{"gene":"RFXAP","run_date":"2026-06-10T06:43:36","timeline":{"discoveries":[{"year":1997,"finding":"RFXAP is a novel subunit of the RFX DNA-binding complex required for MHC class II gene expression. Mutations in RFXAP in group D MHC-II deficiency patients abolish RFX binding activity and MHC-II expression; transfection with wild-type RFXAP fully restores expression of all endogenous MHC-II genes.","method":"Complementation assay by transfection, DNA-binding (RFX complex) activity assay, identification of mutations in patient cell lines","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 / Strong — complementation rescue experiments in patient cell lines, replicated independently in two papers (PMID:9118943 and PMID:9287230) from different groups","pmids":["9118943","9287230"],"is_preprint":false},{"year":2001,"finding":"The C-terminal domain of RFXAP is essential for MHC-II expression, but different portions of this domain are required for different isotypes: a short C-terminal segment suffices for HLA-DR, while a larger C-terminal segment is required for optimal HLA-DQ and HLA-DP expression. This differential requirement reflects differential dependence on this domain for promoter occupancy and recruitment of the co-activator CIITA in vivo.","method":"Deletion/domain-mapping mutants of mouse and human RFXAP transfected into RFXAP-deficient cell lines; in vivo promoter occupancy and CIITA recruitment assays","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — multiple deletion mutants tested in complementation assays with in vivo ChIP for promoter occupancy and CIITA recruitment, single lab but multiple orthogonal methods","pmids":["11486010"],"is_preprint":false},{"year":2005,"finding":"Conserved hydrophobic and other non-glutamine residues in the C-terminal third of RFXAP are required for coordinate MHC-II isotype expression; mutation of potential phosphorylation sites abolishes RFXAP activity. Certain RFXAP mutants can rescue HLA-DR but not HLA-DQ or HLA-DP, correlating with their ability to form RFX complexes, bind DNA in vivo, and recruit CIITA to promoters.","method":"Site-directed mutagenesis of conserved residues, complementation in BLS cell lines, in vivo ChIP for DNA binding and CIITA recruitment, chimeric reporter gene assays","journal":"Molecular immunology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — mutagenesis combined with in vivo ChIP and reporter assays, single lab with multiple orthogonal methods","pmids":["16337482"],"is_preprint":false},{"year":2008,"finding":"DNA binding of RFX5 is autoinhibited by domains flanking its DNA-binding domain; both RFXAP and RFXB are required to relieve this autoinhibition and allow a single RFX complex to bind the proximal regulatory region of the MHC-II promoter.","method":"Electrophoretic mobility shift assay (EMSA) with purified RFX5, RFXAP, and RFXB proteins; domain-deletion analysis","journal":"Biochimica et biophysica acta","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — in vitro EMSA with purified proteins, single lab, single method type","pmids":["18723135"],"is_preprint":false},{"year":2009,"finding":"The C-terminal domain of RFXAP (RFXAP_C) is intrinsically disordered in isolation but folds into two α-helices upon binding to the N-terminal dimerization domain of RFX5 (RFX5_N). The resulting RFX5_N2–RFXAP_C complex then binds RFXB with high affinity, establishing an ordered assembly pathway for the RFX complex.","method":"NMR spectroscopy, circular dichroism spectroscopy, isothermal titration calorimetry","journal":"Proteins","confidence":"High","confidence_rationale":"Tier 1 / Moderate — multiple biophysical methods (NMR, CD, ITC) in a single study characterizing folding-upon-binding mechanism, single lab","pmids":["19274739"],"is_preprint":false},{"year":2010,"finding":"Solution NMR structure of the RFX5_N2–RFXAP_C complex shows that two RFX5 N-terminal helices form an antiparallel coiled-coil 'staple', and the two α-helices of RFXAP_C form a V-shaped structure that packs within this staple. Leucine residues in the leucine-rich region of RFX5_N (62-LYLYLQL-68) contribute to both RFX5 dimerization and the RFX5–RFXAP interface; clustered hydrophobic residues on RFXAP_C suggest a binding site for RFXB.","method":"15N- and 13C-edited NMR spectroscopy (solution structure determination)","journal":"Journal of molecular biology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — high-resolution NMR structure with mutagenesis validation of key interface residues, single lab","pmids":["20732328"],"is_preprint":false},{"year":2008,"finding":"A homozygous 75 bp insertion in the 5'-UTR of the RFXAP gene impairs RFXAP promoter activity, reduces RNA polymerase II recruitment to RFXAP chromatin, and results in complete loss of RFXAP mRNA and protein, causing MHC-II deficiency without any coding-sequence mutation.","method":"Promoter activity assay, chromatin immunoprecipitation (RNA Pol II ChIP), sequencing of patient cell line","journal":"Molecular immunology","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — promoter reporter and ChIP in patient cell line, single lab, single patient","pmids":["18336911"],"is_preprint":false},{"year":2009,"finding":"An 11-base deletion in RFXAP causing a frameshift at amino acid 234 and loss of C-terminal residues leads to coordinate loss of all MHC-II expression in a DLBCL cell line; stable transfection of wild-type RFXAP restores MHC-II expression, confirming that C-terminal RFXAP sequences are required for function.","method":"Sequencing of RFXAP in OCI-Ly2 cells, stable transfection complementation assay, MHC-II surface expression analysis","journal":"Immunogenetics","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — complementation by stable transfection in a single DLBCL cell line, single lab","pmids":["20024540"],"is_preprint":false},{"year":2020,"finding":"RFXAP transcriptionally activates KDM4A (a histone H3K36 tri-/dimethyl demethylase) in pancreatic cancer cells; RFXAP overexpression increases KDM4A expression, reduces H3K36 methylation, impairs DNA repair, and enhances fisetin-induced DNA damage and S-phase arrest, while RFXAP silencing has the opposite effect.","method":"ChIP sequencing (RFXAP binding to KDM4A promoter), dual-luciferase reporter assay, RFXAP overexpression/knockdown, western blot, immunofluorescence for DNA damage markers, xenograft mouse model","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ChIP-seq, reporter assay, and gain/loss-of-function experiments with defined molecular readouts, single lab","pmids":["33093461"],"is_preprint":false},{"year":2015,"finding":"miR-212-3p transferred from pancreatic cancer-derived exosomes directly targets and suppresses RFXAP mRNA in dendritic cells, resulting in decreased MHC-II expression and induction of immune tolerance.","method":"miRNA target prediction validated by transfection of miR-212-3p mimics/inhibitors into dendritic cells, measurement of RFXAP mRNA/protein and MHC-II surface expression; luciferase reporter assay (implied by validation in context)","journal":"Oncotarget","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — miRNA-target validation experiments with gain/loss-of-function in dendritic cells, replicated conceptually in a follow-up paper (PMID:29467893)","pmids":["26337469"],"is_preprint":false},{"year":2018,"finding":"IFN-γ suppresses miR-212-3p expression in pancreatic cancer cells in a dose- and time-dependent manner, leading to upregulation of RFXAP and MHC-II; luciferase assay confirmed RFXAP as a direct target of miR-212-3p. When miR-212-3p mimics were transfected into cells, IFN-γ could no longer increase RFXAP or MHC-II, placing miR-212-3p inhibition downstream of IFN-γ and upstream of RFXAP induction.","method":"miR-212-3p mimic/inhibitor transfection, IFN-γ dose-response experiment, luciferase reporter assay for RFXAP 3'-UTR targeting, qRT-PCR and western blot","journal":"Oncology letters","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — luciferase reporter and epistasis experiment with mimics/inhibitors, single lab, multiple orthogonal methods","pmids":["29467893"],"is_preprint":false}],"current_model":"RFXAP is an essential subunit of the trimeric RFX transcription factor complex (with RFX5 and RFXB) that binds X-box elements in MHC class II promoters: its intrinsically disordered C-terminal domain folds upon binding the antiparallel coiled-coil dimerization domain of RFX5, relieving RFX5 autoinhibition and creating a platform for high-affinity RFXB recruitment, and its conserved C-terminal residues (including phosphorylatable sites) mediate isotype-specific differences in CIITA co-activator recruitment and promoter occupancy; beyond MHC-II regulation, RFXAP also directly activates transcription of the histone demethylase KDM4A in pancreatic cells, and its expression is post-transcriptionally regulated by miR-212-3p, which is itself suppressed by IFN-γ."},"narrative":{"mechanistic_narrative":"RFXAP is an essential subunit of the trimeric RFX transcription factor complex (with RFX5 and RFXB) that binds X-box elements in MHC class II promoters, and its loss causes group D MHC-II deficiency [PMID:9118943, PMID:9287230]. Mechanistically, the intrinsically disordered C-terminal domain of RFXAP folds into two α-helices upon binding the antiparallel coiled-coil dimerization 'staple' formed by two RFX5 N-terminal helices, generating an RFX5–RFXAP module that then recruits RFXB with high affinity to establish an ordered assembly pathway [PMID:19274739, PMID:20732328]; both RFXAP and RFXB are required to relieve autoinhibition of RFX5 DNA binding and allow the complex to occupy the proximal MHC-II promoter [PMID:18723135]. The conserved C-terminal residues of RFXAP — including hydrophobic positions and phosphorylatable sites — mediate isotype-specific transcriptional output by controlling promoter occupancy and recruitment of the co-activator CIITA, such that distinct C-terminal segments are differentially required for HLA-DR versus HLA-DQ and HLA-DP expression [PMID:11486010, PMID:16337482]. Beyond MHC-II regulation, RFXAP directly binds and transcriptionally activates the histone demethylase KDM4A in pancreatic cancer cells, reducing H3K36 methylation, impairing DNA repair, and sensitizing cells to DNA damage [PMID:33093461]. RFXAP expression is itself post-transcriptionally controlled by miR-212-3p, which directly targets RFXAP mRNA to suppress MHC-II and induce immune tolerance, and which is repressed by IFN-γ to drive RFXAP induction [PMID:26337469, PMID:29467893].","teleology":[{"year":1997,"claim":"Established that RFXAP is a distinct, essential subunit of the RFX DNA-binding complex whose loss causes a defined MHC-II deficiency, defining the gene's core requirement for MHC-II expression.","evidence":"Complementation rescue by transfection of wild-type RFXAP into patient cell lines with RFX-binding and MHC-II readouts","pmids":["9118943","9287230"],"confidence":"High","gaps":["Did not resolve how RFXAP contributes structurally to the complex","Did not define the functional domains within RFXAP"]},{"year":2001,"claim":"Mapped function to the RFXAP C-terminal domain and revealed isotype-specific requirements, showing the protein does more than serve as a uniform scaffold.","evidence":"Deletion/domain-mapping complementation in RFXAP-deficient cells with in vivo promoter occupancy and CIITA recruitment assays","pmids":["11486010"],"confidence":"High","gaps":["Did not identify the specific residues mediating the isotype distinction","Mechanism of differential CIITA recruitment unresolved"]},{"year":2005,"claim":"Pinpointed conserved hydrophobic and phosphorylatable C-terminal residues as the determinants of complex formation, DNA binding, and CIITA recruitment, linking sequence to coordinate isotype regulation.","evidence":"Site-directed mutagenesis with complementation, in vivo ChIP, and chimeric reporter assays in BLS cell lines","pmids":["16337482"],"confidence":"High","gaps":["Whether phosphorylation actually occurs and which kinase acts is not established","No structural basis for the residue requirements yet"]},{"year":2008,"claim":"Defined the functional logic of the trimeric complex by showing RFXAP and RFXB together relieve RFX5 autoinhibition to enable promoter binding.","evidence":"EMSA with purified RFX5, RFXAP, and RFXB plus domain-deletion analysis","pmids":["18723135"],"confidence":"Medium","gaps":["In vitro EMSA single method; no structural detail of the autoinhibited state","Did not show conformational changes directly"]},{"year":2008,"claim":"Demonstrated that non-coding regulation of RFXAP itself can cause MHC-II deficiency, expanding the disease mechanism to promoter-level loss of expression.","evidence":"Promoter reporter assay and RNA Pol II ChIP in a patient cell line carrying a 5'-UTR insertion","pmids":["18336911"],"confidence":"Medium","gaps":["Single patient; generalizability unknown","Mechanism by which the insertion blocks Pol II recruitment not defined"]},{"year":2009,"claim":"Resolved the assembly mechanism by showing the disordered RFXAP C-terminus folds upon binding RFX5 and licenses high-affinity RFXB recruitment, defining an ordered pathway.","evidence":"NMR, circular dichroism, and isothermal titration calorimetry on isolated RFXAP_C and RFX5_N","pmids":["19274739"],"confidence":"High","gaps":["Atomic-resolution structure of the interface not yet determined","Role of full-length proteins and DNA in assembly not addressed"]},{"year":2009,"claim":"Confirmed in a lymphoma context that C-terminal frameshift loss abolishes coordinate MHC-II expression and is rescuable, reinforcing the C-terminus as functionally indispensable.","evidence":"Sequencing and stable-transfection complementation with surface MHC-II analysis in OCI-Ly2 DLBCL cells","pmids":["20024540"],"confidence":"Medium","gaps":["Single cell line","Did not dissect which C-terminal interactions were lost"]},{"year":2010,"claim":"Provided the high-resolution structural basis: RFXAP_C forms a V-shaped helix pair packing into the RFX5 antiparallel coiled-coil staple, with a predicted RFXB-binding surface.","evidence":"Solution NMR structure of the RFX5_N2–RFXAP_C complex with mutagenesis of interface leucines","pmids":["20732328"],"confidence":"High","gaps":["The full trimeric RFX5–RFXAP–RFXB structure not solved","Direct structural proof of RFXB contact site lacking"]},{"year":2015,"claim":"Identified post-transcriptional control of RFXAP by tumor-derived exosomal miR-212-3p, linking RFXAP suppression to immune tolerance in dendritic cells.","evidence":"miR-212-3p mimic/inhibitor transfection into dendritic cells with RFXAP and MHC-II readouts","pmids":["26337469"],"confidence":"Medium","gaps":["Direct 3'-UTR targeting not formally validated in this study","In vivo relevance of exosomal transfer not established here"]},{"year":2018,"claim":"Placed miR-212-3p downstream of IFN-γ and upstream of RFXAP, establishing a cytokine-controlled axis governing RFXAP and MHC-II levels.","evidence":"IFN-γ dose-response, luciferase reporter of the RFXAP 3'-UTR, and epistasis with miR-212-3p mimics in pancreatic cancer cells","pmids":["29467893"],"confidence":"Medium","gaps":["Single lab; how IFN-γ represses miR-212-3p not defined","Physiological setting beyond pancreatic cancer cells untested"]},{"year":2020,"claim":"Revealed an MHC-II-independent function: RFXAP directly activates KDM4A transcription, reducing H3K36 methylation and modulating DNA repair and damage sensitivity.","evidence":"ChIP-seq, dual-luciferase reporter, gain/loss-of-function, and xenograft model in pancreatic cancer cells","pmids":["33093461"],"confidence":"Medium","gaps":["Whether RFXAP acts here within the RFX complex or independently is unknown","Generality beyond pancreatic cancer cells not established"]},{"year":null,"claim":"How RFXAP integrates its canonical MHC-II co-activator role with the newly described KDM4A/chromatin and cytokine-miRNA regulatory axes — including whether phosphorylation modulates its activity and whether it engages non-MHC-II targets within or outside the RFX complex — remains open.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No kinase or phosphorylation event on RFXAP directly demonstrated","No full trimeric RFX–DNA structure","Mechanistic link between MHC-II and KDM4A functions undefined"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140110","term_label":"transcription regulator activity","supporting_discovery_ids":[0,8]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[4,5]},{"term_id":"GO:0003677","term_label":"DNA binding","supporting_discovery_ids":[0,3]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[0,8]}],"pathway":[{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[0,9]},{"term_id":"R-HSA-74160","term_label":"Gene expression (Transcription)","supporting_discovery_ids":[0,8]}],"complexes":["RFX complex (RFX5–RFXAP–RFXB)"],"partners":["RFX5","RFXB","CIITA"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"O00287","full_name":"Regulatory factor X-associated protein","aliases":["RFX DNA-binding complex 36 kDa subunit"],"length_aa":272,"mass_kda":28.2,"function":"Part of the RFX complex that binds to the X-box of MHC II promoters","subcellular_location":"Nucleus","url":"https://www.uniprot.org/uniprotkb/O00287/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/RFXAP","classification":"Not Classified","n_dependent_lines":2,"n_total_lines":1208,"dependency_fraction":0.0016556291390728477},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/RFXAP","total_profiled":1310},"omim":[{"mim_id":"620817","title":"MHC CLASS II DEFICIENCY 4; MHC2D4","url":"https://www.omim.org/entry/620817"},{"mim_id":"620815","title":"MHC CLASS II DEFICIENCY 2; MHC2D2","url":"https://www.omim.org/entry/620815"},{"mim_id":"603200","title":"REGULATORY FACTOR X, ANKYRIN REPEAT-CONTAINING; RFXANK","url":"https://www.omim.org/entry/603200"},{"mim_id":"601863","title":"REGULATORY FACTOR X, 5; RFX5","url":"https://www.omim.org/entry/601863"},{"mim_id":"601861","title":"REGULATORY FACTOR X-ASSOCIATED PROTEIN; RFXAP","url":"https://www.omim.org/entry/601861"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Nuclear speckles","reliability":"Supported"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/RFXAP"},"hgnc":{"alias_symbol":[],"prev_symbol":[]},"alphafold":{"accession":"O00287","domains":[{"cath_id":"-","chopping":"229-263","consensus_level":"medium","plddt":95.654,"start":229,"end":263}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/O00287","model_url":"https://alphafold.ebi.ac.uk/files/AF-O00287-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-O00287-F1-predicted_aligned_error_v6.png","plddt_mean":63.97},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=RFXAP","jax_strain_url":"https://www.jax.org/strain/search?query=RFXAP"},"sequence":{"accession":"O00287","fasta_url":"https://rest.uniprot.org/uniprotkb/O00287.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/O00287/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/O00287"}},"corpus_meta":[{"pmid":"26337469","id":"PMC_26337469","title":"Pancreatic cancer-derived exosomes transfer miRNAs to dendritic cells and inhibit RFXAP expression via miR-212-3p.","date":"2015","source":"Oncotarget","url":"https://pubmed.ncbi.nlm.nih.gov/26337469","citation_count":248,"is_preprint":false},{"pmid":"9118943","id":"PMC_9118943","title":"RFXAP, a novel subunit of the RFX DNA binding complex is mutated in MHC class II deficiency.","date":"1997","source":"The EMBO journal","url":"https://pubmed.ncbi.nlm.nih.gov/9118943","citation_count":186,"is_preprint":false},{"pmid":"9287230","id":"PMC_9287230","title":"Mutation of RFXAP, a regulator of MHC class II genes, in primary MHC class II deficiency.","date":"1997","source":"The New England journal of medicine","url":"https://pubmed.ncbi.nlm.nih.gov/9287230","citation_count":53,"is_preprint":false},{"pmid":"33093461","id":"PMC_33093461","title":"Fisetin inhibits proliferation of pancreatic adenocarcinoma by inducing DNA damage via RFXAP/KDM4A-dependent histone H3K36 demethylation.","date":"2020","source":"Cell death & disease","url":"https://pubmed.ncbi.nlm.nih.gov/33093461","citation_count":43,"is_preprint":false},{"pmid":"11486010","id":"PMC_11486010","title":"Expression of the three human major histocompatibility complex class II isotypes exhibits a differential dependence on the transcription factor RFXAP.","date":"2001","source":"Molecular and cellular biology","url":"https://pubmed.ncbi.nlm.nih.gov/11486010","citation_count":28,"is_preprint":false},{"pmid":"18723135","id":"PMC_18723135","title":"Assembly of the RFX complex on the MHCII promoter: role of RFXAP and RFXB in relieving autoinhibition of RFX5.","date":"2008","source":"Biochimica et biophysica acta","url":"https://pubmed.ncbi.nlm.nih.gov/18723135","citation_count":20,"is_preprint":false},{"pmid":"29467893","id":"PMC_29467893","title":"IFN-γ induces the upregulation of RFXAP via inhibition of miR-212-3p in pancreatic cancer cells: A novel mechanism for IFN-γ response.","date":"2018","source":"Oncology letters","url":"https://pubmed.ncbi.nlm.nih.gov/29467893","citation_count":19,"is_preprint":false},{"pmid":"16337482","id":"PMC_16337482","title":"Conserved residues of the bare lymphocyte syndrome transcription factor RFXAP determine coordinate MHC class II expression.","date":"2005","source":"Molecular immunology","url":"https://pubmed.ncbi.nlm.nih.gov/16337482","citation_count":14,"is_preprint":false},{"pmid":"20732328","id":"PMC_20732328","title":"Solution structure of the heterotrimeric complex between the interaction domains of RFX5 and RFXAP from the RFX gene regulatory complex.","date":"2010","source":"Journal of molecular biology","url":"https://pubmed.ncbi.nlm.nih.gov/20732328","citation_count":8,"is_preprint":false},{"pmid":"18336911","id":"PMC_18336911","title":"Transcriptional silencing of RFXAP in MHC class II-deficiency.","date":"2008","source":"Molecular immunology","url":"https://pubmed.ncbi.nlm.nih.gov/18336911","citation_count":8,"is_preprint":false},{"pmid":"22390233","id":"PMC_22390233","title":"Type III bare lymphocyte syndrome associated with a novel RFXAP mutation: a case report.","date":"2012","source":"International journal of immunogenetics","url":"https://pubmed.ncbi.nlm.nih.gov/22390233","citation_count":7,"is_preprint":false},{"pmid":"19274739","id":"PMC_19274739","title":"Formation of the RFX gene regulatory complex induces folding of the interaction domain of RFXAP.","date":"2009","source":"Proteins","url":"https://pubmed.ncbi.nlm.nih.gov/19274739","citation_count":6,"is_preprint":false},{"pmid":"20024540","id":"PMC_20024540","title":"Coordinate loss of MHC class II expression in the diffuse large B cell lymphoma cell line OCI-Ly2 is due to a novel mutation in RFX-AP.","date":"2009","source":"Immunogenetics","url":"https://pubmed.ncbi.nlm.nih.gov/20024540","citation_count":5,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":8018,"output_tokens":3004,"usd":0.034557,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":10275,"output_tokens":3564,"usd":0.070237,"stage2_stop_reason":"end_turn"},"total_usd":0.104794,"stage1_batch_id":"msgbatch_01CNoDbkChHEkgrJnL7BwtGJ","stage2_batch_id":"msgbatch_016Y4x9E9Wg5VQDANj9quSQD","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1997,\n      \"finding\": \"RFXAP is a novel subunit of the RFX DNA-binding complex required for MHC class II gene expression. Mutations in RFXAP in group D MHC-II deficiency patients abolish RFX binding activity and MHC-II expression; transfection with wild-type RFXAP fully restores expression of all endogenous MHC-II genes.\",\n      \"method\": \"Complementation assay by transfection, DNA-binding (RFX complex) activity assay, identification of mutations in patient cell lines\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — complementation rescue experiments in patient cell lines, replicated independently in two papers (PMID:9118943 and PMID:9287230) from different groups\",\n      \"pmids\": [\"9118943\", \"9287230\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"The C-terminal domain of RFXAP is essential for MHC-II expression, but different portions of this domain are required for different isotypes: a short C-terminal segment suffices for HLA-DR, while a larger C-terminal segment is required for optimal HLA-DQ and HLA-DP expression. This differential requirement reflects differential dependence on this domain for promoter occupancy and recruitment of the co-activator CIITA in vivo.\",\n      \"method\": \"Deletion/domain-mapping mutants of mouse and human RFXAP transfected into RFXAP-deficient cell lines; in vivo promoter occupancy and CIITA recruitment assays\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple deletion mutants tested in complementation assays with in vivo ChIP for promoter occupancy and CIITA recruitment, single lab but multiple orthogonal methods\",\n      \"pmids\": [\"11486010\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Conserved hydrophobic and other non-glutamine residues in the C-terminal third of RFXAP are required for coordinate MHC-II isotype expression; mutation of potential phosphorylation sites abolishes RFXAP activity. Certain RFXAP mutants can rescue HLA-DR but not HLA-DQ or HLA-DP, correlating with their ability to form RFX complexes, bind DNA in vivo, and recruit CIITA to promoters.\",\n      \"method\": \"Site-directed mutagenesis of conserved residues, complementation in BLS cell lines, in vivo ChIP for DNA binding and CIITA recruitment, chimeric reporter gene assays\",\n      \"journal\": \"Molecular immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — mutagenesis combined with in vivo ChIP and reporter assays, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"16337482\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"DNA binding of RFX5 is autoinhibited by domains flanking its DNA-binding domain; both RFXAP and RFXB are required to relieve this autoinhibition and allow a single RFX complex to bind the proximal regulatory region of the MHC-II promoter.\",\n      \"method\": \"Electrophoretic mobility shift assay (EMSA) with purified RFX5, RFXAP, and RFXB proteins; domain-deletion analysis\",\n      \"journal\": \"Biochimica et biophysica acta\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — in vitro EMSA with purified proteins, single lab, single method type\",\n      \"pmids\": [\"18723135\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"The C-terminal domain of RFXAP (RFXAP_C) is intrinsically disordered in isolation but folds into two α-helices upon binding to the N-terminal dimerization domain of RFX5 (RFX5_N). The resulting RFX5_N2–RFXAP_C complex then binds RFXB with high affinity, establishing an ordered assembly pathway for the RFX complex.\",\n      \"method\": \"NMR spectroscopy, circular dichroism spectroscopy, isothermal titration calorimetry\",\n      \"journal\": \"Proteins\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — multiple biophysical methods (NMR, CD, ITC) in a single study characterizing folding-upon-binding mechanism, single lab\",\n      \"pmids\": [\"19274739\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Solution NMR structure of the RFX5_N2–RFXAP_C complex shows that two RFX5 N-terminal helices form an antiparallel coiled-coil 'staple', and the two α-helices of RFXAP_C form a V-shaped structure that packs within this staple. Leucine residues in the leucine-rich region of RFX5_N (62-LYLYLQL-68) contribute to both RFX5 dimerization and the RFX5–RFXAP interface; clustered hydrophobic residues on RFXAP_C suggest a binding site for RFXB.\",\n      \"method\": \"15N- and 13C-edited NMR spectroscopy (solution structure determination)\",\n      \"journal\": \"Journal of molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — high-resolution NMR structure with mutagenesis validation of key interface residues, single lab\",\n      \"pmids\": [\"20732328\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"A homozygous 75 bp insertion in the 5'-UTR of the RFXAP gene impairs RFXAP promoter activity, reduces RNA polymerase II recruitment to RFXAP chromatin, and results in complete loss of RFXAP mRNA and protein, causing MHC-II deficiency without any coding-sequence mutation.\",\n      \"method\": \"Promoter activity assay, chromatin immunoprecipitation (RNA Pol II ChIP), sequencing of patient cell line\",\n      \"journal\": \"Molecular immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — promoter reporter and ChIP in patient cell line, single lab, single patient\",\n      \"pmids\": [\"18336911\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"An 11-base deletion in RFXAP causing a frameshift at amino acid 234 and loss of C-terminal residues leads to coordinate loss of all MHC-II expression in a DLBCL cell line; stable transfection of wild-type RFXAP restores MHC-II expression, confirming that C-terminal RFXAP sequences are required for function.\",\n      \"method\": \"Sequencing of RFXAP in OCI-Ly2 cells, stable transfection complementation assay, MHC-II surface expression analysis\",\n      \"journal\": \"Immunogenetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — complementation by stable transfection in a single DLBCL cell line, single lab\",\n      \"pmids\": [\"20024540\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"RFXAP transcriptionally activates KDM4A (a histone H3K36 tri-/dimethyl demethylase) in pancreatic cancer cells; RFXAP overexpression increases KDM4A expression, reduces H3K36 methylation, impairs DNA repair, and enhances fisetin-induced DNA damage and S-phase arrest, while RFXAP silencing has the opposite effect.\",\n      \"method\": \"ChIP sequencing (RFXAP binding to KDM4A promoter), dual-luciferase reporter assay, RFXAP overexpression/knockdown, western blot, immunofluorescence for DNA damage markers, xenograft mouse model\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP-seq, reporter assay, and gain/loss-of-function experiments with defined molecular readouts, single lab\",\n      \"pmids\": [\"33093461\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"miR-212-3p transferred from pancreatic cancer-derived exosomes directly targets and suppresses RFXAP mRNA in dendritic cells, resulting in decreased MHC-II expression and induction of immune tolerance.\",\n      \"method\": \"miRNA target prediction validated by transfection of miR-212-3p mimics/inhibitors into dendritic cells, measurement of RFXAP mRNA/protein and MHC-II surface expression; luciferase reporter assay (implied by validation in context)\",\n      \"journal\": \"Oncotarget\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — miRNA-target validation experiments with gain/loss-of-function in dendritic cells, replicated conceptually in a follow-up paper (PMID:29467893)\",\n      \"pmids\": [\"26337469\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"IFN-γ suppresses miR-212-3p expression in pancreatic cancer cells in a dose- and time-dependent manner, leading to upregulation of RFXAP and MHC-II; luciferase assay confirmed RFXAP as a direct target of miR-212-3p. When miR-212-3p mimics were transfected into cells, IFN-γ could no longer increase RFXAP or MHC-II, placing miR-212-3p inhibition downstream of IFN-γ and upstream of RFXAP induction.\",\n      \"method\": \"miR-212-3p mimic/inhibitor transfection, IFN-γ dose-response experiment, luciferase reporter assay for RFXAP 3'-UTR targeting, qRT-PCR and western blot\",\n      \"journal\": \"Oncology letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — luciferase reporter and epistasis experiment with mimics/inhibitors, single lab, multiple orthogonal methods\",\n      \"pmids\": [\"29467893\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"RFXAP is an essential subunit of the trimeric RFX transcription factor complex (with RFX5 and RFXB) that binds X-box elements in MHC class II promoters: its intrinsically disordered C-terminal domain folds upon binding the antiparallel coiled-coil dimerization domain of RFX5, relieving RFX5 autoinhibition and creating a platform for high-affinity RFXB recruitment, and its conserved C-terminal residues (including phosphorylatable sites) mediate isotype-specific differences in CIITA co-activator recruitment and promoter occupancy; beyond MHC-II regulation, RFXAP also directly activates transcription of the histone demethylase KDM4A in pancreatic cells, and its expression is post-transcriptionally regulated by miR-212-3p, which is itself suppressed by IFN-γ.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"RFXAP is an essential subunit of the trimeric RFX transcription factor complex (with RFX5 and RFXB) that binds X-box elements in MHC class II promoters, and its loss causes group D MHC-II deficiency [#0]. Mechanistically, the intrinsically disordered C-terminal domain of RFXAP folds into two α-helices upon binding the antiparallel coiled-coil dimerization 'staple' formed by two RFX5 N-terminal helices, generating an RFX5–RFXAP module that then recruits RFXB with high affinity to establish an ordered assembly pathway [#4, #5]; both RFXAP and RFXB are required to relieve autoinhibition of RFX5 DNA binding and allow the complex to occupy the proximal MHC-II promoter [#3]. The conserved C-terminal residues of RFXAP — including hydrophobic positions and phosphorylatable sites — mediate isotype-specific transcriptional output by controlling promoter occupancy and recruitment of the co-activator CIITA, such that distinct C-terminal segments are differentially required for HLA-DR versus HLA-DQ and HLA-DP expression [#1, #2]. Beyond MHC-II regulation, RFXAP directly binds and transcriptionally activates the histone demethylase KDM4A in pancreatic cancer cells, reducing H3K36 methylation, impairing DNA repair, and sensitizing cells to DNA damage [#8]. RFXAP expression is itself post-transcriptionally controlled by miR-212-3p, which directly targets RFXAP mRNA to suppress MHC-II and induce immune tolerance, and which is repressed by IFN-γ to drive RFXAP induction [#9, #10].\",\n  \"teleology\": [\n    {\n      \"year\": 1997,\n      \"claim\": \"Established that RFXAP is a distinct, essential subunit of the RFX DNA-binding complex whose loss causes a defined MHC-II deficiency, defining the gene's core requirement for MHC-II expression.\",\n      \"evidence\": \"Complementation rescue by transfection of wild-type RFXAP into patient cell lines with RFX-binding and MHC-II readouts\",\n      \"pmids\": [\"9118943\", \"9287230\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not resolve how RFXAP contributes structurally to the complex\", \"Did not define the functional domains within RFXAP\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Mapped function to the RFXAP C-terminal domain and revealed isotype-specific requirements, showing the protein does more than serve as a uniform scaffold.\",\n      \"evidence\": \"Deletion/domain-mapping complementation in RFXAP-deficient cells with in vivo promoter occupancy and CIITA recruitment assays\",\n      \"pmids\": [\"11486010\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not identify the specific residues mediating the isotype distinction\", \"Mechanism of differential CIITA recruitment unresolved\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Pinpointed conserved hydrophobic and phosphorylatable C-terminal residues as the determinants of complex formation, DNA binding, and CIITA recruitment, linking sequence to coordinate isotype regulation.\",\n      \"evidence\": \"Site-directed mutagenesis with complementation, in vivo ChIP, and chimeric reporter assays in BLS cell lines\",\n      \"pmids\": [\"16337482\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether phosphorylation actually occurs and which kinase acts is not established\", \"No structural basis for the residue requirements yet\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Defined the functional logic of the trimeric complex by showing RFXAP and RFXB together relieve RFX5 autoinhibition to enable promoter binding.\",\n      \"evidence\": \"EMSA with purified RFX5, RFXAP, and RFXB plus domain-deletion analysis\",\n      \"pmids\": [\"18723135\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"In vitro EMSA single method; no structural detail of the autoinhibited state\", \"Did not show conformational changes directly\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Demonstrated that non-coding regulation of RFXAP itself can cause MHC-II deficiency, expanding the disease mechanism to promoter-level loss of expression.\",\n      \"evidence\": \"Promoter reporter assay and RNA Pol II ChIP in a patient cell line carrying a 5'-UTR insertion\",\n      \"pmids\": [\"18336911\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single patient; generalizability unknown\", \"Mechanism by which the insertion blocks Pol II recruitment not defined\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Resolved the assembly mechanism by showing the disordered RFXAP C-terminus folds upon binding RFX5 and licenses high-affinity RFXB recruitment, defining an ordered pathway.\",\n      \"evidence\": \"NMR, circular dichroism, and isothermal titration calorimetry on isolated RFXAP_C and RFX5_N\",\n      \"pmids\": [\"19274739\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Atomic-resolution structure of the interface not yet determined\", \"Role of full-length proteins and DNA in assembly not addressed\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Confirmed in a lymphoma context that C-terminal frameshift loss abolishes coordinate MHC-II expression and is rescuable, reinforcing the C-terminus as functionally indispensable.\",\n      \"evidence\": \"Sequencing and stable-transfection complementation with surface MHC-II analysis in OCI-Ly2 DLBCL cells\",\n      \"pmids\": [\"20024540\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single cell line\", \"Did not dissect which C-terminal interactions were lost\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Provided the high-resolution structural basis: RFXAP_C forms a V-shaped helix pair packing into the RFX5 antiparallel coiled-coil staple, with a predicted RFXB-binding surface.\",\n      \"evidence\": \"Solution NMR structure of the RFX5_N2–RFXAP_C complex with mutagenesis of interface leucines\",\n      \"pmids\": [\"20732328\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"The full trimeric RFX5–RFXAP–RFXB structure not solved\", \"Direct structural proof of RFXB contact site lacking\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Identified post-transcriptional control of RFXAP by tumor-derived exosomal miR-212-3p, linking RFXAP suppression to immune tolerance in dendritic cells.\",\n      \"evidence\": \"miR-212-3p mimic/inhibitor transfection into dendritic cells with RFXAP and MHC-II readouts\",\n      \"pmids\": [\"26337469\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct 3'-UTR targeting not formally validated in this study\", \"In vivo relevance of exosomal transfer not established here\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Placed miR-212-3p downstream of IFN-γ and upstream of RFXAP, establishing a cytokine-controlled axis governing RFXAP and MHC-II levels.\",\n      \"evidence\": \"IFN-γ dose-response, luciferase reporter of the RFXAP 3'-UTR, and epistasis with miR-212-3p mimics in pancreatic cancer cells\",\n      \"pmids\": [\"29467893\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab; how IFN-γ represses miR-212-3p not defined\", \"Physiological setting beyond pancreatic cancer cells untested\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Revealed an MHC-II-independent function: RFXAP directly activates KDM4A transcription, reducing H3K36 methylation and modulating DNA repair and damage sensitivity.\",\n      \"evidence\": \"ChIP-seq, dual-luciferase reporter, gain/loss-of-function, and xenograft model in pancreatic cancer cells\",\n      \"pmids\": [\"33093461\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether RFXAP acts here within the RFX complex or independently is unknown\", \"Generality beyond pancreatic cancer cells not established\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How RFXAP integrates its canonical MHC-II co-activator role with the newly described KDM4A/chromatin and cytokine-miRNA regulatory axes — including whether phosphorylation modulates its activity and whether it engages non-MHC-II targets within or outside the RFX complex — remains open.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No kinase or phosphorylation event on RFXAP directly demonstrated\", \"No full trimeric RFX–DNA structure\", \"Mechanistic link between MHC-II and KDM4A functions undefined\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140110\", \"supporting_discovery_ids\": [0, 8]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [4, 5]},\n      {\"term_id\": \"GO:0003677\", \"supporting_discovery_ids\": [0, 3]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [0, 8]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [0, 9]},\n      {\"term_id\": \"R-HSA-74160\", \"supporting_discovery_ids\": [0, 8]}\n    ],\n    \"complexes\": [\"RFX complex (RFX5–RFXAP–RFXB)\"],\n    \"partners\": [\"RFX5\", \"RFXB\", \"CIITA\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":4,"faith_total":5,"faith_pct":80.0}}