pY445 during G2 to drive centrosome maturation and PLK1 activation, and dephosphorylates RAD51 at pY315 to promote presynaptic filament formation and homologous recombination, with its phosphatase activity stimulated by DNA binding [PMID:38360978, PMID:38084915]. Its serine/threonine phosphatase activity supports replication fork progression and prevents replication stress [PMID:37777742], suppresses NF-κB signaling by blocking IκBα phosphorylation and p65 nuclear translocation [PMID:29764501], and dephosphorylates β-catenin at Ser552 to restrain β-catenin/LEF1-driven MYCBP transcription [PMID:31385398]. In the heart EYA4 regulates a p27kip1/CK2α/HDAC2 cascade required for normal cardiac function [PMID:26499333], and in development it controls Na+/K+-ATPase (atp1b2b) expression needed for sensory hair cell and cardiac formation [PMID:18799547]. EYA4 protein levels are controlled post-transcriptionally and post-translationally, including ERK2-primed, TRIM69-mediated ubiquitylation and proteasomal turnover [PMID:36741265]. Haploinsufficiency from truncating EYA4 mutations causes DFNA10 postlingual progressive autosomal dominant sensorineural hearing loss, and more N-terminal truncations that additionally abolish SIX binding cause dilated cardiomyopathy [PMID:11159937, PMID:15735644].","teleology":[{"year":1999,"claim":"Defined EYA4 at the sequence level and identified a conserved C-terminal domain as the likely interaction module, establishing the structural basis for later functional studies.","evidence":"cDNA cloning and domain characterization of human and mouse genes","pmids":["9887327"],"confidence":"Medium","gaps":["No interaction partner or biochemical activity demonstrated","Function inferred only from homology"]},{"year":2001,"claim":"Established EYA4 as a disease gene by linking truncating mutations to DFNA10 hearing loss, implicating haploinsufficiency as the mechanism.","evidence":"Candidate gene sequencing and co-segregation in two unrelated families","pmids":["11159937"],"confidence":"High","gaps":["Molecular function of EYA4 in the cochlea not addressed","Did not test protein-level consequences of mutations"]},{"year":2004,"claim":"Connected EYA4 to the SIX-EYA network by showing its conserved domain binds SIX1 and is shuttled to the nucleus by SIX1, providing a mechanism for nuclear function.","evidence":"Yeast two-hybrid, immunofluorescence localization, dual Eya constructs","pmids":["15492887"],"confidence":"Medium","gaps":["Direct transcriptional targets not identified","Did not address phosphatase activity"]},{"year":2005,"claim":"Showed that SIX-binding-disrupting truncations cause dilated cardiomyopathy in addition to hearing loss, mechanistically separating cardiac from cochlear phenotypes by interaction competence.","evidence":"Zebrafish morpholino knockdown and truncated-peptide binding assays","pmids":["15735644"],"confidence":"High","gaps":["Cardiac transcriptional targets not defined","How SIX binding maintains cardiac function unresolved"]},{"year":2008,"claim":"Placed Na+/K+-ATPase subunit atp1b2b downstream of EYA4, identifying an effector for its developmental role in hair cell and cardiac formation.","evidence":"Zebrafish morpholino knockdown with rescue/epistasis and phenotypic readouts","pmids":["18799547"],"confidence":"High","gaps":["Whether EYA4 directly regulates atp1b2b transcription not shown","Mammalian relevance not tested"]},{"year":2009,"claim":"Extended the SIX-EYA module to SIX3, demonstrating EYA4 acts as a transcriptional coactivator recruited to the nucleus by a second SIX partner.","evidence":"Co-immunoprecipitation, confocal localization, luciferase reporter","pmids":["19606496"],"confidence":"Medium","gaps":["No endogenous target genes identified","Forebrain role inferred, not directly tested"]},{"year":2015,"claim":"Defined a cardiac transcriptional/signaling mechanism in which EYA4 binds the p27 promoter and acts through CK2α/HDAC2, explaining how dosage changes drive hypertrophy versus dilated cardiomyopathy.","evidence":"Cardiac-specific transgenic mice, ChIP, luciferase, hemodynamic/MRI analysis","pmids":["26499333"],"confidence":"High","gaps":["Whether p27 suppression depends on phosphatase or coactivator activity not separated","Direct CK2α/HDAC2 substrate relationship not biochemically dissected"]},{"year":2016,"claim":"Identified post-transcriptional control of EYA4 by miR-431, showing reduced EYA4 protein causes spiral ganglion loss and hearing loss.","evidence":"Luciferase reporter, western blot, transgenic mice with ABR and SGN counts","pmids":["27545760"],"confidence":"Medium","gaps":["Downstream cochlear effectors of EYA4 not defined","Single regulatory miRNA studied"]},{"year":2018,"claim":"Established EYA4 as a serine/threonine phosphatase that suppresses NF-κB by inhibiting IκBα phosphorylation/ubiquitination, defining a tumor-suppressive signaling role.","evidence":"Stable transfection, xenografts, ChIP, ubiquitination assay, NF-κB reporter, calyculin A inhibition","pmids":["29764501"],"confidence":"High","gaps":["Whether EYA4 directly dephosphorylates IκBα not shown","Specific phospho-site not identified"]},{"year":2018,"claim":"Linked EYA4 to EMT suppression via Akt/GSK-3β/slug and identified TGF-β1/DNMT3A methylation as a route to EYA4 silencing in ESCC.","evidence":"shRNA/overexpression, migration/invasion assays, western blotting, demethylating agents","pmids":["29660222"],"confidence":"Medium","gaps":["Direct phosphatase substrate in the Akt/GSK-3β axis not defined","Mechanism of pathway regulation correlative"]},{"year":2019,"claim":"Identified β-catenin Ser552 as a serine/threonine phosphatase substrate, connecting EYA4 to Wnt/MYCBP-driven proliferation control.","evidence":"Overexpression/KO in HCC lines, siRNA rescue, phospho-β-catenin blotting, nuclear fractionation","pmids":["31385398"],"confidence":"Medium","gaps":["Direct dephosphorylation not shown with purified components","Single cancer context"]},{"year":2021,"claim":"Reported that EYA4 suppresses p27Kip1 in glioma in a SIX1-dependent manner, extending the coactivator-cell cycle link to another tissue.","evidence":"Overexpression, viability/BrdU, flow cytometry, gene expression","pmids":["30231237"],"confidence":"Low","gaps":["No ChIP or direct promoter assay reported for direct p27 suppression claim","Single lab, limited mechanistic depth"]},{"year":2023,"claim":"Demonstrated a genome-maintenance role: EYA4 serine/threonine phosphatase activity supports replication fork progression and prevents replication stress.","evidence":"KD/OE in breast cancer cells/xenografts, γH2AX, ATR markers, HU sensitivity, DNA fiber assay, phosphatase mutant","pmids":["37777742"],"confidence":"Medium","gaps":["Relevant fork-associated phosphatase substrate not identified","Single lab"]},{"year":2023,"claim":"Defined post-translational control of EYA4 stability through an ERK2-priming/TRIM69 ubiquitylation axis, linking MAPK signaling to EYA4 turnover and β-catenin/ID2 activation in PDAC.","evidence":"Co-IP, GST pulldown, in vitro kinase assay (Ser37), CHX chase, ubiquitination assay, ChIP, reporter","pmids":["36741265"],"confidence":"High","gaps":["Physiological contexts of TRIM69 regulation beyond PDAC not tested","Whether turnover affects phosphatase versus coactivator pools unknown"]},{"year":2024,"claim":"Identified PLK1 pY445 as a tyrosine phosphatase substrate, establishing EYA4 as a regulator of centrosome maturation and PLK1 activation in G2.","evidence":"Co-IP, phosphatase substrate assay, molecular dynamics, depletion/inhibition, centrosome and mitosis imaging","pmids":["38360978"],"confidence":"High","gaps":["Relative contributions of EYA4 versus EYA1 not fully separated","Upstream signals controlling this dephosphorylation unclear"]},{"year":2024,"claim":"Identified RAD51 pY315 as a tyrosine phosphatase substrate and showed DNA binding stimulates EYA4 activity, establishing a direct role in homologous recombination repair.","evidence":"In vitro phosphatase assay, HR reporter, RAD51 foci, DNA fiber, ssDNA measurement, KD/OE","pmids":["38084915"],"confidence":"High","gaps":["How DNA recruits/stimulates EYA4 in cells not defined","Coordination with PLK1/replication functions unresolved"]},{"year":2024,"claim":"Reported a direct EYA4-H2AX interaction promoting DSB repair and chemoresistance in osteosarcoma.","evidence":"Co-IP, KD/OE, doxorubicin sensitivity assays, γH2AX measurement","pmids":["38876260"],"confidence":"Low","gaps":["Single co-IP without reciprocal/structural validation","Limited mechanistic detail"]},{"year":2026,"claim":"Showed a phosphatase-independent coactivator function: EYA4-SIX2 activates p21 transcription in a p53-dependent manner to drive senescence, dissociating transcriptional and enzymatic activities.","evidence":"KD, co-IP (EYA4-SIX2), p21 reporter/expression, phosphatase-deficient mutant, p53 KO epistasis, senescence assays","pmids":["41991886"],"confidence":"Medium","gaps":["Direct binding of EYA4-SIX2 to the p21 promoter not established","How phosphatase versus coactivator modes are partitioned in cells unclear"]},{"year":null,"claim":"How EYA4's distinct activities (tyrosine versus serine/threonine phosphatase, SIX-dependent coactivation) are coordinated, partitioned between cytoplasm and nucleus, and selectively deployed across developmental versus genome-maintenance contexts remains unresolved.","evidence":"","pmids":[],"confidence":"Low","gaps":["No structural model integrating phosphatase and coactivator functions","Substrate selectivity rules unknown","Mechanism switching EYA4 between developmental and DNA-repair roles undefined"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[8,10,13,14,15]},{"term_id":"GO:0016787","term_label":"hydrolase activity","supporting_discovery_ids":[14,15]},{"term_id":"GO:0140110","term_label":"transcription regulator activity","supporting_discovery_ids":[5,6,18]},{"term_id":"GO:0003677","term_label":"DNA binding","supporting_discovery_ids":[6,15]},{"term_id":"GO:0060089","term_label":"molecular transducer activity","supporting_discovery_ids":[2,5]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[2]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[2,5]},{"term_id":"GO:0005815","term_label":"microtubule organizing center","supporting_discovery_ids":[14]}],"pathway":[{"term_id":"R-HSA-73894","term_label":"DNA Repair","supporting_discovery_ids":[15]},{"term_id":"R-HSA-69306","term_label":"DNA Replication","supporting_discovery_ids":[13]},{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[14]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[8,10]},{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[3,4]},{"term_id":"R-HSA-74160","term_label":"Gene expression (Transcription)","supporting_discovery_ids":[5,6]}],"complexes":[],"partners":["SIX1","SIX3","SIX2","PLK1","RAD51","TRIM69","H2AFX"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"O95677","full_name":"Protein phosphatase EYA4","aliases":["Eyes absent homolog 4"],"length_aa":639,"mass_kda":69.5,"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. Its function as histone phosphatase probably explains its role in transcription regulation during organogenesis. May be involved in development of the eye (By similarity)","subcellular_location":"Cytoplasm; Nucleus","url":"https://www.uniprot.org/uniprotkb/O95677/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/EYA4","classification":"Not Classified","n_dependent_lines":1,"n_total_lines":1208,"dependency_fraction":0.0008278145695364238},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/EYA4","total_profiled":1310},"omim":[{"mim_id":"620479","title":"CENTRIOLAR SATELLITE-ASSOCIATED TUBULIN POLYGLUTAMYLASE COMPLEX REGULATOR 1; CSTPP1","url":"https://www.omim.org/entry/620479"},{"mim_id":"620475","title":"THROMBOCYTOPENIA 8, WITH DYSMORPHIC FEATURES AND DEVELOPMENTAL DELAY; THC8","url":"https://www.omim.org/entry/620475"},{"mim_id":"620283","title":"DEAFNESS, AUTOSOMAL DOMINANT 88; DFNA88","url":"https://www.omim.org/entry/620283"},{"mim_id":"608389","title":"BRANCHIOOTIC SYNDROME 3; 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characterization\",\n      \"journal\": \"Human molecular genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — domain characterization by sequence homology to Drosophila eya, functional inference from conserved domain, single study\",\n      \"pmids\": [\"9887327\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"EYA4 is the causative gene for postlingual progressive autosomal dominant hearing loss at the DFNA10 locus; mutations creating premature stop codons in two unrelated families established haploinsufficiency as the disease mechanism.\",\n      \"method\": \"Candidate gene sequencing, mutation identification, co-segregation analysis in two families\",\n      \"journal\": \"Human molecular genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — replicated in two independent families with different mutations, co-segregation confirmed\",\n      \"pmids\": [\"11159937\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"EYA4 Eya homologous region (Eya4HR) interacts with SIX1 (demonstrated by yeast two-hybrid), but not with DACH1. Eya4HR localizes to the cytoplasm and is translocated to the nucleus upon co-expression with SIX1. Mutant truncating alleles are not translated, consistent with haploinsufficiency as the mechanism for DFNA10.\",\n      \"method\": \"Yeast two-hybrid, alpha-galactosidase activity assay, immunofluorescence, dual Eya-containing constructs\",\n      \"journal\": \"Journal of the Association for Research in Otolaryngology : JARO\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal methods (Y2H, immunofluorescence, functional assay), single lab\",\n      \"pmids\": [\"15492887\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"EYA4 deletion causes dilated cardiomyopathy in addition to sensorineural hearing loss; eya4 morpholino knockdown in zebrafish produced heart failure. EYA4 peptides associated with SNHL-only mutations bound wild-type EYA4 and SIX proteins, whereas the cardiomyopathy-associated short peptide (193 aa) did not, indicating that SIX protein interaction is required for cardiac function.\",\n      \"method\": \"Zebrafish morpholino knockdown, biochemical interaction assays (co-immunoprecipitation), truncated peptide binding studies\",\n      \"journal\": \"Nature genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (morpholino in vivo, biochemical interaction assays), mechanistic link established between SIX binding and cardiac phenotype\",\n      \"pmids\": [\"15735644\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"EYA4 regulates Na+/K+-ATPase (specifically the atp1b2b subunit) in zebrafish; eya4 morphant fish had reduced atp1b2b levels, reduced hair cells, and heart failure. Morpholino knockdown of atp1b2b recapitulated eya4 deficiency phenotypes, and atp1b2b overexpression rescued eya4 morphant phenotypes, placing atp1b2b downstream of eya4 in sensory and cardiac development.\",\n      \"method\": \"Zebrafish morpholino knockdown, genetic epistasis (rescue by overexpression), hair cell counts, sensory response measurement\",\n      \"journal\": \"Development (Cambridge, England)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — epistasis via morpholino knockdown and rescue overexpression, multiple phenotypic readouts, single lab but multiple orthogonal methods\",\n      \"pmids\": [\"18799547\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"EYA4 physically interacts with SIX3 (co-immunoprecipitation) and is recruited to the nucleus by SIX3. EYA4 cooperates functionally with SIX3 as a transcriptional coactivator (reporter gene assay), suggesting a role in forebrain development.\",\n      \"method\": \"Co-immunoprecipitation, confocal microscopy localization, luciferase reporter assay\",\n      \"journal\": \"Human mutation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal co-IP and reporter assay, single lab, two orthogonal methods\",\n      \"pmids\": [\"19606496\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"EYA4 overexpression in cardiac tissue induces hypertrophy via regulation of p27kip1 expression; chromatin immunoprecipitation and luciferase assays confirmed that EYA4 binds the p27 promoter and suppresses p27 expression. The downstream signaling involves casein kinase-2α and histone deacetylase 2 (HDAC2) phosphorylation. The truncating mutation E193 acts in an opposing manner, promoting dilated cardiomyopathy.\",\n      \"method\": \"Transgenic mice (cardiac-specific overexpression), luciferase reporter assay, chromatin immunoprecipitation, MRI/hemodynamic analysis, in vitro expression studies\",\n      \"journal\": \"Circulation. Cardiovascular genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — ChIP, luciferase, in vivo transgenic models, multiple orthogonal methods in single study\",\n      \"pmids\": [\"26499333\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"miR-431 directly targets Eya4 mRNA; luciferase reporter assay and western blotting confirmed that miR-431 inhibits Eya4 translation. Overexpression of miR-431 in transgenic mice led to reduced EYA4 protein in cochleae, decreased spiral ganglion neuron density, and hearing loss.\",\n      \"method\": \"Luciferase reporter assay, western blotting, transgenic mice, ABR measurement, SGN counting\",\n      \"journal\": \"Biochimica et biophysica acta\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — luciferase and western blot validate direct targeting, in vivo transgenic model, single lab\",\n      \"pmids\": [\"27545760\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"EYA4 suppresses HCC cell growth and invasion by antagonizing NF-κB activity: EYA4 serine/threonine phosphatase activity inhibits phosphorylation and ubiquitination of IκBα, blocks nuclear translocation and transactivation of p65, thereby suppressing RAP1 transcription driven by NF-κB. Calyculin A (phosphatase inhibitor) abolished EYA4's suppression of NF-κB, confirming phosphatase-dependence.\",\n      \"method\": \"Stable transfection, xenograft, chromatin immunoprecipitation, immunofluorescence, cellular ubiquitination assay, NF-κB reporter, pharmacological inhibition\",\n      \"journal\": \"Cancer communications (London, England)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — multiple orthogonal methods (ChIP, ubiquitination assay, phosphatase inhibitor, immunofluorescence, in vivo xenograft), single lab but rigorous\",\n      \"pmids\": [\"29764501\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"EYA4 knockdown in ESCC cells increases migration and invasion; EYA4 overexpression promotes epithelial phenotype and reduces TGF-β1-induced EMT by reducing phosphorylation of Akt and GSK-3β, leading to inactivation of slug. TGF-β1 decreases EYA4 expression through DNMT3A-mediated DNA methylation.\",\n      \"method\": \"shRNA knockdown, overexpression, migration/invasion assays, western blotting for pAkt/pGSK3β/slug, treatment with 5-aza-dC/TSA\",\n      \"journal\": \"Cancer science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — loss-of-function and gain-of-function with defined pathway readouts, single lab\",\n      \"pmids\": [\"29660222\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"EYA4 dephosphorylates β-catenin at Ser552 (serine/threonine phosphatase activity), reducing nuclear translocation of β-catenin and thereby suppressing β-catenin/LEF1-driven transcription of MYCBP. EYA4 KO increased MYCBP; siRNA of MYCBP in EYA4-overexpressing cells rescued proliferation and G2/M arrest phenotypes.\",\n      \"method\": \"EYA4 overexpression/KO in HCC cell lines, siRNA rescue, western blotting for phospho-β-catenin, qRT-PCR, nuclear fractionation\",\n      \"journal\": \"Cancer science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — phosphorylation site identified, epistasis by siRNA rescue, single lab, multiple readouts\",\n      \"pmids\": [\"31385398\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"EYA4 truncating mutations in the EYA-HR domain result in absence of protein expression (confirmed in mammalian COS7 cells), consistent with haploinsufficiency. Missense mutant p.Glu369Asp shows normal expression and nuclear translocation in presence of SIX1. Minigene assays confirmed that synonymous and splice-site variants cause exon skipping leading to frameshifts.\",\n      \"method\": \"Transient expression in COS7 cells, confocal microscopy, minigene splicing assays in NIH3T3 cells, in silico splicing analysis\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — cell expression studies and functional minigene assays, single lab, multiple variant analyses\",\n      \"pmids\": [\"32277154\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"EYA4 promotes glioma cell proliferation by directly suppressing expression of p27Kip1, and SIX1 is required for EYA4 to suppress p27Kip1.\",\n      \"method\": \"EYA4 overexpression, cell viability/BrdU assay, flow cytometry, gene expression analysis\",\n      \"journal\": \"Cellular physiology and biochemistry\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, limited mechanistic depth for direct p27 suppression claim, no ChIP or direct promoter assay described in abstract\",\n      \"pmids\": [\"30231237\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"EYA4 serine/threonine phosphatase domain prevents replication-associated DNA damage and is required for replication fork progression. EYA4 depletion causes spontaneous replication stress (ATR pathway activation, γH2AX accumulation, sensitivity to hydroxyurea, endoreplication/polyploidy). EYA4 overexpression promotes breast cancer cell proliferation, migration, and metastasis.\",\n      \"method\": \"EYA4 KD/OE in breast cancer cells and xenografts, γH2AX staining, ATR pathway markers, hydroxyurea sensitivity, DNA fiber assay (replication fork), phosphatase domain mutant\",\n      \"journal\": \"Molecular cancer\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal methods including phosphatase-domain mutant, in vitro and in vivo, single lab\",\n      \"pmids\": [\"37777742\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"EYA4 (and EYA1) are tyrosine phosphatases that dephosphorylate PLK1 at pY445 during G2 phase of the cell cycle. This dephosphorylation is required for centrosome maturation, PLK1 localization to centrosomes, and polo-box domain (PBD)-dependent interactions between PLK1 and PLK1-activation complexes. Depletion or chemical inhibition of EYA phosphatase activity reduces PLK1 activation, causing mitotic defects and cell death.\",\n      \"method\": \"Co-immunoprecipitation, phosphatase substrate assay, molecular dynamics simulation, EYA4/EYA1 depletion, chemical inhibition, immunofluorescence of centrosome maturation, mitosis assays\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro phosphatase assay identifying specific substrate site (pY445), co-IP, molecular dynamics, cell-based loss-of-function with multiple orthogonal readouts\",\n      \"pmids\": [\"38360978\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"EYA4 tyrosine phosphatase activity dephosphorylates RAD51 at Tyr315, which regulates RAD51 localization, presynaptic filament formation, foci formation, and homologous recombination (HR) at DNA double-strand breaks. DNA binding stimulates EYA4 phosphatase activity. EYA4 depletion decreases ssDNA accumulation after DNA damage and impairs HR; overexpression promotes RAD51 dephosphorylation and nucleoprotein filament formation.\",\n      \"method\": \"In vitro phosphatase assay, HR reporter assay, RAD51 foci formation, DNA fiber assay, ssDNA accumulation measurement, EYA4 KD/OE\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro phosphatase assay with specific substrate site identification, multiple functional readouts (HR assay, foci, ssDNA), single rigorous study with multiple orthogonal methods\",\n      \"pmids\": [\"38084915\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"TRIM69 E3 ligase promotes EYA4 polyubiquitylation and proteasomal turnover. ERK2 directly binds EYA4 at Leu512/514 (MAPK docking groove D-site) and phosphorylates EYA4 at Ser37, which is required for TRIM69-mediated EYA4 polyubiquitylation. Loss of EYA4 via this axis enables β-catenin/ID2 cascade activation promoting PDAC proliferation.\",\n      \"method\": \"Co-immunoprecipitation, GST pulldown, in vitro protein kinase assay, cycloheximide pulse-chase, cellular ubiquitination assay, chromatin immunoprecipitation, luciferase reporter, immunofluorescence\",\n      \"journal\": \"Journal of Cancer\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro kinase assay identifying phosphorylation site, multiple orthogonal methods (GST pulldown, co-IP, ubiquitination, CHX chase), single lab but rigorous\",\n      \"pmids\": [\"36741265\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"EYA4 interacts with histone H2AX directly and facilitates DNA double-strand break repair via DDR pathway activation, reducing osteosarcoma sensitivity to doxorubicin.\",\n      \"method\": \"Co-immunoprecipitation (EYA4-H2AX interaction), EYA4 KD/OE, in vitro and in vivo doxorubicin sensitivity assays, γH2AX measurement\",\n      \"journal\": \"Biochemical pharmacology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single co-IP reported for H2AX interaction, single lab, limited mechanistic detail in abstract\",\n      \"pmids\": [\"38876260\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"EYA4 interacts with transcription factor SIX2 to promote transcription of p21 (CDKN1A), thereby accelerating cellular senescence. This transcriptional activation function is independent of EYA4 phosphatase activity (shown using phosphatase-deficient mutants). EYA4-SIX2-mediated p21 upregulation is p53-dependent.\",\n      \"method\": \"EYA4 KD, co-immunoprecipitation (EYA4-SIX2), reporter/expression assays for p21, phosphatase-deficient mutant, p53 KO epistasis, replicative and stress-induced senescence assays\",\n      \"journal\": \"Advanced biotechnology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP, phosphatase mutant, p53 epistasis, multiple readouts, single lab\",\n      \"pmids\": [\"41991886\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"EYA4 and EYA1 promote PLK1 activation and cell survival in neuroblastoma and glioblastoma; combined chemical inhibition of EYA phosphatase and PLK1 kinase produces synergistic cell death. Mechanism involves decreased PLK1 activity and RAD51 foci formation, mitotic arrest. NuRD complex and SOX9 expression levels correlate with combination sensitivity.\",\n      \"method\": \"Chemical inhibition (EYA inhibitors + PLK1 inhibitors), cancer cell line viability assays, PLK1 activity measurement, RAD51 foci, mitotic arrest quantification, multi-omic correlation\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — preprint, pharmacological inhibition only (no direct EYA4 substrate assay), single study\",\n      \"pmids\": [],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"EYA4 is a dual-function transcriptional coactivator and protein phosphatase (with both serine/threonine and tyrosine phosphatase activities) that operates in a conserved PAX-SIX-EYA-DACH transcriptional network: it resides in the cytoplasm and is translocated to the nucleus by SIX family proteins (SIX1, SIX3), where it activates target gene transcription; its tyrosine phosphatase activity dephosphorylates PLK1 at pY445 to promote centrosome maturation and mitotic fidelity, and dephosphorylates RAD51 at pY315 to promote homologous recombination; its serine/threonine phosphatase activity suppresses NF-κB signaling (via IκBα dephosphorylation) and dephosphorylates β-catenin at Ser552 to suppress MYCBP transcription, and is also required for replication fork progression; in the heart, EYA4 regulates the p27kip1/CK2α/HDAC2 cascade to maintain normal cardiac function, and in the cochlea it regulates Na+/K+-ATPase expression required for sensory hair cell development; haploinsufficiency of EYA4 due to truncating mutations is the primary mechanism underlying DFNA10 sensorineural hearing loss, while more N-terminal truncations additionally disrupt SIX protein binding and cause dilated cardiomyopathy.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"EYA4 is a dual-function protein that acts both as a SIX-dependent transcriptional coactivator and as a phosphatase, operating in developmental gene-regulatory networks and in genome-maintenance pathways [#2, #14]. It is built around a conserved C-terminal Eya homologous region that mediates protein-protein interactions [#0]; through this domain EYA4 binds SIX family transcription factors (SIX1, SIX3), which recruit cytoplasmic EYA4 to the nucleus where the complex activates target gene transcription [#2, #5]. As a tyrosine phosphatase, EYA4 dephosphorylates PLK1 at pY445 during G2 to drive centrosome maturation and PLK1 activation, and dephosphorylates RAD51 at pY315 to promote presynaptic filament formation and homologous recombination, with its phosphatase activity stimulated by DNA binding [#14, #15]. Its serine/threonine phosphatase activity supports replication fork progression and prevents replication stress [#13], suppresses NF-\\u03baB signaling by blocking I\\u03baB\\u03b1 phosphorylation and p65 nuclear translocation [#8], and dephosphorylates \\u03b2-catenin at Ser552 to restrain \\u03b2-catenin/LEF1-driven MYCBP transcription [#10]. In the heart EYA4 regulates a p27kip1/CK2\\u03b1/HDAC2 cascade required for normal cardiac function [#6], and in development it controls Na+/K+-ATPase (atp1b2b) expression needed for sensory hair cell and cardiac formation [#4]. EYA4 protein levels are controlled post-transcriptionally and post-translationally, including ERK2-primed, TRIM69-mediated ubiquitylation and proteasomal turnover [#16]. Haploinsufficiency from truncating EYA4 mutations causes DFNA10 postlingual progressive autosomal dominant sensorineural hearing loss, and more N-terminal truncations that additionally abolish SIX binding cause dilated cardiomyopathy [#1, #3].\",\n  \"teleology\": [\n    {\n      \"year\": 1999,\n      \"claim\": \"Defined EYA4 at the sequence level and identified a conserved C-terminal domain as the likely interaction module, establishing the structural basis for later functional studies.\",\n      \"evidence\": \"cDNA cloning and domain characterization of human and mouse genes\",\n      \"pmids\": [\"9887327\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No interaction partner or biochemical activity demonstrated\", \"Function inferred only from homology\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Established EYA4 as a disease gene by linking truncating mutations to DFNA10 hearing loss, implicating haploinsufficiency as the mechanism.\",\n      \"evidence\": \"Candidate gene sequencing and co-segregation in two unrelated families\",\n      \"pmids\": [\"11159937\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular function of EYA4 in the cochlea not addressed\", \"Did not test protein-level consequences of mutations\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Connected EYA4 to the SIX-EYA network by showing its conserved domain binds SIX1 and is shuttled to the nucleus by SIX1, providing a mechanism for nuclear function.\",\n      \"evidence\": \"Yeast two-hybrid, immunofluorescence localization, dual Eya constructs\",\n      \"pmids\": [\"15492887\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct transcriptional targets not identified\", \"Did not address phosphatase activity\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Showed that SIX-binding-disrupting truncations cause dilated cardiomyopathy in addition to hearing loss, mechanistically separating cardiac from cochlear phenotypes by interaction competence.\",\n      \"evidence\": \"Zebrafish morpholino knockdown and truncated-peptide binding assays\",\n      \"pmids\": [\"15735644\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Cardiac transcriptional targets not defined\", \"How SIX binding maintains cardiac function unresolved\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Placed Na+/K+-ATPase subunit atp1b2b downstream of EYA4, identifying an effector for its developmental role in hair cell and cardiac formation.\",\n      \"evidence\": \"Zebrafish morpholino knockdown with rescue/epistasis and phenotypic readouts\",\n      \"pmids\": [\"18799547\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether EYA4 directly regulates atp1b2b transcription not shown\", \"Mammalian relevance not tested\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Extended the SIX-EYA module to SIX3, demonstrating EYA4 acts as a transcriptional coactivator recruited to the nucleus by a second SIX partner.\",\n      \"evidence\": \"Co-immunoprecipitation, confocal localization, luciferase reporter\",\n      \"pmids\": [\"19606496\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No endogenous target genes identified\", \"Forebrain role inferred, not directly tested\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Defined a cardiac transcriptional/signaling mechanism in which EYA4 binds the p27 promoter and acts through CK2\\u03b1/HDAC2, explaining how dosage changes drive hypertrophy versus dilated cardiomyopathy.\",\n      \"evidence\": \"Cardiac-specific transgenic mice, ChIP, luciferase, hemodynamic/MRI analysis\",\n      \"pmids\": [\"26499333\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether p27 suppression depends on phosphatase or coactivator activity not separated\", \"Direct CK2\\u03b1/HDAC2 substrate relationship not biochemically dissected\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Identified post-transcriptional control of EYA4 by miR-431, showing reduced EYA4 protein causes spiral ganglion loss and hearing loss.\",\n      \"evidence\": \"Luciferase reporter, western blot, transgenic mice with ABR and SGN counts\",\n      \"pmids\": [\"27545760\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Downstream cochlear effectors of EYA4 not defined\", \"Single regulatory miRNA studied\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Established EYA4 as a serine/threonine phosphatase that suppresses NF-\\u03baB by inhibiting I\\u03baB\\u03b1 phosphorylation/ubiquitination, defining a tumor-suppressive signaling role.\",\n      \"evidence\": \"Stable transfection, xenografts, ChIP, ubiquitination assay, NF-\\u03baB reporter, calyculin A inhibition\",\n      \"pmids\": [\"29764501\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether EYA4 directly dephosphorylates I\\u03baB\\u03b1 not shown\", \"Specific phospho-site not identified\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Linked EYA4 to EMT suppression via Akt/GSK-3\\u03b2/slug and identified TGF-\\u03b21/DNMT3A methylation as a route to EYA4 silencing in ESCC.\",\n      \"evidence\": \"shRNA/overexpression, migration/invasion assays, western blotting, demethylating agents\",\n      \"pmids\": [\"29660222\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct phosphatase substrate in the Akt/GSK-3\\u03b2 axis not defined\", \"Mechanism of pathway regulation correlative\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Identified \\u03b2-catenin Ser552 as a serine/threonine phosphatase substrate, connecting EYA4 to Wnt/MYCBP-driven proliferation control.\",\n      \"evidence\": \"Overexpression/KO in HCC lines, siRNA rescue, phospho-\\u03b2-catenin blotting, nuclear fractionation\",\n      \"pmids\": [\"31385398\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct dephosphorylation not shown with purified components\", \"Single cancer context\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Reported that EYA4 suppresses p27Kip1 in glioma in a SIX1-dependent manner, extending the coactivator-cell cycle link to another tissue.\",\n      \"evidence\": \"Overexpression, viability/BrdU, flow cytometry, gene expression\",\n      \"pmids\": [\"30231237\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No ChIP or direct promoter assay reported for direct p27 suppression claim\", \"Single lab, limited mechanistic depth\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Demonstrated a genome-maintenance role: EYA4 serine/threonine phosphatase activity supports replication fork progression and prevents replication stress.\",\n      \"evidence\": \"KD/OE in breast cancer cells/xenografts, \\u03b3H2AX, ATR markers, HU sensitivity, DNA fiber assay, phosphatase mutant\",\n      \"pmids\": [\"37777742\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Relevant fork-associated phosphatase substrate not identified\", \"Single lab\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Defined post-translational control of EYA4 stability through an ERK2-priming/TRIM69 ubiquitylation axis, linking MAPK signaling to EYA4 turnover and \\u03b2-catenin/ID2 activation in PDAC.\",\n      \"evidence\": \"Co-IP, GST pulldown, in vitro kinase assay (Ser37), CHX chase, ubiquitination assay, ChIP, reporter\",\n      \"pmids\": [\"36741265\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Physiological contexts of TRIM69 regulation beyond PDAC not tested\", \"Whether turnover affects phosphatase versus coactivator pools unknown\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Identified PLK1 pY445 as a tyrosine phosphatase substrate, establishing EYA4 as a regulator of centrosome maturation and PLK1 activation in G2.\",\n      \"evidence\": \"Co-IP, phosphatase substrate assay, molecular dynamics, depletion/inhibition, centrosome and mitosis imaging\",\n      \"pmids\": [\"38360978\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Relative contributions of EYA4 versus EYA1 not fully separated\", \"Upstream signals controlling this dephosphorylation unclear\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Identified RAD51 pY315 as a tyrosine phosphatase substrate and showed DNA binding stimulates EYA4 activity, establishing a direct role in homologous recombination repair.\",\n      \"evidence\": \"In vitro phosphatase assay, HR reporter, RAD51 foci, DNA fiber, ssDNA measurement, KD/OE\",\n      \"pmids\": [\"38084915\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How DNA recruits/stimulates EYA4 in cells not defined\", \"Coordination with PLK1/replication functions unresolved\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Reported a direct EYA4-H2AX interaction promoting DSB repair and chemoresistance in osteosarcoma.\",\n      \"evidence\": \"Co-IP, KD/OE, doxorubicin sensitivity assays, \\u03b3H2AX measurement\",\n      \"pmids\": [\"38876260\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Single co-IP without reciprocal/structural validation\", \"Limited mechanistic detail\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Showed a phosphatase-independent coactivator function: EYA4-SIX2 activates p21 transcription in a p53-dependent manner to drive senescence, dissociating transcriptional and enzymatic activities.\",\n      \"evidence\": \"KD, co-IP (EYA4-SIX2), p21 reporter/expression, phosphatase-deficient mutant, p53 KO epistasis, senescence assays\",\n      \"pmids\": [\"41991886\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct binding of EYA4-SIX2 to the p21 promoter not established\", \"How phosphatase versus coactivator modes are partitioned in cells unclear\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How EYA4's distinct activities (tyrosine versus serine/threonine phosphatase, SIX-dependent coactivation) are coordinated, partitioned between cytoplasm and nucleus, and selectively deployed across developmental versus genome-maintenance contexts remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No structural model integrating phosphatase and coactivator functions\", \"Substrate selectivity rules unknown\", \"Mechanism switching EYA4 between developmental and DNA-repair roles undefined\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [8, 10, 13, 14, 15]},\n      {\"term_id\": \"GO:0016787\", \"supporting_discovery_ids\": [14, 15]},\n      {\"term_id\": \"GO:0140110\", \"supporting_discovery_ids\": [5, 6, 18]},\n      {\"term_id\": \"GO:0003677\", \"supporting_discovery_ids\": [6, 15]},\n      {\"term_id\": \"GO:0060089\", \"supporting_discovery_ids\": [2, 5]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [2]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [2, 5]},\n      {\"term_id\": \"GO:0005815\", \"supporting_discovery_ids\": [14]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-73894\", \"supporting_discovery_ids\": [15]},\n      {\"term_id\": \"R-HSA-69306\", \"supporting_discovery_ids\": [13]},\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [14]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [8, 10]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [3, 4]},\n      {\"term_id\": \"R-HSA-74160\", \"supporting_discovery_ids\": [5, 6]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"SIX1\", \"SIX3\", \"SIX2\", \"PLK1\", \"RAD51\", \"TRIM69\", \"H2AFX\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}