{"gene":"MED31","run_date":"2026-04-28T18:30:28","timeline":{"discoveries":[{"year":1999,"finding":"The human SOH1 homolog (MED31) was identified as a component of the SMCC (SRB/MED-containing cofactor) complex, a human Mediator-like complex containing homologs of yeast mediator/holoenzyme components including SOH1 and NUT2, capable of repressing or enhancing activator-dependent transcription and showing direct activator interactions independent of the RNA polymerase II CTD.","method":"Biochemical purification, mass spectrometry identification of complex subunits, in vitro transcription assays","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 1 — biochemical reconstitution and in vitro functional assay with complex purification","pmids":["10024883"],"is_preprint":false},{"year":2001,"finding":"Human Mediator (containing MED31/SOH1) is required for basal RNA polymerase II transcription; antibody depletion of Mediator components abolished transcription on all genes tested, on both TATA-containing and TATA-less promoters, in the presence and absence of activators.","method":"Monoclonal antibody depletion from crude cell extracts, in vitro transcription assays, immunopurification","journal":"EMBO reports","confidence":"High","confidence_rationale":"Tier 1 — in vitro transcription assay with depletion and functional rescue, multiple promoters tested","pmids":["11559591"],"is_preprint":false},{"year":2004,"finding":"Soh1/MED31 is a stable component of the Mediator complex in both Schizosaccharomyces pombe and Saccharomyces cerevisiae, and bioinformatic analysis places the Soh1/MED31 family as among the most evolutionarily ancient Mediator subunits, predating the canonical heptapeptide repeat structure of the RNA Pol II CTD.","method":"Biochemical purification of Mediator complex, co-purification/stable association demonstrated, bioinformatic phylogenetic analysis","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 — stable complex isolation from two independent yeast species, replicated across organisms","pmids":["15356001"],"is_preprint":false},{"year":2004,"finding":"MED31 (NUT2 in prior nomenclature) was identified as one of the consensus mammalian Mediator subunits, present across six independent immunoaffinity-purified Mediator preparations analyzed by MudPIT proteomics.","method":"Multidimensional protein identification technology (MudPIT), six independent Mediator immunopurifications","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 2 — proteomic identification replicated across six independent purifications","pmids":["15175163"],"is_preprint":false},{"year":2007,"finding":"In S. pombe, Med31 (encoded by sep10+) controls large sets of genes involved in cell separation, acting as a coactivator for Sep1-Ace2-dependent cell separation genes; Med31 deletion disrupts cell separation without affecting sep1+ or ace2+ expression, placing Med31 downstream of these transcription factors as a coactivator.","method":"Genome-wide gene expression profiling of sep10 deletion mutants, genetic epistasis analysis","journal":"Molecular genetics and genomics","confidence":"Medium","confidence_rationale":"Tier 2 — genome-wide expression profiling with genetic epistasis, single lab","pmids":["17922236"],"is_preprint":false},{"year":2010,"finding":"Mouse Med31 is required for embryonic growth and cell proliferation; a point mutation causing Med31 protein degradation results in late-gestation lethality, reduced proliferating cells in rapidly expanding tissues, severe proliferation defect in cultured embryonic fibroblasts, reduced Cdc2 (cell cycle protein) levels, and defective chondrogenesis with loss of Sox9 and Col2a1 expression, indicating Med31 promotes transcription of genes required for growth and proliferation.","method":"ENU mutagenesis screen, genetic mapping, protein degradation confirmed by western blot, embryonic fibroblast proliferation assays, immunohistochemistry for cell cycle markers and chondrogenic markers","journal":"Developmental biology","confidence":"High","confidence_rationale":"Tier 2 — loss-of-function mouse model with multiple orthogonal phenotypic readouts, specific molecular targets identified","pmids":["20347762"],"is_preprint":false},{"year":2012,"finding":"MED31 physically interacts with the cytoplasmic engulfment protein Elmo1; this interaction increases the cytoplasmic localization of normally nuclear MED31 and promotes monoubiquitination of Med31, identifying ubiquitination as a novel post-translational modification of Med31. During Salmonella infection in primary macrophages, Elmo1 and Med31 together specifically regulate expression of cytokine genes Il10 and Il33.","method":"Co-immunoprecipitation, subcellular fractionation/localization experiments, ubiquitination assays, gene expression monitoring in primary macrophages during Salmonella infection","journal":"Current biology","confidence":"High","confidence_rationale":"Tier 2 — reciprocal Co-IP, subcellular fractionation with functional consequence, ubiquitination biochemically demonstrated, primary cell infection model","pmids":["23273896"],"is_preprint":false},{"year":2014,"finding":"MED31 is a direct target of miR-1 in osteosarcoma cells, validated by luciferase reporter assay; knockdown of Med31 suppresses osteosarcoma cell proliferation and reduces expression of MET proto-oncogene and blocks downstream HGF-stimulated MET signaling, placing Med31 upstream of MET pathway activation.","method":"Luciferase reporter assay (3'UTR target validation), siRNA knockdown, cell proliferation assays, western blotting for MET and downstream signaling","journal":"Oncology reports","confidence":"Medium","confidence_rationale":"Tier 2-3 — direct target validation by luciferase assay plus KD phenotype with pathway readout, single lab","pmids":["24969180"],"is_preprint":false},{"year":2014,"finding":"Electron microscopy structures of yeast and human Mediator complexes with comprehensive subunit localization experiments provided the first accurate EM-based structural mapping of Mediator subunit positions and revealed that large-scale Mediator conformational rearrangements occur at interfaces between Mediator modules during transcription initiation.","method":"Electron microscopy, subunit localization by antibody labeling and crosslinking, biochemical analyses","journal":"Cell","confidence":"High","confidence_rationale":"Tier 1 — EM structure with subunit localization, biochemical validation, covers MED31 as Mediator subunit","pmids":["24882805"],"is_preprint":false},{"year":2018,"finding":"MED31 knockdown in human mesenchymal stem cells (hMSCs) using siRNA reduces self-renewal capacity (assessed by cell assays and gene expression) and impairs adipogenic differentiation, evidenced by diminished lipid vesicle formation and reduced adipogenic marker expression, demonstrating a role for MED31 in maintaining adult stem cell homeostasis.","method":"siRNA-mediated knockdown, cell proliferation/self-renewal assays, adipogenesis assays (lipid staining), qRT-PCR for lineage markers","journal":"Molecular biology reports","confidence":"Medium","confidence_rationale":"Tier 2-3 — clean KD with multiple defined cellular phenotypes, single lab, no pathway placement","pmids":["30006772"],"is_preprint":false},{"year":2019,"finding":"In Tetrahymena thermophila, endogenously tagged Med31 physically associates with >20 proteins including canonical Mediator subunits identified by AP-MS; Med31 ChIP-seq shows global association with transcribed genes; Med31 knockdown causes ectopic expression of developmental genes for programmed DNA rearrangements; and Med31 localizes to meiotic micronuclei implicating Mediator in ncRNA transcription.","method":"Endogenous tagging, affinity purification coupled with mass spectrometry (AP-MS), ChIP-seq, RNAi knockdown with RNA-seq, indirect immunofluorescence","journal":"Current biology","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods (AP-MS, ChIP-seq, KD + transcriptomics, immunofluorescence) in single study","pmids":["31280994"],"is_preprint":false},{"year":2026,"finding":"ELMO1 methylation interacts with Med31 in H. pylori-infected gastric cancer cells (AGS); Co-immunoprecipitation confirmed ELMO1-Med31 protein interaction; this interaction drives M2 macrophage polarization and promotes epithelial-mesenchymal transition (EMT) and intestinal metaplasia in H. pylori-infected cells.","method":"Co-immunoprecipitation, methylation-specific PCR, co-culture experiments, western blotting, cell viability/migration assays","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 3 — Co-IP confirms interaction, functional co-culture model, single lab, limited mechanistic resolution","pmids":["41535333"],"is_preprint":false}],"current_model":"MED31 is the smallest and most evolutionarily conserved subunit of the Mediator coactivator complex, required for basal and activated RNA polymerase II transcription; it undergoes monoubiquitination regulated by cytoplasmic Elmo1, which also re-localizes MED31 from its predominantly nuclear location to the cytoplasm to modulate cytokine gene expression; in mammalian development, Med31 promotes transcription of genes required for cell proliferation and chondrogenesis, and in stem cells it sustains self-renewal and adipogenic differentiation, while its protein level is itself regulated post-transcriptionally by miR-1 targeting its 3'UTR, upstream of MET oncogene signaling."},"narrative":{"teleology":[{"year":1999,"claim":"Identification of MED31 as a subunit of the human Mediator-like SMCC complex established that a yeast SOH1 homolog is conserved in mammalian transcriptional coactivator machinery capable of both repressing and enhancing activator-dependent transcription.","evidence":"Biochemical purification of SMCC complex with mass spectrometry identification and in vitro transcription assays","pmids":["10024883"],"confidence":"High","gaps":["Specific contribution of MED31 versus other SMCC subunits not dissected","No loss-of-function data for MED31 alone"]},{"year":2001,"claim":"Demonstrating that Mediator depletion abolishes transcription on all tested promoters established MED31-containing Mediator as a general transcription factor required for basal and activated transcription, not merely an activator-specific cofactor.","evidence":"Antibody depletion of Mediator from crude extracts followed by in vitro transcription on TATA-containing and TATA-less promoters","pmids":["11559591"],"confidence":"High","gaps":["Depletion removed entire Mediator rather than MED31 alone","In vitro system may not recapitulate all in vivo regulation"]},{"year":2004,"claim":"Phylogenetic and biochemical analyses across yeast species established MED31 as among the most ancient Mediator subunits, predating the canonical Pol II CTD heptapeptide repeat, and confirmed its stable incorporation into Mediator in multiple organisms.","evidence":"Mediator complex purification from S. pombe and S. cerevisiae; bioinformatic phylogenetic reconstruction","pmids":["15356001","15175163"],"confidence":"High","gaps":["Structural basis for MED31 integration into the complex not resolved","Function of MED31 outside of Mediator context unexplored"]},{"year":2007,"claim":"Genome-wide profiling of Med31 deletion in S. pombe revealed that Med31 acts as a coactivator downstream of Sep1-Ace2 transcription factors for cell separation genes, establishing a specific gene-regulatory role beyond general transcription.","evidence":"Microarray profiling of sep10 (Med31) deletion mutants with genetic epistasis analysis in S. pombe","pmids":["17922236"],"confidence":"Medium","gaps":["Mechanism by which Med31 is recruited to Sep1-Ace2 target promoters not determined","Single organism, single lab"]},{"year":2010,"claim":"A mouse point mutation causing Med31 protein degradation produced late-gestation lethality with reduced cell proliferation and defective chondrogenesis, directly demonstrating that Med31 promotes transcription of cell cycle (Cdc2) and cartilage differentiation (Sox9, Col2a1) genes in vivo.","evidence":"ENU mutagenesis screen, western blot for protein stability, embryonic fibroblast proliferation assays, immunohistochemistry for cell cycle and chondrogenic markers","pmids":["20347762"],"confidence":"High","gaps":["Direct transcriptional targets genome-wide not identified in mammalian context","Whether phenotypes reflect Mediator destabilization versus MED31-specific functions unclear"]},{"year":2012,"claim":"Discovery that cytoplasmic Elmo1 physically interacts with MED31, promotes its monoubiquitination and cytoplasmic relocalization, and together with MED31 regulates Il10 and Il33 expression during Salmonella infection revealed a non-canonical regulatory axis for a Mediator subunit outside the nucleus.","evidence":"Reciprocal Co-IP, subcellular fractionation, ubiquitination assays, gene expression analysis in primary macrophages during bacterial infection","pmids":["23273896"],"confidence":"High","gaps":["Ubiquitin ligase responsible for MED31 monoubiquitination not identified","Whether cytoplasmic MED31 has transcription-independent functions unknown","Mechanism linking MED31 ubiquitination to cytokine gene regulation not resolved"]},{"year":2014,"claim":"MED31 was placed upstream of MET oncogene signaling via miR-1-mediated post-transcriptional regulation, linking Mediator subunit levels to oncogenic pathway activity in osteosarcoma cells; concurrently, EM structural mapping resolved MED31's position within the Mediator architecture.","evidence":"Luciferase 3′UTR reporter assay, siRNA knockdown with MET pathway readouts; EM with antibody labeling for subunit localization","pmids":["24969180","24882805"],"confidence":"Medium","gaps":["Whether MED31 directly regulates MET transcription or acts indirectly through Mediator integrity is unresolved","High-resolution atomic structure of MED31 within Mediator not yet available at this time point"]},{"year":2018,"claim":"MED31 knockdown in human mesenchymal stem cells impaired both self-renewal and adipogenic differentiation, extending its proliferative role to adult stem cell homeostasis.","evidence":"siRNA knockdown with proliferation assays, lipid staining for adipogenesis, qRT-PCR for lineage markers in hMSCs","pmids":["30006772"],"confidence":"Medium","gaps":["Transcriptional targets mediating self-renewal versus differentiation not identified","No genetic rescue performed","Single lab, no in vivo validation"]},{"year":2019,"claim":"In Tetrahymena, Med31 ChIP-seq demonstrated global association with transcribed genes and knockdown caused ectopic developmental gene expression, while Med31 localization to meiotic micronuclei implicated Mediator in ncRNA transcription, extending the functional repertoire beyond protein-coding gene regulation.","evidence":"Endogenous tagging, AP-MS, ChIP-seq, RNAi with RNA-seq, immunofluorescence in Tetrahymena","pmids":["31280994"],"confidence":"High","gaps":["Whether ncRNA transcription role is conserved in metazoans is unknown","Mechanism of Med31 recruitment to specific loci not defined"]},{"year":null,"claim":"Key unresolved questions include the identity of the E3 ubiquitin ligase for MED31 monoubiquitination, the functional significance of cytoplasmic MED31, whether MED31 has Mediator-independent functions, and high-resolution structural details of MED31 contacts within the human Mediator complex.","evidence":"","pmids":[],"confidence":"Low","gaps":["E3 ligase for MED31 monoubiquitination not identified","No Mediator-independent function demonstrated","Genome-wide direct target identification in mammalian cells lacking"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140110","term_label":"transcription regulator activity","supporting_discovery_ids":[0,1,4,5,10]},{"term_id":"GO:0140223","term_label":"general transcription initiation factor activity","supporting_discovery_ids":[1]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[6,10]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[6]}],"pathway":[{"term_id":"R-HSA-74160","term_label":"Gene expression (Transcription)","supporting_discovery_ids":[0,1,4,5,10]},{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[5]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[6]}],"complexes":["Mediator complex"],"partners":["ELMO1","MED7","SOX9"],"other_free_text":[]},"mechanistic_narrative":"MED31 is the smallest and most evolutionarily ancient subunit of the Mediator coactivator complex, essential for both basal and activator-dependent RNA polymerase II transcription across eukaryotes [PMID:10024883, PMID:11559591, PMID:15356001]. In mammals, Med31 promotes transcription of genes required for cell proliferation and chondrogenesis during embryonic development, and sustains self-renewal and adipogenic differentiation in adult mesenchymal stem cells [PMID:20347762, PMID:30006772]. Beyond its nuclear transcriptional role, MED31 undergoes Elmo1-dependent monoubiquitination and cytoplasmic relocalization, which specifically modulates cytokine gene expression (Il10, Il33) during macrophage responses to bacterial infection [PMID:23273896]. MED31 protein levels are post-transcriptionally regulated by miR-1, and MED31 knockdown suppresses MET proto-oncogene expression and downstream HGF signaling in osteosarcoma cells [PMID:24969180]."},"prefetch_data":{"uniprot":{"accession":"Q9Y3C7","full_name":"Mediator of RNA polymerase II transcription subunit 31","aliases":["Mediator complex subunit 31","Mediator complex subunit SOH1","hSOH1"],"length_aa":131,"mass_kda":15.8,"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/Q9Y3C7/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":true,"resolved_as":"","url":"https://depmap.org/portal/gene/MED31","classification":"Common Essential","n_dependent_lines":1158,"n_total_lines":1208,"dependency_fraction":0.9586092715231788},"opencell":{"profiled":true,"resolved_as":"","ensg_id":"ENSG00000108590","cell_line_id":"CID000247","localizations":[{"compartment":"nuclear_punctae","grade":3},{"compartment":"nucleoplasm","grade":3}],"interactors":[{"gene":"MED10","stoichiometry":10.0},{"gene":"MED11","stoichiometry":10.0},{"gene":"MED14","stoichiometry":10.0},{"gene":"MED17","stoichiometry":10.0},{"gene":"MED18","stoichiometry":10.0},{"gene":"MED19","stoichiometry":10.0},{"gene":"MED20","stoichiometry":10.0},{"gene":"MED21","stoichiometry":10.0},{"gene":"MED22","stoichiometry":10.0},{"gene":"MED25","stoichiometry":10.0}],"url":"https://opencell.sf.czbiohub.org/target/CID000247","total_profiled":1310},"omim":[{"mim_id":"621256","title":"ZINC FINGER- AND BTB DOMAIN-CONTAINING PROTEIN 45; ZBTB45","url":"https://www.omim.org/entry/621256"},{"mim_id":"620492","title":"MEDIATOR COMPLEX SUBUNIT 31; MED31","url":"https://www.omim.org/entry/620492"},{"mim_id":"612385","title":"MEDIATOR COMPLEX SUBUNIT 19; MED19","url":"https://www.omim.org/entry/612385"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Nucleoplasm","reliability":"Approved"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/MED31"},"hgnc":{"alias_symbol":["CGI-125","Soh1"],"prev_symbol":[]},"alphafold":{"accession":"Q9Y3C7","domains":[],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9Y3C7","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9Y3C7-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9Y3C7-F1-predicted_aligned_error_v6.png","plddt_mean":92.88},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=MED31","jax_strain_url":"https://www.jax.org/strain/search?query=MED31"},"sequence":{"accession":"Q9Y3C7","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9Y3C7.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9Y3C7/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9Y3C7"}},"corpus_meta":[{"pmid":"15356001","id":"PMC_15356001","title":"The Soh1/MED31 protein is an ancient component of Schizosaccharomyces pombe and Saccharomyces cerevisiae 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reports","url":"https://pubmed.ncbi.nlm.nih.gov/23602568","citation_count":174,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"24882805","id":"PMC_24882805","title":"Subunit architecture and functional modular rearrangements of the transcriptional mediator complex.","date":"2014","source":"Cell","url":"https://pubmed.ncbi.nlm.nih.gov/24882805","citation_count":163,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"18461144","id":"PMC_18461144","title":"Systematic identification of mRNAs recruited to argonaute 2 by specific microRNAs and corresponding changes in transcript abundance.","date":"2008","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/18461144","citation_count":148,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"30554943","id":"PMC_30554943","title":"Interactome Rewiring Following Pharmacological Targeting of BET Bromodomains.","date":"2018","source":"Molecular cell","url":"https://pubmed.ncbi.nlm.nih.gov/30554943","citation_count":136,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"15989967","id":"PMC_15989967","title":"MED1/TRAP220 exists predominantly in a TRAP/ Mediator subpopulation enriched in RNA polymerase II and is required for ER-mediated transcription.","date":"2005","source":"Molecular cell","url":"https://pubmed.ncbi.nlm.nih.gov/15989967","citation_count":122,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"11559591","id":"PMC_11559591","title":"Novel critical role of a human Mediator complex for basal RNA polymerase II transcription.","date":"2001","source":"EMBO reports","url":"https://pubmed.ncbi.nlm.nih.gov/11559591","citation_count":107,"is_preprint":false,"source_track":"gene2pubmed"}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":6239,"output_tokens":2223,"usd":0.026031},"stage2":{"model":"claude-opus-4-6","input_tokens":5511,"output_tokens":2409,"usd":0.13167},"total_usd":0.349816,"stage1_batch_id":"msgbatch_01Mgbec4vM6c9vXht7JVJzss","stage2_batch_id":"msgbatch_01JkuuH5RB2BdJNsgnhjbC6s","note":"batch pricing = 50% of standard","round2_stage1":{"model":"claude-sonnet-4-6","input_tokens":17752,"output_tokens":3505,"usd":0.052915},"round2_rules_fired":"R3","round2_stage2":{"model":"claude-opus-4-6","input_tokens":6435,"output_tokens":2425,"usd":0.1392}},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2004,\n      \"finding\": \"Soh1/MED31 protein is a stable component of the Mediator complex isolated from both Schizosaccharomyces pombe and Saccharomyces cerevisiae, and represents an ancient, conserved Mediator subunit tracing back to the point of major eukaryotic divergence.\",\n      \"method\": \"Biochemical isolation and co-purification of Mediator complex; bioinformatic analysis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — direct biochemical co-purification of endogenous complex, replicated across two yeast species\",\n      \"pmids\": [\"15356001\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"In fission yeast, Med31 (Sep10) controls transcription of cell separation genes downstream of the Sep1-Ace2 transcription factors, acting as a coactivator of Sep1-Ace2-dependent genes required for cell separation during cell division.\",\n      \"method\": \"Genome-wide gene expression profiling of med31/sep10 deletion mutants; genetic epistasis with sep1 and ace2\",\n      \"journal\": \"Molecular genetics and genomics : MGG\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — genome-wide expression profiling plus genetic epistasis in single lab\",\n      \"pmids\": [\"17922236\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Mouse Med31 is required for embryonic cell proliferation; loss-of-function mutation causes degradation of Med31 protein, reduced proliferating cells in rapidly expanding tissues, decreased Cdc2 protein levels, and defective chondrogenesis with loss of Sox9 and Col2a1 expression, indicating Med31 promotes transcription of genes required for growth and cell cycle progression.\",\n      \"method\": \"ENU mutagenesis screen; genetic loss-of-function (Med31 mutant mouse); embryonic fibroblast proliferation assay; Western blot for Cdc2; in situ hybridization for Sox9 and Col2a1\",\n      \"journal\": \"Developmental biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — clean loss-of-function mouse mutant with multiple cellular and molecular readouts, single lab\",\n      \"pmids\": [\"20347762\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"MED31 physically interacts with ELMO1 (cytoplasmic engulfment protein); ELMO1 increases cytoplasmic localization of the predominantly nuclear MED31 and enhances monoubiquitination of MED31; this ELMO1-MED31 interaction specifically affects expression of cytokine genes Il10 and Il33 in primary macrophages during Salmonella infection.\",\n      \"method\": \"Co-immunoprecipitation; subcellular fractionation; immunofluorescence localization; ubiquitination assay; gene expression profiling of >25 genes in primary macrophages\",\n      \"journal\": \"Current biology : CB\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal Co-IP, direct localization experiment with functional consequence, ubiquitination assay, multiple orthogonal methods in single lab\",\n      \"pmids\": [\"23273896\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"MED31 is a direct target of miR-1 in osteosarcoma cells; miR-1 binds the MED31 3'UTR to repress its expression, and downregulation of MED31 suppresses cell proliferation and reduces expression of MET proto-oncogene and downstream HGF/MET signaling.\",\n      \"method\": \"Luciferase reporter assay (3'UTR validation); siRNA knockdown of MED31; cell proliferation assay; Western blot for MET pathway components\",\n      \"journal\": \"Oncology reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — luciferase reporter validates direct miRNA targeting, knockdown with defined molecular and cellular phenotypes, single lab\",\n      \"pmids\": [\"24969180\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"MED31 knockdown in human mesenchymal stem cells reduces self-renewal and impairs adipogenic differentiation, as evidenced by decreased lipid vesicle formation and reduced expression of adipogenic markers, demonstrating MED31 is required for maintaining adult stem cell homeostasis.\",\n      \"method\": \"siRNA-mediated knockdown; cell proliferation/self-renewal assays; Oil Red O staining for lipid vesicles; qRT-PCR for adipogenic markers\",\n      \"journal\": \"Molecular biology reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — knockdown with multiple phenotypic readouts but no direct molecular target identification, single lab\",\n      \"pmids\": [\"30006772\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"In Tetrahymena thermophila, Med31 physically associates with >20 proteins including canonical Mediator subunits and Tetrahymena-specific proteins; Med31 occupies gene promoters genome-wide (ChIP-seq); Med31 knockdown causes ectopic expression of developmental genes for programmed DNA rearrangements; Med31 localizes to meiotic micronuclei, implicating it in ncRNA transcription.\",\n      \"method\": \"Affinity purification coupled with mass spectrometry (AP-MS); ChIP-seq; RNAi knockdown; indirect immunofluorescence\",\n      \"journal\": \"Current biology : CB\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — AP-MS interactome, ChIP-seq, and knockdown phenotype with multiple orthogonal methods, ciliate ortholog context\",\n      \"pmids\": [\"31280994\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"circ-GLIS3 sponges miR-548m to positively upregulate MED31 expression in TMZ-resistant glioma cells, and MED31 overexpression reverses the anti-proliferative and pro-apoptotic effects of circ-GLIS3 knockdown, placing MED31 downstream of the circ-GLIS3/miR-548m axis in chemoresistance.\",\n      \"method\": \"siRNA/shRNA knockdown of circ-GLIS3; miR-548m inhibitor rescue; MED31 mRNA overexpression rescue; cell proliferation, invasion, migration, and apoptosis assays; in vivo xenograft tumor model\",\n      \"journal\": \"Cancer biotherapy & radiopharmaceuticals\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — genetic epistasis rescue experiments with multiple cellular assays, but no direct biochemical validation of miR-548m/MED31 binding, single lab\",\n      \"pmids\": [\"34762494\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"ELMO1 methylation interacts with Med31 (co-immunoprecipitation) in H. pylori-infected gastric cancer cells, and this interaction promotes M2 macrophage polarization, which in turn facilitates epithelial-mesenchymal transition and intestinal metaplasia.\",\n      \"method\": \"Co-immunoprecipitation; methylation-specific PCR; co-culture experiments; Western blot; qRT-PCR; CCK-8, wound healing, and colony formation assays\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — single Co-IP with functional cellular follow-up, single lab, no direct mechanistic reconstitution\",\n      \"pmids\": [\"41535333\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"MED31 is the smallest and most evolutionarily conserved subunit of the Mediator transcriptional coactivator complex, physically incorporated into the Mediator as a stable component; it bridges gene-specific transcription factors (including nuclear receptors and developmental regulators) to the RNA Pol II machinery to promote transcription of genes required for cell proliferation, differentiation, and cell cycle progression; its activity is regulated post-translationally by monoubiquitination, and its nuclear-to-cytoplasmic distribution is modulated by the cytoplasmic GEF protein ELMO1, which also enhances its ubiquitination and influences cytokine gene expression.\"\n}\n```\n\n--- ROUND 2 (R2/R3, augmented corpus) ---\n\nI'll classify each paper first, then extract discoveries from KEEP papers only.\n\n**Classification:**\n\nRETRIEVED PAPERS:\n1. PMID:15356001 — KEEP (Soh1/MED31 in yeast Mediator, ortholog study)\n2. PMID:29844159 — EXCLUDE (Arabidopsis MED31, plant symbol collision)\n3. PMID:20347762 — KEEP (mouse Med31, mammalian ortholog)\n4. PMID:34762494 — EXCLUDE (circ-GLIS3/miR-548m/MED31 axis — circRNA paper, MED31 is only a downstream target with no mechanistic characterization of the protein itself; also the main subject is circ-GLIS3)\n5. PMID:24969180 — KEEP (miR-1 targets Med1 and Med31 in osteosarcoma, includes luciferase validation and MED31 KD with MET signaling)\n6. PMID:17922236 — KEEP (S. pombe Med31/sep10, ortholog)\n7. PMID:23273896 — KEEP (Elmo1-Med31 binding, nuclear localization, ubiquitination)\n8. PMID:31280994 — KEEP (Tetrahymena Med31, ortholog — evolutionarily deep, consistent function)\n9. PMID:30006772 — KEEP (human MED31 in hMSCs, siRNA KD with defined phenotype)\n10. PMID:41535333 — KEEP (ELMO1 methylation-Med31 interaction, Co-IP, functional studies in GC)\n11. bio_10.1101_2024.10.25.24316158 — EXCLUDE (GWAS/GxE interaction study, no mechanistic protein data)\n\nADDITIONAL PAPERS (gene2pubmed):\n- Most are large interactome/proteomics resources or unrelated Mediator subunit papers. I'll include only those with direct MED31-specific mechanistic findings.\n- PMID:10024883 — KEEP (SMCC complex includes SOH1/MED31 homolog, direct biochemical purification)\n- PMID:15175163 — KEEP (MudPIT identifies MED31/NUT2 as consensus Mediator subunit)\n- PMID:24882805 — KEEP (EM structure of Mediator, subunit localization including MED31)\n- PMID:11559591 — KEEP (human Mediator basal transcription function)\n- Others (large interactome screens, cDNA collections, GO annotations) — EXCLUDE as not MED31-specific mechanistic findings\n\n```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1999,\n      \"finding\": \"The human SOH1 homolog (MED31) was identified as a component of the SMCC (SRB/MED-containing cofactor) complex, a human Mediator-like complex containing homologs of yeast mediator/holoenzyme components including SOH1 and NUT2, capable of repressing or enhancing activator-dependent transcription and showing direct activator interactions independent of the RNA polymerase II CTD.\",\n      \"method\": \"Biochemical purification, mass spectrometry identification of complex subunits, in vitro transcription assays\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — biochemical reconstitution and in vitro functional assay with complex purification\",\n      \"pmids\": [\"10024883\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"Human Mediator (containing MED31/SOH1) is required for basal RNA polymerase II transcription; antibody depletion of Mediator components abolished transcription on all genes tested, on both TATA-containing and TATA-less promoters, in the presence and absence of activators.\",\n      \"method\": \"Monoclonal antibody depletion from crude cell extracts, in vitro transcription assays, immunopurification\",\n      \"journal\": \"EMBO reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro transcription assay with depletion and functional rescue, multiple promoters tested\",\n      \"pmids\": [\"11559591\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Soh1/MED31 is a stable component of the Mediator complex in both Schizosaccharomyces pombe and Saccharomyces cerevisiae, and bioinformatic analysis places the Soh1/MED31 family as among the most evolutionarily ancient Mediator subunits, predating the canonical heptapeptide repeat structure of the RNA Pol II CTD.\",\n      \"method\": \"Biochemical purification of Mediator complex, co-purification/stable association demonstrated, bioinformatic phylogenetic analysis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — stable complex isolation from two independent yeast species, replicated across organisms\",\n      \"pmids\": [\"15356001\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"MED31 (NUT2 in prior nomenclature) was identified as one of the consensus mammalian Mediator subunits, present across six independent immunoaffinity-purified Mediator preparations analyzed by MudPIT proteomics.\",\n      \"method\": \"Multidimensional protein identification technology (MudPIT), six independent Mediator immunopurifications\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — proteomic identification replicated across six independent purifications\",\n      \"pmids\": [\"15175163\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"In S. pombe, Med31 (encoded by sep10+) controls large sets of genes involved in cell separation, acting as a coactivator for Sep1-Ace2-dependent cell separation genes; Med31 deletion disrupts cell separation without affecting sep1+ or ace2+ expression, placing Med31 downstream of these transcription factors as a coactivator.\",\n      \"method\": \"Genome-wide gene expression profiling of sep10 deletion mutants, genetic epistasis analysis\",\n      \"journal\": \"Molecular genetics and genomics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — genome-wide expression profiling with genetic epistasis, single lab\",\n      \"pmids\": [\"17922236\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Mouse Med31 is required for embryonic growth and cell proliferation; a point mutation causing Med31 protein degradation results in late-gestation lethality, reduced proliferating cells in rapidly expanding tissues, severe proliferation defect in cultured embryonic fibroblasts, reduced Cdc2 (cell cycle protein) levels, and defective chondrogenesis with loss of Sox9 and Col2a1 expression, indicating Med31 promotes transcription of genes required for growth and proliferation.\",\n      \"method\": \"ENU mutagenesis screen, genetic mapping, protein degradation confirmed by western blot, embryonic fibroblast proliferation assays, immunohistochemistry for cell cycle markers and chondrogenic markers\",\n      \"journal\": \"Developmental biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — loss-of-function mouse model with multiple orthogonal phenotypic readouts, specific molecular targets identified\",\n      \"pmids\": [\"20347762\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"MED31 physically interacts with the cytoplasmic engulfment protein Elmo1; this interaction increases the cytoplasmic localization of normally nuclear MED31 and promotes monoubiquitination of Med31, identifying ubiquitination as a novel post-translational modification of Med31. During Salmonella infection in primary macrophages, Elmo1 and Med31 together specifically regulate expression of cytokine genes Il10 and Il33.\",\n      \"method\": \"Co-immunoprecipitation, subcellular fractionation/localization experiments, ubiquitination assays, gene expression monitoring in primary macrophages during Salmonella infection\",\n      \"journal\": \"Current biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal Co-IP, subcellular fractionation with functional consequence, ubiquitination biochemically demonstrated, primary cell infection model\",\n      \"pmids\": [\"23273896\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"MED31 is a direct target of miR-1 in osteosarcoma cells, validated by luciferase reporter assay; knockdown of Med31 suppresses osteosarcoma cell proliferation and reduces expression of MET proto-oncogene and blocks downstream HGF-stimulated MET signaling, placing Med31 upstream of MET pathway activation.\",\n      \"method\": \"Luciferase reporter assay (3'UTR target validation), siRNA knockdown, cell proliferation assays, western blotting for MET and downstream signaling\",\n      \"journal\": \"Oncology reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — direct target validation by luciferase assay plus KD phenotype with pathway readout, single lab\",\n      \"pmids\": [\"24969180\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Electron microscopy structures of yeast and human Mediator complexes with comprehensive subunit localization experiments provided the first accurate EM-based structural mapping of Mediator subunit positions and revealed that large-scale Mediator conformational rearrangements occur at interfaces between Mediator modules during transcription initiation.\",\n      \"method\": \"Electron microscopy, subunit localization by antibody labeling and crosslinking, biochemical analyses\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — EM structure with subunit localization, biochemical validation, covers MED31 as Mediator subunit\",\n      \"pmids\": [\"24882805\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"MED31 knockdown in human mesenchymal stem cells (hMSCs) using siRNA reduces self-renewal capacity (assessed by cell assays and gene expression) and impairs adipogenic differentiation, evidenced by diminished lipid vesicle formation and reduced adipogenic marker expression, demonstrating a role for MED31 in maintaining adult stem cell homeostasis.\",\n      \"method\": \"siRNA-mediated knockdown, cell proliferation/self-renewal assays, adipogenesis assays (lipid staining), qRT-PCR for lineage markers\",\n      \"journal\": \"Molecular biology reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — clean KD with multiple defined cellular phenotypes, single lab, no pathway placement\",\n      \"pmids\": [\"30006772\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"In Tetrahymena thermophila, endogenously tagged Med31 physically associates with >20 proteins including canonical Mediator subunits identified by AP-MS; Med31 ChIP-seq shows global association with transcribed genes; Med31 knockdown causes ectopic expression of developmental genes for programmed DNA rearrangements; and Med31 localizes to meiotic micronuclei implicating Mediator in ncRNA transcription.\",\n      \"method\": \"Endogenous tagging, affinity purification coupled with mass spectrometry (AP-MS), ChIP-seq, RNAi knockdown with RNA-seq, indirect immunofluorescence\",\n      \"journal\": \"Current biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods (AP-MS, ChIP-seq, KD + transcriptomics, immunofluorescence) in single study\",\n      \"pmids\": [\"31280994\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"ELMO1 methylation interacts with Med31 in H. pylori-infected gastric cancer cells (AGS); Co-immunoprecipitation confirmed ELMO1-Med31 protein interaction; this interaction drives M2 macrophage polarization and promotes epithelial-mesenchymal transition (EMT) and intestinal metaplasia in H. pylori-infected cells.\",\n      \"method\": \"Co-immunoprecipitation, methylation-specific PCR, co-culture experiments, western blotting, cell viability/migration assays\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — Co-IP confirms interaction, functional co-culture model, single lab, limited mechanistic resolution\",\n      \"pmids\": [\"41535333\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"MED31 is the smallest and most evolutionarily conserved subunit of the Mediator coactivator complex, required for basal and activated RNA polymerase II transcription; it undergoes monoubiquitination regulated by cytoplasmic Elmo1, which also re-localizes MED31 from its predominantly nuclear location to the cytoplasm to modulate cytokine gene expression; in mammalian development, Med31 promotes transcription of genes required for cell proliferation and chondrogenesis, and in stem cells it sustains self-renewal and adipogenic differentiation, while its protein level is itself regulated post-transcriptionally by miR-1 targeting its 3'UTR, upstream of MET oncogene signaling.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"MED31 is the smallest and most deeply conserved subunit of the Mediator transcriptional coactivator complex, functioning as a bridge between gene-specific transcription factors and the RNA polymerase II machinery to promote expression of genes governing cell proliferation, differentiation, and cell cycle progression [PMID:15356001, PMID:20347762]. In mouse embryos, MED31 loss reduces proliferating cell populations and abolishes expression of cell-cycle (Cdc2) and chondrogenic (Sox9, Col2a1) genes, while in human mesenchymal stem cells its depletion impairs self-renewal and adipogenic differentiation [PMID:20347762, PMID:30006772]. MED31 is post-translationally regulated by monoubiquitination and its nuclear-to-cytoplasmic distribution is controlled by the cytoplasmic protein ELMO1, an interaction that selectively modulates cytokine gene expression (Il10, Il33) in macrophages [PMID:23273896]. Genome-wide promoter occupancy in Tetrahymena and transcriptome analyses in fission yeast confirm MED31 acts broadly at promoters to control developmental and cell-separation gene programs [PMID:31280994, PMID:17922236].\",\n  \"teleology\": [\n    {\n      \"year\": 2004,\n      \"claim\": \"Establishing that Soh1/MED31 is a bona fide, stably incorporated Mediator subunit — rather than a transient interactor — resolved its molecular identity and placed it within the core transcriptional coactivator machinery across eukaryotes.\",\n      \"evidence\": \"Biochemical co-purification of Mediator complexes from S. cerevisiae and S. pombe with bioinformatic conservation analysis\",\n      \"pmids\": [\"15356001\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Position of MED31 within the Mediator architecture (head, middle, or tail module) not defined\",\n        \"Direct contacts with RNA Pol II or other Mediator subunits not mapped\",\n        \"No target gene specificity determined\"\n      ]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Demonstrating that Med31 is required for transcription of cell-separation genes downstream of Sep1-Ace2 in fission yeast showed it acts as a gene-specific coactivator bridging defined transcription factors to the general machinery.\",\n      \"evidence\": \"Genome-wide expression profiling of med31/sep10 deletion mutants with genetic epistasis analysis in S. pombe\",\n      \"pmids\": [\"17922236\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Whether Med31 physically contacts Sep1 or Ace2 directly was not tested\",\n        \"Applicability of this gene-specific coactivation model to metazoans unknown\"\n      ]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"A loss-of-function mouse mutant revealed that Med31 is essential for embryonic cell proliferation and chondrogenic differentiation, linking Mediator function via Med31 to mammalian development and cell cycle control.\",\n      \"evidence\": \"ENU mutagenesis in mouse; proliferation assays, Western blot for Cdc2, in situ hybridization for Sox9 and Col2a1 in mutant embryos\",\n      \"pmids\": [\"20347762\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Whether Med31 directly coactivates Sox9 or Cdc2 promoters versus acting indirectly was not established\",\n        \"Rescue experiment to confirm specificity of the ENU allele not reported\"\n      ]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Discovery that the cytoplasmic protein ELMO1 physically binds MED31, redistributes it from nucleus to cytoplasm, and enhances its monoubiquitination revealed an unexpected post-translational regulatory mechanism that governs cytokine-selective transcription in macrophages.\",\n      \"evidence\": \"Reciprocal co-immunoprecipitation, subcellular fractionation, immunofluorescence, ubiquitination assay, and profiling of >25 genes in primary macrophages during Salmonella infection\",\n      \"pmids\": [\"23273896\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"The E3 ubiquitin ligase responsible for MED31 monoubiquitination not identified\",\n        \"Functional consequence of monoubiquitination on Mediator assembly or transcription not determined\",\n        \"Whether ELMO1-mediated cytoplasmic sequestration is the primary mechanism regulating Il10/Il33 specificity remains unclear\"\n      ]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Identification of MED31 as a direct miR-1 target linked its expression level to proliferative output and MET/HGF signaling in cancer cells, establishing a post-transcriptional regulatory axis.\",\n      \"evidence\": \"Luciferase 3′UTR reporter assay, siRNA knockdown, cell proliferation assay, and Western blot for MET pathway components in osteosarcoma cells\",\n      \"pmids\": [\"24969180\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Whether MED31 directly coactivates MET transcription or acts indirectly not resolved\",\n        \"In vivo relevance of miR-1/MED31 axis in osteosarcoma not tested\"\n      ]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Knockdown in human mesenchymal stem cells extended MED31's developmental role to adult stem cell self-renewal and adipogenic lineage commitment, broadening its functional scope beyond embryonic contexts.\",\n      \"evidence\": \"siRNA knockdown with proliferation/self-renewal assays, Oil Red O staining, and qRT-PCR for adipogenic markers in hMSCs\",\n      \"pmids\": [\"30006772\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Direct transcriptional targets mediating the adipogenic defect not identified\",\n        \"Whether the phenotype reflects loss of MED31 specifically or Mediator destabilization not distinguished\"\n      ]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Genome-wide ChIP-seq in Tetrahymena established that Med31 occupies gene promoters broadly and restrains ectopic developmental gene expression, providing the first direct evidence of its chromatin occupancy and its role in silencing non-cognate transcriptional programs.\",\n      \"evidence\": \"AP-MS interactome, ChIP-seq, RNAi knockdown, and immunofluorescence in Tetrahymena thermophila\",\n      \"pmids\": [\"31280994\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Whether promoter occupancy reflects Med31's direct DNA or histone contacts versus Mediator recruitment is unknown\",\n        \"Relevance of Tetrahymena-specific interactors to metazoan Med31 function unclear\"\n      ]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"The ELMO1–Med31 interaction was extended to H. pylori-infected gastric cancer, where it promotes M2 macrophage polarization and epithelial-mesenchymal transition, indicating this partnership operates in pathological inflammatory contexts beyond acute infection.\",\n      \"evidence\": \"Co-immunoprecipitation, methylation-specific PCR, co-culture of macrophages and gastric cancer cells, Western blot, qRT-PCR, and functional assays\",\n      \"pmids\": [\"41535333\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Single Co-IP without reciprocal validation in this system\",\n        \"Whether ELMO1 methylation status directly modulates the physical interaction with Med31 not biochemically resolved\",\n        \"Causal direction between Med31 activity and M2 polarization not established\"\n      ]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The E3 ligase catalyzing MED31 monoubiquitination, the structural basis of MED31's position within the Mediator complex, and the mechanism by which ELMO1-mediated cytoplasmic sequestration achieves gene-selective transcriptional effects remain unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"No structural model of MED31 within the Mediator complex in metazoans\",\n        \"E3 ubiquitin ligase for MED31 monoubiquitination unidentified\",\n        \"Genome-wide direct target genes of MED31 in mammalian systems not mapped by ChIP-seq\"\n      ]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140110\", \"supporting_discovery_ids\": [1, 2, 5, 6]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [3, 6]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [3]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-74160\", \"supporting_discovery_ids\": [0, 1, 2, 6]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [2, 5]}\n    ],\n    \"complexes\": [\n      \"Mediator complex\"\n    ],\n    \"partners\": [\n      \"ELMO1\",\n      \"MED7\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```\n\n--- ROUND 2 (R2/R3, augmented corpus) ---\n\n```json\n{\n  \"mechanistic_narrative\": \"MED31 is the smallest and most evolutionarily ancient subunit of the Mediator coactivator complex, essential for both basal and activator-dependent RNA polymerase II transcription across eukaryotes [PMID:10024883, PMID:11559591, PMID:15356001]. In mammals, Med31 promotes transcription of genes required for cell proliferation and chondrogenesis during embryonic development, and sustains self-renewal and adipogenic differentiation in adult mesenchymal stem cells [PMID:20347762, PMID:30006772]. Beyond its nuclear transcriptional role, MED31 undergoes Elmo1-dependent monoubiquitination and cytoplasmic relocalization, which specifically modulates cytokine gene expression (Il10, Il33) during macrophage responses to bacterial infection [PMID:23273896]. MED31 protein levels are post-transcriptionally regulated by miR-1, and MED31 knockdown suppresses MET proto-oncogene expression and downstream HGF signaling in osteosarcoma cells [PMID:24969180].\",\n  \"teleology\": [\n    {\n      \"year\": 1999,\n      \"claim\": \"Identification of MED31 as a subunit of the human Mediator-like SMCC complex established that a yeast SOH1 homolog is conserved in mammalian transcriptional coactivator machinery capable of both repressing and enhancing activator-dependent transcription.\",\n      \"evidence\": \"Biochemical purification of SMCC complex with mass spectrometry identification and in vitro transcription assays\",\n      \"pmids\": [\"10024883\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Specific contribution of MED31 versus other SMCC subunits not dissected\", \"No loss-of-function data for MED31 alone\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Demonstrating that Mediator depletion abolishes transcription on all tested promoters established MED31-containing Mediator as a general transcription factor required for basal and activated transcription, not merely an activator-specific cofactor.\",\n      \"evidence\": \"Antibody depletion of Mediator from crude extracts followed by in vitro transcription on TATA-containing and TATA-less promoters\",\n      \"pmids\": [\"11559591\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Depletion removed entire Mediator rather than MED31 alone\", \"In vitro system may not recapitulate all in vivo regulation\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Phylogenetic and biochemical analyses across yeast species established MED31 as among the most ancient Mediator subunits, predating the canonical Pol II CTD heptapeptide repeat, and confirmed its stable incorporation into Mediator in multiple organisms.\",\n      \"evidence\": \"Mediator complex purification from S. pombe and S. cerevisiae; bioinformatic phylogenetic reconstruction\",\n      \"pmids\": [\"15356001\", \"15175163\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis for MED31 integration into the complex not resolved\", \"Function of MED31 outside of Mediator context unexplored\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Genome-wide profiling of Med31 deletion in S. pombe revealed that Med31 acts as a coactivator downstream of Sep1-Ace2 transcription factors for cell separation genes, establishing a specific gene-regulatory role beyond general transcription.\",\n      \"evidence\": \"Microarray profiling of sep10 (Med31) deletion mutants with genetic epistasis analysis in S. pombe\",\n      \"pmids\": [\"17922236\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism by which Med31 is recruited to Sep1-Ace2 target promoters not determined\", \"Single organism, single lab\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"A mouse point mutation causing Med31 protein degradation produced late-gestation lethality with reduced cell proliferation and defective chondrogenesis, directly demonstrating that Med31 promotes transcription of cell cycle (Cdc2) and cartilage differentiation (Sox9, Col2a1) genes in vivo.\",\n      \"evidence\": \"ENU mutagenesis screen, western blot for protein stability, embryonic fibroblast proliferation assays, immunohistochemistry for cell cycle and chondrogenic markers\",\n      \"pmids\": [\"20347762\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct transcriptional targets genome-wide not identified in mammalian context\", \"Whether phenotypes reflect Mediator destabilization versus MED31-specific functions unclear\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Discovery that cytoplasmic Elmo1 physically interacts with MED31, promotes its monoubiquitination and cytoplasmic relocalization, and together with MED31 regulates Il10 and Il33 expression during Salmonella infection revealed a non-canonical regulatory axis for a Mediator subunit outside the nucleus.\",\n      \"evidence\": \"Reciprocal Co-IP, subcellular fractionation, ubiquitination assays, gene expression analysis in primary macrophages during bacterial infection\",\n      \"pmids\": [\"23273896\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Ubiquitin ligase responsible for MED31 monoubiquitination not identified\", \"Whether cytoplasmic MED31 has transcription-independent functions unknown\", \"Mechanism linking MED31 ubiquitination to cytokine gene regulation not resolved\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"MED31 was placed upstream of MET oncogene signaling via miR-1-mediated post-transcriptional regulation, linking Mediator subunit levels to oncogenic pathway activity in osteosarcoma cells; concurrently, EM structural mapping resolved MED31's position within the Mediator architecture.\",\n      \"evidence\": \"Luciferase 3′UTR reporter assay, siRNA knockdown with MET pathway readouts; EM with antibody labeling for subunit localization\",\n      \"pmids\": [\"24969180\", \"24882805\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether MED31 directly regulates MET transcription or acts indirectly through Mediator integrity is unresolved\", \"High-resolution atomic structure of MED31 within Mediator not yet available at this time point\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"MED31 knockdown in human mesenchymal stem cells impaired both self-renewal and adipogenic differentiation, extending its proliferative role to adult stem cell homeostasis.\",\n      \"evidence\": \"siRNA knockdown with proliferation assays, lipid staining for adipogenesis, qRT-PCR for lineage markers in hMSCs\",\n      \"pmids\": [\"30006772\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Transcriptional targets mediating self-renewal versus differentiation not identified\", \"No genetic rescue performed\", \"Single lab, no in vivo validation\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"In Tetrahymena, Med31 ChIP-seq demonstrated global association with transcribed genes and knockdown caused ectopic developmental gene expression, while Med31 localization to meiotic micronuclei implicated Mediator in ncRNA transcription, extending the functional repertoire beyond protein-coding gene regulation.\",\n      \"evidence\": \"Endogenous tagging, AP-MS, ChIP-seq, RNAi with RNA-seq, immunofluorescence in Tetrahymena\",\n      \"pmids\": [\"31280994\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether ncRNA transcription role is conserved in metazoans is unknown\", \"Mechanism of Med31 recruitment to specific loci not defined\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Key unresolved questions include the identity of the E3 ubiquitin ligase for MED31 monoubiquitination, the functional significance of cytoplasmic MED31, whether MED31 has Mediator-independent functions, and high-resolution structural details of MED31 contacts within the human Mediator complex.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"E3 ligase for MED31 monoubiquitination not identified\", \"No Mediator-independent function demonstrated\", \"Genome-wide direct target identification in mammalian cells lacking\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140110\", \"supporting_discovery_ids\": [0, 1, 4, 5, 10]},\n      {\"term_id\": \"GO:0140223\", \"supporting_discovery_ids\": [1]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [6, 10]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [6]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-74160\", \"supporting_discovery_ids\": [0, 1, 4, 5, 10]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [5]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [6]}\n    ],\n    \"complexes\": [\"Mediator complex\"],\n    \"partners\": [\"ELMO1\", \"MED7\", \"SOX9\"],\n    \"other_free_text\": []\n  }\n}\n```"}