{"gene":"EAPP","run_date":"2026-06-09T23:54:42","timeline":{"discoveries":[{"year":2005,"finding":"EAPP (E2F-associated phosphoprotein) is a nuclear phosphoprotein identified via yeast two-hybrid screen that physically interacts with E2F-1, E2F-2, and E2F-3, but not E2F-4. EAPP is localized in the nucleus, is present throughout the cell cycle but disappears during mitosis, and its overexpression increases the fraction of cells in S-phase while RNAi-mediated knockdown reduces S-phase fraction.","method":"Yeast two-hybrid screen, nuclear localization confirmed by cell fractionation/imaging, transfection reporter assays with E2F-dependent and thymidine kinase promoters, RNAi knockdown with cell cycle analysis","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal interaction confirmed by yeast two-hybrid plus functional reporter assays and RNAi phenotype, multiple orthogonal methods in a single focused study","pmids":["15716352"],"is_preprint":false},{"year":2005,"finding":"EAPP increases E2F-1-driven transcriptional activation from an artificial E2F-dependent promoter and the murine thymidine kinase promoter, but represses E2F-1-driven activation of the p14ARF promoter, demonstrating promoter-context-dependent modulation of E2F transcriptional output.","method":"Transfection reporter assays with E2F-dependent artificial promoter and endogenous promoter constructs","journal":"Molecular biology of the cell","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — clean functional assay in a single lab, single method (reporter assay)","pmids":["15716352"],"is_preprint":false},{"year":2008,"finding":"The EAPP gene promoter is TATA-less and contains functional binding sites for Sp1, Sp3, and Egr-1; Sp1 and Egr-1 activate the EAPP promoter while Sp3 represses it, and reduced Sp3 activity can account for elevated EAPP levels in transformed cells.","method":"Electrophoretic mobility shift assay (EMSA), supershift and competition assays, chromatin immunoprecipitation (ChIP), luciferase reporter assays with promoter truncations","journal":"The international journal of biochemistry & cell biology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal methods (EMSA, ChIP, reporter assays) in a single focused study confirming both in vitro binding and in vivo occupancy","pmids":["18588995"],"is_preprint":false},{"year":2010,"finding":"EAPP and R1 (RAM2/CDCA7L/JPO2) function as transcriptional repressors of the MAO B gene by competing with Sp1 for binding to Sp1 sites in the MAO B core promoter; in response to dexamethasone, EAPP and R1 occupancy at the MAO B promoter decreases while Sp1 occupancy increases, enabling glucocorticoid activation of MAO B.","method":"Yeast one-hybrid screen (using Sp1-binding motifs as bait), EMSA (competition assay for Sp1 site binding), chromatin immunoprecipitation (ChIP) in cells, transfection reporter assays","journal":"Molecular pharmacology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal methods (yeast one-hybrid, EMSA, ChIP, reporter assays) in a single focused study establishing mechanism in vitro and in vivo","pmids":["20980443"],"is_preprint":false},{"year":2011,"finding":"EAPP overexpression in U2OS cells results in G1 arrest and heightened resistance to DNA damage- or E2F1-induced apoptosis in a p21-dependent manner. EAPP levels are upregulated after DNA damage and in confluent cells. EAPP binds directly to the p21 promoter and stimulates p21 expression independently of p53, and appears required for assembly of the transcription initiation complex at the p21 promoter. RNAi knockdown of EAPP increases sensitivity to DNA damage and causes apoptosis even without stress.","method":"Overexpression and RNAi knockdown in U2OS cells, flow cytometry for cell cycle and apoptosis, chromatin immunoprecipitation (ChIP) at p21 promoter, p21 promoter reporter assays, epistasis with p21","journal":"Oncogene","confidence":"High","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal methods (ChIP, reporter assays, genetic epistasis with p21, KD/OE with defined phenotypes) in a single focused mechanistic study","pmids":["21258403"],"is_preprint":false},{"year":2011,"finding":"EAPP regulates the phosphorylation status and activity of Chk2; EAPP binding appears to trigger dephosphorylation of phospho-Chk2, resulting in its inactivation, thereby modulating the DNA damage checkpoint response.","method":"Overexpression and knockdown experiments, western blotting for phospho-Chk2, co-immunoprecipitation/binding assays","journal":"Cell cycle (Georgetown, Tex.)","confidence":"Medium","confidence_rationale":"Tier 3 / Weak — single lab, mechanistic claim based on phosphorylation western blotting with limited orthogonal validation described in the abstract","pmids":["21572256"],"is_preprint":false},{"year":2013,"finding":"EAPP stimulates the MDR1 (ABCB1) promoter resulting in elevated P-glycoprotein (PGP) levels. This activation is independent of E2F1 and is not blocked by co-expression of pRb (which does inhibit E2F1-dependent MDR1 promoter activation), indicating a distinct mechanism for EAPP-driven MDR1 regulation.","method":"Transfection reporter assays with MDR1 promoter, co-expression of pRb as epistasis test, western blotting for PGP protein levels","journal":"FEBS letters","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — reporter assay plus epistasis with pRb distinguishing EAPP from E2F1 mechanism, single lab","pmids":["23542036"],"is_preprint":false},{"year":2015,"finding":"In a rat spinal cord injury model, EAPP protein levels increase and peak at day 3 post-injury in neurons and astrocytes. EAPP co-localizes with active caspase-3 in neurons (suggesting a role in neuronal apoptosis) and with PCNA in astrocytes. In vitro siRNA knockdown of EAPP in astrocytes inhibits proliferation, migration, and CDK4/cyclin D1 expression, while EAPP knockdown in neurons reduces apoptosis and cell cycle protein levels.","method":"Rat SCI model with immunohistochemistry and western blotting, co-localization with caspase-3 and PCNA, siRNA knockdown in vitro with proliferation, migration, and western blot assays","journal":"Journal of cellular biochemistry","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — in vivo expression profiling combined with in vitro siRNA knockdown with defined phenotypic readouts; multiple cell types examined but abstract-level description limits mechanistic depth","pmids":["25704466"],"is_preprint":false},{"year":2016,"finding":"In mouse hypothalamus, Eapp expression is controlled by a cis-acting quantitative trait locus (cis-eQTL). siRNA knockdown of Eapp alters expression of downstream targets Sphk2, Nosip, Mmp9, Npy, Npy5r, and Maob, placing Eapp upstream of these genes in a stress-response gene network.","method":"Western blotting, qPCR, immunohistochemistry in stress-exposed mice, eQTL mapping, siRNA knockdown with expression profiling of downstream targets","journal":"Gene","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — eQTL mapping plus siRNA knockdown with multi-gene expression readout; indirect pathway placement, single lab","pmids":["26802973"],"is_preprint":false},{"year":2005,"finding":"EAPP (referred to as C14ORF11 / BM036) maps to the HPE8 locus on chromosome 14q13 and is expressed in human fetal brain, suggesting candidacy for holoprosencephaly; protein cellular localization was determined experimentally as part of candidate gene characterization.","method":"BAC contig chromosome walking, annotation of minimal critical region, expression analysis in human fetal brain, protein cellular localization assays for candidate genes","journal":"Genomics","confidence":"Low","confidence_rationale":"Tier 3 / Weak — localization and expression data for a candidate gene with no direct functional mechanistic experiment performed on EAPP/C14ORF11 itself","pmids":["15820313"],"is_preprint":false}],"current_model":"EAPP (E2F-associated phosphoprotein) is a nuclear phosphoprotein that interacts selectively with activating E2F family members (E2F-1, -2, -3 but not E2F-4) to modulate E2F-dependent transcription in a promoter-context-dependent manner; it directly binds the p21 promoter to stimulate p21 expression independently of p53, thereby promoting G1 arrest and resistance to apoptosis, and also represses MAO B transcription by competing with Sp1 for promoter occupancy, activates the MDR1/ABCB1 promoter, and modulates DNA damage signaling by promoting dephosphorylation and inactivation of Chk2."},"narrative":{"mechanistic_narrative":"EAPP (E2F-associated phosphoprotein) is a nuclear phosphoprotein that modulates transcriptional programs governing cell-cycle progression and the response to DNA damage [PMID:15716352, PMID:21258403]. Identified through its selective physical interaction with the activating E2F family members E2F-1, -2, and -3 but not E2F-4, EAPP is present throughout the cell cycle, disappears during mitosis, and shifts the population toward S-phase upon overexpression while knockdown reduces S-phase entry [PMID:15716352]. Its effect on E2F output is promoter-context-dependent: it augments E2F-1-driven activation of artificial E2F and thymidine kinase promoters yet represses E2F-1-driven activation of the p14ARF promoter [PMID:15716352]. Beyond E2F, EAPP acts directly at several promoters: it binds the p21 promoter and stimulates p21 expression independently of p53 — and is required for assembly of the transcription initiation complex there — driving G1 arrest and resistance to DNA damage- or E2F1-induced apoptosis in a p21-dependent manner [PMID:21258403]; it represses MAO B transcription by competing with Sp1 for occupancy of Sp1 sites in the core promoter [PMID:20980443]; and it activates the MDR1/ABCB1 promoter by an E2F1-independent, pRb-insensitive mechanism, raising P-glycoprotein levels [PMID:23542036]. EAPP also modulates the DNA damage checkpoint by promoting dephosphorylation and inactivation of Chk2 [PMID:21572256]. EAPP expression is itself controlled at a TATA-less promoter bearing functional Sp1, Sp3, and Egr-1 sites, with Sp1 and Egr-1 activating and Sp3 repressing it [PMID:18588995].","teleology":[{"year":2005,"claim":"Established EAPP as a physical partner of activating E2Fs and a regulator of cell-cycle progression, defining its core identity as an E2F-associated nuclear phosphoprotein.","evidence":"Yeast two-hybrid screen, cell fractionation/imaging, E2F-dependent reporter assays, and RNAi with cell-cycle analysis","pmids":["15716352"],"confidence":"High","gaps":["Interaction surface/domain on EAPP and E2F not mapped","No structural model of the EAPP-E2F complex","Mechanism by which EAPP disappears during mitosis unresolved"]},{"year":2005,"claim":"Showed that EAPP's effect on E2F-1 transcription is not uniformly activating but depends on promoter context, distinguishing it from a simple coactivator.","evidence":"Transfection reporter assays comparing artificial E2F, thymidine kinase, and p14ARF promoters","pmids":["15716352"],"confidence":"Medium","gaps":["Single method (reporter assays) without endogenous gene readout","Basis of context-dependent switch between activation and repression unknown"]},{"year":2005,"claim":"Mapped EAPP (C14ORF11/BM036) to the HPE8 holoprosencephaly locus and documented fetal brain expression, raising it as a developmental candidate gene.","evidence":"BAC contig chromosome walking, fetal brain expression, and cellular localization of candidate genes","pmids":["15820313"],"confidence":"Low","gaps":["No direct functional or mutational evidence linking EAPP to holoprosencephaly","Positional candidacy only, not causation"]},{"year":2008,"claim":"Defined how EAPP itself is transcriptionally controlled, explaining its elevated levels in transformed cells via reduced Sp3 repression.","evidence":"EMSA, supershift/competition assays, ChIP, and luciferase reporter assays with promoter truncations","pmids":["18588995"],"confidence":"High","gaps":["Whether Sp3 loss is sufficient to drive transformation phenotypes not tested","Upstream signals controlling Sp1/Sp3/Egr-1 balance at the EAPP promoter unknown"]},{"year":2010,"claim":"Identified a direct DNA-level mechanism whereby EAPP represses target genes by competing with Sp1 for promoter occupancy, demonstrated at the MAO B promoter.","evidence":"Yeast one-hybrid with Sp1 motifs, EMSA competition, ChIP, and reporter assays with dexamethasone modulation","pmids":["20980443"],"confidence":"High","gaps":["Whether EAPP binds Sp1 sites directly or via a partner not fully resolved","Generality of Sp1-competition mechanism beyond MAO B untested"]},{"year":2011,"claim":"Established EAPP as a p53-independent activator of p21 that promotes G1 arrest and apoptosis resistance, placing it in the DNA damage response.","evidence":"Overexpression/RNAi in U2OS, flow cytometry, ChIP at the p21 promoter, reporter assays, and epistasis with p21","pmids":["21258403"],"confidence":"High","gaps":["How EAPP recruits/assembles the initiation complex at p21 not mechanistically detailed","Reconciliation between S-phase promotion (2005) and G1 arrest (2011) phenotypes unresolved"]},{"year":2011,"claim":"Linked EAPP to checkpoint control by showing it promotes dephosphorylation and inactivation of Chk2.","evidence":"Overexpression/knockdown, phospho-Chk2 western blotting, and co-immunoprecipitation/binding assays","pmids":["21572256"],"confidence":"Medium","gaps":["Phosphatase mediating Chk2 dephosphorylation not identified; limited orthogonal validation","Direct vs indirect EAPP-Chk2 interaction unconfirmed"]},{"year":2013,"claim":"Showed EAPP activates the MDR1/ABCB1 promoter through a mechanism distinct from E2F1, implicating it in multidrug resistance.","evidence":"MDR1 promoter reporter assays, pRb epistasis test, and PGP western blotting","pmids":["23542036"],"confidence":"Medium","gaps":["DNA element and cofactors mediating EAPP activation of MDR1 not defined","Effect on endogenous drug resistance in cells not demonstrated"]},{"year":2015,"claim":"Extended EAPP function to injury and dual cell-type contexts, associating it with neuronal apoptosis and astrocyte proliferation in vivo.","evidence":"Rat spinal cord injury model with IHC/western, caspase-3/PCNA co-localization, and in vitro siRNA with proliferation/migration assays","pmids":["25704466"],"confidence":"Medium","gaps":["Molecular link between EAPP and caspase-3/CDK4/cyclin D1 not mechanistically established","Abstract-level description limits mechanistic depth"]},{"year":2016,"claim":"Placed EAPP upstream of a stress-response gene network in vivo and confirmed cis-genetic control of its own expression.","evidence":"eQTL mapping, qPCR/IHC in stress-exposed mice, and siRNA knockdown with downstream target profiling (including Maob)","pmids":["26802973"],"confidence":"Medium","gaps":["Direct vs indirect regulation of the named downstream genes not distinguished","Mechanistic pathway placement is correlative"]},{"year":null,"claim":"How EAPP integrates its opposing activities — promoting S-phase yet driving p21-dependent G1 arrest, and switching between transcriptional activation and repression at different promoters — into a unified mechanism remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structural or biochemical basis for the activation/repression switch","No identification of the phosphatase or cofactors mediating Chk2 dephosphorylation","Physiological role and any disease causation not established"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140110","term_label":"transcription regulator activity","supporting_discovery_ids":[1,3,4,6]},{"term_id":"GO:0003677","term_label":"DNA binding","supporting_discovery_ids":[3,4]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[0]}],"pathway":[{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[0,4]},{"term_id":"R-HSA-74160","term_label":"Gene expression (Transcription)","supporting_discovery_ids":[1,3,4,6]},{"term_id":"R-HSA-73894","term_label":"DNA Repair","supporting_discovery_ids":[4,5]}],"complexes":[],"partners":["E2F1","E2F2","E2F3","SP1","CDCA7L","CHEK2"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q56P03","full_name":"E2F-associated phosphoprotein","aliases":[],"length_aa":285,"mass_kda":32.8,"function":"May play an important role in the fine-tuning of both major E2F1 activities, the regulation of the cell-cycle and the induction of apoptosis. Promotes S-phase entry, and inhibits p14(ARP) expression","subcellular_location":"Cytoplasm; Nucleus","url":"https://www.uniprot.org/uniprotkb/Q56P03/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":true,"resolved_as":"","url":"https://depmap.org/portal/gene/EAPP","classification":"Common Essential","n_dependent_lines":863,"n_total_lines":1208,"dependency_fraction":0.7144039735099338},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"EFTUD2","stoichiometry":10.0},{"gene":"PRPF8","stoichiometry":10.0},{"gene":"CD2BP2","stoichiometry":0.2},{"gene":"FAM50A","stoichiometry":0.2},{"gene":"SNRPB","stoichiometry":0.2},{"gene":"SNRPD2","stoichiometry":0.2},{"gene":"SNRPF","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/EAPP","total_profiled":1310},"omim":[{"mim_id":"609486","title":"E2F-ASSOCIATED PHOSPHOPROTEIN; EAPP","url":"https://www.omim.org/entry/609486"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Nucleoplasm","reliability":"Supported"},{"location":"Nuclear speckles","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/EAPP"},"hgnc":{"alias_symbol":["BM036","FLJ20578"],"prev_symbol":["C14orf11"]},"alphafold":{"accession":"Q56P03","domains":[{"cath_id":"-","chopping":"179-238_247-284","consensus_level":"high","plddt":84.9994,"start":179,"end":284}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q56P03","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q56P03-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q56P03-F1-predicted_aligned_error_v6.png","plddt_mean":74.69},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=EAPP","jax_strain_url":"https://www.jax.org/strain/search?query=EAPP"},"sequence":{"accession":"Q56P03","fasta_url":"https://rest.uniprot.org/uniprotkb/Q56P03.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q56P03/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q56P03"}},"corpus_meta":[{"pmid":"16060537","id":"PMC_16060537","title":"Osteoblast elastic modulus measured by atomic force microscopy is substrate dependent.","date":"2005","source":"Annals of biomedical engineering","url":"https://pubmed.ncbi.nlm.nih.gov/16060537","citation_count":110,"is_preprint":false},{"pmid":"27336447","id":"PMC_27336447","title":"Cooperative Effect of miR-141-3p and miR-145-5p in the Regulation of Targets in Clear Cell Renal Cell Carcinoma.","date":"2016","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/27336447","citation_count":69,"is_preprint":false},{"pmid":"15352875","id":"PMC_15352875","title":"Protein interaction quantified in vivo by spectrally resolved fluorescence resonance energy transfer.","date":"2005","source":"The Biochemical journal","url":"https://pubmed.ncbi.nlm.nih.gov/15352875","citation_count":64,"is_preprint":false},{"pmid":"11679249","id":"PMC_11679249","title":"An electrochemical immunosensor for milk progesterone using a continuous flow system.","date":"2001","source":"Biosensors & bioelectronics","url":"https://pubmed.ncbi.nlm.nih.gov/11679249","citation_count":52,"is_preprint":false},{"pmid":"24209838","id":"PMC_24209838","title":"FRET spectrometry: a new tool for the determination of protein quaternary structure in living cells.","date":"2013","source":"Biophysical journal","url":"https://pubmed.ncbi.nlm.nih.gov/24209838","citation_count":48,"is_preprint":false},{"pmid":"15044149","id":"PMC_15044149","title":"Stretch-induced nitric oxide modulates mechanical properties of skeletal muscle cells.","date":"2004","source":"American journal of physiology. 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Application to the analysis of gluconic acid in musts and wines.","date":"2007","source":"Journal of agricultural and food chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/17323970","citation_count":5,"is_preprint":false},{"pmid":"15683230","id":"PMC_15683230","title":"Folding of the SARS coronavirus spike glycoprotein immunological fragment (SARS_S1b): thermodynamic and kinetic investigation correlating with three-dimensional structural modeling.","date":"2005","source":"Biochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/15683230","citation_count":4,"is_preprint":false},{"pmid":"33875994","id":"PMC_33875994","title":"The effect of Cu dopants on electron transfer to O2 and the connection with acetone photocatalytic oxidations over nano-TiO2.","date":"2021","source":"Physical chemistry chemical physics : PCCP","url":"https://pubmed.ncbi.nlm.nih.gov/33875994","citation_count":4,"is_preprint":false},{"pmid":"17192923","id":"PMC_17192923","title":"Induced and repressed genes after irradiation sensitizing by pentoxyphylline.","date":"2007","source":"International journal of cancer","url":"https://pubmed.ncbi.nlm.nih.gov/17192923","citation_count":3,"is_preprint":false},{"pmid":"36196853","id":"PMC_36196853","title":"Interplay between β-Diimino and β-Diketiminato Ligands in Nickel Complexes Active in the Proton Reduction Reaction.","date":"2022","source":"Inorganic chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/36196853","citation_count":3,"is_preprint":false},{"pmid":"26802973","id":"PMC_26802973","title":"Genetic expression analysis of E2F-associated phosphoprotein in stress responses in the mouse.","date":"2016","source":"Gene","url":"https://pubmed.ncbi.nlm.nih.gov/26802973","citation_count":2,"is_preprint":false},{"pmid":"38422872","id":"PMC_38422872","title":"Mechanical loading of ex vivo bovine trabecular bone in 3D printed bioreactor chambers.","date":"2024","source":"Journal of the mechanical behavior of biomedical materials","url":"https://pubmed.ncbi.nlm.nih.gov/38422872","citation_count":2,"is_preprint":false},{"pmid":"33399139","id":"PMC_33399139","title":"Electric field controlled uphill electron migration along α-helical oligopeptides.","date":"2021","source":"Physical chemistry chemical physics : PCCP","url":"https://pubmed.ncbi.nlm.nih.gov/33399139","citation_count":1,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":26220,"output_tokens":2736,"usd":0.05985,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":9971,"output_tokens":3430,"usd":0.067803,"stage2_stop_reason":"end_turn"},"total_usd":0.127653,"stage1_batch_id":"msgbatch_01AsR7g8dGTEiEw9U83bJeWn","stage2_batch_id":"msgbatch_01YV5ZyxtFNUZEjxRcpd9jzb","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2005,\n      \"finding\": \"EAPP (E2F-associated phosphoprotein) is a nuclear phosphoprotein identified via yeast two-hybrid screen that physically interacts with E2F-1, E2F-2, and E2F-3, but not E2F-4. EAPP is localized in the nucleus, is present throughout the cell cycle but disappears during mitosis, and its overexpression increases the fraction of cells in S-phase while RNAi-mediated knockdown reduces S-phase fraction.\",\n      \"method\": \"Yeast two-hybrid screen, nuclear localization confirmed by cell fractionation/imaging, transfection reporter assays with E2F-dependent and thymidine kinase promoters, RNAi knockdown with cell cycle analysis\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal interaction confirmed by yeast two-hybrid plus functional reporter assays and RNAi phenotype, multiple orthogonal methods in a single focused study\",\n      \"pmids\": [\"15716352\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"EAPP increases E2F-1-driven transcriptional activation from an artificial E2F-dependent promoter and the murine thymidine kinase promoter, but represses E2F-1-driven activation of the p14ARF promoter, demonstrating promoter-context-dependent modulation of E2F transcriptional output.\",\n      \"method\": \"Transfection reporter assays with E2F-dependent artificial promoter and endogenous promoter constructs\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — clean functional assay in a single lab, single method (reporter assay)\",\n      \"pmids\": [\"15716352\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"The EAPP gene promoter is TATA-less and contains functional binding sites for Sp1, Sp3, and Egr-1; Sp1 and Egr-1 activate the EAPP promoter while Sp3 represses it, and reduced Sp3 activity can account for elevated EAPP levels in transformed cells.\",\n      \"method\": \"Electrophoretic mobility shift assay (EMSA), supershift and competition assays, chromatin immunoprecipitation (ChIP), luciferase reporter assays with promoter truncations\",\n      \"journal\": \"The international journal of biochemistry & cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal methods (EMSA, ChIP, reporter assays) in a single focused study confirming both in vitro binding and in vivo occupancy\",\n      \"pmids\": [\"18588995\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"EAPP and R1 (RAM2/CDCA7L/JPO2) function as transcriptional repressors of the MAO B gene by competing with Sp1 for binding to Sp1 sites in the MAO B core promoter; in response to dexamethasone, EAPP and R1 occupancy at the MAO B promoter decreases while Sp1 occupancy increases, enabling glucocorticoid activation of MAO B.\",\n      \"method\": \"Yeast one-hybrid screen (using Sp1-binding motifs as bait), EMSA (competition assay for Sp1 site binding), chromatin immunoprecipitation (ChIP) in cells, transfection reporter assays\",\n      \"journal\": \"Molecular pharmacology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal methods (yeast one-hybrid, EMSA, ChIP, reporter assays) in a single focused study establishing mechanism in vitro and in vivo\",\n      \"pmids\": [\"20980443\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"EAPP overexpression in U2OS cells results in G1 arrest and heightened resistance to DNA damage- or E2F1-induced apoptosis in a p21-dependent manner. EAPP levels are upregulated after DNA damage and in confluent cells. EAPP binds directly to the p21 promoter and stimulates p21 expression independently of p53, and appears required for assembly of the transcription initiation complex at the p21 promoter. RNAi knockdown of EAPP increases sensitivity to DNA damage and causes apoptosis even without stress.\",\n      \"method\": \"Overexpression and RNAi knockdown in U2OS cells, flow cytometry for cell cycle and apoptosis, chromatin immunoprecipitation (ChIP) at p21 promoter, p21 promoter reporter assays, epistasis with p21\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal methods (ChIP, reporter assays, genetic epistasis with p21, KD/OE with defined phenotypes) in a single focused mechanistic study\",\n      \"pmids\": [\"21258403\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"EAPP regulates the phosphorylation status and activity of Chk2; EAPP binding appears to trigger dephosphorylation of phospho-Chk2, resulting in its inactivation, thereby modulating the DNA damage checkpoint response.\",\n      \"method\": \"Overexpression and knockdown experiments, western blotting for phospho-Chk2, co-immunoprecipitation/binding assays\",\n      \"journal\": \"Cell cycle (Georgetown, Tex.)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, mechanistic claim based on phosphorylation western blotting with limited orthogonal validation described in the abstract\",\n      \"pmids\": [\"21572256\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"EAPP stimulates the MDR1 (ABCB1) promoter resulting in elevated P-glycoprotein (PGP) levels. This activation is independent of E2F1 and is not blocked by co-expression of pRb (which does inhibit E2F1-dependent MDR1 promoter activation), indicating a distinct mechanism for EAPP-driven MDR1 regulation.\",\n      \"method\": \"Transfection reporter assays with MDR1 promoter, co-expression of pRb as epistasis test, western blotting for PGP protein levels\",\n      \"journal\": \"FEBS letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — reporter assay plus epistasis with pRb distinguishing EAPP from E2F1 mechanism, single lab\",\n      \"pmids\": [\"23542036\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"In a rat spinal cord injury model, EAPP protein levels increase and peak at day 3 post-injury in neurons and astrocytes. EAPP co-localizes with active caspase-3 in neurons (suggesting a role in neuronal apoptosis) and with PCNA in astrocytes. In vitro siRNA knockdown of EAPP in astrocytes inhibits proliferation, migration, and CDK4/cyclin D1 expression, while EAPP knockdown in neurons reduces apoptosis and cell cycle protein levels.\",\n      \"method\": \"Rat SCI model with immunohistochemistry and western blotting, co-localization with caspase-3 and PCNA, siRNA knockdown in vitro with proliferation, migration, and western blot assays\",\n      \"journal\": \"Journal of cellular biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — in vivo expression profiling combined with in vitro siRNA knockdown with defined phenotypic readouts; multiple cell types examined but abstract-level description limits mechanistic depth\",\n      \"pmids\": [\"25704466\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"In mouse hypothalamus, Eapp expression is controlled by a cis-acting quantitative trait locus (cis-eQTL). siRNA knockdown of Eapp alters expression of downstream targets Sphk2, Nosip, Mmp9, Npy, Npy5r, and Maob, placing Eapp upstream of these genes in a stress-response gene network.\",\n      \"method\": \"Western blotting, qPCR, immunohistochemistry in stress-exposed mice, eQTL mapping, siRNA knockdown with expression profiling of downstream targets\",\n      \"journal\": \"Gene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — eQTL mapping plus siRNA knockdown with multi-gene expression readout; indirect pathway placement, single lab\",\n      \"pmids\": [\"26802973\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"EAPP (referred to as C14ORF11 / BM036) maps to the HPE8 locus on chromosome 14q13 and is expressed in human fetal brain, suggesting candidacy for holoprosencephaly; protein cellular localization was determined experimentally as part of candidate gene characterization.\",\n      \"method\": \"BAC contig chromosome walking, annotation of minimal critical region, expression analysis in human fetal brain, protein cellular localization assays for candidate genes\",\n      \"journal\": \"Genomics\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — localization and expression data for a candidate gene with no direct functional mechanistic experiment performed on EAPP/C14ORF11 itself\",\n      \"pmids\": [\"15820313\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"EAPP (E2F-associated phosphoprotein) is a nuclear phosphoprotein that interacts selectively with activating E2F family members (E2F-1, -2, -3 but not E2F-4) to modulate E2F-dependent transcription in a promoter-context-dependent manner; it directly binds the p21 promoter to stimulate p21 expression independently of p53, thereby promoting G1 arrest and resistance to apoptosis, and also represses MAO B transcription by competing with Sp1 for promoter occupancy, activates the MDR1/ABCB1 promoter, and modulates DNA damage signaling by promoting dephosphorylation and inactivation of Chk2.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"EAPP (E2F-associated phosphoprotein) is a nuclear phosphoprotein that modulates transcriptional programs governing cell-cycle progression and the response to DNA damage [#0, #4]. Identified through its selective physical interaction with the activating E2F family members E2F-1, -2, and -3 but not E2F-4, EAPP is present throughout the cell cycle, disappears during mitosis, and shifts the population toward S-phase upon overexpression while knockdown reduces S-phase entry [#0]. Its effect on E2F output is promoter-context-dependent: it augments E2F-1-driven activation of artificial E2F and thymidine kinase promoters yet represses E2F-1-driven activation of the p14ARF promoter [#1]. Beyond E2F, EAPP acts directly at several promoters: it binds the p21 promoter and stimulates p21 expression independently of p53 — and is required for assembly of the transcription initiation complex there — driving G1 arrest and resistance to DNA damage- or E2F1-induced apoptosis in a p21-dependent manner [#4]; it represses MAO B transcription by competing with Sp1 for occupancy of Sp1 sites in the core promoter [#3]; and it activates the MDR1/ABCB1 promoter by an E2F1-independent, pRb-insensitive mechanism, raising P-glycoprotein levels [#6]. EAPP also modulates the DNA damage checkpoint by promoting dephosphorylation and inactivation of Chk2 [#5]. EAPP expression is itself controlled at a TATA-less promoter bearing functional Sp1, Sp3, and Egr-1 sites, with Sp1 and Egr-1 activating and Sp3 repressing it [#2].\",\n  \"teleology\": [\n    {\n      \"year\": 2005,\n      \"claim\": \"Established EAPP as a physical partner of activating E2Fs and a regulator of cell-cycle progression, defining its core identity as an E2F-associated nuclear phosphoprotein.\",\n      \"evidence\": \"Yeast two-hybrid screen, cell fractionation/imaging, E2F-dependent reporter assays, and RNAi with cell-cycle analysis\",\n      \"pmids\": [\"15716352\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Interaction surface/domain on EAPP and E2F not mapped\",\n        \"No structural model of the EAPP-E2F complex\",\n        \"Mechanism by which EAPP disappears during mitosis unresolved\"\n      ]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Showed that EAPP's effect on E2F-1 transcription is not uniformly activating but depends on promoter context, distinguishing it from a simple coactivator.\",\n      \"evidence\": \"Transfection reporter assays comparing artificial E2F, thymidine kinase, and p14ARF promoters\",\n      \"pmids\": [\"15716352\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Single method (reporter assays) without endogenous gene readout\",\n        \"Basis of context-dependent switch between activation and repression unknown\"\n      ]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Mapped EAPP (C14ORF11/BM036) to the HPE8 holoprosencephaly locus and documented fetal brain expression, raising it as a developmental candidate gene.\",\n      \"evidence\": \"BAC contig chromosome walking, fetal brain expression, and cellular localization of candidate genes\",\n      \"pmids\": [\"15820313\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\n        \"No direct functional or mutational evidence linking EAPP to holoprosencephaly\",\n        \"Positional candidacy only, not causation\"\n      ]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Defined how EAPP itself is transcriptionally controlled, explaining its elevated levels in transformed cells via reduced Sp3 repression.\",\n      \"evidence\": \"EMSA, supershift/competition assays, ChIP, and luciferase reporter assays with promoter truncations\",\n      \"pmids\": [\"18588995\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Whether Sp3 loss is sufficient to drive transformation phenotypes not tested\",\n        \"Upstream signals controlling Sp1/Sp3/Egr-1 balance at the EAPP promoter unknown\"\n      ]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Identified a direct DNA-level mechanism whereby EAPP represses target genes by competing with Sp1 for promoter occupancy, demonstrated at the MAO B promoter.\",\n      \"evidence\": \"Yeast one-hybrid with Sp1 motifs, EMSA competition, ChIP, and reporter assays with dexamethasone modulation\",\n      \"pmids\": [\"20980443\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Whether EAPP binds Sp1 sites directly or via a partner not fully resolved\",\n        \"Generality of Sp1-competition mechanism beyond MAO B untested\"\n      ]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Established EAPP as a p53-independent activator of p21 that promotes G1 arrest and apoptosis resistance, placing it in the DNA damage response.\",\n      \"evidence\": \"Overexpression/RNAi in U2OS, flow cytometry, ChIP at the p21 promoter, reporter assays, and epistasis with p21\",\n      \"pmids\": [\"21258403\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"How EAPP recruits/assembles the initiation complex at p21 not mechanistically detailed\",\n        \"Reconciliation between S-phase promotion (2005) and G1 arrest (2011) phenotypes unresolved\"\n      ]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Linked EAPP to checkpoint control by showing it promotes dephosphorylation and inactivation of Chk2.\",\n      \"evidence\": \"Overexpression/knockdown, phospho-Chk2 western blotting, and co-immunoprecipitation/binding assays\",\n      \"pmids\": [\"21572256\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Phosphatase mediating Chk2 dephosphorylation not identified; limited orthogonal validation\",\n        \"Direct vs indirect EAPP-Chk2 interaction unconfirmed\"\n      ]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Showed EAPP activates the MDR1/ABCB1 promoter through a mechanism distinct from E2F1, implicating it in multidrug resistance.\",\n      \"evidence\": \"MDR1 promoter reporter assays, pRb epistasis test, and PGP western blotting\",\n      \"pmids\": [\"23542036\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"DNA element and cofactors mediating EAPP activation of MDR1 not defined\",\n        \"Effect on endogenous drug resistance in cells not demonstrated\"\n      ]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Extended EAPP function to injury and dual cell-type contexts, associating it with neuronal apoptosis and astrocyte proliferation in vivo.\",\n      \"evidence\": \"Rat spinal cord injury model with IHC/western, caspase-3/PCNA co-localization, and in vitro siRNA with proliferation/migration assays\",\n      \"pmids\": [\"25704466\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Molecular link between EAPP and caspase-3/CDK4/cyclin D1 not mechanistically established\",\n        \"Abstract-level description limits mechanistic depth\"\n      ]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Placed EAPP upstream of a stress-response gene network in vivo and confirmed cis-genetic control of its own expression.\",\n      \"evidence\": \"eQTL mapping, qPCR/IHC in stress-exposed mice, and siRNA knockdown with downstream target profiling (including Maob)\",\n      \"pmids\": [\"26802973\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Direct vs indirect regulation of the named downstream genes not distinguished\",\n        \"Mechanistic pathway placement is correlative\"\n      ]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How EAPP integrates its opposing activities — promoting S-phase yet driving p21-dependent G1 arrest, and switching between transcriptional activation and repression at different promoters — into a unified mechanism remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"No structural or biochemical basis for the activation/repression switch\",\n        \"No identification of the phosphatase or cofactors mediating Chk2 dephosphorylation\",\n        \"Physiological role and any disease causation not established\"\n      ]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140110\", \"supporting_discovery_ids\": [1, 3, 4, 6]},\n      {\"term_id\": \"GO:0003677\", \"supporting_discovery_ids\": [3, 4]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [0]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [0, 4]},\n      {\"term_id\": \"R-HSA-74160\", \"supporting_discovery_ids\": [1, 3, 4, 6]},\n      {\"term_id\": \"R-HSA-73894\", \"supporting_discovery_ids\": [4, 5]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"E2F1\", \"E2F2\", \"E2F3\", \"Sp1\", \"CDCA7L\", \"CHEK2\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}