{"gene":"MED25","run_date":"2026-06-10T02:59:50","timeline":{"discoveries":[{"year":2004,"finding":"ARC92 (MED25) is a direct and specific binding target of the VP16 transcriptional activation domain. It associates with VP16 activation domain both in vitro (affinity chromatography) and in vivo (co-IP), and siRNA-mediated knockdown of ARC92 selectively inhibits Gal4-VP16 gene activation, establishing MED25 as a functionally important transducer of VP16 activating signals to the RNA Pol II machinery.","method":"Affinity chromatography, peptide microsequencing, co-immunoprecipitation, siRNA knockdown with reporter gene assay","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal biochemical pulldown plus in vivo co-IP plus functional siRNA knockdown, single lab but multiple orthogonal methods","pmids":["14983011"],"is_preprint":false},{"year":2007,"finding":"MED25 directly associates with retinoic acid receptor (RAR/RXR) through its C-terminal LxxLL (NR box) motif in a ligand-dependent manner, recruits CBP to the RARβ2 promoter, and enhances RAR/RXR-mediated transcription. Its PTOV domain mediates intrinsic transcriptional activity via direct association with CBP. MED25 knockdown selectively reduces RAR (but not thyroid hormone receptor) activity.","method":"Co-immunoprecipitation, chromatin immunoprecipitation (ChIP), reporter assays, dominant-negative constructs, siRNA knockdown","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (Co-IP, ChIP, reporter assays, siRNA), single lab, clear functional consequence","pmids":["17641689"],"is_preprint":false},{"year":2010,"finding":"The NMR structure of the MED25 ACID domain reveals a seven-stranded β-barrel with three α-helices, architecturally related to the SPOC domain superfamily. VP16 H2 (VP16C) interacts with MED25 ACID through one face of the β-barrel (strands B4-B7-B6).","method":"NMR structure determination, NMR chemical shift mapping","journal":"Journal of structural biology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — NMR structure with binding-interface mapping, replicated by independent structural study (PMID:21378965)","pmids":["20974256"],"is_preprint":false},{"year":2011,"finding":"NMR structure of the MED25 ACID domain (also solved independently) confirms a seven-stranded β-barrel framed by three helices. The VP16 subdomains H1 and H2 bind to opposite faces of ACID and cooperate during promoter-dependent activated transcription in an in vitro transcription system. The activator-binding ACID faces are functionally required (mutation of these faces abolishes activation) and conserved among higher eukaryotes.","method":"NMR structure determination, in vitro transcription assay, site-directed mutagenesis of ACID binding faces","journal":"Nature structural & molecular biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — NMR structure plus in vitro functional reconstitution plus mutagenesis, independently replicated structural result","pmids":["21378965"],"is_preprint":false},{"year":2011,"finding":"Solution NMR structure of MED25(391-543) ACID domain is similar to β-barrel domains of human Ku and the SPOC domain of SHARP. The domain interacts with acidic transactivation domains of HSV-1 VP16 and VZV IE62.","method":"Solution NMR structure determination, backbone heteronuclear 15N-{1H} NOE measurements","journal":"Journal of structural and functional genomics","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — NMR structure, single lab, no functional mutagenesis reported in abstract","pmids":["21785987"],"is_preprint":false},{"year":2010,"finding":"MED25 (Med25) is required for HNF4α to associate with the Mediator complex and with RNA Pol II at target promoters. Modulation of Med25 levels alters composition of the transcriptional complex and Pol II recruitment, selectively affecting a subset of HNF4α target genes involved in drug and lipid metabolism.","method":"Chromatin immunoprecipitation, co-immunoprecipitation, siRNA knockdown and overexpression with gene expression analysis","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP, ChIP, gain- and loss-of-function, multiple target genes confirmed","pmids":["21135126"],"is_preprint":false},{"year":2013,"finding":"MED25 plays a critical role in recruiting Mediator to ER stress response gene promoters via the transcription factor ATF6α. A specific MED25 domain serves as a docking site on Mediator for the ATF6α transcription activation domain.","method":"Co-immunoprecipitation, chromatin immunoprecipitation, domain-mapping experiments","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — Co-IP and ChIP with domain mapping, single lab, single study","pmids":["23864652"],"is_preprint":false},{"year":2013,"finding":"The N-terminal acidic transactivation domain (TAD) of PEA3 group ETS factors (PEA3, ERM, ER81) directly contacts the ACID/PTOV domain of MED25 to activate transcription. MED25 ACID domain depletion disrupts ERM-Mediator association in vitro. siRNA knockdown of MED25 inhibits PEA3-driven MMP-1 expression and Mediator recruitment. Mutations preventing MED25-ERM binding strongly reduce ERM transactivation.","method":"Pulldown, siRNA knockdown, reporter assay, chromatin immunoprecipitation, in vitro binding assays, mutagenesis","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (pulldown, ChIP, reporter, mutagenesis, siRNA), single lab","pmids":["23531547"],"is_preprint":false},{"year":2014,"finding":"MED25 is required for HNF4α-mediated epigenetic regulation of CYP2C9. MED25 promotes H3K27 acetylation (via CREBBP/CBP) at the CYP2C9 promoter and its silencing leads to H3K27 trimethylation via Polycomb repressive complex 2 (PRC2). Silencing MED25 increases association of PRC2 with both CYP2C9 promoter chromatin and HNF4α itself. MED25 was shown to be required for full preinitiation complex assembly in vitro.","method":"ChIP, siRNA/shRNA knockdown, FAIRE chromatin accessibility assay, in vitro transcription reconstitution, co-immunoprecipitation","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — in vitro reconstitution combined with ChIP, FAIRE, Co-IP, and loss-of-function, multiple orthogonal methods","pmids":["25391650"],"is_preprint":false},{"year":2012,"finding":"MED25 interacts with HNF4α in pancreatic β-cells via an LXXLL motif in a ligand-independent manner, and this interaction is required for full activation of HNF4α-mediated transcription of genes driving glucose-stimulated insulin secretion. MODY mutations at the LXXLL-binding pocket of HNF4α disrupt this interaction and impair insulin secretion.","method":"Yeast two-hybrid, co-immunoprecipitation (in vivo and in vitro), reporter assays, siRNA/shRNA silencing, functional insulin secretion assay","journal":"PloS one","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP, yeast two-hybrid, reporter assays, siRNA knockdown, mutagenesis, functional assay; single lab but highly orthogonal","pmids":["22952853"],"is_preprint":false},{"year":2015,"finding":"The ERM/ETV5 TAD (ERM38-68) folds from a disordered state upon binding to MED25 ACID domain, forming a fuzzy complex. Two aromatic residues in ERM TAD (F47 and W57) are critical for binding and transactivation. The ERM TAD shares a common binding interface (VP16 H1 pocket, involving MED25 Q451) with VP16 H1.","method":"NMR spectroscopy (chemical shift perturbation, NOE), mutagenesis, competition binding experiments","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 1 / Moderate — NMR structure mapping plus mutagenesis plus competition assay, single lab, multiple orthogonal NMR methods","pmids":["26130716"],"is_preprint":false},{"year":2016,"finding":"Med25 acts as a coactivator of ligand-activated ERα, interacting with ERα through its C-terminal LXXLL motif after BPA exposure, and is functionally required for BPA-induced transcriptional activation of CYP2C9.","method":"Co-immunoprecipitation, reporter assays, gene expression analysis, ChIP","journal":"Environmental toxicology","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — Co-IP, reporter assays, single lab, single study on this specific ligand/receptor combination","pmids":["27273787"],"is_preprint":false},{"year":2014,"finding":"Med25 interacts with ERα through its C-terminal LXXLL motif in a ligand-dependent (17β-estradiol) manner and is required for ERα-mediated transcriptional activation of CYP2C9.","method":"Co-immunoprecipitation, reporter assays, gene expression analysis","journal":"Biochemical pharmacology","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — Co-IP and reporter assay, single lab, single method combination","pmids":["24960263"],"is_preprint":false},{"year":2017,"finding":"ETV4 activation domain (AD) and DNA-binding domain (DBD) each independently bind MED25, with the DBD interacting with three MED25 sites allowing simultaneous binding of both domains. High-affinity MED25 interaction is specific to the ETV1/4/5 subfamily. MED25 stimulates ETV4 DNA binding in vitro by relieving autoinhibition. FOS strongly binds the same MED25 site as ETV4 AD; JUN interacts with the other two MED25 sites.","method":"NMR/biophysical binding assays, in vitro DNA binding assays, ChIP-seq, reporter assays, mutagenesis","journal":"Journal of molecular biology","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — in vitro biochemical binding assays with mutagenesis, in vitro DNA binding relief, ChIP-seq, reporter assays, multiple orthogonal methods","pmids":["28728983"],"is_preprint":false},{"year":2018,"finding":"p53 transactivation domain (TAD) interacts with MED25 ACID domain primarily through the p53TAD2 sequence motif. An amphipathic α-helix of p53TAD2 binds an elongated hydrophobic groove of MED25 ACID, using a conserved mechanism shared with ERM TAD and VP16.","method":"NMR chemical shift perturbation, isothermal titration calorimetry (ITC), mutagenesis, structural modeling","journal":"Molecules (Basel, Switzerland)","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — NMR and ITC with mutagenesis, single lab, no in vivo functional validation described in abstract","pmids":["30360415"],"is_preprint":false},{"year":2009,"finding":"A missense mutation (p.A335V) in the proline-rich, SH3-binding region of MED25 causes decreased binding specificity, leading to recognition of a broader range of SH3 domain-containing proteins, implicating this domain in protein-protein interaction selectivity.","method":"Biochemical binding assays for SH3 domain interaction specificity, genetic linkage analysis","journal":"Neurogenetics","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single binding assay with mutant, no full mechanistic reconstitution, single study","pmids":["19290556"],"is_preprint":false},{"year":2015,"finding":"A homozygous mutation p.(Tyr39Cys) in the von Willebrand factor type A (VWA) domain of MED25 dramatically impairs MED25 interaction with the Mediator complex in mammalian cells, establishing that the VWA domain mediates MED25 recruitment into the Mediator complex.","method":"Co-immunoprecipitation in mammalian cells with patient-derived mutation","journal":"Human genetics","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — Co-IP with disease-causing mutation in mammalian cells, single method, single lab","pmids":["25792360"],"is_preprint":false},{"year":2010,"finding":"PTOV1 competes with MED25 for binding to CBP, and the two proteins reciprocally regulate RAR transcriptional activity through competitive binding to CBP and opposite regulation of CBP recruitment to RA-responsive gene promoters.","method":"Co-immunoprecipitation, chromatin immunoprecipitation, reporter assays, competition binding experiments","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — Co-IP and ChIP with competition assay, single lab","pmids":["21110951"],"is_preprint":false},{"year":2019,"finding":"MED25 physically interacts with MYC2 and recruits it to JAZ gene promoters where it promotes alternative splicing of JAZ genes by recruiting the splicing factors PRP39a and PRP40a. JA-induced generation of dominant JAZ splice variants depends on MED25 and this MED25-PRP39a-PRP40a module prevents excessive desensitization of JA signaling.","method":"Co-immunoprecipitation, chromatin immunoprecipitation, genetic analysis, alternative splicing assays, loss-of-function mutants","journal":"The Plant cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP, ChIP, genetic epistasis, splicing assays; multiple orthogonal methods, single lab","pmids":["31852773"],"is_preprint":false},{"year":2017,"finding":"Plant MED25 physically interacts with COI1 (jasmonate receptor F-box protein), brings COI1 to MYC2 target promoters, facilitates COI1-dependent JAZ repressor degradation, and physically and functionally interacts with histone acetyltransferase HAC1 to selectively promote H3K9 acetylation at MYC2 target promoters. MED25 and COI1 mutually influence each other's promoter enrichment.","method":"Co-immunoprecipitation, chromatin immunoprecipitation, genetic analysis, loss-of-function mutants, histone modification assays","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP, ChIP, genetic epistasis, histone modification readout; multiple orthogonal methods","pmids":["28973940"],"is_preprint":false},{"year":2019,"finding":"MED25 is required for JA-regulated dynamic chromatin looping between jasmonate enhancers (JAEs) and their target gene promoters in a MED25-dependent manner. MED25 and MYC2 co-occupy JAEs genome-wide.","method":"ChIP-seq profiling of MYC2 and MED25, chromatin conformation capture (3C/Hi-C-type), loss-of-function mutants","journal":"Nature plants","confidence":"High","confidence_rationale":"Tier 2 / Strong — ChIP-seq genome-wide profiling plus chromatin looping assay plus loss-of-function; multiple orthogonal methods","pmids":["31182849"],"is_preprint":false},{"year":2019,"finding":"LEUNIG_HOMOLOG (LUH), a Gro/Tup1 family coregulator, is physically recruited by MED25 to MYC2 target promoters; LUH then links MYC2 with HAC1-dependent H3K9 acetylation to activate JAZ2 and LOX2. LUH promotes hormone-dependent enhancement of MYC2-MED25 and MYC2-HAC1 protein interactions.","method":"Co-immunoprecipitation, chromatin immunoprecipitation, bimolecular fluorescence complementation, loss-of-function mutants, reporter assays","journal":"The Plant cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP, ChIP, BiFC, genetic analysis, multiple orthogonal methods","pmids":["31320481"],"is_preprint":false},{"year":2021,"finding":"A short binding domain of MED25 (CMIDM) is responsible for interaction with MYC3. The MYC3-MED25 interaction is bipartite and critical for stable complex formation. Binding affinity order established: JAZJas < MED25CMIDM < JAZCMID, providing a mechanism for transcriptional activation versus negative feedback regulation in JA signaling.","method":"Biochemical binding assays (cell-free protein synthesis), quantitative affinity measurements, domain mapping","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — quantitative biochemical binding with domain mapping and mutagenesis, single lab, no in vivo ChIP","pmids":["34929168"],"is_preprint":false},{"year":2022,"finding":"EIN3/EIL1 (ethylene signaling master transcription factors) interact with the same short binding domain of MED25 (CMIDM) as MYC3, suggesting that EIN3/EIL1 and MYC compete for binding to MED25, providing a molecular basis for coordination between ethylene and jasmonate signaling.","method":"Biochemical binding assays, domain mapping, competition binding experiments","journal":"Bioscience, biotechnology, and biochemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — biochemical binding assays with domain mapping, single lab, no in vivo validation described","pmids":["35876657"],"is_preprint":false},{"year":2025,"finding":"MED16 stabilizes MED25 protein by competing with E3 ubiquitin ligases MBR1 and MBR2 for binding to the VWA domain of MED25, thereby antagonizing MBR1/2-mediated ubiquitin-proteasomal degradation of MED25. MED16 also promotes hormone-induced MYC2-MED25 interactions. Thus, the MED16-MBR1/2 module controls MED25 homeostasis to tune JA transcriptional output.","method":"Co-immunoprecipitation, ubiquitination assay, genetic epistasis, protein stability assays, domain mapping","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — Co-IP, ubiquitination assay, genetic epistasis, multiple orthogonal methods establishing molecular mechanism","pmids":["39824838"],"is_preprint":false},{"year":2022,"finding":"RSV nonstructural protein NS1 directly interacts with MED25, confirmed by co-immunoprecipitation during natural RSV infection with endogenous NS1. MED25 knockout A549 cells show enhanced RSV replication, indicating an antiviral role for MED25 during RSV infection.","method":"Three complementary PPI screens (BioID, MAPPIT, KISS), co-immunoprecipitation (overexpression and endogenous), MED25 knockout with viral replication assay","journal":"Journal of virology","confidence":"High","confidence_rationale":"Tier 2 / Strong — three orthogonal PPI screens plus endogenous Co-IP plus functional knockout, single lab but highly orthogonal methods","pmids":["36102648"],"is_preprint":false},{"year":2022,"finding":"RSV NS1 directly interacts with the MED25 ACID domain in vitro and in cellula. The interaction involves the MED25 ACID H2 face (similarly to VP16 and ATF6α TADs) and the NS1 C-terminal α3 helix plus globular domain. NS1 competes with ATF6α TAD for binding to MED25 ACID.","method":"Yeast two-hybrid, in vitro binding assay, NMR chemical shift mapping, co-immunoprecipitation, competition binding assay","journal":"Journal of molecular biology","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — NMR structure mapping plus in vitro binding plus yeast two-hybrid plus Co-IP plus competition assay; multiple orthogonal methods","pmids":["35907573"],"is_preprint":false},{"year":2025,"finding":"X-ray crystal structure of the NS1-MED25 ACID complex reveals that NS1 straddles and binds two faces of MED25 ACID, a binding mode distinct from previously known viral activators. NS1 single mutations (E110A, I54A) reduce affinity for MED25 ACID, attenuate RSV replication, and enhance antiviral ISG expression. ChIP-seq and RNA-seq identified ATF3 as a mediator of NS1/Mediator/ATF3-dependent host gene regulation during RSV infection.","method":"X-ray crystallography, NMR, mutagenesis, recombinant virus replication assay, ChIP-seq, RNA-seq","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1 / Strong — X-ray crystal structure plus NMR plus mutagenesis plus viral functional assays plus genomic studies; multiple orthogonal methods in single rigorous study","pmids":["40128225"],"is_preprint":false},{"year":2025,"finding":"NS1 α/β core domain binds MED25 ACID cooperatively with NS1 α3 helix to achieve nanomolar affinity. The dual NS1 binding site on MED25 ACID overlaps with the two canonical TAD binding interfaces, confirmed by NMR and AlphaFold prediction. NS1 E110A mutation reduces NS1-MED25 affinity, attenuates RSV replication, and upregulates antiviral ISG15. In MED25 knockdown cells, differences between WT and NS1 mutant RSV are partially lost, supporting functional relevance of the NS1-MED25 interaction in controlling antiviral responses.","method":"NMR chemical shift perturbation, AlphaFold structural prediction, mutagenesis, in vitro binding assay, recombinant virus replication assay, MED25 knockdown with ISG expression readout","journal":"PLoS pathogens","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — NMR plus mutagenesis plus in vitro binding plus functional viral assays plus knockdown, multiple orthogonal methods","pmids":["40920851"],"is_preprint":false},{"year":2024,"finding":"A lipopeptidomimetic (LPPM-8) selectively inhibits MED25 protein-protein interactions by engaging the H2 face of the MED25 ACID domain with >20-fold improved affinity over the peptide alone (Ki ~4 μM). This interaction stabilizes full-length MED25 in cells and inhibits expression of Med25-activator PPI-regulated genes in a triple-negative breast cancer cell model.","method":"Binding affinity assays, NMR/structural mapping, cellular proteome stabilization assay, gene expression analysis","journal":"Angewandte Chemie (International ed. in English)","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — biochemical binding assay with structural mapping and cellular functional readout, single lab, single study","pmids":["38527936"],"is_preprint":false},{"year":2023,"finding":"Tomato MED25 physically interacts with EIN3/EIL (EIL1) transcription factors and is required for preinitiation complex (PIC) formation during ethylene-induced gene transcription. The MED25-EIL1 module co-occupies promoters of ripening-related genes and orchestrates positive and negative feedback transcriptional circuits for ethylene homeostasis during fruit ripening.","method":"Co-immunoprecipitation, chromatin immunoprecipitation, ChIP-seq, loss-of-function mutants, reporter assays","journal":"The Plant cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — Co-IP, ChIP-seq, loss-of-function, multiple orthogonal methods","pmids":["36471914"],"is_preprint":false},{"year":2023,"finding":"MED25 physically interacts with both PIF4 and HDA9 (histone deacetylase 9) in Arabidopsis. MED25 is required for efficient binding of PIF4 to the YUCCA8 locus under warm temperatures (thermomorphogenesis) and for HDA9-mediated H2A.Z depletion at YUCCA8. MED25 also destabilizes HDA9 protein. Genetic analysis shows MED25 and HDA9 operate in the same pathway for hypocotyl elongation.","method":"Co-immunoprecipitation, chromatin immunoprecipitation, genetic epistasis analysis (double mutants), protein stability assay","journal":"Plant physiology","confidence":"High","confidence_rationale":"Tier 2 / Strong — Co-IP, ChIP, genetic epistasis, protein stability, multiple orthogonal methods","pmids":["36537119"],"is_preprint":false},{"year":2020,"finding":"Tomato MED25 physically interacts with PIF4 at promoter regions of PIF4 target genes and recruits RNA Pol II to induce gene transcription during shade-induced hypocotyl elongation. MED25 is required for PIF4-dependent transcriptional regulation of auxin biosynthesis and signaling genes under shade.","method":"Co-immunoprecipitation, chromatin immunoprecipitation, loss-of-function analysis, gene expression assays","journal":"Plant physiology","confidence":"High","confidence_rationale":"Tier 2 / Strong — Co-IP, ChIP, loss-of-function with specific gene expression readout","pmids":["32938743"],"is_preprint":false},{"year":2022,"finding":"Rice OsMED25 physically interacts with the zinc finger transcription factor DST at the promoter region of OsCKX2 and recruits RNA Pol II to activate OsCKX2 transcription. Genetic analysis shows OsMED25 acts in the same pathway as the DST-OsCKX2 module to regulate spikelet number. OsMED25 RNAi and osmed25 loss-of-function plants phenocopy dst mutants.","method":"Co-immunoprecipitation, chromatin immunoprecipitation, genetic epistasis, RNAi loss-of-function","journal":"Journal of integrative plant biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — Co-IP, ChIP, genetic epistasis, loss-of-function, single lab, multiple orthogonal methods","pmids":["35212455"],"is_preprint":false},{"year":2023,"finding":"Med25 silencing in 3T3-L1 preadipocytes enhances lipid accumulation and super-induces the master adipogenic regulators C/EBPα and PPARγ during adipogenesis, establishing Med25 as a limiter of adipogenic potential.","method":"siRNA knockdown, adipogenesis induction assay, lipid accumulation measurement, gene expression analysis","journal":"International journal of molecular sciences","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — siRNA knockdown with functional phenotype, single lab, single study, no direct mechanistic pathway placement beyond target TF identification","pmids":["37047128"],"is_preprint":false}],"current_model":"MED25 is a structurally characterized subunit of the Mediator transcriptional coactivator complex whose central ACID/PTOV domain adopts a seven-stranded β-barrel (NMR/X-ray structures solved) that directly binds the acidic transactivation domains of diverse activators—including VP16, ATF6α, p53, ETS factors (ETV1/4/5, PEA3 group), JUN/FOS, plant MYC2/MYC3, EIN3/EIL1, PIF4, and viral NS1—on two distinct faces to transduce activating signals to RNA Pol II; its N-terminal VWA domain mediates integration into the Mediator complex, its C-terminal LxxLL motif engages ligand-activated nuclear receptors (RAR, HNF4α, ERα), and its PTOV/ACID domain recruits co-regulators including CBP/CREBBP and splicing factors (PRP39a/PRP40a), enabling MED25 to orchestrate preinitiation complex assembly, histone H3K9 acetylation, H3K27 methylation status (via PRC2 antagonism), chromatin looping between enhancers and promoters, alternative splicing, and transcriptional termination; MED25 protein stability itself is controlled by E3 ubiquitin ligases MBR1/2 (which promote its degradation via the VWA domain) antagonized by MED16, providing a regulatory module that tunes transcriptional output strength."},"narrative":{"mechanistic_narrative":"MED25 is a subunit of the Mediator transcriptional coactivator that functions as a signal-specific docking hub, transducing activating signals from diverse DNA-bound transcription factors to RNA Polymerase II [PMID:14983011, PMID:21135126]. Its central ACID/PTOV domain is a seven-stranded β-barrel framed by three helices that engages the acidic transactivation domains of activators on two distinct faces (the VP16 H1 and H2 pockets), a binding mode shared by VP16, the PEA3/ETS factors ERM/ETV4/ETV5, p53, ATF6α, and the viral protein NS1 [PMID:20974256, PMID:21378965, PMID:26130716, PMID:28728983, PMID:30360415]. Activator TADs are intrinsically disordered and fold upon binding ACID to form fuzzy complexes [PMID:26130716], and MED25 can stimulate factor activity directly, as it relieves ETV4 autoinhibition to promote its DNA binding [PMID:28728983]. The N-terminal VWA domain integrates MED25 into the Mediator complex [PMID:25792360], while a C-terminal LxxLL/NR-box motif recruits ligand-activated nuclear receptors including RAR/RXR, HNF4α, and ERα [PMID:17641689, PMID:22952853, PMID:24960263]. Through these contacts MED25 enables preinitiation complex assembly and couples activators to chromatin modification, recruiting CBP/CREBBP to drive H3K27/H3K9 acetylation while its loss promotes PRC2-mediated repressive methylation [PMID:17641689, PMID:25391650]. In plants, MED25 serves as an integrating node for jasmonate, ethylene, and light signaling, physically bridging MYC2/MYC3, EIN3/EIL1, and PIF4 transcription factors to chromatin where it directs histone acetylation, dynamic enhancer–promoter looping, and alternative splicing of target genes [PMID:31852773, PMID:28973940, PMID:31182849, PMID:36471914, PMID:32938743]. MED25 also contributes to host antiviral defense, as it restricts respiratory syncytial virus replication and is targeted by RSV NS1, which competes with cellular activators for the ACID domain [PMID:36102648, PMID:35907573]. MED25 abundance is itself tuned by a regulatory module in which the E3 ligases MBR1/2 promote VWA-dependent ubiquitin-proteasomal degradation, antagonized by MED16 [PMID:39824838].","teleology":[{"year":2004,"claim":"Established that MED25 is a direct, functionally required transducer of an activator signal, defining its role as a Mediator-linked coactivator rather than a passive structural subunit.","evidence":"Affinity chromatography, co-IP, and siRNA knockdown with VP16 reporter assays in human cells","pmids":["14983011"],"confidence":"High","gaps":["Did not resolve the structural basis of VP16 recognition","Did not define which other activators use MED25"]},{"year":2007,"claim":"Showed MED25 uses distinct domains for distinct partners, with a C-terminal LxxLL motif engaging ligand-activated nuclear receptors and the PTOV domain recruiting CBP for coactivation.","evidence":"Co-IP, ChIP, reporter assays, dominant-negative constructs, and siRNA in human cells (RAR/RXR)","pmids":["17641689"],"confidence":"High","gaps":["Selectivity for RAR over thyroid receptor not structurally explained","CBP recruitment mechanism not mapped at residue level"]},{"year":2010,"claim":"Defined the ACID domain fold as a seven-stranded β-barrel and located the VP16 binding surface, providing the structural framework for activator recognition.","evidence":"NMR structure determination and chemical shift mapping; independently corroborated by a parallel NMR study and in vitro transcription","pmids":["20974256","21378965","21785987"],"confidence":"High","gaps":["How two activator faces cooperate kinetically not fully resolved","Structures of full-length MED25 not determined"]},{"year":2010,"claim":"Demonstrated MED25 is required for HNF4α to assemble Mediator and recruit Pol II at a defined subset of metabolic target genes, linking the coactivator to selective gene programs.","evidence":"ChIP, co-IP, and gain/loss-of-function with expression profiling in human cells","pmids":["21135126"],"confidence":"High","gaps":["Basis for gene-subset selectivity not defined","Direct vs Mediator-bridged contact not separated"]},{"year":2013,"claim":"Extended the activator repertoire to ETS/PEA3 factors and ATF6α and showed MED25 depletion disrupts their Mediator association, generalizing the ACID docking-site model across activator families.","evidence":"Pulldown, ChIP, reporter assays, mutagenesis, and siRNA; domain mapping for ATF6α","pmids":["23531547","23864652"],"confidence":"High","gaps":["ATF6α study was a single lab with limited orthogonal validation","In vivo specificity among ETS family members not fully resolved"]},{"year":2014,"claim":"Connected MED25 activator function to chromatin state, showing it drives CBP-dependent H3K27 acetylation and that its loss shifts loci toward PRC2-mediated repressive methylation, plus a direct PIC-assembly requirement.","evidence":"ChIP, FAIRE, in vitro transcription reconstitution, and knockdown in human cells (HNF4α/CYP2C9)","pmids":["25391650"],"confidence":"High","gaps":["Direct vs indirect PRC2 antagonism not separated","Generality beyond CYP2C9 not established"]},{"year":2012,"claim":"Showed MED25 is required for HNF4α-driven insulin secretion gene programs and that MODY-associated HNF4α mutations disrupt the interaction, linking the coactivator to physiology and disease.","evidence":"Yeast two-hybrid, co-IP, reporter assays, knockdown, mutagenesis, and insulin secretion assays in β-cells","pmids":["22952853"],"confidence":"High","gaps":["MED25's own genetic contribution to MODY not tested","Ligand-independence vs nuclear-receptor LxxLL paradigm not reconciled"]},{"year":2015,"claim":"Resolved the molecular nature of activator binding as coupled folding into a fuzzy complex, identifying critical aromatic anchor residues and a shared binding pocket with VP16.","evidence":"NMR, mutagenesis, and competition binding for ERM/ETV5 TAD on MED25 ACID","pmids":["26130716"],"confidence":"High","gaps":["Dynamics under transcription conditions not measured","Affinity differences among activators not quantified here"]},{"year":2014,"claim":"Established ERα as an additional ligand-dependent LxxLL partner required for receptor-driven transcription, reinforcing the C-terminal nuclear-receptor module.","evidence":"Co-IP, reporter assays, and expression analysis in human cells (estradiol and later BPA)","pmids":["24960263","27273787"],"confidence":"Medium","gaps":["Single-lab Co-IP/reporter combinations without structural validation","Ligand-dependent vs independent binding modes not unified"]},{"year":2017,"claim":"Showed MED25 actively stimulates factor activity—relieving ETV4 autoinhibition to enhance DNA binding—and that ETS, FOS and JUN engage overlapping or adjacent ACID sites, revealing combinatorial activator integration.","evidence":"Biophysical binding, in vitro DNA binding, ChIP-seq, reporter assays, and mutagenesis","pmids":["28728983"],"confidence":"High","gaps":["Stoichiometry of multi-factor co-occupancy in vivo not resolved","Mechanism of autoinhibition relief not structurally detailed"]},{"year":2018,"claim":"Added p53 to the set of activators recognized through the conserved ACID hydrophobic groove via its TAD2 amphipathic helix, supporting a unified recognition mechanism.","evidence":"NMR chemical shift perturbation, ITC, mutagenesis, and structural modeling","pmids":["30360415"],"confidence":"Medium","gaps":["No in vivo functional validation of the p53-MED25 axis","Single-lab biophysical study"]},{"year":2015,"claim":"Pinpointed the VWA domain as the determinant of MED25 incorporation into Mediator using a disease-associated mutation that abolishes the interaction.","evidence":"Co-IP with a patient-derived p.(Tyr39Cys) VWA mutation in mammalian cells","pmids":["25792360"],"confidence":"Medium","gaps":["Single-method evidence","Which Mediator subunits VWA contacts not identified"]},{"year":2019,"claim":"Defined MED25 as the central activator-integration node of plant jasmonate signaling, bridging MYC2 to chromatin and coupling it to histone acetylation, enhancer-promoter looping, and alternative splicing.","evidence":"Co-IP, ChIP/ChIP-seq, chromatin conformation capture, splicing assays, and loss-of-function mutants in Arabidopsis (MYC2, HAC1, COI1, LUH, PRP39a/PRP40a)","pmids":["28973940","31182849","31320481","31852773"],"confidence":"High","gaps":["Whether splicing and looping functions are mechanistically coupled is unresolved","Conservation of these chromatin functions in mammalian MED25 not tested"]},{"year":2022,"claim":"Mapped a short MED25 binding domain (CMIDM) recognized by both MYC3 and EIN3/EIL1, providing a competition-based mechanism for cross-talk between jasmonate and ethylene signaling, and extended the activator-bridging role across light and developmental pathways.","evidence":"Quantitative biochemical binding, domain mapping, competition assays, plus Co-IP/ChIP-seq for PIF4 and DST modules across plant species","pmids":["34929168","35876657","32938743","36471914","36537119","35212455"],"confidence":"High","gaps":["In vivo competition between EIN3/EIL1 and MYC not directly demonstrated","Whether one MED25 surface or several mediate these distinct factors not unified"]},{"year":2025,"claim":"Established a homeostatic control loop for MED25 abundance in which MBR1/2 E3 ligases drive VWA-dependent degradation antagonized by MED16, defining how coactivator dosage tunes transcriptional output.","evidence":"Co-IP, ubiquitination assays, genetic epistasis, and protein stability assays in Arabidopsis","pmids":["39824838"],"confidence":"High","gaps":["Whether mammalian MED25 is regulated by an analogous module unknown","Signals controlling MBR1/2 activity not defined"]},{"year":2025,"claim":"Defined MED25 as a host antiviral factor and the target of RSV NS1, which straddles two ACID faces to competitively block cellular activators and reprogram host transcription via ATF3, attenuating interferon-stimulated gene responses.","evidence":"PPI screens, endogenous Co-IP, X-ray crystallography, NMR, mutagenesis, recombinant virus replication, ChIP-seq and RNA-seq in human cells","pmids":["36102648","35907573","40128225","40920851"],"confidence":"High","gaps":["Full set of MED25-dependent antiviral target genes not defined","Whether NS1 binding alters Mediator integration not resolved"]},{"year":2024,"claim":"Provided proof of concept that the MED25 ACID H2 face is a druggable activator-interaction surface, with a lipopeptidomimetic that stabilizes MED25 and suppresses PPI-regulated genes in cancer cells.","evidence":"Binding affinity assays, structural mapping, proteome stabilization, and expression analysis in a triple-negative breast cancer model","pmids":["38527936"],"confidence":"Medium","gaps":["Single-lab study without in vivo efficacy","Selectivity across the activator repertoire not fully characterized"]},{"year":2023,"claim":"Implicated MED25 as a negative regulator of adipogenesis, limiting C/EBPα and PPARγ induction, broadening its physiological roles beyond defined activator pathways.","evidence":"siRNA knockdown with adipogenesis and lipid accumulation assays in 3T3-L1 cells","pmids":["37047128"],"confidence":"Medium","gaps":["Direct transcription factor target through which MED25 acts not identified","Mechanistic placement in adipogenic transcription circuit absent"]},{"year":null,"claim":"How MED25 selects among its many competing activator partners in a given cell, and whether its abundance-control and chromatin-remodeling functions are conserved between plant and mammalian systems, remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structure of full-length MED25 within assembled Mediator","Rules governing competitive occupancy of ACID faces in vivo unknown","Conservation of MED16/MBR1-2 dosage control outside plants untested"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140110","term_label":"transcription regulator activity","supporting_discovery_ids":[0,1,5,7,13]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[0,5,6,7,16]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[13,1,8]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[1,5,8,20]},{"term_id":"GO:0000228","term_label":"nuclear chromosome","supporting_discovery_ids":[8,20,19]}],"pathway":[{"term_id":"R-HSA-74160","term_label":"Gene expression (Transcription)","supporting_discovery_ids":[0,3,5,8]},{"term_id":"R-HSA-4839726","term_label":"Chromatin organization","supporting_discovery_ids":[8,19,20,31]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[1,6,9,18,30]},{"term_id":"R-HSA-168256","term_label":"Immune 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Diaminoamides.","date":"2023","source":"Angewandte Chemie (International ed. in English)","url":"https://pubmed.ncbi.nlm.nih.gov/37358186","citation_count":6,"is_preprint":false},{"pmid":"35876657","id":"PMC_35876657","title":"Coordinately regulated transcription factors EIN3/EIL1 and MYCs in ethylene and jasmonate signaling interact with the same domain of MED25.","date":"2022","source":"Bioscience, biotechnology, and biochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/35876657","citation_count":5,"is_preprint":false},{"pmid":"37047128","id":"PMC_37047128","title":"Med25 Limits Master Regulators That Govern Adipogenesis.","date":"2023","source":"International journal of molecular sciences","url":"https://pubmed.ncbi.nlm.nih.gov/37047128","citation_count":4,"is_preprint":false},{"pmid":"30410084","id":"PMC_30410084","title":"Origin and age of the causative mutations in KLC2, IMPA1, MED25 and WNT7A unravelled through Brazilian admixed populations.","date":"2018","source":"Scientific 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chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/38389893","citation_count":2,"is_preprint":false},{"pmid":"40920851","id":"PMC_40920851","title":"A dual interaction between RSV NS1 and MED25 ACID domain reshapes antiviral responses.","date":"2025","source":"PLoS pathogens","url":"https://pubmed.ncbi.nlm.nih.gov/40920851","citation_count":0,"is_preprint":false},{"pmid":"36993479","id":"PMC_36993479","title":"A lipopeptidomimetic of transcriptional activation domains selectively disrupts Med25 PPIs.","date":"2023","source":"bioRxiv : the preprint server for biology","url":"https://pubmed.ncbi.nlm.nih.gov/36993479","citation_count":0,"is_preprint":false},{"pmid":"40909439","id":"PMC_40909439","title":"Unraveling the pathogenicity role of the novel compound heterozygous mutations of MED25 gene in a Chinese patient with BVSYS.","date":"2025","source":"Frontiers in 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It associates with VP16 activation domain both in vitro (affinity chromatography) and in vivo (co-IP), and siRNA-mediated knockdown of ARC92 selectively inhibits Gal4-VP16 gene activation, establishing MED25 as a functionally important transducer of VP16 activating signals to the RNA Pol II machinery.\",\n      \"method\": \"Affinity chromatography, peptide microsequencing, co-immunoprecipitation, siRNA knockdown with reporter gene assay\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal biochemical pulldown plus in vivo co-IP plus functional siRNA knockdown, single lab but multiple orthogonal methods\",\n      \"pmids\": [\"14983011\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"MED25 directly associates with retinoic acid receptor (RAR/RXR) through its C-terminal LxxLL (NR box) motif in a ligand-dependent manner, recruits CBP to the RARβ2 promoter, and enhances RAR/RXR-mediated transcription. Its PTOV domain mediates intrinsic transcriptional activity via direct association with CBP. MED25 knockdown selectively reduces RAR (but not thyroid hormone receptor) activity.\",\n      \"method\": \"Co-immunoprecipitation, chromatin immunoprecipitation (ChIP), reporter assays, dominant-negative constructs, siRNA knockdown\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (Co-IP, ChIP, reporter assays, siRNA), single lab, clear functional consequence\",\n      \"pmids\": [\"17641689\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"The NMR structure of the MED25 ACID domain reveals a seven-stranded β-barrel with three α-helices, architecturally related to the SPOC domain superfamily. VP16 H2 (VP16C) interacts with MED25 ACID through one face of the β-barrel (strands B4-B7-B6).\",\n      \"method\": \"NMR structure determination, NMR chemical shift mapping\",\n      \"journal\": \"Journal of structural biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — NMR structure with binding-interface mapping, replicated by independent structural study (PMID:21378965)\",\n      \"pmids\": [\"20974256\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"NMR structure of the MED25 ACID domain (also solved independently) confirms a seven-stranded β-barrel framed by three helices. The VP16 subdomains H1 and H2 bind to opposite faces of ACID and cooperate during promoter-dependent activated transcription in an in vitro transcription system. The activator-binding ACID faces are functionally required (mutation of these faces abolishes activation) and conserved among higher eukaryotes.\",\n      \"method\": \"NMR structure determination, in vitro transcription assay, site-directed mutagenesis of ACID binding faces\",\n      \"journal\": \"Nature structural & molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — NMR structure plus in vitro functional reconstitution plus mutagenesis, independently replicated structural result\",\n      \"pmids\": [\"21378965\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Solution NMR structure of MED25(391-543) ACID domain is similar to β-barrel domains of human Ku and the SPOC domain of SHARP. The domain interacts with acidic transactivation domains of HSV-1 VP16 and VZV IE62.\",\n      \"method\": \"Solution NMR structure determination, backbone heteronuclear 15N-{1H} NOE measurements\",\n      \"journal\": \"Journal of structural and functional genomics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — NMR structure, single lab, no functional mutagenesis reported in abstract\",\n      \"pmids\": [\"21785987\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"MED25 (Med25) is required for HNF4α to associate with the Mediator complex and with RNA Pol II at target promoters. Modulation of Med25 levels alters composition of the transcriptional complex and Pol II recruitment, selectively affecting a subset of HNF4α target genes involved in drug and lipid metabolism.\",\n      \"method\": \"Chromatin immunoprecipitation, co-immunoprecipitation, siRNA knockdown and overexpression with gene expression analysis\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP, ChIP, gain- and loss-of-function, multiple target genes confirmed\",\n      \"pmids\": [\"21135126\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"MED25 plays a critical role in recruiting Mediator to ER stress response gene promoters via the transcription factor ATF6α. A specific MED25 domain serves as a docking site on Mediator for the ATF6α transcription activation domain.\",\n      \"method\": \"Co-immunoprecipitation, chromatin immunoprecipitation, domain-mapping experiments\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — Co-IP and ChIP with domain mapping, single lab, single study\",\n      \"pmids\": [\"23864652\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"The N-terminal acidic transactivation domain (TAD) of PEA3 group ETS factors (PEA3, ERM, ER81) directly contacts the ACID/PTOV domain of MED25 to activate transcription. MED25 ACID domain depletion disrupts ERM-Mediator association in vitro. siRNA knockdown of MED25 inhibits PEA3-driven MMP-1 expression and Mediator recruitment. Mutations preventing MED25-ERM binding strongly reduce ERM transactivation.\",\n      \"method\": \"Pulldown, siRNA knockdown, reporter assay, chromatin immunoprecipitation, in vitro binding assays, mutagenesis\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (pulldown, ChIP, reporter, mutagenesis, siRNA), single lab\",\n      \"pmids\": [\"23531547\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"MED25 is required for HNF4α-mediated epigenetic regulation of CYP2C9. MED25 promotes H3K27 acetylation (via CREBBP/CBP) at the CYP2C9 promoter and its silencing leads to H3K27 trimethylation via Polycomb repressive complex 2 (PRC2). Silencing MED25 increases association of PRC2 with both CYP2C9 promoter chromatin and HNF4α itself. MED25 was shown to be required for full preinitiation complex assembly in vitro.\",\n      \"method\": \"ChIP, siRNA/shRNA knockdown, FAIRE chromatin accessibility assay, in vitro transcription reconstitution, co-immunoprecipitation\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — in vitro reconstitution combined with ChIP, FAIRE, Co-IP, and loss-of-function, multiple orthogonal methods\",\n      \"pmids\": [\"25391650\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"MED25 interacts with HNF4α in pancreatic β-cells via an LXXLL motif in a ligand-independent manner, and this interaction is required for full activation of HNF4α-mediated transcription of genes driving glucose-stimulated insulin secretion. MODY mutations at the LXXLL-binding pocket of HNF4α disrupt this interaction and impair insulin secretion.\",\n      \"method\": \"Yeast two-hybrid, co-immunoprecipitation (in vivo and in vitro), reporter assays, siRNA/shRNA silencing, functional insulin secretion assay\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP, yeast two-hybrid, reporter assays, siRNA knockdown, mutagenesis, functional assay; single lab but highly orthogonal\",\n      \"pmids\": [\"22952853\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"The ERM/ETV5 TAD (ERM38-68) folds from a disordered state upon binding to MED25 ACID domain, forming a fuzzy complex. Two aromatic residues in ERM TAD (F47 and W57) are critical for binding and transactivation. The ERM TAD shares a common binding interface (VP16 H1 pocket, involving MED25 Q451) with VP16 H1.\",\n      \"method\": \"NMR spectroscopy (chemical shift perturbation, NOE), mutagenesis, competition binding experiments\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — NMR structure mapping plus mutagenesis plus competition assay, single lab, multiple orthogonal NMR methods\",\n      \"pmids\": [\"26130716\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Med25 acts as a coactivator of ligand-activated ERα, interacting with ERα through its C-terminal LXXLL motif after BPA exposure, and is functionally required for BPA-induced transcriptional activation of CYP2C9.\",\n      \"method\": \"Co-immunoprecipitation, reporter assays, gene expression analysis, ChIP\",\n      \"journal\": \"Environmental toxicology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — Co-IP, reporter assays, single lab, single study on this specific ligand/receptor combination\",\n      \"pmids\": [\"27273787\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Med25 interacts with ERα through its C-terminal LXXLL motif in a ligand-dependent (17β-estradiol) manner and is required for ERα-mediated transcriptional activation of CYP2C9.\",\n      \"method\": \"Co-immunoprecipitation, reporter assays, gene expression analysis\",\n      \"journal\": \"Biochemical pharmacology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — Co-IP and reporter assay, single lab, single method combination\",\n      \"pmids\": [\"24960263\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"ETV4 activation domain (AD) and DNA-binding domain (DBD) each independently bind MED25, with the DBD interacting with three MED25 sites allowing simultaneous binding of both domains. High-affinity MED25 interaction is specific to the ETV1/4/5 subfamily. MED25 stimulates ETV4 DNA binding in vitro by relieving autoinhibition. FOS strongly binds the same MED25 site as ETV4 AD; JUN interacts with the other two MED25 sites.\",\n      \"method\": \"NMR/biophysical binding assays, in vitro DNA binding assays, ChIP-seq, reporter assays, mutagenesis\",\n      \"journal\": \"Journal of molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — in vitro biochemical binding assays with mutagenesis, in vitro DNA binding relief, ChIP-seq, reporter assays, multiple orthogonal methods\",\n      \"pmids\": [\"28728983\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"p53 transactivation domain (TAD) interacts with MED25 ACID domain primarily through the p53TAD2 sequence motif. An amphipathic α-helix of p53TAD2 binds an elongated hydrophobic groove of MED25 ACID, using a conserved mechanism shared with ERM TAD and VP16.\",\n      \"method\": \"NMR chemical shift perturbation, isothermal titration calorimetry (ITC), mutagenesis, structural modeling\",\n      \"journal\": \"Molecules (Basel, Switzerland)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — NMR and ITC with mutagenesis, single lab, no in vivo functional validation described in abstract\",\n      \"pmids\": [\"30360415\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"A missense mutation (p.A335V) in the proline-rich, SH3-binding region of MED25 causes decreased binding specificity, leading to recognition of a broader range of SH3 domain-containing proteins, implicating this domain in protein-protein interaction selectivity.\",\n      \"method\": \"Biochemical binding assays for SH3 domain interaction specificity, genetic linkage analysis\",\n      \"journal\": \"Neurogenetics\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single binding assay with mutant, no full mechanistic reconstitution, single study\",\n      \"pmids\": [\"19290556\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"A homozygous mutation p.(Tyr39Cys) in the von Willebrand factor type A (VWA) domain of MED25 dramatically impairs MED25 interaction with the Mediator complex in mammalian cells, establishing that the VWA domain mediates MED25 recruitment into the Mediator complex.\",\n      \"method\": \"Co-immunoprecipitation in mammalian cells with patient-derived mutation\",\n      \"journal\": \"Human genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — Co-IP with disease-causing mutation in mammalian cells, single method, single lab\",\n      \"pmids\": [\"25792360\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"PTOV1 competes with MED25 for binding to CBP, and the two proteins reciprocally regulate RAR transcriptional activity through competitive binding to CBP and opposite regulation of CBP recruitment to RA-responsive gene promoters.\",\n      \"method\": \"Co-immunoprecipitation, chromatin immunoprecipitation, reporter assays, competition binding experiments\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — Co-IP and ChIP with competition assay, single lab\",\n      \"pmids\": [\"21110951\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"MED25 physically interacts with MYC2 and recruits it to JAZ gene promoters where it promotes alternative splicing of JAZ genes by recruiting the splicing factors PRP39a and PRP40a. JA-induced generation of dominant JAZ splice variants depends on MED25 and this MED25-PRP39a-PRP40a module prevents excessive desensitization of JA signaling.\",\n      \"method\": \"Co-immunoprecipitation, chromatin immunoprecipitation, genetic analysis, alternative splicing assays, loss-of-function mutants\",\n      \"journal\": \"The Plant cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP, ChIP, genetic epistasis, splicing assays; multiple orthogonal methods, single lab\",\n      \"pmids\": [\"31852773\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Plant MED25 physically interacts with COI1 (jasmonate receptor F-box protein), brings COI1 to MYC2 target promoters, facilitates COI1-dependent JAZ repressor degradation, and physically and functionally interacts with histone acetyltransferase HAC1 to selectively promote H3K9 acetylation at MYC2 target promoters. MED25 and COI1 mutually influence each other's promoter enrichment.\",\n      \"method\": \"Co-immunoprecipitation, chromatin immunoprecipitation, genetic analysis, loss-of-function mutants, histone modification assays\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP, ChIP, genetic epistasis, histone modification readout; multiple orthogonal methods\",\n      \"pmids\": [\"28973940\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"MED25 is required for JA-regulated dynamic chromatin looping between jasmonate enhancers (JAEs) and their target gene promoters in a MED25-dependent manner. MED25 and MYC2 co-occupy JAEs genome-wide.\",\n      \"method\": \"ChIP-seq profiling of MYC2 and MED25, chromatin conformation capture (3C/Hi-C-type), loss-of-function mutants\",\n      \"journal\": \"Nature plants\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — ChIP-seq genome-wide profiling plus chromatin looping assay plus loss-of-function; multiple orthogonal methods\",\n      \"pmids\": [\"31182849\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"LEUNIG_HOMOLOG (LUH), a Gro/Tup1 family coregulator, is physically recruited by MED25 to MYC2 target promoters; LUH then links MYC2 with HAC1-dependent H3K9 acetylation to activate JAZ2 and LOX2. LUH promotes hormone-dependent enhancement of MYC2-MED25 and MYC2-HAC1 protein interactions.\",\n      \"method\": \"Co-immunoprecipitation, chromatin immunoprecipitation, bimolecular fluorescence complementation, loss-of-function mutants, reporter assays\",\n      \"journal\": \"The Plant cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP, ChIP, BiFC, genetic analysis, multiple orthogonal methods\",\n      \"pmids\": [\"31320481\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"A short binding domain of MED25 (CMIDM) is responsible for interaction with MYC3. The MYC3-MED25 interaction is bipartite and critical for stable complex formation. Binding affinity order established: JAZJas < MED25CMIDM < JAZCMID, providing a mechanism for transcriptional activation versus negative feedback regulation in JA signaling.\",\n      \"method\": \"Biochemical binding assays (cell-free protein synthesis), quantitative affinity measurements, domain mapping\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — quantitative biochemical binding with domain mapping and mutagenesis, single lab, no in vivo ChIP\",\n      \"pmids\": [\"34929168\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"EIN3/EIL1 (ethylene signaling master transcription factors) interact with the same short binding domain of MED25 (CMIDM) as MYC3, suggesting that EIN3/EIL1 and MYC compete for binding to MED25, providing a molecular basis for coordination between ethylene and jasmonate signaling.\",\n      \"method\": \"Biochemical binding assays, domain mapping, competition binding experiments\",\n      \"journal\": \"Bioscience, biotechnology, and biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — biochemical binding assays with domain mapping, single lab, no in vivo validation described\",\n      \"pmids\": [\"35876657\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"MED16 stabilizes MED25 protein by competing with E3 ubiquitin ligases MBR1 and MBR2 for binding to the VWA domain of MED25, thereby antagonizing MBR1/2-mediated ubiquitin-proteasomal degradation of MED25. MED16 also promotes hormone-induced MYC2-MED25 interactions. Thus, the MED16-MBR1/2 module controls MED25 homeostasis to tune JA transcriptional output.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, genetic epistasis, protein stability assays, domain mapping\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — Co-IP, ubiquitination assay, genetic epistasis, multiple orthogonal methods establishing molecular mechanism\",\n      \"pmids\": [\"39824838\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"RSV nonstructural protein NS1 directly interacts with MED25, confirmed by co-immunoprecipitation during natural RSV infection with endogenous NS1. MED25 knockout A549 cells show enhanced RSV replication, indicating an antiviral role for MED25 during RSV infection.\",\n      \"method\": \"Three complementary PPI screens (BioID, MAPPIT, KISS), co-immunoprecipitation (overexpression and endogenous), MED25 knockout with viral replication assay\",\n      \"journal\": \"Journal of virology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — three orthogonal PPI screens plus endogenous Co-IP plus functional knockout, single lab but highly orthogonal methods\",\n      \"pmids\": [\"36102648\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"RSV NS1 directly interacts with the MED25 ACID domain in vitro and in cellula. The interaction involves the MED25 ACID H2 face (similarly to VP16 and ATF6α TADs) and the NS1 C-terminal α3 helix plus globular domain. NS1 competes with ATF6α TAD for binding to MED25 ACID.\",\n      \"method\": \"Yeast two-hybrid, in vitro binding assay, NMR chemical shift mapping, co-immunoprecipitation, competition binding assay\",\n      \"journal\": \"Journal of molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — NMR structure mapping plus in vitro binding plus yeast two-hybrid plus Co-IP plus competition assay; multiple orthogonal methods\",\n      \"pmids\": [\"35907573\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"X-ray crystal structure of the NS1-MED25 ACID complex reveals that NS1 straddles and binds two faces of MED25 ACID, a binding mode distinct from previously known viral activators. NS1 single mutations (E110A, I54A) reduce affinity for MED25 ACID, attenuate RSV replication, and enhance antiviral ISG expression. ChIP-seq and RNA-seq identified ATF3 as a mediator of NS1/Mediator/ATF3-dependent host gene regulation during RSV infection.\",\n      \"method\": \"X-ray crystallography, NMR, mutagenesis, recombinant virus replication assay, ChIP-seq, RNA-seq\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — X-ray crystal structure plus NMR plus mutagenesis plus viral functional assays plus genomic studies; multiple orthogonal methods in single rigorous study\",\n      \"pmids\": [\"40128225\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"NS1 α/β core domain binds MED25 ACID cooperatively with NS1 α3 helix to achieve nanomolar affinity. The dual NS1 binding site on MED25 ACID overlaps with the two canonical TAD binding interfaces, confirmed by NMR and AlphaFold prediction. NS1 E110A mutation reduces NS1-MED25 affinity, attenuates RSV replication, and upregulates antiviral ISG15. In MED25 knockdown cells, differences between WT and NS1 mutant RSV are partially lost, supporting functional relevance of the NS1-MED25 interaction in controlling antiviral responses.\",\n      \"method\": \"NMR chemical shift perturbation, AlphaFold structural prediction, mutagenesis, in vitro binding assay, recombinant virus replication assay, MED25 knockdown with ISG expression readout\",\n      \"journal\": \"PLoS pathogens\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — NMR plus mutagenesis plus in vitro binding plus functional viral assays plus knockdown, multiple orthogonal methods\",\n      \"pmids\": [\"40920851\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"A lipopeptidomimetic (LPPM-8) selectively inhibits MED25 protein-protein interactions by engaging the H2 face of the MED25 ACID domain with >20-fold improved affinity over the peptide alone (Ki ~4 μM). This interaction stabilizes full-length MED25 in cells and inhibits expression of Med25-activator PPI-regulated genes in a triple-negative breast cancer cell model.\",\n      \"method\": \"Binding affinity assays, NMR/structural mapping, cellular proteome stabilization assay, gene expression analysis\",\n      \"journal\": \"Angewandte Chemie (International ed. in English)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — biochemical binding assay with structural mapping and cellular functional readout, single lab, single study\",\n      \"pmids\": [\"38527936\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Tomato MED25 physically interacts with EIN3/EIL (EIL1) transcription factors and is required for preinitiation complex (PIC) formation during ethylene-induced gene transcription. The MED25-EIL1 module co-occupies promoters of ripening-related genes and orchestrates positive and negative feedback transcriptional circuits for ethylene homeostasis during fruit ripening.\",\n      \"method\": \"Co-immunoprecipitation, chromatin immunoprecipitation, ChIP-seq, loss-of-function mutants, reporter assays\",\n      \"journal\": \"The Plant cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — Co-IP, ChIP-seq, loss-of-function, multiple orthogonal methods\",\n      \"pmids\": [\"36471914\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"MED25 physically interacts with both PIF4 and HDA9 (histone deacetylase 9) in Arabidopsis. MED25 is required for efficient binding of PIF4 to the YUCCA8 locus under warm temperatures (thermomorphogenesis) and for HDA9-mediated H2A.Z depletion at YUCCA8. MED25 also destabilizes HDA9 protein. Genetic analysis shows MED25 and HDA9 operate in the same pathway for hypocotyl elongation.\",\n      \"method\": \"Co-immunoprecipitation, chromatin immunoprecipitation, genetic epistasis analysis (double mutants), protein stability assay\",\n      \"journal\": \"Plant physiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — Co-IP, ChIP, genetic epistasis, protein stability, multiple orthogonal methods\",\n      \"pmids\": [\"36537119\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Tomato MED25 physically interacts with PIF4 at promoter regions of PIF4 target genes and recruits RNA Pol II to induce gene transcription during shade-induced hypocotyl elongation. MED25 is required for PIF4-dependent transcriptional regulation of auxin biosynthesis and signaling genes under shade.\",\n      \"method\": \"Co-immunoprecipitation, chromatin immunoprecipitation, loss-of-function analysis, gene expression assays\",\n      \"journal\": \"Plant physiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — Co-IP, ChIP, loss-of-function with specific gene expression readout\",\n      \"pmids\": [\"32938743\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Rice OsMED25 physically interacts with the zinc finger transcription factor DST at the promoter region of OsCKX2 and recruits RNA Pol II to activate OsCKX2 transcription. Genetic analysis shows OsMED25 acts in the same pathway as the DST-OsCKX2 module to regulate spikelet number. OsMED25 RNAi and osmed25 loss-of-function plants phenocopy dst mutants.\",\n      \"method\": \"Co-immunoprecipitation, chromatin immunoprecipitation, genetic epistasis, RNAi loss-of-function\",\n      \"journal\": \"Journal of integrative plant biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — Co-IP, ChIP, genetic epistasis, loss-of-function, single lab, multiple orthogonal methods\",\n      \"pmids\": [\"35212455\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Med25 silencing in 3T3-L1 preadipocytes enhances lipid accumulation and super-induces the master adipogenic regulators C/EBPα and PPARγ during adipogenesis, establishing Med25 as a limiter of adipogenic potential.\",\n      \"method\": \"siRNA knockdown, adipogenesis induction assay, lipid accumulation measurement, gene expression analysis\",\n      \"journal\": \"International journal of molecular sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — siRNA knockdown with functional phenotype, single lab, single study, no direct mechanistic pathway placement beyond target TF identification\",\n      \"pmids\": [\"37047128\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"MED25 is a structurally characterized subunit of the Mediator transcriptional coactivator complex whose central ACID/PTOV domain adopts a seven-stranded β-barrel (NMR/X-ray structures solved) that directly binds the acidic transactivation domains of diverse activators—including VP16, ATF6α, p53, ETS factors (ETV1/4/5, PEA3 group), JUN/FOS, plant MYC2/MYC3, EIN3/EIL1, PIF4, and viral NS1—on two distinct faces to transduce activating signals to RNA Pol II; its N-terminal VWA domain mediates integration into the Mediator complex, its C-terminal LxxLL motif engages ligand-activated nuclear receptors (RAR, HNF4α, ERα), and its PTOV/ACID domain recruits co-regulators including CBP/CREBBP and splicing factors (PRP39a/PRP40a), enabling MED25 to orchestrate preinitiation complex assembly, histone H3K9 acetylation, H3K27 methylation status (via PRC2 antagonism), chromatin looping between enhancers and promoters, alternative splicing, and transcriptional termination; MED25 protein stability itself is controlled by E3 ubiquitin ligases MBR1/2 (which promote its degradation via the VWA domain) antagonized by MED16, providing a regulatory module that tunes transcriptional output strength.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"MED25 is a subunit of the Mediator transcriptional coactivator that functions as a signal-specific docking hub, transducing activating signals from diverse DNA-bound transcription factors to RNA Polymerase II [#0, #5]. Its central ACID/PTOV domain is a seven-stranded β-barrel framed by three helices that engages the acidic transactivation domains of activators on two distinct faces (the VP16 H1 and H2 pockets), a binding mode shared by VP16, the PEA3/ETS factors ERM/ETV4/ETV5, p53, ATF6α, and the viral protein NS1 [#2, #3, #10, #13, #14]. Activator TADs are intrinsically disordered and fold upon binding ACID to form fuzzy complexes [#10], and MED25 can stimulate factor activity directly, as it relieves ETV4 autoinhibition to promote its DNA binding [#13]. The N-terminal VWA domain integrates MED25 into the Mediator complex [#16], while a C-terminal LxxLL/NR-box motif recruits ligand-activated nuclear receptors including RAR/RXR, HNF4α, and ERα [#1, #9, #12]. Through these contacts MED25 enables preinitiation complex assembly and couples activators to chromatin modification, recruiting CBP/CREBBP to drive H3K27/H3K9 acetylation while its loss promotes PRC2-mediated repressive methylation [#1, #8]. In plants, MED25 serves as an integrating node for jasmonate, ethylene, and light signaling, physically bridging MYC2/MYC3, EIN3/EIL1, and PIF4 transcription factors to chromatin where it directs histone acetylation, dynamic enhancer–promoter looping, and alternative splicing of target genes [#18, #19, #20, #30, #32]. MED25 also contributes to host antiviral defense, as it restricts respiratory syncytial virus replication and is targeted by RSV NS1, which competes with cellular activators for the ACID domain [#25, #26]. MED25 abundance is itself tuned by a regulatory module in which the E3 ligases MBR1/2 promote VWA-dependent ubiquitin-proteasomal degradation, antagonized by MED16 [#24].\",\n  \"teleology\": [\n    {\n      \"year\": 2004,\n      \"claim\": \"Established that MED25 is a direct, functionally required transducer of an activator signal, defining its role as a Mediator-linked coactivator rather than a passive structural subunit.\",\n      \"evidence\": \"Affinity chromatography, co-IP, and siRNA knockdown with VP16 reporter assays in human cells\",\n      \"pmids\": [\"14983011\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not resolve the structural basis of VP16 recognition\", \"Did not define which other activators use MED25\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Showed MED25 uses distinct domains for distinct partners, with a C-terminal LxxLL motif engaging ligand-activated nuclear receptors and the PTOV domain recruiting CBP for coactivation.\",\n      \"evidence\": \"Co-IP, ChIP, reporter assays, dominant-negative constructs, and siRNA in human cells (RAR/RXR)\",\n      \"pmids\": [\"17641689\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Selectivity for RAR over thyroid receptor not structurally explained\", \"CBP recruitment mechanism not mapped at residue level\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Defined the ACID domain fold as a seven-stranded β-barrel and located the VP16 binding surface, providing the structural framework for activator recognition.\",\n      \"evidence\": \"NMR structure determination and chemical shift mapping; independently corroborated by a parallel NMR study and in vitro transcription\",\n      \"pmids\": [\"20974256\", \"21378965\", \"21785987\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How two activator faces cooperate kinetically not fully resolved\", \"Structures of full-length MED25 not determined\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Demonstrated MED25 is required for HNF4α to assemble Mediator and recruit Pol II at a defined subset of metabolic target genes, linking the coactivator to selective gene programs.\",\n      \"evidence\": \"ChIP, co-IP, and gain/loss-of-function with expression profiling in human cells\",\n      \"pmids\": [\"21135126\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Basis for gene-subset selectivity not defined\", \"Direct vs Mediator-bridged contact not separated\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Extended the activator repertoire to ETS/PEA3 factors and ATF6α and showed MED25 depletion disrupts their Mediator association, generalizing the ACID docking-site model across activator families.\",\n      \"evidence\": \"Pulldown, ChIP, reporter assays, mutagenesis, and siRNA; domain mapping for ATF6α\",\n      \"pmids\": [\"23531547\", \"23864652\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"ATF6α study was a single lab with limited orthogonal validation\", \"In vivo specificity among ETS family members not fully resolved\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Connected MED25 activator function to chromatin state, showing it drives CBP-dependent H3K27 acetylation and that its loss shifts loci toward PRC2-mediated repressive methylation, plus a direct PIC-assembly requirement.\",\n      \"evidence\": \"ChIP, FAIRE, in vitro transcription reconstitution, and knockdown in human cells (HNF4α/CYP2C9)\",\n      \"pmids\": [\"25391650\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct vs indirect PRC2 antagonism not separated\", \"Generality beyond CYP2C9 not established\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Showed MED25 is required for HNF4α-driven insulin secretion gene programs and that MODY-associated HNF4α mutations disrupt the interaction, linking the coactivator to physiology and disease.\",\n      \"evidence\": \"Yeast two-hybrid, co-IP, reporter assays, knockdown, mutagenesis, and insulin secretion assays in β-cells\",\n      \"pmids\": [\"22952853\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"MED25's own genetic contribution to MODY not tested\", \"Ligand-independence vs nuclear-receptor LxxLL paradigm not reconciled\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Resolved the molecular nature of activator binding as coupled folding into a fuzzy complex, identifying critical aromatic anchor residues and a shared binding pocket with VP16.\",\n      \"evidence\": \"NMR, mutagenesis, and competition binding for ERM/ETV5 TAD on MED25 ACID\",\n      \"pmids\": [\"26130716\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Dynamics under transcription conditions not measured\", \"Affinity differences among activators not quantified here\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Established ERα as an additional ligand-dependent LxxLL partner required for receptor-driven transcription, reinforcing the C-terminal nuclear-receptor module.\",\n      \"evidence\": \"Co-IP, reporter assays, and expression analysis in human cells (estradiol and later BPA)\",\n      \"pmids\": [\"24960263\", \"27273787\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-lab Co-IP/reporter combinations without structural validation\", \"Ligand-dependent vs independent binding modes not unified\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Showed MED25 actively stimulates factor activity—relieving ETV4 autoinhibition to enhance DNA binding—and that ETS, FOS and JUN engage overlapping or adjacent ACID sites, revealing combinatorial activator integration.\",\n      \"evidence\": \"Biophysical binding, in vitro DNA binding, ChIP-seq, reporter assays, and mutagenesis\",\n      \"pmids\": [\"28728983\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Stoichiometry of multi-factor co-occupancy in vivo not resolved\", \"Mechanism of autoinhibition relief not structurally detailed\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Added p53 to the set of activators recognized through the conserved ACID hydrophobic groove via its TAD2 amphipathic helix, supporting a unified recognition mechanism.\",\n      \"evidence\": \"NMR chemical shift perturbation, ITC, mutagenesis, and structural modeling\",\n      \"pmids\": [\"30360415\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No in vivo functional validation of the p53-MED25 axis\", \"Single-lab biophysical study\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Pinpointed the VWA domain as the determinant of MED25 incorporation into Mediator using a disease-associated mutation that abolishes the interaction.\",\n      \"evidence\": \"Co-IP with a patient-derived p.(Tyr39Cys) VWA mutation in mammalian cells\",\n      \"pmids\": [\"25792360\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-method evidence\", \"Which Mediator subunits VWA contacts not identified\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Defined MED25 as the central activator-integration node of plant jasmonate signaling, bridging MYC2 to chromatin and coupling it to histone acetylation, enhancer-promoter looping, and alternative splicing.\",\n      \"evidence\": \"Co-IP, ChIP/ChIP-seq, chromatin conformation capture, splicing assays, and loss-of-function mutants in Arabidopsis (MYC2, HAC1, COI1, LUH, PRP39a/PRP40a)\",\n      \"pmids\": [\"28973940\", \"31182849\", \"31320481\", \"31852773\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether splicing and looping functions are mechanistically coupled is unresolved\", \"Conservation of these chromatin functions in mammalian MED25 not tested\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Mapped a short MED25 binding domain (CMIDM) recognized by both MYC3 and EIN3/EIL1, providing a competition-based mechanism for cross-talk between jasmonate and ethylene signaling, and extended the activator-bridging role across light and developmental pathways.\",\n      \"evidence\": \"Quantitative biochemical binding, domain mapping, competition assays, plus Co-IP/ChIP-seq for PIF4 and DST modules across plant species\",\n      \"pmids\": [\"34929168\", \"35876657\", \"32938743\", \"36471914\", \"36537119\", \"35212455\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo competition between EIN3/EIL1 and MYC not directly demonstrated\", \"Whether one MED25 surface or several mediate these distinct factors not unified\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Established a homeostatic control loop for MED25 abundance in which MBR1/2 E3 ligases drive VWA-dependent degradation antagonized by MED16, defining how coactivator dosage tunes transcriptional output.\",\n      \"evidence\": \"Co-IP, ubiquitination assays, genetic epistasis, and protein stability assays in Arabidopsis\",\n      \"pmids\": [\"39824838\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether mammalian MED25 is regulated by an analogous module unknown\", \"Signals controlling MBR1/2 activity not defined\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Defined MED25 as a host antiviral factor and the target of RSV NS1, which straddles two ACID faces to competitively block cellular activators and reprogram host transcription via ATF3, attenuating interferon-stimulated gene responses.\",\n      \"evidence\": \"PPI screens, endogenous Co-IP, X-ray crystallography, NMR, mutagenesis, recombinant virus replication, ChIP-seq and RNA-seq in human cells\",\n      \"pmids\": [\"36102648\", \"35907573\", \"40128225\", \"40920851\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Full set of MED25-dependent antiviral target genes not defined\", \"Whether NS1 binding alters Mediator integration not resolved\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Provided proof of concept that the MED25 ACID H2 face is a druggable activator-interaction surface, with a lipopeptidomimetic that stabilizes MED25 and suppresses PPI-regulated genes in cancer cells.\",\n      \"evidence\": \"Binding affinity assays, structural mapping, proteome stabilization, and expression analysis in a triple-negative breast cancer model\",\n      \"pmids\": [\"38527936\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-lab study without in vivo efficacy\", \"Selectivity across the activator repertoire not fully characterized\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Implicated MED25 as a negative regulator of adipogenesis, limiting C/EBPα and PPARγ induction, broadening its physiological roles beyond defined activator pathways.\",\n      \"evidence\": \"siRNA knockdown with adipogenesis and lipid accumulation assays in 3T3-L1 cells\",\n      \"pmids\": [\"37047128\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct transcription factor target through which MED25 acts not identified\", \"Mechanistic placement in adipogenic transcription circuit absent\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How MED25 selects among its many competing activator partners in a given cell, and whether its abundance-control and chromatin-remodeling functions are conserved between plant and mammalian systems, remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structure of full-length MED25 within assembled Mediator\", \"Rules governing competitive occupancy of ACID faces in vivo unknown\", \"Conservation of MED16/MBR1-2 dosage control outside plants untested\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140110\", \"supporting_discovery_ids\": [0, 1, 5, 7, 13]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [0, 5, 6, 7, 16]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [13, 1, 8]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [1, 5, 8, 20]},\n      {\"term_id\": \"GO:0000228\", \"supporting_discovery_ids\": [8, 20, 19]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-74160\", \"supporting_discovery_ids\": [0, 3, 5, 8]},\n      {\"term_id\": \"R-HSA-4839726\", \"supporting_discovery_ids\": [8, 19, 20, 31]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [1, 6, 9, 18, 30]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [25, 26, 27]},\n      {\"term_id\": \"R-HSA-8953854\", \"supporting_discovery_ids\": [18]}\n    ],\n    \"complexes\": [\"Mediator complex\"],\n    \"partners\": [\"HNF4A\", \"CREBBP\", \"ESR1\", \"ETV4\", \"MYC2\", \"EIN3\", \"PIF4\", \"MED16\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":8,"faith_total":8,"faith_pct":100.0}}