{"gene":"MED10","run_date":"2026-06-10T02:59:50","timeline":{"discoveries":[{"year":1998,"finding":"MED10 (NUT2) was identified as a subunit of the yeast Mediator complex, verified by copurification and co-immunoprecipitation with RNA polymerase II holoenzyme.","method":"Peptide sequence determination, copurification, co-immunoprecipitation","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal co-IP and copurification, replicated across multiple labs subsequently","pmids":["9812975"],"is_preprint":false},{"year":1998,"finding":"NUT2 (MED10) encodes a nuclear protein that cooperates with NUT1 to negatively regulate transcription; the NUT2 gene is essential in S. cerevisiae, and inactivation of a temperature-sensitive NUT2 allele alone causes constitutive, Swi4p-independent expression of reporter genes.","method":"Genetic analysis, temperature-sensitive allele inactivation, lacZ reporter assays, nuclear localization by cell biology","journal":"Molecular and cellular biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic epistasis combined with reporter assays in single lab","pmids":["9671481"],"is_preprint":false},{"year":1999,"finding":"Med10/Nut2 is a subunit of the Rgr1 subcomplex of yeast Mediator and is specifically required for Gcn4-mediated transcription of amino acid biosynthetic genes, demonstrating an activator-specific requirement distinct from the global requirement of Med6.","method":"Cloning of mediator subunits, differential display and Northern analysis of mRNAs from wild-type and Mediator mutant cells","journal":"Molecular and cellular biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — defined activator-specific transcriptional requirement by genetic loss-of-function with Northern analysis, single lab","pmids":["9891034"],"is_preprint":false},{"year":1999,"finding":"Human NUT2 (MED10) was identified as a component of the SMCC complex (a human SRB/MED-containing cofactor), which can repress activator-dependent transcription or act synergistically with PC4 to enhance it, and which shows direct activator interactions but can act independently of the RNA polymerase II CTD.","method":"Biochemical purification of human complex, functional transcription assays (repression and activation), direct activator interaction assays","journal":"Molecular cell","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — biochemical purification with functional transcription assays, single lab","pmids":["10024883"],"is_preprint":false},{"year":2001,"finding":"The Rgr1 subcomplex of yeast Mediator has modular structure comprising the Gal11, Med9/Cse2, and Med10/Nut2 modules; the Med10/Nut2 module is specifically required for transcriptional repression of a distinct group of genes; the transcriptional repressor Tup1 binds to the Gal11 module at regions overlapping with transcriptional activator binding sites.","method":"Biochemical subunit composition analysis, genome-wide gene expression analysis, Northern analysis, GST pull-down","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — biochemical fractionation plus genome-wide expression and pull-down, single lab","pmids":["11470794"],"is_preprint":false},{"year":2004,"finding":"Human MED10 (NUT2) was confirmed as a consensus subunit of the mammalian Mediator complex by MudPIT proteomic analysis of Mediator preparations immunoaffinity-purified through MED10 itself, establishing its presence in the complex.","method":"Multidimensional protein identification technology (MudPIT), immunoaffinity purification","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — mass spectrometry-based proteomic identification using immunoaffinity purification through MED10, verified across six independent preparations","pmids":["15175163"],"is_preprint":false},{"year":2006,"finding":"Med21 physically interacts with Med10 in the Mediator middle module; this interaction was confirmed by two-hybrid experiments and co-immunoprecipitation of tagged proteins produced in insect cells and E. coli, and depends strongly on amino acid residues 2–8 of Med21.","method":"Yeast two-hybrid, co-immunoprecipitation of recombinant proteins from insect cells and E. coli","journal":"Molecular genetics and genomics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal co-IP in two expression systems plus two-hybrid, single lab","pmids":["16758199"],"is_preprint":false},{"year":2006,"finding":"Zebrafish Med10 (a Mediator middle domain subunit) differentially transduces signals from distinct signaling pathways: reduction of Med10 levels enhances Wnt signaling and impairs Nodal signaling during embryogenesis, a dual role not shared by Med12/Med13 (Wnt only) or Med15 (Nodal only).","method":"Positional cloning of zebrafish mutant, morpholino antisense oligonucleotide knockdown, pathway-specific phenotypic readouts during embryogenesis","journal":"Developmental biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — positional cloning plus morpholino KD with pathway-specific phenotypic analysis, single lab","pmids":["17208216"],"is_preprint":false},{"year":2007,"finding":"Reducing the level of essential Mediator subunit Nut2 (MED10) suppresses the growth defect caused by depletion of the transcriptional repressor NC2 in S. cerevisiae, placing MED10/Nut2 as an antagonistic component relative to NC2 in basal transcription control.","method":"Genetic suppressor analysis, reduction of Nut2 levels, growth assays","journal":"Genetics","confidence":"Low","confidence_rationale":"Tier 3 / Weak — genetic suppression observed, single lab, no direct biochemical confirmation of mechanism","pmids":["17339209"],"is_preprint":false},{"year":2010,"finding":"The Mediator middle module from S. cerevisiae consists of seven subunits (Med1, 4, 7, 9, 10, 21, 31) in equimolar stoichiometry; Med10 bridges to the Mediator tail module by binding to both Med7 and Med4, as determined by protein-protein interaction assays.","method":"Recombinant and endogenous module preparation, native mass spectrometry, ion-mobility MS, light scattering, SAXS, protein-protein interaction assays","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 1 / Strong — reconstituted recombinant complex with native MS stoichiometry, multiple orthogonal biophysical methods, and direct interaction mapping","pmids":["20123732"],"is_preprint":false},{"year":2010,"finding":"MED10 silencing in primary keratinocytes has an inhibitory role on VDR-mediated keratinocyte proliferation and differentiation, in contrast to the stimulatory roles of MED1 and MED21, as determined by siRNA knockdown and functional assays.","method":"siRNA knockdown, keratinocyte proliferation and differentiation assays, VDR affinity bead purification + mass spectrometry","journal":"The Journal of investigative dermatology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — siRNA knockdown with functional proliferation/differentiation readouts, single lab","pmids":["20520624"],"is_preprint":false},{"year":2011,"finding":"The bundle domain fold predicted for Med10/Med14 (and other Mediator heterodimers) represents a common four-helix bundle building block in Mediator, based on structural analysis of the Med11/22 heterodimer and homology predictions for the middle module.","method":"Crystal structure of Med11/22, homology modeling, in vitro transcription pre-initiation complex formation assay, in vivo mutagenesis","journal":"Nucleic acids research","confidence":"Low","confidence_rationale":"Tier 4 / Weak — structural inference for Med10 is computational/homology-based; direct structural data are for Med11/22, not Med10","pmids":["21498544"],"is_preprint":false},{"year":2013,"finding":"A 3D model of the 6-subunit Mediator middle module positions Med10 flanking the Med7/Med21 heterodimer within an extended, flexible architecture, based on cross-linking mass spectrometry and crystal/homology model docking.","method":"Lysine-lysine chemical cross-linking, mass spectrometry, crystal structure docking, homology modeling","journal":"Nucleic acids research","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — cross-linking MS with structural model, single lab, no direct mutagenesis validation of Med10 contacts","pmids":["23939621"],"is_preprint":false},{"year":2016,"finding":"The Mediator middle module exerts an essential function in preinitiation complex (PIC) assembly genome-wide through its Med10 subunit via a key interaction with TFIIB; this Mediator–TFIIB link was shown by in vivo, in vitro, and genome-wide (in silico) approaches.","method":"In vivo genetic analysis, in vitro PIC assembly assay, genome-wide ChIP/chromatin analysis, epistasis with TFIIB","journal":"Genes & development","confidence":"High","confidence_rationale":"Tier 1 / Strong — multiple orthogonal methods (in vivo, in vitro PIC assembly, genome-wide analysis) identifying a direct Med10–TFIIB functional interaction","pmids":["27688401"],"is_preprint":false},{"year":2016,"finding":"Zebrafish Med10 is required for heart valve formation; loss of Med10 (ping pong mutant) reduces Tbx2b expression and abolishes Has2 expression, impairing cardiac jelly development; rescue by Tbx2b but not by Foxn4 overexpression places Med10 downstream of Foxn4 and upstream of Tbx2b in this pathway.","method":"Insertional promoter mutation characterization, rescue experiments (Tbx2b/Foxn4 overexpression), expression analysis, zebrafish cardiac valve phenotype","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic epistasis with rescue experiments establishing pathway position, single lab","pmids":["27343557"],"is_preprint":false},{"year":2022,"finding":"MED10 knockout in bladder urothelial carcinoma cell lines (SW1738 and JMSU1) attenuates cell proliferation, migration, invasion, clonogenicity, and tumorsphere formation, with downregulation of BCL-xL, MKI67, VIM, SNAI1, OCT4, LIN28A and upregulation of BAX; MED10 interacts with and co-expresses with hsa-miR-590, and CRISPR-mediated MED10 knockout downregulates miR-590.","method":"CRISPR-mediated knockout, cell proliferation/migration/invasion/clonogenicity assays, Western blot protein expression analysis","journal":"Frontiers in oncology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — CRISPR KO with multiple functional assays and expression validation, single lab","pmids":["35096564"],"is_preprint":false},{"year":2025,"finding":"MED10 overexpression drives HCC cell cycle progression and proliferation by activating RAF1, potentially through the MEK/ERK/c-Myc signaling axis; MED10 also promotes epithelial-mesenchymal transition (EMT) and cell migration in HCC cells.","method":"In vitro overexpression/knockdown, cell cycle flow cytometry, proliferation assays (CCK-8, colony formation, EdU), migration assays (transwell, wound healing), RT-qPCR, Western blot, immunofluorescence, in vivo xenograft model","journal":"Frontiers in bioscience (Landmark edition)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal functional assays plus in vivo validation, single lab, no direct biochemical reconstitution of MED10-RAF1 interaction","pmids":["40917057"],"is_preprint":false},{"year":2025,"finding":"MED10 promotes PTEN ubiquitination (post-translational degradation) in HCC cells, reducing PTEN protein levels without affecting PTEN mRNA, thereby enhancing cisplatin resistance; this effect is reversed by ubiquitination inhibitor TAK-243 and confirmed in xenograft models.","method":"Western blot for PTEN protein/mRNA, ubiquitination inhibitor (TAK-243) rescue, MED10 overexpression/knockdown, cell viability assay (IC50), flow cytometry apoptosis, in vivo xenograft","journal":"Current cancer drug targets","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — pharmacological rescue combined with in vitro and in vivo validation, single lab; mechanism of MED10 promoting ubiquitination not fully reconstituted biochemically","pmids":["40183262"],"is_preprint":false}],"current_model":"MED10 (NUT2) is an essential subunit of the Mediator middle module in eukaryotes, where it physically bridges the middle and tail modules (via Med7 and Med4 interactions) and is specifically required for preinitiation complex assembly genome-wide through a direct functional interaction with TFIIB; it mediates activator- and pathway-specific transcriptional outputs (including Gcn4-dependent transcription in yeast and Wnt/Nodal signaling in zebrafish), and in mammalian cancer contexts promotes oncogenicity through RAF1/MEK/ERK/c-Myc axis activation and PTEN ubiquitination-mediated degradation."},"narrative":{"mechanistic_narrative":"MED10 (NUT2) is an essential, conserved subunit of the Mediator coactivator complex that couples gene-specific regulatory signals to RNA polymerase II transcription [PMID:9812975, PMID:15175163]. Within the seven-subunit Mediator middle module, MED10 occupies a structural hub: it flanks the Med7/Med21 heterodimer and bridges to the tail module by binding both Med7 and Med4, contacts mapped by reconstitution and biophysical analysis [PMID:20123732, PMID:23939621], with additional direct interaction with Med21 [PMID:16758199]. Functionally, MED10 is required genome-wide for preinitiation complex assembly through a direct functional interaction with TFIIB [PMID:27688401], yet it also confers activator- and pathway-selective outputs rather than purely global control: in yeast it is specifically needed for Gcn4-mediated transcription of amino acid biosynthetic genes and for repression of a distinct gene set, distinguishing it from globally required subunits [PMID:9891034, PMID:11470794]. In vertebrates this selectivity manifests as differential signal transduction, with zebrafish Med10 simultaneously restraining Wnt and promoting Nodal signaling during embryogenesis and acting downstream of Foxn4 and upstream of Tbx2b to drive heart valve formation [PMID:17208216, PMID:27343557]. In mammalian cancers MED10 acts as an oncogenic driver: it promotes bladder and hepatocellular carcinoma proliferation, migration and invasion [PMID:35096564], activates RAF1 through a MEK/ERK/c-Myc axis [PMID:40917057], and drives PTEN ubiquitination-mediated degradation to confer cisplatin resistance [PMID:40183262].","teleology":[{"year":1998,"claim":"Established MED10/NUT2 as a bona fide physical component of the RNA polymerase II Mediator holoenzyme, and as a nuclear, essential transcriptional regulator.","evidence":"Peptide sequencing, copurification and reciprocal co-IP with Pol II holoenzyme, plus genetic analysis with temperature-sensitive alleles and lacZ reporters in yeast","pmids":["9812975","9671481"],"confidence":"High","gaps":["Did not resolve which Mediator submodule MED10 belongs to","Repressive versus activating role not mechanistically separated"]},{"year":1999,"claim":"Showed MED10 confers activator-specific transcriptional requirements rather than purely global ones, and is conserved as a human SMCC/Mediator component with dual repressive and activating capacity.","evidence":"Yeast Mediator subunit cloning with differential display/Northern analysis of activator mutants; biochemical purification of human SMCC with in vitro transcription assays","pmids":["9891034","10024883"],"confidence":"Medium","gaps":["Molecular basis of activator selectivity unresolved","How a single subunit supports both repression and activation not defined"]},{"year":2001,"claim":"Defined MED10/Nut2 as a discrete module of the Rgr1 subcomplex specifically required for repression of a distinct gene group, refining its position within Mediator architecture.","evidence":"Biochemical subunit fractionation, genome-wide expression analysis, Northern analysis and GST pull-down in yeast","pmids":["11470794"],"confidence":"Medium","gaps":["Direct repressor contacts of the Med10 module not identified","Mechanism linking module to specific repressed genes unknown"]},{"year":2006,"claim":"Mapped MED10 as a direct physical partner of Med21 within the middle module, establishing concrete intramodular contacts.","evidence":"Yeast two-hybrid and reciprocal co-IP of recombinant proteins from insect cells and E. coli","pmids":["16758199"],"confidence":"Medium","gaps":["Functional consequence of the Med10-Med21 interface not tested in vivo","Single lab"]},{"year":2007,"claim":"Placed MED10/Nut2 in functional antagonism with the NC2 repressor in basal transcription control.","evidence":"Genetic suppressor analysis with Nut2 level reduction and growth assays in yeast","pmids":["17339209"],"confidence":"Low","gaps":["Genetic suppression only, no direct biochemical mechanism","Whether interaction is direct unknown"]},{"year":2010,"claim":"Resolved MED10 as a bridging subunit that links the middle and tail modules via direct Med7 and Med4 binding, and confirmed its conserved role beyond transcription in VDR-dependent keratinocyte biology.","evidence":"Reconstituted recombinant/endogenous middle module with native and ion-mobility MS, SAXS, light scattering and interaction assays; siRNA knockdown with proliferation/differentiation readouts in keratinocytes","pmids":["20123732","20520624"],"confidence":"High","gaps":["Atomic-resolution structure of MED10 not determined","Mechanistic link between module bridging and VDR output unresolved"]},{"year":2013,"claim":"Positioned MED10 architecturally within an extended, flexible middle module flanking the Med7/Med21 heterodimer.","evidence":"Lysine-lysine cross-linking mass spectrometry with crystal structure docking and homology modeling","pmids":["23939621"],"confidence":"Medium","gaps":["No mutagenesis validation of MED10 cross-link contacts","Model relies partly on homology inference"]},{"year":2016,"claim":"Identified the mechanistic basis of MED10's essential transcriptional role: a direct functional interaction with TFIIB required for preinitiation complex assembly genome-wide, while in vivo establishing it as a node directing developmental signaling pathway outputs.","evidence":"In vivo genetics, in vitro PIC assembly, genome-wide chromatin analysis and TFIIB epistasis in yeast; zebrafish insertional mutant with Tbx2b/Foxn4 rescue and expression analysis","pmids":["27688401","27343557"],"confidence":"High","gaps":["Structural detail of the Med10-TFIIB interface not solved","How PIC-assembly function relates to activator selectivity not integrated"]},{"year":2025,"claim":"Extended MED10 to an oncogenic driver in carcinoma, acting through RAF1/MEK/ERK/c-Myc activation and PTEN ubiquitination-mediated degradation that confers chemoresistance.","evidence":"CRISPR knockout and overexpression with proliferation/migration/invasion assays, Western blot, RT-qPCR, flow cytometry, TAK-243 ubiquitination rescue, and xenograft models in bladder and hepatocellular carcinoma","pmids":["35096564","40917057","40183262"],"confidence":"Medium","gaps":["Direct biochemical MED10-RAF1 interaction not reconstituted","Mechanism by which MED10 promotes PTEN ubiquitination not defined","Whether oncogenic effects depend on Mediator transcriptional function unresolved"]},{"year":null,"claim":"How MED10's structural bridging and TFIIB-dependent PIC-assembly function mechanistically translate into activator/pathway-selective and oncogenic outputs remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No high-resolution structure of MED10 itself","Direct enzymatic/recruitment mechanism for cancer-context PTEN degradation unknown","Connection between Mediator role and RAF1 axis activation undefined"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140110","term_label":"transcription regulator activity","supporting_discovery_ids":[2,4,13]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[9,13]},{"term_id":"GO:0005198","term_label":"structural molecule activity","supporting_discovery_ids":[9,12]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[1]}],"pathway":[{"term_id":"R-HSA-74160","term_label":"Gene expression (Transcription)","supporting_discovery_ids":[0,5,13]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[7,16]},{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[7,14]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[15,16,17]}],"complexes":["Mediator complex (middle module)","Rgr1 subcomplex","SMCC complex"],"partners":["MED7","MED4","MED21","TFIIB"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q9BTT4","full_name":"Mediator of RNA polymerase II transcription subunit 10","aliases":["Mediator complex subunit 10","Transformation-related gene 17 protein","TRG-17","Transformation-related gene 20 protein","TRG-20"],"length_aa":135,"mass_kda":15.7,"function":"Component of the Mediator complex, a coactivator involved in the regulated transcription of nearly all RNA polymerase II-dependent genes. Mediator functions as a bridge to convey information from gene-specific regulatory proteins to the basal RNA polymerase II transcription machinery. Mediator is recruited to promoters by direct interactions with regulatory proteins and serves as a scaffold for the assembly of a functional preinitiation complex with RNA polymerase II and the general transcription factors","subcellular_location":"Nucleus","url":"https://www.uniprot.org/uniprotkb/Q9BTT4/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/MED10","classification":"Not Classified","n_dependent_lines":619,"n_total_lines":1208,"dependency_fraction":0.5124172185430463},"opencell":{"profiled":true,"resolved_as":"","ensg_id":"ENSG00000133398","cell_line_id":"CID000235","localizations":[{"compartment":"nuclear_punctae","grade":3},{"compartment":"nucleoplasm","grade":3}],"interactors":[{"gene":"MED7","stoichiometry":10.0},{"gene":"MED14","stoichiometry":10.0},{"gene":"IXL;MED29","stoichiometry":10.0},{"gene":"MED25","stoichiometry":10.0},{"gene":"MED24","stoichiometry":10.0},{"gene":"MED6","stoichiometry":10.0},{"gene":"MED17","stoichiometry":10.0},{"gene":"MED22","stoichiometry":10.0},{"gene":"MED21","stoichiometry":10.0},{"gene":"MED18","stoichiometry":10.0}],"url":"https://opencell.sf.czbiohub.org/target/CID000235","total_profiled":1310},"omim":[{"mim_id":"612385","title":"MEDIATOR COMPLEX SUBUNIT 19; MED19","url":"https://www.omim.org/entry/612385"},{"mim_id":"612384","title":"MEDIATOR COMPLEX SUBUNIT 18; MED18","url":"https://www.omim.org/entry/612384"},{"mim_id":"612383","title":"MEDIATOR COMPLEX SUBUNIT 11; MED11","url":"https://www.omim.org/entry/612383"},{"mim_id":"612382","title":"MEDIATOR COMPLEX SUBUNIT 10; MED10","url":"https://www.omim.org/entry/612382"},{"mim_id":"607956","title":"MEDIATOR COMPLEX SUBUNIT 8; MED8","url":"https://www.omim.org/entry/607956"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Enhanced","locations":[{"location":"Nucleoplasm","reliability":"Enhanced"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/MED10"},"hgnc":{"alias_symbol":["TRG20","L6","MGC5309","NUT2"],"prev_symbol":[]},"alphafold":{"accession":"Q9BTT4","domains":[{"cath_id":"1.10.287","chopping":"1-61","consensus_level":"medium","plddt":94.0302,"start":1,"end":61},{"cath_id":"1.20.5","chopping":"64-135","consensus_level":"medium","plddt":91.7411,"start":64,"end":135}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9BTT4","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9BTT4-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9BTT4-F1-predicted_aligned_error_v6.png","plddt_mean":92.75},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=MED10","jax_strain_url":"https://www.jax.org/strain/search?query=MED10"},"sequence":{"accession":"Q9BTT4","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9BTT4.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9BTT4/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9BTT4"}},"corpus_meta":[{"pmid":"15175163","id":"PMC_15175163","title":"A 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research","url":"https://pubmed.ncbi.nlm.nih.gov/8554379","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":18977,"output_tokens":4075,"usd":0.059028,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":11936,"output_tokens":3276,"usd":0.07079,"stage2_stop_reason":"end_turn"},"total_usd":0.129818,"stage1_batch_id":"msgbatch_01BbTCMyrRPyhtEq6b1MdwCN","stage2_batch_id":"msgbatch_011dEed5bbxYvTAaQWKJevxu","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1998,\n      \"finding\": \"MED10 (NUT2) was identified as a subunit of the yeast Mediator complex, verified by copurification and co-immunoprecipitation with RNA polymerase II holoenzyme.\",\n      \"method\": \"Peptide sequence determination, copurification, co-immunoprecipitation\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal co-IP and copurification, replicated across multiple labs subsequently\",\n      \"pmids\": [\"9812975\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"NUT2 (MED10) encodes a nuclear protein that cooperates with NUT1 to negatively regulate transcription; the NUT2 gene is essential in S. cerevisiae, and inactivation of a temperature-sensitive NUT2 allele alone causes constitutive, Swi4p-independent expression of reporter genes.\",\n      \"method\": \"Genetic analysis, temperature-sensitive allele inactivation, lacZ reporter assays, nuclear localization by cell biology\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis combined with reporter assays in single lab\",\n      \"pmids\": [\"9671481\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"Med10/Nut2 is a subunit of the Rgr1 subcomplex of yeast Mediator and is specifically required for Gcn4-mediated transcription of amino acid biosynthetic genes, demonstrating an activator-specific requirement distinct from the global requirement of Med6.\",\n      \"method\": \"Cloning of mediator subunits, differential display and Northern analysis of mRNAs from wild-type and Mediator mutant cells\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — defined activator-specific transcriptional requirement by genetic loss-of-function with Northern analysis, single lab\",\n      \"pmids\": [\"9891034\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"Human NUT2 (MED10) was identified as a component of the SMCC complex (a human SRB/MED-containing cofactor), which can repress activator-dependent transcription or act synergistically with PC4 to enhance it, and which shows direct activator interactions but can act independently of the RNA polymerase II CTD.\",\n      \"method\": \"Biochemical purification of human complex, functional transcription assays (repression and activation), direct activator interaction assays\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — biochemical purification with functional transcription assays, single lab\",\n      \"pmids\": [\"10024883\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"The Rgr1 subcomplex of yeast Mediator has modular structure comprising the Gal11, Med9/Cse2, and Med10/Nut2 modules; the Med10/Nut2 module is specifically required for transcriptional repression of a distinct group of genes; the transcriptional repressor Tup1 binds to the Gal11 module at regions overlapping with transcriptional activator binding sites.\",\n      \"method\": \"Biochemical subunit composition analysis, genome-wide gene expression analysis, Northern analysis, GST pull-down\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — biochemical fractionation plus genome-wide expression and pull-down, single lab\",\n      \"pmids\": [\"11470794\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Human MED10 (NUT2) was confirmed as a consensus subunit of the mammalian Mediator complex by MudPIT proteomic analysis of Mediator preparations immunoaffinity-purified through MED10 itself, establishing its presence in the complex.\",\n      \"method\": \"Multidimensional protein identification technology (MudPIT), immunoaffinity purification\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — mass spectrometry-based proteomic identification using immunoaffinity purification through MED10, verified across six independent preparations\",\n      \"pmids\": [\"15175163\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"Med21 physically interacts with Med10 in the Mediator middle module; this interaction was confirmed by two-hybrid experiments and co-immunoprecipitation of tagged proteins produced in insect cells and E. coli, and depends strongly on amino acid residues 2–8 of Med21.\",\n      \"method\": \"Yeast two-hybrid, co-immunoprecipitation of recombinant proteins from insect cells and E. coli\",\n      \"journal\": \"Molecular genetics and genomics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal co-IP in two expression systems plus two-hybrid, single lab\",\n      \"pmids\": [\"16758199\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"Zebrafish Med10 (a Mediator middle domain subunit) differentially transduces signals from distinct signaling pathways: reduction of Med10 levels enhances Wnt signaling and impairs Nodal signaling during embryogenesis, a dual role not shared by Med12/Med13 (Wnt only) or Med15 (Nodal only).\",\n      \"method\": \"Positional cloning of zebrafish mutant, morpholino antisense oligonucleotide knockdown, pathway-specific phenotypic readouts during embryogenesis\",\n      \"journal\": \"Developmental biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — positional cloning plus morpholino KD with pathway-specific phenotypic analysis, single lab\",\n      \"pmids\": [\"17208216\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Reducing the level of essential Mediator subunit Nut2 (MED10) suppresses the growth defect caused by depletion of the transcriptional repressor NC2 in S. cerevisiae, placing MED10/Nut2 as an antagonistic component relative to NC2 in basal transcription control.\",\n      \"method\": \"Genetic suppressor analysis, reduction of Nut2 levels, growth assays\",\n      \"journal\": \"Genetics\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — genetic suppression observed, single lab, no direct biochemical confirmation of mechanism\",\n      \"pmids\": [\"17339209\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"The Mediator middle module from S. cerevisiae consists of seven subunits (Med1, 4, 7, 9, 10, 21, 31) in equimolar stoichiometry; Med10 bridges to the Mediator tail module by binding to both Med7 and Med4, as determined by protein-protein interaction assays.\",\n      \"method\": \"Recombinant and endogenous module preparation, native mass spectrometry, ion-mobility MS, light scattering, SAXS, protein-protein interaction assays\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — reconstituted recombinant complex with native MS stoichiometry, multiple orthogonal biophysical methods, and direct interaction mapping\",\n      \"pmids\": [\"20123732\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"MED10 silencing in primary keratinocytes has an inhibitory role on VDR-mediated keratinocyte proliferation and differentiation, in contrast to the stimulatory roles of MED1 and MED21, as determined by siRNA knockdown and functional assays.\",\n      \"method\": \"siRNA knockdown, keratinocyte proliferation and differentiation assays, VDR affinity bead purification + mass spectrometry\",\n      \"journal\": \"The Journal of investigative dermatology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — siRNA knockdown with functional proliferation/differentiation readouts, single lab\",\n      \"pmids\": [\"20520624\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"The bundle domain fold predicted for Med10/Med14 (and other Mediator heterodimers) represents a common four-helix bundle building block in Mediator, based on structural analysis of the Med11/22 heterodimer and homology predictions for the middle module.\",\n      \"method\": \"Crystal structure of Med11/22, homology modeling, in vitro transcription pre-initiation complex formation assay, in vivo mutagenesis\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 4 / Weak — structural inference for Med10 is computational/homology-based; direct structural data are for Med11/22, not Med10\",\n      \"pmids\": [\"21498544\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"A 3D model of the 6-subunit Mediator middle module positions Med10 flanking the Med7/Med21 heterodimer within an extended, flexible architecture, based on cross-linking mass spectrometry and crystal/homology model docking.\",\n      \"method\": \"Lysine-lysine chemical cross-linking, mass spectrometry, crystal structure docking, homology modeling\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — cross-linking MS with structural model, single lab, no direct mutagenesis validation of Med10 contacts\",\n      \"pmids\": [\"23939621\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"The Mediator middle module exerts an essential function in preinitiation complex (PIC) assembly genome-wide through its Med10 subunit via a key interaction with TFIIB; this Mediator–TFIIB link was shown by in vivo, in vitro, and genome-wide (in silico) approaches.\",\n      \"method\": \"In vivo genetic analysis, in vitro PIC assembly assay, genome-wide ChIP/chromatin analysis, epistasis with TFIIB\",\n      \"journal\": \"Genes & development\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — multiple orthogonal methods (in vivo, in vitro PIC assembly, genome-wide analysis) identifying a direct Med10–TFIIB functional interaction\",\n      \"pmids\": [\"27688401\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Zebrafish Med10 is required for heart valve formation; loss of Med10 (ping pong mutant) reduces Tbx2b expression and abolishes Has2 expression, impairing cardiac jelly development; rescue by Tbx2b but not by Foxn4 overexpression places Med10 downstream of Foxn4 and upstream of Tbx2b in this pathway.\",\n      \"method\": \"Insertional promoter mutation characterization, rescue experiments (Tbx2b/Foxn4 overexpression), expression analysis, zebrafish cardiac valve phenotype\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis with rescue experiments establishing pathway position, single lab\",\n      \"pmids\": [\"27343557\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"MED10 knockout in bladder urothelial carcinoma cell lines (SW1738 and JMSU1) attenuates cell proliferation, migration, invasion, clonogenicity, and tumorsphere formation, with downregulation of BCL-xL, MKI67, VIM, SNAI1, OCT4, LIN28A and upregulation of BAX; MED10 interacts with and co-expresses with hsa-miR-590, and CRISPR-mediated MED10 knockout downregulates miR-590.\",\n      \"method\": \"CRISPR-mediated knockout, cell proliferation/migration/invasion/clonogenicity assays, Western blot protein expression analysis\",\n      \"journal\": \"Frontiers in oncology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — CRISPR KO with multiple functional assays and expression validation, single lab\",\n      \"pmids\": [\"35096564\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"MED10 overexpression drives HCC cell cycle progression and proliferation by activating RAF1, potentially through the MEK/ERK/c-Myc signaling axis; MED10 also promotes epithelial-mesenchymal transition (EMT) and cell migration in HCC cells.\",\n      \"method\": \"In vitro overexpression/knockdown, cell cycle flow cytometry, proliferation assays (CCK-8, colony formation, EdU), migration assays (transwell, wound healing), RT-qPCR, Western blot, immunofluorescence, in vivo xenograft model\",\n      \"journal\": \"Frontiers in bioscience (Landmark edition)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal functional assays plus in vivo validation, single lab, no direct biochemical reconstitution of MED10-RAF1 interaction\",\n      \"pmids\": [\"40917057\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"MED10 promotes PTEN ubiquitination (post-translational degradation) in HCC cells, reducing PTEN protein levels without affecting PTEN mRNA, thereby enhancing cisplatin resistance; this effect is reversed by ubiquitination inhibitor TAK-243 and confirmed in xenograft models.\",\n      \"method\": \"Western blot for PTEN protein/mRNA, ubiquitination inhibitor (TAK-243) rescue, MED10 overexpression/knockdown, cell viability assay (IC50), flow cytometry apoptosis, in vivo xenograft\",\n      \"journal\": \"Current cancer drug targets\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — pharmacological rescue combined with in vitro and in vivo validation, single lab; mechanism of MED10 promoting ubiquitination not fully reconstituted biochemically\",\n      \"pmids\": [\"40183262\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"MED10 (NUT2) is an essential subunit of the Mediator middle module in eukaryotes, where it physically bridges the middle and tail modules (via Med7 and Med4 interactions) and is specifically required for preinitiation complex assembly genome-wide through a direct functional interaction with TFIIB; it mediates activator- and pathway-specific transcriptional outputs (including Gcn4-dependent transcription in yeast and Wnt/Nodal signaling in zebrafish), and in mammalian cancer contexts promotes oncogenicity through RAF1/MEK/ERK/c-Myc axis activation and PTEN ubiquitination-mediated degradation.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"MED10 (NUT2) is an essential, conserved subunit of the Mediator coactivator complex that couples gene-specific regulatory signals to RNA polymerase II transcription [#0, #5]. Within the seven-subunit Mediator middle module, MED10 occupies a structural hub: it flanks the Med7/Med21 heterodimer and bridges to the tail module by binding both Med7 and Med4, contacts mapped by reconstitution and biophysical analysis [#9, #12], with additional direct interaction with Med21 [#6]. Functionally, MED10 is required genome-wide for preinitiation complex assembly through a direct functional interaction with TFIIB [#13], yet it also confers activator- and pathway-selective outputs rather than purely global control: in yeast it is specifically needed for Gcn4-mediated transcription of amino acid biosynthetic genes and for repression of a distinct gene set, distinguishing it from globally required subunits [#2, #4]. In vertebrates this selectivity manifests as differential signal transduction, with zebrafish Med10 simultaneously restraining Wnt and promoting Nodal signaling during embryogenesis and acting downstream of Foxn4 and upstream of Tbx2b to drive heart valve formation [#7, #14]. In mammalian cancers MED10 acts as an oncogenic driver: it promotes bladder and hepatocellular carcinoma proliferation, migration and invasion [#15], activates RAF1 through a MEK/ERK/c-Myc axis [#16], and drives PTEN ubiquitination-mediated degradation to confer cisplatin resistance [#17].\",\n  \"teleology\": [\n    {\n      \"year\": 1998,\n      \"claim\": \"Established MED10/NUT2 as a bona fide physical component of the RNA polymerase II Mediator holoenzyme, and as a nuclear, essential transcriptional regulator.\",\n      \"evidence\": \"Peptide sequencing, copurification and reciprocal co-IP with Pol II holoenzyme, plus genetic analysis with temperature-sensitive alleles and lacZ reporters in yeast\",\n      \"pmids\": [\"9812975\", \"9671481\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not resolve which Mediator submodule MED10 belongs to\", \"Repressive versus activating role not mechanistically separated\"]\n    },\n    {\n      \"year\": 1999,\n      \"claim\": \"Showed MED10 confers activator-specific transcriptional requirements rather than purely global ones, and is conserved as a human SMCC/Mediator component with dual repressive and activating capacity.\",\n      \"evidence\": \"Yeast Mediator subunit cloning with differential display/Northern analysis of activator mutants; biochemical purification of human SMCC with in vitro transcription assays\",\n      \"pmids\": [\"9891034\", \"10024883\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular basis of activator selectivity unresolved\", \"How a single subunit supports both repression and activation not defined\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Defined MED10/Nut2 as a discrete module of the Rgr1 subcomplex specifically required for repression of a distinct gene group, refining its position within Mediator architecture.\",\n      \"evidence\": \"Biochemical subunit fractionation, genome-wide expression analysis, Northern analysis and GST pull-down in yeast\",\n      \"pmids\": [\"11470794\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct repressor contacts of the Med10 module not identified\", \"Mechanism linking module to specific repressed genes unknown\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Mapped MED10 as a direct physical partner of Med21 within the middle module, establishing concrete intramodular contacts.\",\n      \"evidence\": \"Yeast two-hybrid and reciprocal co-IP of recombinant proteins from insect cells and E. coli\",\n      \"pmids\": [\"16758199\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Functional consequence of the Med10-Med21 interface not tested in vivo\", \"Single lab\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Placed MED10/Nut2 in functional antagonism with the NC2 repressor in basal transcription control.\",\n      \"evidence\": \"Genetic suppressor analysis with Nut2 level reduction and growth assays in yeast\",\n      \"pmids\": [\"17339209\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Genetic suppression only, no direct biochemical mechanism\", \"Whether interaction is direct unknown\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Resolved MED10 as a bridging subunit that links the middle and tail modules via direct Med7 and Med4 binding, and confirmed its conserved role beyond transcription in VDR-dependent keratinocyte biology.\",\n      \"evidence\": \"Reconstituted recombinant/endogenous middle module with native and ion-mobility MS, SAXS, light scattering and interaction assays; siRNA knockdown with proliferation/differentiation readouts in keratinocytes\",\n      \"pmids\": [\"20123732\", \"20520624\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Atomic-resolution structure of MED10 not determined\", \"Mechanistic link between module bridging and VDR output unresolved\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Positioned MED10 architecturally within an extended, flexible middle module flanking the Med7/Med21 heterodimer.\",\n      \"evidence\": \"Lysine-lysine cross-linking mass spectrometry with crystal structure docking and homology modeling\",\n      \"pmids\": [\"23939621\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No mutagenesis validation of MED10 cross-link contacts\", \"Model relies partly on homology inference\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Identified the mechanistic basis of MED10's essential transcriptional role: a direct functional interaction with TFIIB required for preinitiation complex assembly genome-wide, while in vivo establishing it as a node directing developmental signaling pathway outputs.\",\n      \"evidence\": \"In vivo genetics, in vitro PIC assembly, genome-wide chromatin analysis and TFIIB epistasis in yeast; zebrafish insertional mutant with Tbx2b/Foxn4 rescue and expression analysis\",\n      \"pmids\": [\"27688401\", \"27343557\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural detail of the Med10-TFIIB interface not solved\", \"How PIC-assembly function relates to activator selectivity not integrated\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Extended MED10 to an oncogenic driver in carcinoma, acting through RAF1/MEK/ERK/c-Myc activation and PTEN ubiquitination-mediated degradation that confers chemoresistance.\",\n      \"evidence\": \"CRISPR knockout and overexpression with proliferation/migration/invasion assays, Western blot, RT-qPCR, flow cytometry, TAK-243 ubiquitination rescue, and xenograft models in bladder and hepatocellular carcinoma\",\n      \"pmids\": [\"35096564\", \"40917057\", \"40183262\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct biochemical MED10-RAF1 interaction not reconstituted\", \"Mechanism by which MED10 promotes PTEN ubiquitination not defined\", \"Whether oncogenic effects depend on Mediator transcriptional function unresolved\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How MED10's structural bridging and TFIIB-dependent PIC-assembly function mechanistically translate into activator/pathway-selective and oncogenic outputs remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No high-resolution structure of MED10 itself\", \"Direct enzymatic/recruitment mechanism for cancer-context PTEN degradation unknown\", \"Connection between Mediator role and RAF1 axis activation undefined\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140110\", \"supporting_discovery_ids\": [2, 4, 13]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [9, 13]},\n      {\"term_id\": \"GO:0005198\", \"supporting_discovery_ids\": [9, 12]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [1]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-74160\", \"supporting_discovery_ids\": [0, 5, 13]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [7, 16]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [7, 14]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [15, 16, 17]}\n    ],\n    \"complexes\": [\"Mediator complex (middle module)\", \"Rgr1 subcomplex\", \"SMCC complex\"],\n    \"partners\": [\"MED7\", \"MED4\", \"MED21\", \"TFIIB\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":5,"faith_total":5,"faith_pct":100.0}}