{"gene":"RFX5","run_date":"2026-06-10T06:43:36","timeline":{"discoveries":[{"year":1997,"finding":"RFX5 directly interacts with CIITA to form a transcriptional complex. RFX5 provides promoter specificity via its DNA binding domain, while CIITA recruits the general transcription apparatus via its acidic activation domain. The direct interaction was demonstrated by yeast two-hybrid and far-Western blot assays.","method":"Yeast two-hybrid, far-Western blot, cotransfection reporter assay in CIITA-deficient cells","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal yeast two-hybrid and far-Western blot, replicated by functional rescue in CIITA-deficient cells, independently confirmed in later studies","pmids":["9177217"],"is_preprint":false},{"year":1995,"finding":"RFX5 is required for both constitutive and IFN-gamma-inducible transcription of HLA-DMA and HLA-DMB genes, as demonstrated by complementation of regulatory mutant cell lines with RFX5 cDNA.","method":"Complementation assay using cDNA transfection into MHC class II regulatory mutant cells","journal":"International immunology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean genetic complementation with defined phenotypic readout (DM gene expression restoration), single lab","pmids":["7495736"],"is_preprint":false},{"year":2000,"finding":"The C-terminal domain of RFX5 mediates cooperative binding between the RFX complex and NF-Y (which binds the Y box), and this cooperative interaction is essential for transcriptional activation of MHC-II genes. The N-terminal region of RFX5 is required for association with RFXANK and RFXAP and for RFX complex assembly and X-box DNA binding, but is insufficient alone for activation.","method":"Domain deletion/mutagenesis, in vitro and in vivo RFX complex assembly assays, MHC-II promoter reporter assays","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — mutagenesis combined with in vitro reconstitution and in vivo transcriptional assays, multiple orthogonal methods in one rigorous study","pmids":["10779326"],"is_preprint":false},{"year":2000,"finding":"In chlamydia-infected cells, RFX5 is degraded by a lactacystin-sensitive proteasome-like activity found in the cytosolic fraction of infected cells, dependent on chlamydial (not host) protein synthesis. This degradation correlates with suppression of both constitutive and IFN-gamma-inducible MHC class I expression.","method":"Immunoblot of RFX5 protein levels in infected cells, pharmacological inhibition with lactacystin, cell fractionation, inhibition of chlamydial vs. host protein synthesis","journal":"The Journal of experimental medicine","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — biochemical fractionation and pharmacological dissection, single lab, correlative but mechanistically grounded","pmids":["10790427"],"is_preprint":false},{"year":2002,"finding":"RFX5 forms homodimers in vivo and in vitro via a leucine-rich stretch N-terminal to its DNA binding domain; leucine 66 is critical for self-association. Mutant RFX5 unable to dimerize fails to support formation of higher-order DNA-protein complexes on MHC-II conserved upstream sequences in vitro and fails to activate MHC-II transcription in vivo.","method":"In vivo and in vitro dimerization assays, site-directed mutagenesis (L66), electrophoretic mobility shift assay (EMSA), MHC-II transcription assay","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — mutagenesis combined with in vitro EMSA and in vivo transcription assay, multiple orthogonal methods","pmids":["12101253"],"is_preprint":false},{"year":2003,"finding":"The RFX5 complex (RFX5, RFXB/RFXANK, and RFXAP) mediates IFN-gamma-induced repression of the COL1A2 (collagen type I) gene. All three subunits are required for maximum repression; RFX5 dominant-negative mutants reverse IFN-gamma-induced collagen repression. IFN-gamma increases RFX5 nuclear translocation and recruitment of all three complex subunits to the COL1A2 transcription start site, as shown by ChIP.","method":"Promoter reporter assay, dominant-negative overexpression, IFN-gamma treatment, chromatin immunoprecipitation (ChIP), immunofluorescence for nuclear translocation","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (reporter assay, dominant-negative genetics, ChIP), single lab but comprehensive","pmids":["12968017"],"is_preprint":false},{"year":2008,"finding":"DNA binding of RFX5 alone is autoinhibited by domains flanking its DNA binding domain. Both RFXAP and RFXB are required to relieve this autoinhibition and allow high-affinity binding of the RFX complex to the MHC-II promoter. A single RFX complex binds the proximal regulatory region.","method":"Electrophoretic mobility shift assay (EMSA) with recombinant proteins and MHC-II promoter DNA fragments","journal":"Biochimica et biophysica acta","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution with defined recombinant proteins, single lab","pmids":["18723135"],"is_preprint":false},{"year":2010,"finding":"The solution NMR structure of the RFX5 N-terminal dimerization domain (RFX5(N)) in complex with the C-terminal domain of RFXAP reveals that two RFX5(N) molecules form an antiparallel coiled coil (staple shape), with RFXAP(C) adopting two α-helices that pack within this staple. Leucine residues in the leucine-rich region (62-LYLYLQL-68) contribute to both the dimer interface (Leu64, Leu68) and the RFX5-RFXAP interface (Leu62, Leu66). RFXAP is unstructured alone but folds upon binding RFX5.","method":"Solution NMR structure determination (15N- and 13C-edited), in vitro binding assays","journal":"Journal of molecular biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — NMR structure with residue-level functional validation, comprehensive structural and biochemical analysis","pmids":["20732328"],"is_preprint":false},{"year":2010,"finding":"The DNA binding domain of RFX5 (RFX5DBD) binds X-box DNA with an entropy-driven, enthalpy-favorable interaction; the dissociation constant was determined by fluorescence quenching. RFX5DBD also interacts directly with RFXANK in the absence of RFXAP, with an apparent Kd of 128 nM, as measured by FRET and chemical cross-linking/mass spectrometry.","method":"Steady-state fluorescence quenching, circular dichroism, FRET, chemical cross-linking followed by tandem mass spectrometry","journal":"Biochimica et biophysica acta","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — multiple biophysical methods in vitro, single lab, thermodynamic parameters determined","pmids":["20637319"],"is_preprint":false},{"year":2012,"finding":"SIRT1 forms a complex with RFX5 and deacetylates it. SIRT1-mediated deacetylation promotes nuclear expulsion and proteasomal degradation of RFX5, thereby reducing RFX5 binding to the COL1A2 promoter and antagonizing RFX5-mediated repression of collagen transcription. IFN-gamma represses COL1A2 by downregulating SIRT1, which leads to increased RFX5 acetylation and enhanced collagen repression.","method":"Co-immunoprecipitation, overexpression and knockdown of SIRT1/NAMPT, SIRT1 agonist/inhibitor treatment, acetylation assays, subcellular fractionation, promoter reporter assay, ChIP","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP, multiple genetic and pharmacological perturbations, single lab","pmids":["23079621"],"is_preprint":false},{"year":2013,"finding":"RFX5 contains an AT-hook motif that is involved in regulating transcription of the HLA-DQ (but not HLA-DR) MHC-II isotype. PRMT6, an arginine methyltransferase, methylates this AT-hook motif and specifically downregulates HLA-DQ expression in an AT-hook-dependent manner, providing isotype-specific fine-tuning of MHC-II transcription.","method":"AT-hook motif mutagenesis, PRMT6 overexpression/knockdown, MHC-II isotype-specific expression assays","journal":"Molecular immunology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — mutagenesis combined with functional reporter assays and endogenous gene expression, single lab","pmids":["23911394"],"is_preprint":false},{"year":2006,"finding":"CREB interacts with the RFX5 subunit of the RFX complex through the C-terminal portion of CREB, as demonstrated by co-immunoprecipitation. Phospho-CREB is present at the HLA-DRA promoter (shown by ChIP) and enhances MHC-II transcription, though phosphorylation is not required for basal transcription.","method":"Co-immunoprecipitation, MHC-II promoter reporter assay, chromatin immunoprecipitation (ChIP)","journal":"Molecular immunology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP and ChIP with functional reporter assay, single lab","pmids":["16730065"],"is_preprint":false},{"year":2016,"finding":"RFX5 binds directly to the TPP1 promoter and transcriptionally activates TPP1 gene expression in hepatocellular carcinoma cells; manipulation of RFX5 expression levels correspondingly affects TPP1 expression in HepG2 cells.","method":"ChIP-PCR, luciferase reporter assay, RFX5 overexpression/knockdown with qRT-PCR","journal":"Oncology reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ChIP and luciferase assay with functional perturbation, single lab","pmids":["27840983"],"is_preprint":false},{"year":2020,"finding":"RFX5 binds directly to the KDM4A promoter and transcriptionally activates KDM4A in hepatocellular carcinoma cells. The RFX5-KDM4A pathway promotes cell cycle progression from G0/G1 to S phase and inhibits apoptosis through regulation of p53 and downstream genes; KDM4A overexpression rescues growth inhibitory effects of RFX5 deletion.","method":"ChIP-PCR, luciferase reporter assay, ChIP-seq (ENCODE), RFX5 knockout/rescue with KDM4A overexpression, cell cycle and apoptosis assays","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ChIP, luciferase, and epistasis rescue experiment, single lab","pmids":["32883983"],"is_preprint":false},{"year":2022,"finding":"In synovial macrophages, CCL18 signaling induces RFX5, which selectively upregulates glutamate dehydrogenase 1 (GLUD1) to enable glutamate-based energy production under glucose withdrawal. In parallel, RFX5 enhances surface HLA-DR expression, coupling metabolic adaptation to antigen-presenting function.","method":"RFX5 knockdown/overexpression, CCL18 stimulation, GLUD1 expression analysis, HLA-DR surface expression, metabolic assays in primary synovial macrophages","journal":"Nature metabolism","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — loss- and gain-of-function with defined metabolic and immunological phenotypic readouts, single lab, primary cells","pmids":["35739396"],"is_preprint":false},{"year":1998,"finding":"RFX5-deficient mice lack MHC-II expression in thymic cortex, resting B cells, and macrophages, causing failure of CD4+ T cell positive selection and severe immunodeficiency. However, residual MHC-II expression persists in thymic medulla, mature dendritic cells, and activated B cells, revealing that CIITA and RFX5 have differential requirements in different antigen-presenting cell subsets.","method":"RFX5 knockout mouse (conditional gene targeting), flow cytometry for MHC-II expression, lymphocyte subset analysis","journal":"Immunity","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean KO mouse with defined cellular phenotypes and subset-specific mechanistic dissection","pmids":["9491996"],"is_preprint":false},{"year":2025,"finding":"Cryo-EM structure of the RFX5-nucleosome complex reveals that the extended DNA binding domain (eDBD) of RFX5 binds the nucleosome at superhelical location +2, engaging both nucleosomal DNA and histone proteins. RFX5 eDBD induces localized distortion of the bound DNA gyre and detachment of an adjacent DNA gyre, increasing DNA accessibility and potentially enhancing transcriptional activity.","method":"Cryo-EM structure determination of RFX5 eDBD-nucleosome complex","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 1 / Moderate — cryo-EM structure with detailed structural analysis and in vivo functional correlates, single study but high-quality structural data","pmids":["40744500"],"is_preprint":false},{"year":2025,"finding":"RFX5 transcriptionally activates JAG1 by directly binding the JAG1 promoter (at the -1890/+15 or -1359/+15 region), thereby activating the Notch signaling pathway (Notch1, NICD, Hes1) in triple-negative breast cancer cells.","method":"Chromatin immunoprecipitation, luciferase reporter assay, JAG1 knockdown rescue experiment, RFX5 overexpression/knockdown","journal":"Human cell","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ChIP and luciferase assay with epistasis rescue, single lab","pmids":["40220043"],"is_preprint":false},{"year":2026,"finding":"IER3 functions as a transcriptional co-activator for RFX5, forming a complex with RFX5 (demonstrated by co-immunoprecipitation) that upregulates AKR1B10, which subsequently suppresses p53 in hepatocellular carcinoma cells.","method":"Co-immunoprecipitation, dual-luciferase reporter assay, siRNA knockdown, qPCR and western blot","journal":"Discover oncology","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single Co-IP with reporter assay, single lab, in vitro only with no in vivo validation","pmids":["42209919"],"is_preprint":false},{"year":2024,"finding":"RFX5 exhibits methylation-sensitive DNA binding: it discriminates between specific CpG hydroxymethylation (hmC) symmetries in CpG dyads, as identified by meSMiLE-seq assay.","method":"meSMiLE-seq (microfluidic selective ligand enrichment followed by sequencing with methylated vs unmethylated DNA probes)","journal":"bioRxiv","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single in vitro biochemical screen, preprint, no mutagenesis or functional validation of the methylation-sensitive binding","pmids":[],"is_preprint":true}],"current_model":"RFX5 is the largest subunit of the heterotrimeric RFX complex (with RFXANK/B and RFXAP), where it homodimerizes via an antiparallel coiled-coil N-terminal domain that also mediates RFXAP binding (NMR structure resolved), and its DNA binding domain—subject to autoinhibition relieved by RFXANK and RFXAP—engages X-box sequences in MHC-II promoters and can bind and destabilize nucleosomes (cryo-EM structure resolved); the C-terminal domain of RFX5 mediates cooperative interaction with NF-Y to activate MHC-II transcription, while RFX5 also directly interacts with CIITA (which recruits the general transcription apparatus), with CREB, and is subject to acetylation (erased by SIRT1) that controls its nuclear localization and proteasomal degradation; beyond MHC-II regulation, the RFX5 complex represses COL1A2 collagen transcription in response to IFN-gamma, and RFX5 transcriptionally activates additional targets including KDM4A, TPP1, and JAG1 in cancer contexts."},"narrative":{"mechanistic_narrative":"RFX5 is the DNA-binding subunit of the heterotrimeric RFX complex that confers promoter specificity for X-box sequences in MHC class II genes, where it cooperates with the master coactivator CIITA to drive antigen-presentation gene transcription [PMID:9177217, PMID:9491996]. RFX5 contributes promoter recognition through its DNA binding domain while CIITA, recruited via direct protein-protein interaction, brings in the general transcription apparatus; loss of RFX5 in mice abolishes MHC-II expression in thymic cortex, resting B cells and macrophages and blocks CD4+ T cell positive selection, establishing its central immunological role [PMID:9177217, PMID:9491996]. Complex assembly requires the RFX5 N-terminal region, which homodimerizes through a leucine-rich segment (Leu66 critical) that forms an antiparallel coiled-coil staple and simultaneously engages RFXAP, while the C-terminal domain mediates cooperative interaction with NF-Y to activate transcription [PMID:10779326, PMID:12101253, PMID:20732328]. The isolated RFX5 DNA binding domain is autoinhibited by flanking sequences; both RFXAP and RFXANK are required to relieve this autoinhibition and permit high-affinity X-box binding, and its extended DNA binding domain engages nucleosomes directly, distorting DNA gyres to increase accessibility [PMID:18723135, PMID:40744500]. RFX5 activity is tuned post-translationally: SIRT1 deacetylation controls its nuclear retention and proteasomal turnover, and PRMT6 methylation of an AT-hook motif selectively downregulates the HLA-DQ isotype [PMID:23079621, PMID:23911394]. Beyond MHC-II, the RFX complex mediates IFN-gamma-induced repression of COL1A2 collagen [PMID:12968017], and RFX5 directly activates KDM4A, TPP1 and JAG1 to promote proliferation and Notch signaling in cancer contexts [PMID:27840983, PMID:32883983, PMID:40220043].","teleology":[{"year":1995,"claim":"Established that RFX5 is genetically required for MHC class II gene expression, identifying it as a non-redundant regulator of antigen presentation.","evidence":"cDNA complementation of MHC-II regulatory mutant cell lines restoring HLA-DMA/DMB expression","pmids":["7495736"],"confidence":"Medium","gaps":["Did not define the molecular interaction or DNA-binding mechanism","Single lab, restricted to DM isotype genes"]},{"year":1997,"claim":"Defined the molecular partnership underlying MHC-II activation by showing RFX5 supplies promoter specificity and CIITA supplies the activation function through a direct interaction.","evidence":"Yeast two-hybrid, far-Western blot, and cotransfection rescue in CIITA-deficient cells","pmids":["9177217"],"confidence":"High","gaps":["Did not map the structural basis of the RFX5-CIITA interface","Did not address contribution of other complex subunits"]},{"year":1998,"claim":"Demonstrated the physiological consequence of RFX5 loss in vivo, linking it to CD4+ T cell selection and revealing cell-type-specific requirements for MHC-II control.","evidence":"RFX5 knockout mouse with flow cytometry of MHC-II and lymphocyte subset analysis","pmids":["9491996"],"confidence":"High","gaps":["Did not explain the residual MHC-II expression in medulla/dendritic cells mechanistically","Did not test non-immune RFX5 functions"]},{"year":2002,"claim":"Mapped the self-association mechanism of RFX5, showing homodimerization via a leucine-rich N-terminal segment is required for higher-order promoter complex formation and transcription.","evidence":"In vivo/in vitro dimerization assays, L66 site-directed mutagenesis, EMSA, and transcription assays","pmids":["12101253"],"confidence":"High","gaps":["Atomic structure of the dimer not yet resolved","Did not address how dimerization couples to RFXAP/RFXANK binding"]},{"year":2000,"claim":"Assigned distinct functions to RFX5 domains, showing the C-terminus mediates cooperative NF-Y binding for activation while the N-terminus is needed for complex assembly and X-box binding.","evidence":"Domain deletion/mutagenesis with in vitro and in vivo complex assembly and reporter assays","pmids":["10779326"],"confidence":"High","gaps":["Did not resolve atomic structure of the RFX5-NF-Y contact","Did not quantify cooperativity thermodynamically"]},{"year":2008,"claim":"Revealed that RFX5 DNA binding is autoinhibited and relieved only by RFXAP and RFXANK, explaining why complex assembly is obligatory for high-affinity promoter engagement.","evidence":"EMSA with recombinant proteins and MHC-II promoter DNA","pmids":["18723135"],"confidence":"Medium","gaps":["Structural basis of autoinhibition not defined","In vitro only, single lab"]},{"year":2010,"claim":"Provided the structural and thermodynamic basis for RFX5 dimerization, RFXAP folding-upon-binding, and X-box recognition.","evidence":"Solution NMR structure of RFX5(N)-RFXAP(C) and biophysical (fluorescence/FRET/crosslinking-MS) DNA and RFXANK binding measurements","pmids":["20732328","20637319"],"confidence":"High","gaps":["No structure of the full trimer on DNA","DNA binding domain structure with DNA not resolved at this stage"]},{"year":2003,"claim":"Extended RFX5 function beyond activation by showing the complex mediates IFN-gamma-induced repression of COL1A2 collagen, requiring all three subunits.","evidence":"Reporter assays, dominant-negative RFX5, IFN-gamma treatment, ChIP, and immunofluorescence","pmids":["12968017"],"confidence":"High","gaps":["Mechanism of repression versus activation at distinct promoters not resolved","Corepressor recruitment not identified"]},{"year":2012,"claim":"Identified post-translational control of RFX5 stability and localization by SIRT1 deacetylation, linking it to the IFN-gamma collagen repression circuit.","evidence":"Reciprocal Co-IP, SIRT1/NAMPT perturbation, acetylation assays, fractionation, reporter and ChIP","pmids":["23079621"],"confidence":"Medium","gaps":["Acetyltransferase that adds the mark not identified","Specific lysine residues not mapped","Single lab"]},{"year":2013,"claim":"Showed isotype-specific tuning of MHC-II via PRMT6 methylation of an RFX5 AT-hook motif that selectively represses HLA-DQ.","evidence":"AT-hook mutagenesis, PRMT6 overexpression/knockdown, isotype-specific expression assays","pmids":["23911394"],"confidence":"Medium","gaps":["Methylated residue not directly mapped at structural level","Mechanism of DQ-versus-DR selectivity unclear"]},{"year":2006,"claim":"Added CREB as a direct RFX5 partner that enhances MHC-II transcription, broadening the activator network at these promoters.","evidence":"Co-IP, MHC-II reporter assay, and ChIP for phospho-CREB at HLA-DRA","pmids":["16730065"],"confidence":"Medium","gaps":["CREB binding region on RFX5 not mapped","Functional importance relative to CIITA/NF-Y unquantified"]},{"year":2020,"claim":"Established a non-immune oncogenic role: RFX5 directly activates target genes (TPP1, KDM4A) to drive cell cycle progression and suppress apoptosis in hepatocellular carcinoma.","evidence":"ChIP-PCR, luciferase, RFX5 knockout/rescue with KDM4A overexpression, cell cycle and apoptosis assays","pmids":["27840983","32883983"],"confidence":"Medium","gaps":["Whether RFX5 acts as the trimeric complex or independently at these promoters not resolved","Single cancer lineage tested per study"]},{"year":2022,"claim":"Connected RFX5 to metabolic-immune coupling, showing CCL18-induced RFX5 upregulates GLUD1 for glutamate energy metabolism while enhancing HLA-DR in synovial macrophages.","evidence":"RFX5 knockdown/overexpression, CCL18 stimulation, metabolic and surface HLA-DR assays in primary cells","pmids":["35739396"],"confidence":"Medium","gaps":["Direct RFX5 binding at the GLUD1 locus not structurally validated","In vivo relevance beyond primary cells untested"]},{"year":2025,"claim":"Resolved how RFX5 engages chromatin, showing its extended DNA binding domain binds nucleosomes and distorts DNA to increase accessibility, and identified JAG1/Notch activation as another oncogenic target.","evidence":"Cryo-EM of RFX5 eDBD-nucleosome complex; ChIP, luciferase and rescue for JAG1 in TNBC","pmids":["40744500","40220043"],"confidence":"High","gaps":["Cryo-EM used isolated eDBD, not the full trimer","Pioneer-factor-like chromatin opening not tested genome-wide"]},{"year":null,"claim":"How RFX5's collection of partners and post-translational marks is selected to switch between MHC-II activation, COL1A2 repression, and oncogenic target activation in a context-dependent manner remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unifying model for activation-versus-repression decision","Whether cancer target activation requires the full RFX trimer is untested","Methylation-sensitive DNA binding (preprint) lacks functional validation"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0003677","term_label":"DNA binding","supporting_discovery_ids":[2,4,6,8,16]},{"term_id":"GO:0140110","term_label":"transcription regulator activity","supporting_discovery_ids":[0,2,5,13]},{"term_id":"GO:0042393","term_label":"histone binding","supporting_discovery_ids":[16]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[5,9]},{"term_id":"GO:0000228","term_label":"nuclear chromosome","supporting_discovery_ids":[16]}],"pathway":[{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[0,1,15]},{"term_id":"R-HSA-74160","term_label":"Gene expression (Transcription)","supporting_discovery_ids":[0,2,5]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[12,13,17]}],"complexes":["RFX complex (RFX5/RFXANK/RFXAP)"],"partners":["CIITA","RFXAP","RFXANK","NF-Y","CREB","SIRT1","PRMT6","IER3"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"P48382","full_name":"DNA-binding protein RFX5","aliases":["Regulatory factor X 5"],"length_aa":616,"mass_kda":65.3,"function":"Activates transcription from class II MHC promoters. Recognizes X-boxes. Mediates cooperative binding between RFX and NF-Y. RFX binds the X1 box of MHC-II promoters","subcellular_location":"Nucleus","url":"https://www.uniprot.org/uniprotkb/P48382/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/RFX5","classification":"Not Classified","n_dependent_lines":77,"n_total_lines":1208,"dependency_fraction":0.06374172185430464},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/RFX5","total_profiled":1310},"omim":[{"mim_id":"620818","title":"MHC CLASS II DEFICIENCY 5; MHC2D5","url":"https://www.omim.org/entry/620818"},{"mim_id":"620816","title":"MHC CLASS II DEFICIENCY 3; MHC2D3","url":"https://www.omim.org/entry/620816"},{"mim_id":"613591","title":"BUTYROPHILIN, SUBFAMILY 2, MEMBER A2; BTN2A2","url":"https://www.omim.org/entry/613591"},{"mim_id":"612660","title":"REGULATORY FACTOR X, 7; RFX7","url":"https://www.omim.org/entry/612660"},{"mim_id":"604167","title":"CCCTC-BINDING FACTOR; CTCF","url":"https://www.omim.org/entry/604167"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Nucleoplasm","reliability":"Supported"},{"location":"Vesicles","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/RFX5"},"hgnc":{"alias_symbol":[],"prev_symbol":[]},"alphafold":{"accession":"P48382","domains":[{"cath_id":"1.10.10.10","chopping":"87-167","consensus_level":"high","plddt":92.1374,"start":87,"end":167},{"cath_id":"-","chopping":"198-249","consensus_level":"high","plddt":87.1802,"start":198,"end":249}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/P48382","model_url":"https://alphafold.ebi.ac.uk/files/AF-P48382-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-P48382-F1-predicted_aligned_error_v6.png","plddt_mean":57.12},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=RFX5","jax_strain_url":"https://www.jax.org/strain/search?query=RFX5"},"sequence":{"accession":"P48382","fasta_url":"https://rest.uniprot.org/uniprotkb/P48382.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/P48382/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/P48382"}},"corpus_meta":[{"pmid":"10790427","id":"PMC_10790427","title":"Degradation of transcription factor RFX5 during the inhibition of both constitutive and interferon gamma-inducible major histocompatibility complex class I expression in chlamydia-infected cells.","date":"2000","source":"The Journal of experimental medicine","url":"https://pubmed.ncbi.nlm.nih.gov/10790427","citation_count":153,"is_preprint":false},{"pmid":"9177217","id":"PMC_9177217","title":"Specific complex formation between the type II bare lymphocyte syndrome-associated transactivators CIITA and RFX5.","date":"1997","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/9177217","citation_count":111,"is_preprint":false},{"pmid":"7495736","id":"PMC_7495736","title":"The two novel MHC class II transactivators RFX5 and CIITA both control expression of HLA-DM genes.","date":"1995","source":"International immunology","url":"https://pubmed.ncbi.nlm.nih.gov/7495736","citation_count":91,"is_preprint":false},{"pmid":"10779326","id":"PMC_10779326","title":"A functionally essential domain of RFX5 mediates activation of major histocompatibility complex class II promoters by promoting cooperative binding between RFX and NF-Y.","date":"2000","source":"Molecular and cellular biology","url":"https://pubmed.ncbi.nlm.nih.gov/10779326","citation_count":70,"is_preprint":false},{"pmid":"35739396","id":"PMC_35739396","title":"The transcription factor RFX5 coordinates antigen-presenting function and resistance to nutrient stress in synovial macrophages.","date":"2022","source":"Nature metabolism","url":"https://pubmed.ncbi.nlm.nih.gov/35739396","citation_count":65,"is_preprint":false},{"pmid":"9491996","id":"PMC_9491996","title":"Residual MHC class II expression on mature dendritic cells and activated B cells in RFX5-deficient 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RFX5 provides promoter specificity via its DNA binding domain, while CIITA recruits the general transcription apparatus via its acidic activation domain. The direct interaction was demonstrated by yeast two-hybrid and far-Western blot assays.\",\n      \"method\": \"Yeast two-hybrid, far-Western blot, cotransfection reporter assay in CIITA-deficient cells\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal yeast two-hybrid and far-Western blot, replicated by functional rescue in CIITA-deficient cells, independently confirmed in later studies\",\n      \"pmids\": [\"9177217\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1995,\n      \"finding\": \"RFX5 is required for both constitutive and IFN-gamma-inducible transcription of HLA-DMA and HLA-DMB genes, as demonstrated by complementation of regulatory mutant cell lines with RFX5 cDNA.\",\n      \"method\": \"Complementation assay using cDNA transfection into MHC class II regulatory mutant cells\",\n      \"journal\": \"International immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean genetic complementation with defined phenotypic readout (DM gene expression restoration), single lab\",\n      \"pmids\": [\"7495736\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"The C-terminal domain of RFX5 mediates cooperative binding between the RFX complex and NF-Y (which binds the Y box), and this cooperative interaction is essential for transcriptional activation of MHC-II genes. The N-terminal region of RFX5 is required for association with RFXANK and RFXAP and for RFX complex assembly and X-box DNA binding, but is insufficient alone for activation.\",\n      \"method\": \"Domain deletion/mutagenesis, in vitro and in vivo RFX complex assembly assays, MHC-II promoter reporter assays\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — mutagenesis combined with in vitro reconstitution and in vivo transcriptional assays, multiple orthogonal methods in one rigorous study\",\n      \"pmids\": [\"10779326\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"In chlamydia-infected cells, RFX5 is degraded by a lactacystin-sensitive proteasome-like activity found in the cytosolic fraction of infected cells, dependent on chlamydial (not host) protein synthesis. This degradation correlates with suppression of both constitutive and IFN-gamma-inducible MHC class I expression.\",\n      \"method\": \"Immunoblot of RFX5 protein levels in infected cells, pharmacological inhibition with lactacystin, cell fractionation, inhibition of chlamydial vs. host protein synthesis\",\n      \"journal\": \"The Journal of experimental medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — biochemical fractionation and pharmacological dissection, single lab, correlative but mechanistically grounded\",\n      \"pmids\": [\"10790427\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"RFX5 forms homodimers in vivo and in vitro via a leucine-rich stretch N-terminal to its DNA binding domain; leucine 66 is critical for self-association. Mutant RFX5 unable to dimerize fails to support formation of higher-order DNA-protein complexes on MHC-II conserved upstream sequences in vitro and fails to activate MHC-II transcription in vivo.\",\n      \"method\": \"In vivo and in vitro dimerization assays, site-directed mutagenesis (L66), electrophoretic mobility shift assay (EMSA), MHC-II transcription assay\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — mutagenesis combined with in vitro EMSA and in vivo transcription assay, multiple orthogonal methods\",\n      \"pmids\": [\"12101253\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"The RFX5 complex (RFX5, RFXB/RFXANK, and RFXAP) mediates IFN-gamma-induced repression of the COL1A2 (collagen type I) gene. All three subunits are required for maximum repression; RFX5 dominant-negative mutants reverse IFN-gamma-induced collagen repression. IFN-gamma increases RFX5 nuclear translocation and recruitment of all three complex subunits to the COL1A2 transcription start site, as shown by ChIP.\",\n      \"method\": \"Promoter reporter assay, dominant-negative overexpression, IFN-gamma treatment, chromatin immunoprecipitation (ChIP), immunofluorescence for nuclear translocation\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (reporter assay, dominant-negative genetics, ChIP), single lab but comprehensive\",\n      \"pmids\": [\"12968017\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"DNA binding of RFX5 alone is autoinhibited by domains flanking its DNA binding domain. Both RFXAP and RFXB are required to relieve this autoinhibition and allow high-affinity binding of the RFX complex to the MHC-II promoter. A single RFX complex binds the proximal regulatory region.\",\n      \"method\": \"Electrophoretic mobility shift assay (EMSA) with recombinant proteins and MHC-II promoter DNA fragments\",\n      \"journal\": \"Biochimica et biophysica acta\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution with defined recombinant proteins, single lab\",\n      \"pmids\": [\"18723135\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"The solution NMR structure of the RFX5 N-terminal dimerization domain (RFX5(N)) in complex with the C-terminal domain of RFXAP reveals that two RFX5(N) molecules form an antiparallel coiled coil (staple shape), with RFXAP(C) adopting two α-helices that pack within this staple. Leucine residues in the leucine-rich region (62-LYLYLQL-68) contribute to both the dimer interface (Leu64, Leu68) and the RFX5-RFXAP interface (Leu62, Leu66). RFXAP is unstructured alone but folds upon binding RFX5.\",\n      \"method\": \"Solution NMR structure determination (15N- and 13C-edited), in vitro binding assays\",\n      \"journal\": \"Journal of molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — NMR structure with residue-level functional validation, comprehensive structural and biochemical analysis\",\n      \"pmids\": [\"20732328\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"The DNA binding domain of RFX5 (RFX5DBD) binds X-box DNA with an entropy-driven, enthalpy-favorable interaction; the dissociation constant was determined by fluorescence quenching. RFX5DBD also interacts directly with RFXANK in the absence of RFXAP, with an apparent Kd of 128 nM, as measured by FRET and chemical cross-linking/mass spectrometry.\",\n      \"method\": \"Steady-state fluorescence quenching, circular dichroism, FRET, chemical cross-linking followed by tandem mass spectrometry\",\n      \"journal\": \"Biochimica et biophysica acta\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — multiple biophysical methods in vitro, single lab, thermodynamic parameters determined\",\n      \"pmids\": [\"20637319\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"SIRT1 forms a complex with RFX5 and deacetylates it. SIRT1-mediated deacetylation promotes nuclear expulsion and proteasomal degradation of RFX5, thereby reducing RFX5 binding to the COL1A2 promoter and antagonizing RFX5-mediated repression of collagen transcription. IFN-gamma represses COL1A2 by downregulating SIRT1, which leads to increased RFX5 acetylation and enhanced collagen repression.\",\n      \"method\": \"Co-immunoprecipitation, overexpression and knockdown of SIRT1/NAMPT, SIRT1 agonist/inhibitor treatment, acetylation assays, subcellular fractionation, promoter reporter assay, ChIP\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP, multiple genetic and pharmacological perturbations, single lab\",\n      \"pmids\": [\"23079621\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"RFX5 contains an AT-hook motif that is involved in regulating transcription of the HLA-DQ (but not HLA-DR) MHC-II isotype. PRMT6, an arginine methyltransferase, methylates this AT-hook motif and specifically downregulates HLA-DQ expression in an AT-hook-dependent manner, providing isotype-specific fine-tuning of MHC-II transcription.\",\n      \"method\": \"AT-hook motif mutagenesis, PRMT6 overexpression/knockdown, MHC-II isotype-specific expression assays\",\n      \"journal\": \"Molecular immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — mutagenesis combined with functional reporter assays and endogenous gene expression, single lab\",\n      \"pmids\": [\"23911394\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"CREB interacts with the RFX5 subunit of the RFX complex through the C-terminal portion of CREB, as demonstrated by co-immunoprecipitation. Phospho-CREB is present at the HLA-DRA promoter (shown by ChIP) and enhances MHC-II transcription, though phosphorylation is not required for basal transcription.\",\n      \"method\": \"Co-immunoprecipitation, MHC-II promoter reporter assay, chromatin immunoprecipitation (ChIP)\",\n      \"journal\": \"Molecular immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP and ChIP with functional reporter assay, single lab\",\n      \"pmids\": [\"16730065\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"RFX5 binds directly to the TPP1 promoter and transcriptionally activates TPP1 gene expression in hepatocellular carcinoma cells; manipulation of RFX5 expression levels correspondingly affects TPP1 expression in HepG2 cells.\",\n      \"method\": \"ChIP-PCR, luciferase reporter assay, RFX5 overexpression/knockdown with qRT-PCR\",\n      \"journal\": \"Oncology reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP and luciferase assay with functional perturbation, single lab\",\n      \"pmids\": [\"27840983\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"RFX5 binds directly to the KDM4A promoter and transcriptionally activates KDM4A in hepatocellular carcinoma cells. The RFX5-KDM4A pathway promotes cell cycle progression from G0/G1 to S phase and inhibits apoptosis through regulation of p53 and downstream genes; KDM4A overexpression rescues growth inhibitory effects of RFX5 deletion.\",\n      \"method\": \"ChIP-PCR, luciferase reporter assay, ChIP-seq (ENCODE), RFX5 knockout/rescue with KDM4A overexpression, cell cycle and apoptosis assays\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP, luciferase, and epistasis rescue experiment, single lab\",\n      \"pmids\": [\"32883983\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"In synovial macrophages, CCL18 signaling induces RFX5, which selectively upregulates glutamate dehydrogenase 1 (GLUD1) to enable glutamate-based energy production under glucose withdrawal. In parallel, RFX5 enhances surface HLA-DR expression, coupling metabolic adaptation to antigen-presenting function.\",\n      \"method\": \"RFX5 knockdown/overexpression, CCL18 stimulation, GLUD1 expression analysis, HLA-DR surface expression, metabolic assays in primary synovial macrophages\",\n      \"journal\": \"Nature metabolism\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — loss- and gain-of-function with defined metabolic and immunological phenotypic readouts, single lab, primary cells\",\n      \"pmids\": [\"35739396\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"RFX5-deficient mice lack MHC-II expression in thymic cortex, resting B cells, and macrophages, causing failure of CD4+ T cell positive selection and severe immunodeficiency. However, residual MHC-II expression persists in thymic medulla, mature dendritic cells, and activated B cells, revealing that CIITA and RFX5 have differential requirements in different antigen-presenting cell subsets.\",\n      \"method\": \"RFX5 knockout mouse (conditional gene targeting), flow cytometry for MHC-II expression, lymphocyte subset analysis\",\n      \"journal\": \"Immunity\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean KO mouse with defined cellular phenotypes and subset-specific mechanistic dissection\",\n      \"pmids\": [\"9491996\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Cryo-EM structure of the RFX5-nucleosome complex reveals that the extended DNA binding domain (eDBD) of RFX5 binds the nucleosome at superhelical location +2, engaging both nucleosomal DNA and histone proteins. RFX5 eDBD induces localized distortion of the bound DNA gyre and detachment of an adjacent DNA gyre, increasing DNA accessibility and potentially enhancing transcriptional activity.\",\n      \"method\": \"Cryo-EM structure determination of RFX5 eDBD-nucleosome complex\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — cryo-EM structure with detailed structural analysis and in vivo functional correlates, single study but high-quality structural data\",\n      \"pmids\": [\"40744500\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"RFX5 transcriptionally activates JAG1 by directly binding the JAG1 promoter (at the -1890/+15 or -1359/+15 region), thereby activating the Notch signaling pathway (Notch1, NICD, Hes1) in triple-negative breast cancer cells.\",\n      \"method\": \"Chromatin immunoprecipitation, luciferase reporter assay, JAG1 knockdown rescue experiment, RFX5 overexpression/knockdown\",\n      \"journal\": \"Human cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP and luciferase assay with epistasis rescue, single lab\",\n      \"pmids\": [\"40220043\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"IER3 functions as a transcriptional co-activator for RFX5, forming a complex with RFX5 (demonstrated by co-immunoprecipitation) that upregulates AKR1B10, which subsequently suppresses p53 in hepatocellular carcinoma cells.\",\n      \"method\": \"Co-immunoprecipitation, dual-luciferase reporter assay, siRNA knockdown, qPCR and western blot\",\n      \"journal\": \"Discover oncology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single Co-IP with reporter assay, single lab, in vitro only with no in vivo validation\",\n      \"pmids\": [\"42209919\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"RFX5 exhibits methylation-sensitive DNA binding: it discriminates between specific CpG hydroxymethylation (hmC) symmetries in CpG dyads, as identified by meSMiLE-seq assay.\",\n      \"method\": \"meSMiLE-seq (microfluidic selective ligand enrichment followed by sequencing with methylated vs unmethylated DNA probes)\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single in vitro biochemical screen, preprint, no mutagenesis or functional validation of the methylation-sensitive binding\",\n      \"pmids\": [],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"RFX5 is the largest subunit of the heterotrimeric RFX complex (with RFXANK/B and RFXAP), where it homodimerizes via an antiparallel coiled-coil N-terminal domain that also mediates RFXAP binding (NMR structure resolved), and its DNA binding domain—subject to autoinhibition relieved by RFXANK and RFXAP—engages X-box sequences in MHC-II promoters and can bind and destabilize nucleosomes (cryo-EM structure resolved); the C-terminal domain of RFX5 mediates cooperative interaction with NF-Y to activate MHC-II transcription, while RFX5 also directly interacts with CIITA (which recruits the general transcription apparatus), with CREB, and is subject to acetylation (erased by SIRT1) that controls its nuclear localization and proteasomal degradation; beyond MHC-II regulation, the RFX5 complex represses COL1A2 collagen transcription in response to IFN-gamma, and RFX5 transcriptionally activates additional targets including KDM4A, TPP1, and JAG1 in cancer contexts.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"RFX5 is the DNA-binding subunit of the heterotrimeric RFX complex that confers promoter specificity for X-box sequences in MHC class II genes, where it cooperates with the master coactivator CIITA to drive antigen-presentation gene transcription [#0, #15]. RFX5 contributes promoter recognition through its DNA binding domain while CIITA, recruited via direct protein-protein interaction, brings in the general transcription apparatus; loss of RFX5 in mice abolishes MHC-II expression in thymic cortex, resting B cells and macrophages and blocks CD4+ T cell positive selection, establishing its central immunological role [#0, #15]. Complex assembly requires the RFX5 N-terminal region, which homodimerizes through a leucine-rich segment (Leu66 critical) that forms an antiparallel coiled-coil staple and simultaneously engages RFXAP, while the C-terminal domain mediates cooperative interaction with NF-Y to activate transcription [#2, #4, #7]. The isolated RFX5 DNA binding domain is autoinhibited by flanking sequences; both RFXAP and RFXANK are required to relieve this autoinhibition and permit high-affinity X-box binding, and its extended DNA binding domain engages nucleosomes directly, distorting DNA gyres to increase accessibility [#6, #16]. RFX5 activity is tuned post-translationally: SIRT1 deacetylation controls its nuclear retention and proteasomal turnover, and PRMT6 methylation of an AT-hook motif selectively downregulates the HLA-DQ isotype [#9, #10]. Beyond MHC-II, the RFX complex mediates IFN-gamma-induced repression of COL1A2 collagen [#5], and RFX5 directly activates KDM4A, TPP1 and JAG1 to promote proliferation and Notch signaling in cancer contexts [#12, #13, #17].\",\n  \"teleology\": [\n    {\n      \"year\": 1995,\n      \"claim\": \"Established that RFX5 is genetically required for MHC class II gene expression, identifying it as a non-redundant regulator of antigen presentation.\",\n      \"evidence\": \"cDNA complementation of MHC-II regulatory mutant cell lines restoring HLA-DMA/DMB expression\",\n      \"pmids\": [\"7495736\"],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Did not define the molecular interaction or DNA-binding mechanism\", \"Single lab, restricted to DM isotype genes\"]\n    },\n    {\n      \"year\": 1997,\n      \"claim\": \"Defined the molecular partnership underlying MHC-II activation by showing RFX5 supplies promoter specificity and CIITA supplies the activation function through a direct interaction.\",\n      \"evidence\": \"Yeast two-hybrid, far-Western blot, and cotransfection rescue in CIITA-deficient cells\",\n      \"pmids\": [\"9177217\"],\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Did not map the structural basis of the RFX5-CIITA interface\", \"Did not address contribution of other complex subunits\"]\n    },\n    {\n      \"year\": 1998,\n      \"claim\": \"Demonstrated the physiological consequence of RFX5 loss in vivo, linking it to CD4+ T cell selection and revealing cell-type-specific requirements for MHC-II control.\",\n      \"evidence\": \"RFX5 knockout mouse with flow cytometry of MHC-II and lymphocyte subset analysis\",\n      \"pmids\": [\"9491996\"],\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Did not explain the residual MHC-II expression in medulla/dendritic cells mechanistically\", \"Did not test non-immune RFX5 functions\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Mapped the self-association mechanism of RFX5, showing homodimerization via a leucine-rich N-terminal segment is required for higher-order promoter complex formation and transcription.\",\n      \"evidence\": \"In vivo/in vitro dimerization assays, L66 site-directed mutagenesis, EMSA, and transcription assays\",\n      \"pmids\": [\"12101253\"],\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Atomic structure of the dimer not yet resolved\", \"Did not address how dimerization couples to RFXAP/RFXANK binding\"]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"Assigned distinct functions to RFX5 domains, showing the C-terminus mediates cooperative NF-Y binding for activation while the N-terminus is needed for complex assembly and X-box binding.\",\n      \"evidence\": \"Domain deletion/mutagenesis with in vitro and in vivo complex assembly and reporter assays\",\n      \"pmids\": [\"10779326\"],\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Did not resolve atomic structure of the RFX5-NF-Y contact\", \"Did not quantify cooperativity thermodynamically\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Revealed that RFX5 DNA binding is autoinhibited and relieved only by RFXAP and RFXANK, explaining why complex assembly is obligatory for high-affinity promoter engagement.\",\n      \"evidence\": \"EMSA with recombinant proteins and MHC-II promoter DNA\",\n      \"pmids\": [\"18723135\"],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Structural basis of autoinhibition not defined\", \"In vitro only, single lab\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Provided the structural and thermodynamic basis for RFX5 dimerization, RFXAP folding-upon-binding, and X-box recognition.\",\n      \"evidence\": \"Solution NMR structure of RFX5(N)-RFXAP(C) and biophysical (fluorescence/FRET/crosslinking-MS) DNA and RFXANK binding measurements\",\n      \"pmids\": [\"20732328\", \"20637319\"],\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"No structure of the full trimer on DNA\", \"DNA binding domain structure with DNA not resolved at this stage\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Extended RFX5 function beyond activation by showing the complex mediates IFN-gamma-induced repression of COL1A2 collagen, requiring all three subunits.\",\n      \"evidence\": \"Reporter assays, dominant-negative RFX5, IFN-gamma treatment, ChIP, and immunofluorescence\",\n      \"pmids\": [\"12968017\"],\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Mechanism of repression versus activation at distinct promoters not resolved\", \"Corepressor recruitment not identified\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Identified post-translational control of RFX5 stability and localization by SIRT1 deacetylation, linking it to the IFN-gamma collagen repression circuit.\",\n      \"evidence\": \"Reciprocal Co-IP, SIRT1/NAMPT perturbation, acetylation assays, fractionation, reporter and ChIP\",\n      \"pmids\": [\"23079621\"],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Acetyltransferase that adds the mark not identified\", \"Specific lysine residues not mapped\", \"Single lab\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Showed isotype-specific tuning of MHC-II via PRMT6 methylation of an RFX5 AT-hook motif that selectively represses HLA-DQ.\",\n      \"evidence\": \"AT-hook mutagenesis, PRMT6 overexpression/knockdown, isotype-specific expression assays\",\n      \"pmids\": [\"23911394\"],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Methylated residue not directly mapped at structural level\", \"Mechanism of DQ-versus-DR selectivity unclear\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Added CREB as a direct RFX5 partner that enhances MHC-II transcription, broadening the activator network at these promoters.\",\n      \"evidence\": \"Co-IP, MHC-II reporter assay, and ChIP for phospho-CREB at HLA-DRA\",\n      \"pmids\": [\"16730065\"],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"CREB binding region on RFX5 not mapped\", \"Functional importance relative to CIITA/NF-Y unquantified\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Established a non-immune oncogenic role: RFX5 directly activates target genes (TPP1, KDM4A) to drive cell cycle progression and suppress apoptosis in hepatocellular carcinoma.\",\n      \"evidence\": \"ChIP-PCR, luciferase, RFX5 knockout/rescue with KDM4A overexpression, cell cycle and apoptosis assays\",\n      \"pmids\": [\"27840983\", \"32883983\"],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Whether RFX5 acts as the trimeric complex or independently at these promoters not resolved\", \"Single cancer lineage tested per study\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Connected RFX5 to metabolic-immune coupling, showing CCL18-induced RFX5 upregulates GLUD1 for glutamate energy metabolism while enhancing HLA-DR in synovial macrophages.\",\n      \"evidence\": \"RFX5 knockdown/overexpression, CCL18 stimulation, metabolic and surface HLA-DR assays in primary cells\",\n      \"pmids\": [\"35739396\"],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Direct RFX5 binding at the GLUD1 locus not structurally validated\", \"In vivo relevance beyond primary cells untested\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Resolved how RFX5 engages chromatin, showing its extended DNA binding domain binds nucleosomes and distorts DNA to increase accessibility, and identified JAG1/Notch activation as another oncogenic target.\",\n      \"evidence\": \"Cryo-EM of RFX5 eDBD-nucleosome complex; ChIP, luciferase and rescue for JAG1 in TNBC\",\n      \"pmids\": [\"40744500\", \"40220043\"],\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Cryo-EM used isolated eDBD, not the full trimer\", \"Pioneer-factor-like chromatin opening not tested genome-wide\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How RFX5's collection of partners and post-translational marks is selected to switch between MHC-II activation, COL1A2 repression, and oncogenic target activation in a context-dependent manner remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"No unifying model for activation-versus-repression decision\", \"Whether cancer target activation requires the full RFX trimer is untested\", \"Methylation-sensitive DNA binding (preprint) lacks functional validation\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0003677\", \"supporting_discovery_ids\": [2, 4, 6, 8, 16]},\n      {\"term_id\": \"GO:0140110\", \"supporting_discovery_ids\": [0, 2, 5, 13]},\n      {\"term_id\": \"GO:0042393\", \"supporting_discovery_ids\": [16]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [5, 9]},\n      {\"term_id\": \"GO:0000228\", \"supporting_discovery_ids\": [16]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [0, 1, 15]},\n      {\"term_id\": \"R-HSA-74160\", \"supporting_discovery_ids\": [0, 2, 5]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [12, 13, 17]}\n    ],\n    \"complexes\": [\"RFX complex (RFX5/RFXANK/RFXAP)\"],\n    \"partners\": [\"CIITA\", \"RFXAP\", \"RFXANK\", \"NF-Y\", \"CREB\", \"SIRT1\", \"PRMT6\", \"IER3\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}