{"gene":"EYA4","run_date":"2026-04-28T17:46:03","timeline":{"discoveries":[{"year":1999,"finding":"EYA4 encodes a protein containing a highly conserved C-terminal Eya homologous domain (eya-HR) of 271 amino acids, which in Drosophila eya mediates developmentally important protein-protein interactions.","method":"Molecular cloning and sequence analysis of human and mouse EYA4","journal":"Human molecular genetics","confidence":"Medium","confidence_rationale":"Tier 3 — domain identification by sequence homology, foundational characterization paper","pmids":["9887327"],"is_preprint":false},{"year":2001,"finding":"EYA4 functions as a transcriptional activator and its haploinsufficiency (via premature stop codons) causes late-onset progressive sensorineural hearing loss (DFNA10), demonstrating a post-developmental role in maintenance of the organ of Corti.","method":"Mutation identification in two unrelated families; genetic linkage and sequencing","journal":"Human molecular genetics","confidence":"High","confidence_rationale":"Tier 2 — replicated across two unrelated families, loss-of-function with defined phenotype","pmids":["11159937"],"is_preprint":false},{"year":2004,"finding":"EYA4 Eya-homologous region (Eya4HR) interacts with SIX1 (but not DACH1) in yeast two-hybrid assays; Eya4HR localizes to the cytoplasm alone but is translocated to the nucleus upon co-expression with SIX1.","method":"Yeast two-hybrid system; immunofluorescence staining; coexpression in mammalian cells","journal":"Journal of the Association for Research in Otolaryngology : JARO","confidence":"Medium","confidence_rationale":"Tier 2 — direct protein interaction and localization with functional consequence demonstrated in single lab","pmids":["15492887"],"is_preprint":false},{"year":2005,"finding":"EYA4 peptides associated with sensorineural hearing loss (C-terminal Eya domain truncations) bind wild-type EYA4 and SIX proteins, whereas the shortened 193-amino acid peptide associated with dilated cardiomyopathy does not; zebrafish eya4 morphants display heart failure phenotype, and eya4 regulates cardiac function via EYA4-SIX-mediated transcriptional regulation.","method":"Biochemical interaction assays of mutant EYA4 peptides; antisense morpholino knockdown in zebrafish; hemodynamic analysis","journal":"Nature genetics","confidence":"High","confidence_rationale":"Tier 1-2 — multiple orthogonal methods (biochemical binding, in vivo morpholino, hemodynamics), replicated across model systems","pmids":["15735644"],"is_preprint":false},{"year":2008,"finding":"Eya4 regulates expression of Na+/K+-ATPase (atp1b2b beta2b subunit) in zebrafish; loss of eya4 reduces atp1b2b levels and causes hair cell loss and heart failure, and atp1b2b overexpression rescues these eya4 morphant phenotypes.","method":"Morpholino knockdown of eya4 and atp1b2b in zebrafish; rescue experiments with atp1b2b overexpression; sensory response assays","journal":"Development (Cambridge, England)","confidence":"High","confidence_rationale":"Tier 2 — epistasis established by rescue experiment, multiple phenotypic readouts in ortholog model","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 in reporter gene assays.","method":"Co-immunoprecipitation; confocal microscopy; luciferase reporter assays","journal":"Human mutation","confidence":"Medium","confidence_rationale":"Tier 2 — reciprocal Co-IP and functional reporter assay in single study","pmids":["19606496"],"is_preprint":false},{"year":2015,"finding":"EYA4 and its truncation mutant E193 regulate p27Kip1 expression by binding and regulating the p27 promoter (ChIP assay); cardiac-specific overexpression of Eya4 in mice causes age-dependent hypertrophy via p27/casein kinase-2α/HDAC2 signaling, whereas E193 overexpression causes dilated cardiomyopathy.","method":"Luciferase reporter assays; chromatin immunoprecipitation; transgenic mouse overexpression; MRI and hemodynamic analysis","journal":"Circulation. Cardiovascular genetics","confidence":"High","confidence_rationale":"Tier 1-2 — ChIP, reporter assay, and in vivo transgenic model with multiple readouts in single study","pmids":["26499333"],"is_preprint":false},{"year":2016,"finding":"miR-431 directly targets Eya4 mRNA in cochlear spiral ganglion neurons; overexpression of miR-431 in transgenic mice inhibits EYA4 translation, reduces spiral ganglion neuron density, and causes hearing loss.","method":"Luciferase reporter assay; western blotting; transgenic mouse model; ABR testing; primary SGN culture","journal":"Biochimica et biophysica acta","confidence":"Medium","confidence_rationale":"Tier 2 — direct target validation by luciferase assay and in vivo transgenic model","pmids":["27545760"],"is_preprint":false},{"year":2018,"finding":"EYA4 suppresses HCC proliferation and invasion by antagonizing TNF-α-induced NF-κB activation: EYA4's serine/threonine phosphatase activity blocks phosphorylation and ubiquitination of IκBα, inhibiting p65 nuclear translocation and RAP1A transactivation.","method":"Stable transfection; ChIP; immunofluorescence; ubiquitination assay; calyculin A (phosphatase inhibitor) treatment; xenograft","journal":"Cancer communications (London, England)","confidence":"Medium","confidence_rationale":"Tier 2 — multiple mechanistic assays including ChIP and phosphatase inhibition in a single lab study","pmids":["29764501"],"is_preprint":false},{"year":2018,"finding":"EYA4 overexpression reduces phosphorylation of AKT and GSK-3β, leading to inactivation of Slug and suppression of TGF-β1-induced epithelial-mesenchymal transition in esophageal squamous cell carcinoma cells.","method":"EYA4 overexpression and knockdown; western blotting for phospho-AKT, phospho-GSK-3β, Slug; migration/invasion assays","journal":"Cancer science","confidence":"Medium","confidence_rationale":"Tier 3 — pathway placement by western blot in overexpression/knockdown context, single lab","pmids":["29660222"],"is_preprint":false},{"year":2018,"finding":"EYA4 promotes glioma cell proliferation by suppressing p27Kip1 expression in a SIX1-dependent manner.","method":"EYA4 overexpression in glioma cells; CCK-8, BrdU proliferation assays; western blotting for p27Kip1; SIX1 co-expression experiments","journal":"Cellular physiology and biochemistry","confidence":"Low","confidence_rationale":"Tier 3 — single lab, overexpression with western blot, no reconstitution or ChIP","pmids":["30231237"],"is_preprint":false},{"year":2019,"finding":"EYA4 dephosphorylates β-catenin at Ser552 (using its serine/threonine phosphatase activity), reducing nuclear translocation of β-catenin and thereby suppressing β-catenin/LEF1-driven transcription of MYCBP, inhibiting HCC proliferation.","method":"EYA4 overexpression/KO; rescue with MYCBP siRNA or overexpression; western blot for p-β-catenin S552; gene microarray","journal":"Cancer science","confidence":"Medium","confidence_rationale":"Tier 2 — phosphorylation site identified with rescue epistasis, multiple functional readouts in single lab","pmids":["31385398"],"is_preprint":false},{"year":2020,"finding":"Truncating EYA4 mutations lead to absence of mutant protein expression (consistent with nonsense-mediated decay), and haploinsufficiency is the common disease mechanism for DFNA10; nuclear translocation of missense EYA4 mutant p.Glu369Asp requires SIX1 co-expression, as for wild-type EYA4.","method":"Transient expression of c-myc-tagged EYA4 mutants in COS7 cells; western blot; immunofluorescence; minigene splicing assays in NIH3T3 cells","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 — direct expression and localization experiments with functional validation of splicing variants","pmids":["32277154"],"is_preprint":false},{"year":2023,"finding":"TRIM69 (an E3 ubiquitin ligase) promotes EYA4 polyubiquitylation and proteasomal degradation; ERK2 directly binds a D-site docking groove in EYA4 (Leu512/514) and phosphorylates EYA4 at Ser37, which is required for TRIM69-mediated ubiquitylation and turnover of EYA4 in pancreatic cancer.","method":"Co-immunoprecipitation; GST-pulldown; in vitro kinase assay; CHX pulse-chase; cellular ubiquitination assay; ChIP; luciferase reporter assay; xenograft","journal":"Journal of Cancer","confidence":"High","confidence_rationale":"Tier 1-2 — in vitro kinase assay plus multiple orthogonal biochemical methods establishing PTM mechanism","pmids":["36741265"],"is_preprint":false},{"year":2023,"finding":"EYA4 serine/threonine phosphatase activity promotes replication fork progression and prevents replication stress; depletion of EYA4 activates the ATR pathway, causes endoreplication and polyploidy, and reduces breast cancer cell tumorigenic properties in vitro and in vivo.","method":"EYA4 knockdown/overexpression; hydroxyurea sensitivity assay; γH2AX immunofluorescence; ATR pathway activation assays; in vivo xenograft","journal":"Molecular cancer","confidence":"Medium","confidence_rationale":"Tier 2 — multiple orthogonal assays in single lab, phosphatase domain specificity demonstrated","pmids":["37777742"],"is_preprint":false},{"year":2024,"finding":"EYA4 (and EYA1) are tyrosine phosphatases that dephosphorylate PLK1 at pY445 during G2 phase; dephosphorylation of pY445 is required for centrosome maturation, PLK1 localization to centrosomes, polo-box domain (PBD)-dependent interactions with PLK1-activation complexes, and successful mitosis. Chemical inhibition of EYA phosphatase activity reduces PLK1 activation and causes mitotic defects and cell death.","method":"Co-immunoprecipitation; in vitro phosphatase assay; molecular dynamics simulation; EYA4/EYA1 depletion; centrosome maturation and PLK1 localization imaging; chemical inhibitor studies","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1 — in vitro phosphatase assay, structural modeling, multiple orthogonal cell biology readouts, with pharmacological validation","pmids":["38360978"],"is_preprint":false},{"year":2024,"finding":"EYA4 tyrosine phosphatase activity dephosphorylates RAD51 at Tyr315, promoting RAD51 localization, presynaptic filament formation, foci formation, and homologous recombination at DNA double-strand breaks; DNA binding stimulates EYA4 phosphatase activity.","method":"In vitro phosphatase assay with RAD51; RAD51 foci formation assay; HR reporter assay; EYA4 overexpression/depletion; single-stranded DNA accumulation assay","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 1 — in vitro reconstitution of phosphatase activity on defined substrate plus multiple cellular functional assays","pmids":["38084915"],"is_preprint":false},{"year":2024,"finding":"EYA4 directly interacts with histone H2AX and facilitates DNA double-strand break repair, reducing osteosarcoma sensitivity to doxorubicin.","method":"Co-immunoprecipitation (EYA4-H2AX); EYA4 knockdown/overexpression; in vitro and in vivo doxorubicin sensitivity assays","journal":"Biochemical pharmacology","confidence":"Low","confidence_rationale":"Tier 3 — single Co-IP with partial mechanistic follow-up, single lab","pmids":["38876260"],"is_preprint":false},{"year":2024,"finding":"EYA4 interacts with SIX2 and promotes transcription of p21 (CDKN1A) in a p53-dependent manner to drive cellular senescence; this function requires transcriptional activation activity but is independent of EYA4's phosphatase activity.","method":"EYA4 knockdown; co-immunoprecipitation (EYA4-SIX2); luciferase reporter for p21 promoter; phosphatase-deficient mutant; p53 knockdown epistasis; replicative and stress-induced senescence assays","journal":"Advanced biotechnology","confidence":"Medium","confidence_rationale":"Tier 2 — Co-IP, reporter assay, epistasis with p53, and phosphatase-dead mutant in single lab","pmids":["41991886"],"is_preprint":false},{"year":2024,"finding":"lncRNA EYA4-au1 recruits the mediator subunit Med11 to the EYA4 promoter to activate EYA4 transcription, subsequently activating the EYA4/p27Kip1/CK2α/HDAC2 cascade to drive cardiomyocyte hypertrophy.","method":"AngII-induced in vitro hypertrophy model; EYA4-au1 knockdown/overexpression; ChIP for Med11 at EYA4 promoter; pathway component analysis","journal":"Ecotoxicology and environmental safety","confidence":"Medium","confidence_rationale":"Tier 2 — ChIP for transcriptional mechanism, multiple pathway readouts, single lab","pmids":["39471666"],"is_preprint":false},{"year":2025,"finding":"Combined inhibition of EYA phosphatase activity and PLK1 shows synergistic lethality in neuroblastoma and glioblastoma cells overexpressing EYA1/EYA4, mediated by decreased PLK1 activity, reduced RAD51 foci, and mitotic arrest.","method":"Chemical inhibitor combination studies in cancer cell lines and glioblastoma stem cell models; PLK1 activity assay; RAD51 foci quantification; multi-omic correlational analysis","journal":"bioRxiv","confidence":"Low","confidence_rationale":"Tier 3 — preprint, pharmacological studies without in vitro reconstitution","pmids":["bio_10.1101_2025.01.19.633804"],"is_preprint":true}],"current_model":"EYA4 is a dual-function protein acting as a transcriptional coactivator (via interaction with SIX and DACH family members, with nuclear translocation dependent on SIX proteins) and as a tyrosine and serine/threonine phosphatase; its phosphatase activities dephosphorylate substrates including PLK1 (pY445) to promote centrosome maturation and mitosis, RAD51 (pY315) to promote homologous recombination, and β-catenin (pS552) to suppress oncogenic signaling, while its transcriptional coactivator function regulates p27Kip1/CK2α/HDAC2 and p21 cascades affecting cardiac hypertrophy, cardiomyopathy, and cellular senescence; haploinsufficiency of EYA4 due to truncating mutations causes DFNA10 sensorineural hearing loss, and more severe N-terminal truncations additionally impair EYA4–SIX interactions and cardiac function, causing dilated cardiomyopathy."},"narrative":{"teleology":[{"year":1999,"claim":"Identification of EYA4 as a member of the EYA gene family with a conserved C-terminal Eya-homologous region (eya-HR) established the structural framework for subsequent functional studies.","evidence":"Molecular cloning and sequence analysis of human and mouse EYA4","pmids":["9887327"],"confidence":"Medium","gaps":["No functional activity was demonstrated for the protein at this stage","Expression pattern in specific tissues not fully resolved"]},{"year":2001,"claim":"Demonstration that EYA4 haploinsufficiency causes DFNA10 sensorineural hearing loss established a post-developmental, maintenance role for EYA4 in the organ of Corti — the first disease link.","evidence":"Mutation identification in two unrelated DFNA10 families with premature stop codons","pmids":["11159937"],"confidence":"High","gaps":["Molecular targets of EYA4 in cochlear maintenance unknown","Whether residual protein is produced from truncating alleles was not tested"]},{"year":2004,"claim":"Showing that EYA4 interacts with SIX1 and requires SIX1 for nuclear translocation resolved how EYA4 reaches transcriptional targets, establishing the SIX-dependent shuttling mechanism.","evidence":"Yeast two-hybrid and immunofluorescence in mammalian cells","pmids":["15492887"],"confidence":"Medium","gaps":["DACH1 interaction was negative but other DACH family members untested","Endogenous interaction not confirmed"]},{"year":2005,"claim":"Correlation of mutation severity with disease spectrum — C-terminal truncations preserving SIX-binding cause hearing loss alone, whereas the short 193-aa peptide losing SIX-binding causes dilated cardiomyopathy — established SIX-EYA interaction as the critical determinant of cardiac function, validated by zebrafish morphant heart failure.","evidence":"Biochemical binding assays of EYA4 mutant peptides; morpholino knockdown in zebrafish with hemodynamic analysis","pmids":["15735644"],"confidence":"High","gaps":["Downstream cardiac transcriptional targets of EYA4-SIX unidentified","Mammalian in vivo cardiac model not yet available"]},{"year":2008,"claim":"Identification of Na+/K+-ATPase β2b subunit (atp1b2b) as a downstream effector whose overexpression rescues eya4-morphant hair cell loss and heart failure provided the first defined transcriptional target linking EYA4 to ion homeostasis.","evidence":"Morpholino knockdown and rescue overexpression in zebrafish","pmids":["18799547"],"confidence":"High","gaps":["Whether EYA4 directly binds the atp1b2b promoter was not tested","Relevance to mammalian hearing not confirmed"]},{"year":2009,"claim":"EYA4 interaction with SIX3 broadened the coactivator partnership beyond SIX1, showing that EYA4 uses multiple SIX family members for nuclear recruitment and transcriptional activation.","evidence":"Co-immunoprecipitation, confocal microscopy, and luciferase reporter assays","pmids":["19606496"],"confidence":"Medium","gaps":["Endogenous tissue context for EYA4-SIX3 cooperation not defined","SIX3 target genes regulated by EYA4 not identified"]},{"year":2015,"claim":"ChIP demonstration that EYA4 binds the p27Kip1 promoter and drives p27/CK2α/HDAC2 signaling in the heart, with transgenic mice showing dose-dependent cardiac pathology (hypertrophy vs. dilated cardiomyopathy), provided the first defined mammalian transcriptional cascade and in vivo cardiac model.","evidence":"ChIP, luciferase reporter, cardiac-specific transgenic mouse overexpression with MRI and hemodynamics","pmids":["26499333"],"confidence":"High","gaps":["Whether phosphatase activity contributes to cardiac phenotype was not tested","Direct vs. indirect regulation of p27 promoter not resolved"]},{"year":2018,"claim":"Discovery of EYA4's serine/threonine phosphatase activity toward IκBα (blocking NF-κB signaling) and β-catenin Ser552 (suppressing Wnt signaling) established EYA4 as a dual-specificity phosphatase with tumor-suppressive roles in hepatocellular carcinoma.","evidence":"EYA4 overexpression/KO with phosphatase inhibitor treatment, ubiquitination assays, western blots for phospho-substrates, and xenograft models","pmids":["29764501","31385398"],"confidence":"Medium","gaps":["In vitro phosphatase reconstitution on IκBα not performed","Whether EYA4 directly dephosphorylates IκBα or acts upstream is ambiguous"]},{"year":2020,"claim":"Confirmation that truncating EYA4 mutations produce no detectable protein (consistent with nonsense-mediated decay) formally established haploinsufficiency as the universal DFNA10 disease mechanism.","evidence":"Transient expression of tagged EYA4 mutants in COS7 cells with western blot and immunofluorescence","pmids":["32277154"],"confidence":"Medium","gaps":["Patient-derived cells not examined","Residual mRNA levels not quantified in human cochlear tissue"]},{"year":2023,"claim":"Identification of the ERK2-TRIM69 axis that phosphorylates EYA4 at Ser37 and drives its polyubiquitylation and proteasomal degradation revealed how EYA4 protein levels are post-translationally controlled.","evidence":"In vitro kinase assay, GST-pulldown, CHX pulse-chase, ubiquitination assay, and xenograft in pancreatic cancer","pmids":["36741265"],"confidence":"High","gaps":["Physiological contexts beyond pancreatic cancer not explored","Whether Ser37 phosphorylation affects EYA4 phosphatase or coactivator activity is unknown"]},{"year":2023,"claim":"EYA4's serine/threonine phosphatase activity was shown to promote replication fork progression and prevent ATR-dependent replication stress, extending its phosphatase role to S-phase genome integrity.","evidence":"EYA4 knockdown/overexpression with hydroxyurea sensitivity, γH2AX immunofluorescence, ATR activation assays, and xenograft","pmids":["37777742"],"confidence":"Medium","gaps":["Direct replication fork substrate(s) not identified","Separation of S/T vs. tyrosine phosphatase contributions not fully resolved"]},{"year":2024,"claim":"In vitro reconstitution proved EYA4 is a tyrosine phosphatase for PLK1 pY445, and that this dephosphorylation is required for centrosome maturation, PLK1 centrosomal localization, and mitotic entry — establishing a cell-cycle-essential catalytic function.","evidence":"In vitro phosphatase assay, molecular dynamics simulation, EYA4 depletion with centrosome imaging, chemical inhibitor validation","pmids":["38360978"],"confidence":"High","gaps":["Crystal structure of EYA4-PLK1 complex not available","Relative contributions of EYA4 vs. EYA1 in different tissues unknown"]},{"year":2024,"claim":"Reconstitution of EYA4 tyrosine phosphatase activity on RAD51 pY315 showed it promotes presynaptic filament formation and homologous recombination, with the novel finding that DNA binding stimulates EYA4's phosphatase activity.","evidence":"In vitro phosphatase assay on RAD51, HR reporter assay, RAD51 foci formation, ssDNA accumulation assay","pmids":["38084915"],"confidence":"High","gaps":["Structural basis for DNA-stimulated phosphatase activity unknown","Whether EYA4 acts on RAD51 before or after loading onto ssDNA is unclear"]},{"year":2024,"claim":"EYA4 interaction with SIX2 and phosphatase-independent transcriptional activation of p21/CDKN1A in a p53-dependent manner established a role for EYA4 in cellular senescence, functionally separating its coactivator and phosphatase activities.","evidence":"Co-IP (EYA4-SIX2), p21 promoter reporter, phosphatase-dead mutant, p53 knockdown epistasis, senescence assays","pmids":["41991886"],"confidence":"Medium","gaps":["Physiological tissue context for EYA4-driven senescence not defined","Whether EYA4-SIX2 binds the p21 promoter directly (ChIP) was not shown"]},{"year":null,"claim":"Key open questions include: the structural basis of EYA4's dual phosphatase specificity, the tissue-specific division of labor between its coactivator and phosphatase functions, and the identity of replication-fork substrates mediating its role in S-phase genome stability.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No crystal structure of EYA4 catalytic domain available","Relative contribution of EYA4 vs. other EYA family members in each tissue context unclear","Replication fork substrate(s) for S/T phosphatase activity unidentified"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[8,11,14,15,16]},{"term_id":"GO:0140110","term_label":"transcription regulator activity","supporting_discovery_ids":[1,5,6,18]},{"term_id":"GO:0003677","term_label":"DNA binding","supporting_discovery_ids":[16]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[2,12]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[2,5,6,12]},{"term_id":"GO:0005815","term_label":"microtubule organizing center","supporting_discovery_ids":[15]}],"pathway":[{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[15]},{"term_id":"R-HSA-73894","term_label":"DNA Repair","supporting_discovery_ids":[16,17]},{"term_id":"R-HSA-69306","term_label":"DNA Replication","supporting_discovery_ids":[14]},{"term_id":"R-HSA-74160","term_label":"Gene expression (Transcription)","supporting_discovery_ids":[1,6,18]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[8,9,11]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[1,3]}],"complexes":[],"partners":["SIX1","SIX2","SIX3","PLK1","RAD51","TRIM69","ERK2","H2AX"],"other_free_text":[]},"mechanistic_narrative":"EYA4 is a dual-function protein that acts both as a transcriptional coactivator in partnership with SIX-family homeodomain proteins and as an intrinsic tyrosine and serine/threonine phosphatase, coupling gene regulation to cell cycle control, DNA repair, and organ maintenance. As a coactivator, EYA4 is recruited to the nucleus by SIX1, SIX2, or SIX3, where it regulates targets including p27Kip1 (via the p27/CK2α/HDAC2 cascade in cardiomyocytes) and p21/CDKN1A (in a p53-dependent senescence program) [PMID:26499333, PMID:41991886, PMID:15492887]. Its tyrosine phosphatase activity dephosphorylates PLK1 at pY445 to enable centrosome maturation and mitotic entry, and RAD51 at pY315 to promote homologous recombination, while its serine/threonine phosphatase activity dephosphorylates β-catenin at Ser552 to restrain Wnt signaling and maintains replication fork stability [PMID:38360978, PMID:38084915, PMID:31385398, PMID:37777742]. Haploinsufficiency caused by truncating mutations underlies DFNA10 progressive sensorineural hearing loss, and more severe N-terminal truncations that abolish SIX-binding cause dilated cardiomyopathy [PMID:11159937, PMID:15735644]."},"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; BOS3","url":"https://www.omim.org/entry/608389"},{"mim_id":"607371","title":"DYSTONIA-DEAFNESS SYNDROME 1; DDS1","url":"https://www.omim.org/entry/607371"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Nucleoplasm","reliability":"Approved"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in many","driving_tissues":[{"tissue":"skeletal muscle","ntpm":29.0},{"tissue":"tongue","ntpm":18.7}],"url":"https://www.proteinatlas.org/search/EYA4"},"hgnc":{"alias_symbol":[],"prev_symbol":["DFNA10","CMD1J"]},"alphafold":{"accession":"O95677","domains":[{"cath_id":"3.40.50.12350","chopping":"371-637","consensus_level":"medium","plddt":95.2098,"start":371,"end":637}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/O95677","model_url":"https://alphafold.ebi.ac.uk/files/AF-O95677-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-O95677-F1-predicted_aligned_error_v6.png","plddt_mean":62.72},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=EYA4","jax_strain_url":"https://www.jax.org/strain/search?query=EYA4"},"sequence":{"accession":"O95677","fasta_url":"https://rest.uniprot.org/uniprotkb/O95677.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/O95677/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/O95677"}},"corpus_meta":[{"pmid":"11159937","id":"PMC_11159937","title":"Mutations in the transcriptional activator EYA4 cause late-onset deafness at the DFNA10 locus.","date":"2001","source":"Human molecular genetics","url":"https://pubmed.ncbi.nlm.nih.gov/11159937","citation_count":171,"is_preprint":false},{"pmid":"15735644","id":"PMC_15735644","title":"Mutation in the transcriptional coactivator EYA4 causes dilated cardiomyopathy and sensorineural hearing loss.","date":"2005","source":"Nature genetics","url":"https://pubmed.ncbi.nlm.nih.gov/15735644","citation_count":153,"is_preprint":false},{"pmid":"31101108","id":"PMC_31101108","title":"Circular RNA ACVR2A suppresses bladder cancer cells proliferation and metastasis through miR-626/EYA4 axis.","date":"2019","source":"Molecular cancer","url":"https://pubmed.ncbi.nlm.nih.gov/31101108","citation_count":133,"is_preprint":false},{"pmid":"9887327","id":"PMC_9887327","title":"EYA4, a novel vertebrate gene related to Drosophila eyes absent.","date":"1999","source":"Human molecular genetics","url":"https://pubmed.ncbi.nlm.nih.gov/9887327","citation_count":129,"is_preprint":false},{"pmid":"8776603","id":"PMC_8776603","title":"A gene for autosomal dominant late-onset progressive non-syndromic hearing loss, DFNA10, maps to chromosome 6.","date":"1996","source":"Human molecular genetics","url":"https://pubmed.ncbi.nlm.nih.gov/8776603","citation_count":53,"is_preprint":false},{"pmid":"18799547","id":"PMC_18799547","title":"Eya4 regulation of Na+/K+-ATPase is required for sensory system development in zebrafish.","date":"2008","source":"Development (Cambridge, England)","url":"https://pubmed.ncbi.nlm.nih.gov/18799547","citation_count":52,"is_preprint":false},{"pmid":"15492887","id":"PMC_15492887","title":"A comparative study of Eya1 and Eya4 protein function and its implication in branchio-oto-renal syndrome and DFNA10.","date":"2004","source":"Journal of the Association for Research in Otolaryngology : JARO","url":"https://pubmed.ncbi.nlm.nih.gov/15492887","citation_count":46,"is_preprint":false},{"pmid":"12477971","id":"PMC_12477971","title":"A 4-bp insertion in the eya-homologous region (eyaHR) of EYA4 causes hearing impairment in a Hungarian family linked to DFNA10.","date":"2002","source":"Molecular medicine (Cambridge, Mass.)","url":"https://pubmed.ncbi.nlm.nih.gov/12477971","citation_count":45,"is_preprint":false},{"pmid":"24096489","id":"PMC_24096489","title":"EYA4 is inactivated biallelically at a high frequency in sporadic lung cancer and is associated with familial lung cancer risk.","date":"2013","source":"Oncogene","url":"https://pubmed.ncbi.nlm.nih.gov/24096489","citation_count":37,"is_preprint":false},{"pmid":"17567890","id":"PMC_17567890","title":"Nonsyndromic hearing loss DFNA10 and a novel mutation of EYA4: evidence for correlation of normal cardiac phenotype with truncating mutations of the Eya domain.","date":"2007","source":"American journal of medical genetics. 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in Drosophila eya mediates developmentally important protein-protein interactions.\",\n      \"method\": \"Molecular cloning and sequence analysis of human and mouse EYA4\",\n      \"journal\": \"Human molecular genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — domain identification by sequence homology, foundational characterization paper\",\n      \"pmids\": [\"9887327\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"EYA4 functions as a transcriptional activator and its haploinsufficiency (via premature stop codons) causes late-onset progressive sensorineural hearing loss (DFNA10), demonstrating a post-developmental role in maintenance of the organ of Corti.\",\n      \"method\": \"Mutation identification in two unrelated families; genetic linkage and sequencing\",\n      \"journal\": \"Human molecular genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — replicated across two unrelated families, loss-of-function with defined phenotype\",\n      \"pmids\": [\"11159937\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"EYA4 Eya-homologous region (Eya4HR) interacts with SIX1 (but not DACH1) in yeast two-hybrid assays; Eya4HR localizes to the cytoplasm alone but is translocated to the nucleus upon co-expression with SIX1.\",\n      \"method\": \"Yeast two-hybrid system; immunofluorescence staining; coexpression in mammalian cells\",\n      \"journal\": \"Journal of the Association for Research in Otolaryngology : JARO\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — direct protein interaction and localization with functional consequence demonstrated in single lab\",\n      \"pmids\": [\"15492887\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"EYA4 peptides associated with sensorineural hearing loss (C-terminal Eya domain truncations) bind wild-type EYA4 and SIX proteins, whereas the shortened 193-amino acid peptide associated with dilated cardiomyopathy does not; zebrafish eya4 morphants display heart failure phenotype, and eya4 regulates cardiac function via EYA4-SIX-mediated transcriptional regulation.\",\n      \"method\": \"Biochemical interaction assays of mutant EYA4 peptides; antisense morpholino knockdown in zebrafish; hemodynamic analysis\",\n      \"journal\": \"Nature genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — multiple orthogonal methods (biochemical binding, in vivo morpholino, hemodynamics), replicated across model systems\",\n      \"pmids\": [\"15735644\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Eya4 regulates expression of Na+/K+-ATPase (atp1b2b beta2b subunit) in zebrafish; loss of eya4 reduces atp1b2b levels and causes hair cell loss and heart failure, and atp1b2b overexpression rescues these eya4 morphant phenotypes.\",\n      \"method\": \"Morpholino knockdown of eya4 and atp1b2b in zebrafish; rescue experiments with atp1b2b overexpression; sensory response assays\",\n      \"journal\": \"Development (Cambridge, England)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — epistasis established by rescue experiment, multiple phenotypic readouts in ortholog model\",\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 in reporter gene assays.\",\n      \"method\": \"Co-immunoprecipitation; confocal microscopy; luciferase reporter assays\",\n      \"journal\": \"Human mutation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal Co-IP and functional reporter assay in single study\",\n      \"pmids\": [\"19606496\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"EYA4 and its truncation mutant E193 regulate p27Kip1 expression by binding and regulating the p27 promoter (ChIP assay); cardiac-specific overexpression of Eya4 in mice causes age-dependent hypertrophy via p27/casein kinase-2α/HDAC2 signaling, whereas E193 overexpression causes dilated cardiomyopathy.\",\n      \"method\": \"Luciferase reporter assays; chromatin immunoprecipitation; transgenic mouse overexpression; MRI and hemodynamic analysis\",\n      \"journal\": \"Circulation. Cardiovascular genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — ChIP, reporter assay, and in vivo transgenic model with multiple readouts in single study\",\n      \"pmids\": [\"26499333\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"miR-431 directly targets Eya4 mRNA in cochlear spiral ganglion neurons; overexpression of miR-431 in transgenic mice inhibits EYA4 translation, reduces spiral ganglion neuron density, and causes hearing loss.\",\n      \"method\": \"Luciferase reporter assay; western blotting; transgenic mouse model; ABR testing; primary SGN culture\",\n      \"journal\": \"Biochimica et biophysica acta\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — direct target validation by luciferase assay and in vivo transgenic model\",\n      \"pmids\": [\"27545760\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"EYA4 suppresses HCC proliferation and invasion by antagonizing TNF-α-induced NF-κB activation: EYA4's serine/threonine phosphatase activity blocks phosphorylation and ubiquitination of IκBα, inhibiting p65 nuclear translocation and RAP1A transactivation.\",\n      \"method\": \"Stable transfection; ChIP; immunofluorescence; ubiquitination assay; calyculin A (phosphatase inhibitor) treatment; xenograft\",\n      \"journal\": \"Cancer communications (London, England)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple mechanistic assays including ChIP and phosphatase inhibition in a single lab study\",\n      \"pmids\": [\"29764501\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"EYA4 overexpression reduces phosphorylation of AKT and GSK-3β, leading to inactivation of Slug and suppression of TGF-β1-induced epithelial-mesenchymal transition in esophageal squamous cell carcinoma cells.\",\n      \"method\": \"EYA4 overexpression and knockdown; western blotting for phospho-AKT, phospho-GSK-3β, Slug; migration/invasion assays\",\n      \"journal\": \"Cancer science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — pathway placement by western blot in overexpression/knockdown context, single lab\",\n      \"pmids\": [\"29660222\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"EYA4 promotes glioma cell proliferation by suppressing p27Kip1 expression in a SIX1-dependent manner.\",\n      \"method\": \"EYA4 overexpression in glioma cells; CCK-8, BrdU proliferation assays; western blotting for p27Kip1; SIX1 co-expression experiments\",\n      \"journal\": \"Cellular physiology and biochemistry\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — single lab, overexpression with western blot, no reconstitution or ChIP\",\n      \"pmids\": [\"30231237\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"EYA4 dephosphorylates β-catenin at Ser552 (using its serine/threonine phosphatase activity), reducing nuclear translocation of β-catenin and thereby suppressing β-catenin/LEF1-driven transcription of MYCBP, inhibiting HCC proliferation.\",\n      \"method\": \"EYA4 overexpression/KO; rescue with MYCBP siRNA or overexpression; western blot for p-β-catenin S552; gene microarray\",\n      \"journal\": \"Cancer science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — phosphorylation site identified with rescue epistasis, multiple functional readouts in single lab\",\n      \"pmids\": [\"31385398\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Truncating EYA4 mutations lead to absence of mutant protein expression (consistent with nonsense-mediated decay), and haploinsufficiency is the common disease mechanism for DFNA10; nuclear translocation of missense EYA4 mutant p.Glu369Asp requires SIX1 co-expression, as for wild-type EYA4.\",\n      \"method\": \"Transient expression of c-myc-tagged EYA4 mutants in COS7 cells; western blot; immunofluorescence; minigene splicing assays in NIH3T3 cells\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — direct expression and localization experiments with functional validation of splicing variants\",\n      \"pmids\": [\"32277154\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"TRIM69 (an E3 ubiquitin ligase) promotes EYA4 polyubiquitylation and proteasomal degradation; ERK2 directly binds a D-site docking groove in EYA4 (Leu512/514) and phosphorylates EYA4 at Ser37, which is required for TRIM69-mediated ubiquitylation and turnover of EYA4 in pancreatic cancer.\",\n      \"method\": \"Co-immunoprecipitation; GST-pulldown; in vitro kinase assay; CHX pulse-chase; cellular ubiquitination assay; ChIP; luciferase reporter assay; xenograft\",\n      \"journal\": \"Journal of Cancer\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — in vitro kinase assay plus multiple orthogonal biochemical methods establishing PTM mechanism\",\n      \"pmids\": [\"36741265\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"EYA4 serine/threonine phosphatase activity promotes replication fork progression and prevents replication stress; depletion of EYA4 activates the ATR pathway, causes endoreplication and polyploidy, and reduces breast cancer cell tumorigenic properties in vitro and in vivo.\",\n      \"method\": \"EYA4 knockdown/overexpression; hydroxyurea sensitivity assay; γH2AX immunofluorescence; ATR pathway activation assays; in vivo xenograft\",\n      \"journal\": \"Molecular cancer\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal assays in single lab, phosphatase domain specificity demonstrated\",\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; dephosphorylation of pY445 is required for centrosome maturation, PLK1 localization to centrosomes, polo-box domain (PBD)-dependent interactions with PLK1-activation complexes, and successful mitosis. Chemical inhibition of EYA phosphatase activity reduces PLK1 activation and causes mitotic defects and cell death.\",\n      \"method\": \"Co-immunoprecipitation; in vitro phosphatase assay; molecular dynamics simulation; EYA4/EYA1 depletion; centrosome maturation and PLK1 localization imaging; chemical inhibitor studies\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro phosphatase assay, structural modeling, multiple orthogonal cell biology readouts, with pharmacological validation\",\n      \"pmids\": [\"38360978\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"EYA4 tyrosine phosphatase activity dephosphorylates RAD51 at Tyr315, promoting RAD51 localization, presynaptic filament formation, foci formation, and homologous recombination at DNA double-strand breaks; DNA binding stimulates EYA4 phosphatase activity.\",\n      \"method\": \"In vitro phosphatase assay with RAD51; RAD51 foci formation assay; HR reporter assay; EYA4 overexpression/depletion; single-stranded DNA accumulation assay\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro reconstitution of phosphatase activity on defined substrate plus multiple cellular functional assays\",\n      \"pmids\": [\"38084915\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"EYA4 directly interacts with histone H2AX and facilitates DNA double-strand break repair, reducing osteosarcoma sensitivity to doxorubicin.\",\n      \"method\": \"Co-immunoprecipitation (EYA4-H2AX); EYA4 knockdown/overexpression; in vitro and in vivo doxorubicin sensitivity assays\",\n      \"journal\": \"Biochemical pharmacology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — single Co-IP with partial mechanistic follow-up, single lab\",\n      \"pmids\": [\"38876260\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"EYA4 interacts with SIX2 and promotes transcription of p21 (CDKN1A) in a p53-dependent manner to drive cellular senescence; this function requires transcriptional activation activity but is independent of EYA4's phosphatase activity.\",\n      \"method\": \"EYA4 knockdown; co-immunoprecipitation (EYA4-SIX2); luciferase reporter for p21 promoter; phosphatase-deficient mutant; p53 knockdown epistasis; replicative and stress-induced senescence assays\",\n      \"journal\": \"Advanced biotechnology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — Co-IP, reporter assay, epistasis with p53, and phosphatase-dead mutant in single lab\",\n      \"pmids\": [\"41991886\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"lncRNA EYA4-au1 recruits the mediator subunit Med11 to the EYA4 promoter to activate EYA4 transcription, subsequently activating the EYA4/p27Kip1/CK2α/HDAC2 cascade to drive cardiomyocyte hypertrophy.\",\n      \"method\": \"AngII-induced in vitro hypertrophy model; EYA4-au1 knockdown/overexpression; ChIP for Med11 at EYA4 promoter; pathway component analysis\",\n      \"journal\": \"Ecotoxicology and environmental safety\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — ChIP for transcriptional mechanism, multiple pathway readouts, single lab\",\n      \"pmids\": [\"39471666\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Combined inhibition of EYA phosphatase activity and PLK1 shows synergistic lethality in neuroblastoma and glioblastoma cells overexpressing EYA1/EYA4, mediated by decreased PLK1 activity, reduced RAD51 foci, and mitotic arrest.\",\n      \"method\": \"Chemical inhibitor combination studies in cancer cell lines and glioblastoma stem cell models; PLK1 activity assay; RAD51 foci quantification; multi-omic correlational analysis\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — preprint, pharmacological studies without in vitro reconstitution\",\n      \"pmids\": [\"bio_10.1101_2025.01.19.633804\"],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"EYA4 is a dual-function protein acting as a transcriptional coactivator (via interaction with SIX and DACH family members, with nuclear translocation dependent on SIX proteins) and as a tyrosine and serine/threonine phosphatase; its phosphatase activities dephosphorylate substrates including PLK1 (pY445) to promote centrosome maturation and mitosis, RAD51 (pY315) to promote homologous recombination, and β-catenin (pS552) to suppress oncogenic signaling, while its transcriptional coactivator function regulates p27Kip1/CK2α/HDAC2 and p21 cascades affecting cardiac hypertrophy, cardiomyopathy, and cellular senescence; haploinsufficiency of EYA4 due to truncating mutations causes DFNA10 sensorineural hearing loss, and more severe N-terminal truncations additionally impair EYA4–SIX interactions and cardiac function, causing dilated cardiomyopathy.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"EYA4 is a dual-function protein that acts both as a transcriptional coactivator in partnership with SIX-family homeodomain proteins and as an intrinsic tyrosine and serine/threonine phosphatase, coupling gene regulation to cell cycle control, DNA repair, and organ maintenance. As a coactivator, EYA4 is recruited to the nucleus by SIX1, SIX2, or SIX3, where it regulates targets including p27Kip1 (via the p27/CK2α/HDAC2 cascade in cardiomyocytes) and p21/CDKN1A (in a p53-dependent senescence program) [PMID:26499333, PMID:41991886, PMID:15492887]. Its tyrosine phosphatase activity dephosphorylates PLK1 at pY445 to enable centrosome maturation and mitotic entry, and RAD51 at pY315 to promote homologous recombination, while its serine/threonine phosphatase activity dephosphorylates β-catenin at Ser552 to restrain Wnt signaling and maintains replication fork stability [PMID:38360978, PMID:38084915, PMID:31385398, PMID:37777742]. Haploinsufficiency caused by truncating mutations underlies DFNA10 progressive sensorineural hearing loss, and more severe N-terminal truncations that abolish SIX-binding cause dilated cardiomyopathy [PMID:11159937, PMID:15735644].\",\n  \"teleology\": [\n    {\n      \"year\": 1999,\n      \"claim\": \"Identification of EYA4 as a member of the EYA gene family with a conserved C-terminal Eya-homologous region (eya-HR) established the structural framework for subsequent functional studies.\",\n      \"evidence\": \"Molecular cloning and sequence analysis of human and mouse EYA4\",\n      \"pmids\": [\"9887327\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No functional activity was demonstrated for the protein at this stage\", \"Expression pattern in specific tissues not fully resolved\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Demonstration that EYA4 haploinsufficiency causes DFNA10 sensorineural hearing loss established a post-developmental, maintenance role for EYA4 in the organ of Corti — the first disease link.\",\n      \"evidence\": \"Mutation identification in two unrelated DFNA10 families with premature stop codons\",\n      \"pmids\": [\"11159937\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular targets of EYA4 in cochlear maintenance unknown\", \"Whether residual protein is produced from truncating alleles was not tested\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Showing that EYA4 interacts with SIX1 and requires SIX1 for nuclear translocation resolved how EYA4 reaches transcriptional targets, establishing the SIX-dependent shuttling mechanism.\",\n      \"evidence\": \"Yeast two-hybrid and immunofluorescence in mammalian cells\",\n      \"pmids\": [\"15492887\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"DACH1 interaction was negative but other DACH family members untested\", \"Endogenous interaction not confirmed\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Correlation of mutation severity with disease spectrum — C-terminal truncations preserving SIX-binding cause hearing loss alone, whereas the short 193-aa peptide losing SIX-binding causes dilated cardiomyopathy — established SIX-EYA interaction as the critical determinant of cardiac function, validated by zebrafish morphant heart failure.\",\n      \"evidence\": \"Biochemical binding assays of EYA4 mutant peptides; morpholino knockdown in zebrafish with hemodynamic analysis\",\n      \"pmids\": [\"15735644\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Downstream cardiac transcriptional targets of EYA4-SIX unidentified\", \"Mammalian in vivo cardiac model not yet available\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Identification of Na+/K+-ATPase β2b subunit (atp1b2b) as a downstream effector whose overexpression rescues eya4-morphant hair cell loss and heart failure provided the first defined transcriptional target linking EYA4 to ion homeostasis.\",\n      \"evidence\": \"Morpholino knockdown and rescue overexpression in zebrafish\",\n      \"pmids\": [\"18799547\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether EYA4 directly binds the atp1b2b promoter was not tested\", \"Relevance to mammalian hearing not confirmed\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"EYA4 interaction with SIX3 broadened the coactivator partnership beyond SIX1, showing that EYA4 uses multiple SIX family members for nuclear recruitment and transcriptional activation.\",\n      \"evidence\": \"Co-immunoprecipitation, confocal microscopy, and luciferase reporter assays\",\n      \"pmids\": [\"19606496\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Endogenous tissue context for EYA4-SIX3 cooperation not defined\", \"SIX3 target genes regulated by EYA4 not identified\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"ChIP demonstration that EYA4 binds the p27Kip1 promoter and drives p27/CK2α/HDAC2 signaling in the heart, with transgenic mice showing dose-dependent cardiac pathology (hypertrophy vs. dilated cardiomyopathy), provided the first defined mammalian transcriptional cascade and in vivo cardiac model.\",\n      \"evidence\": \"ChIP, luciferase reporter, cardiac-specific transgenic mouse overexpression with MRI and hemodynamics\",\n      \"pmids\": [\"26499333\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether phosphatase activity contributes to cardiac phenotype was not tested\", \"Direct vs. indirect regulation of p27 promoter not resolved\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Discovery of EYA4's serine/threonine phosphatase activity toward IκBα (blocking NF-κB signaling) and β-catenin Ser552 (suppressing Wnt signaling) established EYA4 as a dual-specificity phosphatase with tumor-suppressive roles in hepatocellular carcinoma.\",\n      \"evidence\": \"EYA4 overexpression/KO with phosphatase inhibitor treatment, ubiquitination assays, western blots for phospho-substrates, and xenograft models\",\n      \"pmids\": [\"29764501\", \"31385398\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"In vitro phosphatase reconstitution on IκBα not performed\", \"Whether EYA4 directly dephosphorylates IκBα or acts upstream is ambiguous\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Confirmation that truncating EYA4 mutations produce no detectable protein (consistent with nonsense-mediated decay) formally established haploinsufficiency as the universal DFNA10 disease mechanism.\",\n      \"evidence\": \"Transient expression of tagged EYA4 mutants in COS7 cells with western blot and immunofluorescence\",\n      \"pmids\": [\"32277154\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Patient-derived cells not examined\", \"Residual mRNA levels not quantified in human cochlear tissue\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Identification of the ERK2-TRIM69 axis that phosphorylates EYA4 at Ser37 and drives its polyubiquitylation and proteasomal degradation revealed how EYA4 protein levels are post-translationally controlled.\",\n      \"evidence\": \"In vitro kinase assay, GST-pulldown, CHX pulse-chase, ubiquitination assay, and xenograft in pancreatic cancer\",\n      \"pmids\": [\"36741265\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Physiological contexts beyond pancreatic cancer not explored\", \"Whether Ser37 phosphorylation affects EYA4 phosphatase or coactivator activity is unknown\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"EYA4's serine/threonine phosphatase activity was shown to promote replication fork progression and prevent ATR-dependent replication stress, extending its phosphatase role to S-phase genome integrity.\",\n      \"evidence\": \"EYA4 knockdown/overexpression with hydroxyurea sensitivity, γH2AX immunofluorescence, ATR activation assays, and xenograft\",\n      \"pmids\": [\"37777742\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct replication fork substrate(s) not identified\", \"Separation of S/T vs. tyrosine phosphatase contributions not fully resolved\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"In vitro reconstitution proved EYA4 is a tyrosine phosphatase for PLK1 pY445, and that this dephosphorylation is required for centrosome maturation, PLK1 centrosomal localization, and mitotic entry — establishing a cell-cycle-essential catalytic function.\",\n      \"evidence\": \"In vitro phosphatase assay, molecular dynamics simulation, EYA4 depletion with centrosome imaging, chemical inhibitor validation\",\n      \"pmids\": [\"38360978\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Crystal structure of EYA4-PLK1 complex not available\", \"Relative contributions of EYA4 vs. EYA1 in different tissues unknown\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Reconstitution of EYA4 tyrosine phosphatase activity on RAD51 pY315 showed it promotes presynaptic filament formation and homologous recombination, with the novel finding that DNA binding stimulates EYA4's phosphatase activity.\",\n      \"evidence\": \"In vitro phosphatase assay on RAD51, HR reporter assay, RAD51 foci formation, ssDNA accumulation assay\",\n      \"pmids\": [\"38084915\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis for DNA-stimulated phosphatase activity unknown\", \"Whether EYA4 acts on RAD51 before or after loading onto ssDNA is unclear\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"EYA4 interaction with SIX2 and phosphatase-independent transcriptional activation of p21/CDKN1A in a p53-dependent manner established a role for EYA4 in cellular senescence, functionally separating its coactivator and phosphatase activities.\",\n      \"evidence\": \"Co-IP (EYA4-SIX2), p21 promoter reporter, phosphatase-dead mutant, p53 knockdown epistasis, senescence assays\",\n      \"pmids\": [\"41991886\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Physiological tissue context for EYA4-driven senescence not defined\", \"Whether EYA4-SIX2 binds the p21 promoter directly (ChIP) was not shown\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Key open questions include: the structural basis of EYA4's dual phosphatase specificity, the tissue-specific division of labor between its coactivator and phosphatase functions, and the identity of replication-fork substrates mediating its role in S-phase genome stability.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No crystal structure of EYA4 catalytic domain available\", \"Relative contribution of EYA4 vs. other EYA family members in each tissue context unclear\", \"Replication fork substrate(s) for S/T phosphatase activity unidentified\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [8, 11, 14, 15, 16]},\n      {\"term_id\": \"GO:0140110\", \"supporting_discovery_ids\": [1, 5, 6, 18]},\n      {\"term_id\": \"GO:0003677\", \"supporting_discovery_ids\": [16]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [2, 12]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [2, 5, 6, 12]},\n      {\"term_id\": \"GO:0005815\", \"supporting_discovery_ids\": [15]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [15]},\n      {\"term_id\": \"R-HSA-73894\", \"supporting_discovery_ids\": [16, 17]},\n      {\"term_id\": \"R-HSA-69306\", \"supporting_discovery_ids\": [14]},\n      {\"term_id\": \"R-HSA-74160\", \"supporting_discovery_ids\": [1, 6, 18]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [8, 9, 11]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [1, 3]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"SIX1\", \"SIX2\", \"SIX3\", \"PLK1\", \"RAD51\", \"TRIM69\", \"ERK2\", \"H2AX\"],\n    \"other_free_text\": []\n  }\n}\n```"}