{"gene":"MED26","run_date":"2026-06-10T02:59:50","timeline":{"discoveries":[{"year":2011,"finding":"The conserved N-terminal domain (NTD) of MED26 contains overlapping docking sites for the super elongation complex (SEC, containing ELL/EAF and P-TEFb) and general initiation factor TFIID. MED26 functions as a molecular switch, interacting first with TFIID during Pol II initiation and then exchanging TFIID for SEC/ELL/EAF-containing complexes to facilitate transition of Pol II into productive elongation.","method":"Co-immunoprecipitation, domain mapping/mutational analysis, chromatin immunoprecipitation, functional transcription assays","journal":"Cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP, domain mapping with mutants, multiple orthogonal methods, highly cited foundational paper","pmids":["21729782"],"is_preprint":false},{"year":2015,"finding":"MED26 recruits the little elongation complex (LEC) to a subset of snRNA genes through direct interaction of EAF with the N-terminal domain (NTD) of MED26. Loss of MED26 decreases LEC occupancy at snRNA genes and reduces their transcription. The MED26-NTD functions as a molecular switch exchanging TAF7 for LEC to facilitate the initiation-to-elongation transition at snRNA genes.","method":"Co-immunoprecipitation, ChIP-seq, siRNA knockdown, transcription assays","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP, ChIP-seq, loss-of-function with defined transcriptional phenotype, multiple orthogonal methods","pmids":["25575120"],"is_preprint":false},{"year":2000,"finding":"The N-terminal domain (domain I) of TFIIS, which is homologous to CRSP70 (MED26), forms a compact four-helix bundle structure. Modeling of the homologous domain from CRSP70 identified a conserved positively charged surface patch potentially involved in interactions with the transcriptional machinery.","method":"NMR structure determination of yeast TFIIS domain I; homology modeling of CRSP70 domain","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — NMR structure solved but functional validation of CRSP70 model is limited to structural homology inference, single study","pmids":["10811649"],"is_preprint":false},{"year":2014,"finding":"Drosophila MED26 interacts with other core Mediator components but not with the kinase module, and is recruited to genes upon activation. MED26 interacts with heterochromatin protein 1 (HP1); this interaction is mediated through the chromoshadow domain of HP1 and a conserved motif in the C-terminus of MED26. A fraction of MED26 localizes to pericentric heterochromatin (chromocenter) in polytene chromosomes.","method":"Co-immunoprecipitation, domain mapping with deletion/mutation constructs, immunofluorescence on polytene chromosomes, null allele genetic analysis","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP with domain mapping, direct localization by immunostaining, loss-of-function phenotype, multiple orthogonal methods in single study","pmids":["24820420"],"is_preprint":false},{"year":2017,"finding":"The NMR solution structure of MED26-NTD reveals a 4-helix bundle. EAF1 and TAF7 peptides both bind to the same groove formed by H3 and H4 helices of MED26-NTD, with dissociation constants in the 10-μM range. Binding involves a folding-upon-binding mechanism; residues I222/F223 of TAF7 anchor to a hydrophobic pocket in MED26-NTD (L48, W80, I84). Alanine mutations of charged residues in EAF1 and TAF7 peptides reduce binding ~10-fold.","method":"NMR structure determination, NMR chemical shift perturbation mapping, NOE contacts, alanine mutagenesis, binding affinity measurements","journal":"Journal of molecular biology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — NMR structure with mutagenesis and quantitative binding data, multiple orthogonal methods, single lab","pmids":["28893534"],"is_preprint":false},{"year":2021,"finding":"MED26 (CRSP70) co-purifies with TFIID under high salt conditions and interacts strongly with TFIID; this interaction was identified during development of a purification protocol that required an additional DE52 chromatography step to separate MED26 from TFIID.","method":"Biochemical co-purification, chromatography fractionation, immunopurification","journal":"Protein expression and purification","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — direct biochemical co-purification demonstrating strong MED26-TFIID interaction, single lab, single method","pmids":["33836240"],"is_preprint":false},{"year":2024,"finding":"Cryo-EM structure of the human complete Mediator complex reveals that the CKM inhibits transcription by occluding the binding site for Pol II and MED26 on the core Mediator (cMED) through an intrinsically disordered region in MED13, placing MED26 binding to cMED as a site sterically blocked by the CKM.","method":"Cryo-electron microscopy structure determination of human complete Mediator and CKM","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — cryo-EM structure with direct visualization of MED26 binding site, but preprint and single study without functional mutagenesis validation of MED26-specific interaction","pmids":[],"is_preprint":true},{"year":2024,"finding":"During erythropoiesis, MED26 remains relatively abundant while other Mediator subunits decrease. MED26 preferentially occupies loci associated with Pol II pausing and recruits pausing-related factors. MED26 forms condensates, and this condensate-forming capability is required for its function in promoting erythropoiesis and recruiting pausing-related factors. More than half of MED26 occupancy sites do not co-localize with MED1, indicating context-dependent gene regulation.","method":"ChIP-seq, knockdown/knockout with erythroid differentiation assays, live-cell imaging of condensates, co-immunoprecipitation of pausing factors","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — multiple orthogonal methods (ChIP-seq, condensate imaging, KO phenotype) but preprint, single lab, not yet peer-reviewed","pmids":[],"is_preprint":true}],"current_model":"MED26 is a metazoan-specific Mediator subunit whose conserved 4-helix-bundle N-terminal domain (NTD) acts as a molecular switch: it first docks TFIID (via TAF7) at the initiation complex, then exchanges TAF7 for ELL/EAF-containing elongation complexes—either the super elongation complex (SEC) at polyadenylated genes or the little elongation complex (LEC) at snRNA genes—to facilitate Pol II pause-release and the transition into productive elongation; additionally, the C-terminal region of MED26 mediates interaction with HP1 at heterochromatin, and the CKM inhibits transcription by sterically occluding the MED26 binding site on core Mediator."},"narrative":{"mechanistic_narrative":"MED26 is a metazoan Mediator subunit that controls the transition of RNA polymerase II from initiation into productive elongation, acting through its conserved N-terminal domain (NTD) as a molecular switch [PMID:21729782]. The NTD folds into a four-helix bundle whose H3/H4 groove provides overlapping docking sites for the general initiation factor TFIID (via TAF7) and for ELL/EAF-containing elongation complexes, with both TAF7 and EAF1 peptides binding the same groove by a folding-upon-binding mechanism [PMID:28893534]. By first engaging TFIID at the initiation complex and then exchanging TAF7 for ELL/EAF complexes, MED26 facilitates Pol II pause-release: it recruits the super elongation complex (SEC, containing ELL/EAF and P-TEFb) at protein-coding genes [PMID:21729782] and, through direct EAF–NTD contact, recruits the little elongation complex (LEC) to a subset of snRNA genes, where MED26 loss reduces LEC occupancy and snRNA transcription [PMID:25575120]. Beyond its elongation role, a conserved C-terminal motif of MED26 binds the chromoshadow domain of heterochromatin protein 1 (HP1), and a fraction of MED26 localizes to pericentric heterochromatin [PMID:24820420]. MED26 integrates into core Mediator at a site occluded by the CKM, which sterically blocks both Pol II and MED26 binding via a disordered region of MED13.","teleology":[{"year":2000,"claim":"Before MED26's mechanism was known, structural analysis of a homologous domain established the architectural template for its functional region.","evidence":"NMR structure of yeast TFIIS domain I with homology modeling of the CRSP70 (MED26) domain","pmids":["10811649"],"confidence":"Medium","gaps":["CRSP70/MED26 structure inferred only by homology, not directly solved here","Functional role of the conserved charged surface patch untested","No partner interactions demonstrated"]},{"year":2011,"claim":"Established the central function of MED26 as a molecular switch coupling initiation to elongation by exchanging TFIID for the super elongation complex on its NTD.","evidence":"Co-IP, domain mapping with mutants, ChIP, and transcription assays in human cells","pmids":["21729782"],"confidence":"High","gaps":["Structural basis of the competitive exchange not resolved","Kinetics and trigger of the switch in vivo unknown","Generality across gene classes not addressed"]},{"year":2014,"claim":"Extended MED26 function beyond Mediator/elongation by showing a heterochromatin connection through its C-terminus, implying a role outside active transcription.","evidence":"Co-IP, domain mapping, immunofluorescence on Drosophila polytene chromosomes, and null allele analysis","pmids":["24820420"],"confidence":"High","gaps":["Functional consequence of MED26-HP1 binding at heterochromatin unresolved","Whether HP1 interaction is conserved in mammals untested","Relationship between heterochromatin pool and elongation pool unclear"]},{"year":2015,"claim":"Generalized the switch model to snRNA genes by showing the NTD recruits the little elongation complex via EAF, distinguishing gene-class-specific elongation factor choice.","evidence":"Co-IP, ChIP-seq, siRNA knockdown, and transcription assays","pmids":["25575120"],"confidence":"High","gaps":["What determines SEC versus LEC selection at a given gene is unknown","Direct competition between LEC and TFIID not structurally defined"]},{"year":2017,"claim":"Provided the structural and energetic basis for the switch by showing TAF7 and EAF1 compete for the same NTD groove with comparable micromolar affinities.","evidence":"NMR solution structure, chemical shift perturbation, alanine mutagenesis, and binding affinity measurements","pmids":["28893534"],"confidence":"High","gaps":["How comparable affinities are biased toward exchange in vivo unclear","Full-length protein contributions beyond peptides untested"]},{"year":2021,"claim":"Confirmed a robust biochemical association between MED26 and TFIID, reinforcing the initiation-stage interaction inferred from earlier functional work.","evidence":"Biochemical co-purification and chromatography fractionation of TFIID","pmids":["33836240"],"confidence":"Medium","gaps":["Interaction characterized only biochemically, not structurally here","Stoichiometry and regulation not addressed"]},{"year":2024,"claim":"Defined how MED26 incorporation into core Mediator is regulated, showing the CKM sterically occludes the MED26/Pol II binding site.","evidence":"Cryo-EM of human complete Mediator and CKM (preprint)","pmids":[],"confidence":"Medium","gaps":["Preprint, no functional mutagenesis of the occluded MED26 site","Dynamics of CKM displacement to permit MED26 binding not shown"]},{"year":2024,"claim":"Revealed a context-dependent, condensate-based mode of MED26 action during differentiation, distinct from canonical Mediator co-occupancy.","evidence":"ChIP-seq, knockdown/knockout erythroid differentiation assays, live-cell condensate imaging, and Co-IP of pausing factors (preprint)","pmids":[],"confidence":"Medium","gaps":["Preprint, single lab","Molecular determinants of condensate formation undefined","Why MED26 occupancy diverges from MED1 mechanistically unclear"]},{"year":null,"claim":"How MED26 integrates its initiation-to-elongation switching, heterochromatin association, and condensate formation into context-specific gene regulatory outcomes remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unified model linking the three activities","In vivo trigger of the TFIID-to-elongation-complex exchange unknown","Mammalian relevance of HP1 interaction untested"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[0,1,4]},{"term_id":"GO:0140110","term_label":"transcription regulator activity","supporting_discovery_ids":[0,1]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[0,3]},{"term_id":"GO:0000228","term_label":"nuclear chromosome","supporting_discovery_ids":[3]}],"pathway":[{"term_id":"R-HSA-74160","term_label":"Gene expression (Transcription)","supporting_discovery_ids":[0,1]}],"complexes":["Mediator","super elongation complex (SEC)","little elongation complex (LEC)"],"partners":["TAF7","TFIID","ELL","EAF1","HP1","MED13"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"O95402","full_name":"Mediator of RNA polymerase II transcription subunit 26","aliases":["Activator-recruited cofactor 70 kDa component","ARC70","Cofactor required for Sp1 transcriptional activation subunit 7","CRSP complex subunit 7","Mediator complex subunit 26","Transcriptional coactivator CRSP70"],"length_aa":600,"mass_kda":65.4,"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 pre-initiation complex with RNA polymerase II and the general transcription factors","subcellular_location":"Nucleus","url":"https://www.uniprot.org/uniprotkb/O95402/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":true,"resolved_as":"","url":"https://depmap.org/portal/gene/MED26","classification":"Common Essential","n_dependent_lines":1135,"n_total_lines":1208,"dependency_fraction":0.9395695364238411},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"MED14","stoichiometry":10.0},{"gene":"MED19","stoichiometry":10.0},{"gene":"MED4","stoichiometry":10.0},{"gene":"KPNA4","stoichiometry":0.2},{"gene":"MED10","stoichiometry":0.2},{"gene":"MED11","stoichiometry":0.2},{"gene":"MED21","stoichiometry":0.2},{"gene":"MED27","stoichiometry":0.2},{"gene":"MED28","stoichiometry":0.2},{"gene":"MED29","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/MED26","total_profiled":1310},"omim":[{"mim_id":"605043","title":"MEDIATOR COMPLEX SUBUNIT 26; MED26","url":"https://www.omim.org/entry/605043"},{"mim_id":"300182","title":"MEDIATOR COMPLEX SUBUNIT 14; MED14","url":"https://www.omim.org/entry/300182"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Nucleoplasm","reliability":"Approved"},{"location":"Mitotic chromosome","reliability":"Additional"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in all","driving_tissues":[{"tissue":"testis","ntpm":34.0}],"url":"https://www.proteinatlas.org/search/MED26"},"hgnc":{"alias_symbol":["CRSP70"],"prev_symbol":["CRSP7"]},"alphafold":{"accession":"O95402","domains":[{"cath_id":"1.20.930.10","chopping":"9-85","consensus_level":"high","plddt":87.6365,"start":9,"end":85},{"cath_id":"-","chopping":"527-600","consensus_level":"medium","plddt":83.5377,"start":527,"end":600}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/O95402","model_url":"https://alphafold.ebi.ac.uk/files/AF-O95402-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-O95402-F1-predicted_aligned_error_v6.png","plddt_mean":58.03},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=MED26","jax_strain_url":"https://www.jax.org/strain/search?query=MED26"},"sequence":{"accession":"O95402","fasta_url":"https://rest.uniprot.org/uniprotkb/O95402.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/O95402/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/O95402"}},"corpus_meta":[{"pmid":"21729782","id":"PMC_21729782","title":"Human mediator subunit MED26 functions as a docking site for transcription elongation factors.","date":"2011","source":"Cell","url":"https://pubmed.ncbi.nlm.nih.gov/21729782","citation_count":282,"is_preprint":false},{"pmid":"10811649","id":"PMC_10811649","title":"Structure of a conserved domain common to the transcription factors TFIIS, elongin A, and CRSP70.","date":"2000","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/10811649","citation_count":53,"is_preprint":false},{"pmid":"25575120","id":"PMC_25575120","title":"MED26 regulates the transcription of snRNA genes through the recruitment of little elongation complex.","date":"2015","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/25575120","citation_count":44,"is_preprint":false},{"pmid":"24820420","id":"PMC_24820420","title":"The metazoan-specific mediator subunit 26 (Med26) is essential for viability and is found at both active genes and pericentric heterochromatin in Drosophila melanogaster.","date":"2014","source":"Molecular and cellular biology","url":"https://pubmed.ncbi.nlm.nih.gov/24820420","citation_count":17,"is_preprint":false},{"pmid":"28893534","id":"PMC_28893534","title":"Solution Structure of the N-Terminal Domain of Mediator Subunit MED26 and Molecular Characterization of Its Interaction with EAF1 and TAF7.","date":"2017","source":"Journal of molecular biology","url":"https://pubmed.ncbi.nlm.nih.gov/28893534","citation_count":12,"is_preprint":false},{"pmid":"22215486","id":"PMC_22215486","title":"Identification of mediator complex 26 (Crsp7) gametologs on platypus X1 and Y5 sex chromosomes: a candidate testis-determining gene in monotremes?","date":"2012","source":"Chromosome research : an international journal on the molecular, supramolecular and evolutionary aspects of chromosome biology","url":"https://pubmed.ncbi.nlm.nih.gov/22215486","citation_count":8,"is_preprint":false},{"pmid":"36852638","id":"PMC_36852638","title":"MED26-containing Mediator may orchestrate multiple transcription processes through organization of nuclear bodies.","date":"2023","source":"BioEssays : news and reviews in molecular, cellular and developmental biology","url":"https://pubmed.ncbi.nlm.nih.gov/36852638","citation_count":3,"is_preprint":false},{"pmid":"26861138","id":"PMC_26861138","title":"1H, 15N and 13C assignments of the N-terminal domain of the Mediator complex subunit MED26.","date":"2016","source":"Biomolecular NMR assignments","url":"https://pubmed.ncbi.nlm.nih.gov/26861138","citation_count":3,"is_preprint":false},{"pmid":"33836240","id":"PMC_33836240","title":"A simple protocol to purify human TFIID free of the MED26 subunit of mediator complex.","date":"2021","source":"Protein expression and purification","url":"https://pubmed.ncbi.nlm.nih.gov/33836240","citation_count":0,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2024.08.26.609654","title":"MED26-enriched condensates drive erythropoiesis through modulating transcription pausing","date":"2024-08-26","source":"bioRxiv","url":"https://doi.org/10.1101/2024.08.26.609654","citation_count":0,"is_preprint":true},{"pmid":null,"id":"bio_10.1101_2025.01.11.632546","title":"Dynamic interactions of retroviral Gag condensates with nascent viral RNA at transcriptional burst sites: implications for genomic RNA packaging","date":"2025-01-12","source":"bioRxiv","url":"https://doi.org/10.1101/2025.01.11.632546","citation_count":0,"is_preprint":true},{"pmid":null,"id":"bio_10.1101_2024.07.01.601608","title":"Structural basis of the human transcriptional Mediator complex modulated by its dissociable Kinase module","date":"2024-07-03","source":"bioRxiv","url":"https://doi.org/10.1101/2024.07.01.601608","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":7513,"output_tokens":2161,"usd":0.027477,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":9094,"output_tokens":2649,"usd":0.055848,"stage2_stop_reason":"end_turn"},"total_usd":0.083325,"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\": 2011,\n      \"finding\": \"The conserved N-terminal domain (NTD) of MED26 contains overlapping docking sites for the super elongation complex (SEC, containing ELL/EAF and P-TEFb) and general initiation factor TFIID. MED26 functions as a molecular switch, interacting first with TFIID during Pol II initiation and then exchanging TFIID for SEC/ELL/EAF-containing complexes to facilitate transition of Pol II into productive elongation.\",\n      \"method\": \"Co-immunoprecipitation, domain mapping/mutational analysis, chromatin immunoprecipitation, functional transcription assays\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP, domain mapping with mutants, multiple orthogonal methods, highly cited foundational paper\",\n      \"pmids\": [\"21729782\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"MED26 recruits the little elongation complex (LEC) to a subset of snRNA genes through direct interaction of EAF with the N-terminal domain (NTD) of MED26. Loss of MED26 decreases LEC occupancy at snRNA genes and reduces their transcription. The MED26-NTD functions as a molecular switch exchanging TAF7 for LEC to facilitate the initiation-to-elongation transition at snRNA genes.\",\n      \"method\": \"Co-immunoprecipitation, ChIP-seq, siRNA knockdown, transcription assays\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP, ChIP-seq, loss-of-function with defined transcriptional phenotype, multiple orthogonal methods\",\n      \"pmids\": [\"25575120\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"The N-terminal domain (domain I) of TFIIS, which is homologous to CRSP70 (MED26), forms a compact four-helix bundle structure. Modeling of the homologous domain from CRSP70 identified a conserved positively charged surface patch potentially involved in interactions with the transcriptional machinery.\",\n      \"method\": \"NMR structure determination of yeast TFIIS domain I; homology modeling of CRSP70 domain\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — NMR structure solved but functional validation of CRSP70 model is limited to structural homology inference, single study\",\n      \"pmids\": [\"10811649\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Drosophila MED26 interacts with other core Mediator components but not with the kinase module, and is recruited to genes upon activation. MED26 interacts with heterochromatin protein 1 (HP1); this interaction is mediated through the chromoshadow domain of HP1 and a conserved motif in the C-terminus of MED26. A fraction of MED26 localizes to pericentric heterochromatin (chromocenter) in polytene chromosomes.\",\n      \"method\": \"Co-immunoprecipitation, domain mapping with deletion/mutation constructs, immunofluorescence on polytene chromosomes, null allele genetic analysis\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP with domain mapping, direct localization by immunostaining, loss-of-function phenotype, multiple orthogonal methods in single study\",\n      \"pmids\": [\"24820420\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"The NMR solution structure of MED26-NTD reveals a 4-helix bundle. EAF1 and TAF7 peptides both bind to the same groove formed by H3 and H4 helices of MED26-NTD, with dissociation constants in the 10-μM range. Binding involves a folding-upon-binding mechanism; residues I222/F223 of TAF7 anchor to a hydrophobic pocket in MED26-NTD (L48, W80, I84). Alanine mutations of charged residues in EAF1 and TAF7 peptides reduce binding ~10-fold.\",\n      \"method\": \"NMR structure determination, NMR chemical shift perturbation mapping, NOE contacts, alanine mutagenesis, binding affinity measurements\",\n      \"journal\": \"Journal of molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — NMR structure with mutagenesis and quantitative binding data, multiple orthogonal methods, single lab\",\n      \"pmids\": [\"28893534\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"MED26 (CRSP70) co-purifies with TFIID under high salt conditions and interacts strongly with TFIID; this interaction was identified during development of a purification protocol that required an additional DE52 chromatography step to separate MED26 from TFIID.\",\n      \"method\": \"Biochemical co-purification, chromatography fractionation, immunopurification\",\n      \"journal\": \"Protein expression and purification\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — direct biochemical co-purification demonstrating strong MED26-TFIID interaction, single lab, single method\",\n      \"pmids\": [\"33836240\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Cryo-EM structure of the human complete Mediator complex reveals that the CKM inhibits transcription by occluding the binding site for Pol II and MED26 on the core Mediator (cMED) through an intrinsically disordered region in MED13, placing MED26 binding to cMED as a site sterically blocked by the CKM.\",\n      \"method\": \"Cryo-electron microscopy structure determination of human complete Mediator and CKM\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — cryo-EM structure with direct visualization of MED26 binding site, but preprint and single study without functional mutagenesis validation of MED26-specific interaction\",\n      \"pmids\": [],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"During erythropoiesis, MED26 remains relatively abundant while other Mediator subunits decrease. MED26 preferentially occupies loci associated with Pol II pausing and recruits pausing-related factors. MED26 forms condensates, and this condensate-forming capability is required for its function in promoting erythropoiesis and recruiting pausing-related factors. More than half of MED26 occupancy sites do not co-localize with MED1, indicating context-dependent gene regulation.\",\n      \"method\": \"ChIP-seq, knockdown/knockout with erythroid differentiation assays, live-cell imaging of condensates, co-immunoprecipitation of pausing factors\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — multiple orthogonal methods (ChIP-seq, condensate imaging, KO phenotype) but preprint, single lab, not yet peer-reviewed\",\n      \"pmids\": [],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"MED26 is a metazoan-specific Mediator subunit whose conserved 4-helix-bundle N-terminal domain (NTD) acts as a molecular switch: it first docks TFIID (via TAF7) at the initiation complex, then exchanges TAF7 for ELL/EAF-containing elongation complexes—either the super elongation complex (SEC) at polyadenylated genes or the little elongation complex (LEC) at snRNA genes—to facilitate Pol II pause-release and the transition into productive elongation; additionally, the C-terminal region of MED26 mediates interaction with HP1 at heterochromatin, and the CKM inhibits transcription by sterically occluding the MED26 binding site on core Mediator.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"MED26 is a metazoan Mediator subunit that controls the transition of RNA polymerase II from initiation into productive elongation, acting through its conserved N-terminal domain (NTD) as a molecular switch [#0]. The NTD folds into a four-helix bundle whose H3/H4 groove provides overlapping docking sites for the general initiation factor TFIID (via TAF7) and for ELL/EAF-containing elongation complexes, with both TAF7 and EAF1 peptides binding the same groove by a folding-upon-binding mechanism [#4]. By first engaging TFIID at the initiation complex and then exchanging TAF7 for ELL/EAF complexes, MED26 facilitates Pol II pause-release: it recruits the super elongation complex (SEC, containing ELL/EAF and P-TEFb) at protein-coding genes [#0] and, through direct EAF–NTD contact, recruits the little elongation complex (LEC) to a subset of snRNA genes, where MED26 loss reduces LEC occupancy and snRNA transcription [#1]. Beyond its elongation role, a conserved C-terminal motif of MED26 binds the chromoshadow domain of heterochromatin protein 1 (HP1), and a fraction of MED26 localizes to pericentric heterochromatin [#3]. MED26 integrates into core Mediator at a site occluded by the CKM, which sterically blocks both Pol II and MED26 binding via a disordered region of MED13 [#6].\",\n  \"teleology\": [\n    {\n      \"year\": 2000,\n      \"claim\": \"Before MED26's mechanism was known, structural analysis of a homologous domain established the architectural template for its functional region.\",\n      \"evidence\": \"NMR structure of yeast TFIIS domain I with homology modeling of the CRSP70 (MED26) domain\",\n      \"pmids\": [\"10811649\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"CRSP70/MED26 structure inferred only by homology, not directly solved here\", \"Functional role of the conserved charged surface patch untested\", \"No partner interactions demonstrated\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Established the central function of MED26 as a molecular switch coupling initiation to elongation by exchanging TFIID for the super elongation complex on its NTD.\",\n      \"evidence\": \"Co-IP, domain mapping with mutants, ChIP, and transcription assays in human cells\",\n      \"pmids\": [\"21729782\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of the competitive exchange not resolved\", \"Kinetics and trigger of the switch in vivo unknown\", \"Generality across gene classes not addressed\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Extended MED26 function beyond Mediator/elongation by showing a heterochromatin connection through its C-terminus, implying a role outside active transcription.\",\n      \"evidence\": \"Co-IP, domain mapping, immunofluorescence on Drosophila polytene chromosomes, and null allele analysis\",\n      \"pmids\": [\"24820420\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional consequence of MED26-HP1 binding at heterochromatin unresolved\", \"Whether HP1 interaction is conserved in mammals untested\", \"Relationship between heterochromatin pool and elongation pool unclear\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Generalized the switch model to snRNA genes by showing the NTD recruits the little elongation complex via EAF, distinguishing gene-class-specific elongation factor choice.\",\n      \"evidence\": \"Co-IP, ChIP-seq, siRNA knockdown, and transcription assays\",\n      \"pmids\": [\"25575120\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"What determines SEC versus LEC selection at a given gene is unknown\", \"Direct competition between LEC and TFIID not structurally defined\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Provided the structural and energetic basis for the switch by showing TAF7 and EAF1 compete for the same NTD groove with comparable micromolar affinities.\",\n      \"evidence\": \"NMR solution structure, chemical shift perturbation, alanine mutagenesis, and binding affinity measurements\",\n      \"pmids\": [\"28893534\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How comparable affinities are biased toward exchange in vivo unclear\", \"Full-length protein contributions beyond peptides untested\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Confirmed a robust biochemical association between MED26 and TFIID, reinforcing the initiation-stage interaction inferred from earlier functional work.\",\n      \"evidence\": \"Biochemical co-purification and chromatography fractionation of TFIID\",\n      \"pmids\": [\"33836240\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Interaction characterized only biochemically, not structurally here\", \"Stoichiometry and regulation not addressed\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Defined how MED26 incorporation into core Mediator is regulated, showing the CKM sterically occludes the MED26/Pol II binding site.\",\n      \"evidence\": \"Cryo-EM of human complete Mediator and CKM (preprint)\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Preprint, no functional mutagenesis of the occluded MED26 site\", \"Dynamics of CKM displacement to permit MED26 binding not shown\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Revealed a context-dependent, condensate-based mode of MED26 action during differentiation, distinct from canonical Mediator co-occupancy.\",\n      \"evidence\": \"ChIP-seq, knockdown/knockout erythroid differentiation assays, live-cell condensate imaging, and Co-IP of pausing factors (preprint)\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Preprint, single lab\", \"Molecular determinants of condensate formation undefined\", \"Why MED26 occupancy diverges from MED1 mechanistically unclear\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How MED26 integrates its initiation-to-elongation switching, heterochromatin association, and condensate formation into context-specific gene regulatory outcomes remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No unified model linking the three activities\", \"In vivo trigger of the TFIID-to-elongation-complex exchange unknown\", \"Mammalian relevance of HP1 interaction untested\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [0, 1, 4]},\n      {\"term_id\": \"GO:0140110\", \"supporting_discovery_ids\": [0, 1]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [0, 3]},\n      {\"term_id\": \"GO:0000228\", \"supporting_discovery_ids\": [3]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-74160\", \"supporting_discovery_ids\": [0, 1]}\n    ],\n    \"complexes\": [\"Mediator\", \"super elongation complex (SEC)\", \"little elongation complex (LEC)\"],\n    \"partners\": [\"TAF7\", \"TFIID\", \"ELL\", \"EAF1\", \"HP1\", \"MED13\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":4,"faith_total":4,"faith_pct":100.0}}