{"gene":"RFX5","run_date":"2026-04-28T19:45:45","timeline":{"discoveries":[{"year":1997,"finding":"RFX5 directly interacts with CIITA to form a complex capable of activating transcription from MHC class II promoters; in this complex, promoter specificity is determined by the DNA binding domain of RFX5 while the general transcription apparatus is recruited by the acidic activation domain of CIITA.","method":"Yeast two-hybrid, far-Western blot, GAL4 fusion reporter assays, cotransfection in CIITA-deficient cells","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 — reciprocal interaction assays (Y2H + far-Western) plus functional reporter rescue, replicated context","pmids":["9177217"],"is_preprint":false},{"year":1995,"finding":"RFX5 and CIITA are both essential regulators of HLA-DMA and HLA-DMB gene expression, controlling both constitutive and IFN-γ-inducible expression of DM genes in addition to classical MHC class II genes.","method":"Complementation of regulatory mutants by cDNA transfection; genetic epistasis in cell lines deficient for CIITA or RFX5","journal":"International immunology","confidence":"High","confidence_rationale":"Tier 2 — clean genetic complementation with defined molecular readout, foundational paper","pmids":["7495736"],"is_preprint":false},{"year":2000,"finding":"The N-terminal region of RFX5 is required for association with RFXANK and RFXAP and for assembly of the trimeric RFX complex; a separate C-terminal domain of RFX5 mediates cooperative binding between the RFX complex and NF-Y at the Y box of MHC-II promoters, and this cooperative binding is essential for transcriptional activation.","method":"Domain deletion/mutagenesis, in vitro and in vivo complex assembly assays, electrophoretic mobility shift assays (EMSA), reporter assays","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 1-2 — mutagenesis combined with in vitro binding assays and functional reporter assays in multiple cell lines","pmids":["10779326"],"is_preprint":false},{"year":2000,"finding":"Chlamydia infection degrades RFX5 via a lactacystin-sensitive, chlamydia-dependent proteasome-like activity present in the cytosolic fraction of infected cells, leading to suppression of both constitutive and IFN-γ-inducible MHC class I expression.","method":"Immunoblot for RFX5 degradation, lactacystin inhibition, cell fractionation, chlamydial protein synthesis inhibition","journal":"The Journal of experimental medicine","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods (pharmacological inhibition, fractionation, protein synthesis block) in a single study","pmids":["10790427"],"is_preprint":false},{"year":2002,"finding":"RFX5 forms homodimers in vivo and in vitro through a leucine-rich stretch (residues 62-68) N-terminal to its DNA binding domain; leucine at position 66 is critical for self-association, and dimerization-deficient RFX5 mutants fail to support higher-order DNA-protein complex formation on MHC-II conserved upstream sequences or MHC-II transcription in vivo.","method":"Yeast two-hybrid, in vitro binding, site-directed mutagenesis, EMSA, reporter assays in cells","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 1-2 — mutagenesis combined with in vitro and in vivo functional validation","pmids":["12101253"],"is_preprint":false},{"year":1998,"finding":"RFX5 knockout mice lack MHC-II expression in thymic cortex, resting B cells, and macrophages, causing failure of positive selection of CD4+ T cells; however, some residual MHC-II expression persists in mature dendritic cells and activated B cells, demonstrating a cell-type-specific requirement for RFX5.","method":"Conditional gene knockout (RFX5-/- mice), flow cytometry, immunohistochemistry, T cell selection assays","journal":"Immunity","confidence":"High","confidence_rationale":"Tier 2 — clean knockout mouse with defined cellular and immunological phenotypes","pmids":["9491996"],"is_preprint":false},{"year":2003,"finding":"The intact RFX5 trimeric complex (RFX5, RFXB/RFXANK, RFXAP) is required for maximum repression of collagen COL1A2 transcription; IFN-γ increases nuclear translocation of all three RFX complex subunits and their occupancy at the collagen transcription start site, while dominant-negative RFX5 mutants reverse IFN-γ-mediated collagen repression.","method":"Reporter assays with dominant-negative mutants, chromatin immunoprecipitation (ChIP), immunofluorescence for nuclear translocation, overexpression in human lung fibroblasts","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 — ChIP plus dominant-negative rescue and reporter assays, multiple orthogonal methods","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 overcome this autoinhibition, and a single RFX trimeric complex binds the proximal regulatory region of the MHC-II promoter.","method":"Electrophoretic mobility shift assays (EMSA) with purified recombinant proteins and mutant constructs","journal":"Biochimica et biophysica acta","confidence":"Medium","confidence_rationale":"Tier 1 — in vitro reconstitution with purified proteins, single lab","pmids":["18723135"],"is_preprint":false},{"year":2010,"finding":"The NMR solution structure of the RFX5(N)2-RFXAP(C) heterotrimeric complex reveals that two RFX5 N-terminal domains form an antiparallel coiled-coil 'staple' structure, with RFXAP(C) adopting a V-shaped helical structure that packs within the RFX5 dimer; leucine residues in the leucine-rich region (62-LYLYLQL-68) contribute to both the RFX5 dimer interface and the RFX5-RFXAP interface.","method":"Solution NMR (15N- and 13C-edited), structural determination of heterotrimeric complex","journal":"Journal of molecular biology","confidence":"High","confidence_rationale":"Tier 1 — atomic-resolution structure with residue-level functional mapping, validated against prior mutagenesis data","pmids":["20732328"],"is_preprint":false},{"year":2010,"finding":"The DNA binding domain (DBD) of RFX5 directly interacts with X-box DNA in an entropy-driven, enthalpy-favorable manner, and directly binds RFXANK in vitro (Kd ~128 nM) even in the absence of RFXAP, as measured by fluorescence and FRET methods.","method":"Steady-state fluorescence quenching, circular dichroism, FRET, chemical cross-linking with tandem mass spectrometry","journal":"Biochimica et biophysica acta","confidence":"Medium","confidence_rationale":"Tier 1-2 — multiple biophysical methods in vitro, single lab","pmids":["20637319"],"is_preprint":false},{"year":2012,"finding":"SIRT1 forms a complex with RFX5, deacetylates it, promotes its nuclear expulsion and proteasomal degradation, thereby antagonizing RFX5-mediated repression of COL1A2 transcription in smooth muscle cells; IFN-γ represses COL1A2 by downregulating SIRT1, increasing RFX5 acetylation.","method":"Co-immunoprecipitation, overexpression/knockdown of SIRT1, NAMPT, resveratrol/inhibitor treatment, reporter assays, nuclear/cytoplasmic fractionation","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2-3 — co-IP plus pharmacological and genetic manipulation, single lab","pmids":["23079621"],"is_preprint":false},{"year":2013,"finding":"An AT-hook motif in RFX5 is involved in regulating transcription of HLA-DQ but not HLA-DR MHC-II genes; PRMT6, an arginine methyltransferase, methylates this AT-hook motif and selectively downregulates HLA-DQ expression in an AT-hook-dependent manner, providing isotype-specific regulation of MHC-II.","method":"Site-directed mutagenesis of AT-hook motif, PRMT6 overexpression/knockdown, reporter assays, ChIP","journal":"Molecular immunology","confidence":"Medium","confidence_rationale":"Tier 2 — mutagenesis combined with functional reporter assays and ChIP, single lab","pmids":["23911394"],"is_preprint":false},{"year":2006,"finding":"CREB and phospho-CREB interact directly with RFX5 (through the C-terminal portion of CREB) and with CIITA to form part of the MHC-II transcriptional regulatory complex; phosphorylation of CREB enhances transcription from MHC-II promoters and phospho-CREB is found at the HLA-DRA promoter by ChIP.","method":"Co-immunoprecipitation with CREB mutants, reporter assays, chromatin immunoprecipitation (ChIP)","journal":"Molecular immunology","confidence":"Medium","confidence_rationale":"Tier 2 — reciprocal co-IP with multiple mutants plus ChIP, single lab","pmids":["16730065"],"is_preprint":false},{"year":2022,"finding":"CCL18 induces RFX5 expression in synovial macrophages; RFX5 selectively upregulates GLUD1 transcription to enable glutamate utilization for energy under glucose withdrawal, and simultaneously enhances surface HLA-DR expression to promote antigen-specific T cell expansion.","method":"RFX5 knockdown/overexpression in primary macrophages, ChIP, metabolic assays (glutamate utilization), flow cytometry for HLA-DR and T cell activation","journal":"Nature metabolism","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods (ChIP, metabolic assays, flow cytometry) with genetic loss-of-function in primary cells","pmids":["35739396"],"is_preprint":false},{"year":2016,"finding":"RFX5 binds directly to the TPP1 promoter region and transcriptionally activates TPP1 expression in hepatocellular carcinoma cells, as demonstrated by ChIP and reporter assays.","method":"ChIP, luciferase reporter assay, RFX5 overexpression/knockdown in HepG2 cells","journal":"Oncology reports","confidence":"Medium","confidence_rationale":"Tier 2 — ChIP plus reporter assay, single lab","pmids":["27840983"],"is_preprint":false},{"year":2020,"finding":"RFX5 directly binds the KDM4A promoter and transcriptionally activates KDM4A expression in hepatocellular carcinoma; the RFX5-KDM4A pathway promotes cell cycle progression from G0/G1 to S phase and inhibits apoptosis through regulation of p53 and downstream genes.","method":"ChIP-seq (ENCODE), ChIP-PCR, luciferase reporter assay, RFX5 deletion/overexpression, cell cycle analysis","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 — ChIP-PCR plus luciferase reporter and functional rescue, single lab","pmids":["32883983"],"is_preprint":false},{"year":2025,"finding":"Cryo-EM structure of the RFX5 extended DNA binding domain (eDBD) bound to a nucleosome reveals that eDBD engages nucleosomal DNA at superhelical location +2 and contacts histones; RFX5 eDBD induces localized distortion of the bound DNA gyre and detachment of the adjacent DNA gyre, increasing DNA accessibility.","method":"Cryo-EM structure determination of RFX5 eDBD-nucleosome complex","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 1 — atomic-resolution cryo-EM structure with mechanistic characterization of nucleosome distortion","pmids":["40744500"],"is_preprint":false},{"year":2025,"finding":"RFX5 discriminates between strand-symmetric and strand-asymmetric 5-hydroxymethylcytosine (hmC) modifications in CpG dyads, identified as a reader of specific hmC symmetries in the nuclear proteome.","method":"Comparative enrichment proteomics with promoter probes bearing symmetric or asymmetric hmC modifications in human and mouse nuclear lysates","journal":"bioRxiv","confidence":"Low","confidence_rationale":"Tier 3 — single pulldown/enrichment proteomics method, preprint, no functional validation of hmC reading","pmids":["bio_10.1101_2025.06.27.661915"],"is_preprint":true},{"year":2025,"finding":"RFX5 transcriptionally activates JAG1 by binding to its promoter (−1890/+15 or −1359/+15 region), thereby activating Notch signaling (Notch1, NICD, Hes1) in triple-negative breast cancer cells.","method":"Chromatin immunoprecipitation, luciferase reporter assay, RFX5 knockdown/overexpression, JAG1 knockdown rescue","journal":"Human cell","confidence":"Medium","confidence_rationale":"Tier 2 — ChIP plus luciferase reporter and epistasis rescue, single lab","pmids":["40220043"],"is_preprint":false}],"current_model":"RFX5 is the largest subunit of the trimeric RFX transcription factor complex (with RFXANK and RFXAP), which binds X-box sequences in MHC class II promoters through an autoinhibited DNA binding domain that is activated upon complex assembly; RFX5 homodimerizes via a leucine-rich N-terminal coiled-coil that also recruits RFXAP (as resolved by NMR and cryo-EM), its C-terminal domain mediates cooperative binding with NF-Y to activate MHC-II transcription, it directly interacts with CIITA (which provides the transcriptional activation function), and it is subject to post-translational regulation by SIRT1-mediated deacetylation and PRMT6-mediated arginine methylation that control its stability, localization, and target-gene selectivity; beyond MHC-II regulation, RFX5 represses collagen (COL1A2) and activates non-immune targets including GLUD1, TPP1, KDM4A, and JAG1 in a context-dependent manner, and its extended DNA binding domain can directly bind and destabilize nucleosomes to increase chromatin accessibility."},"narrative":{"teleology":[{"year":1995,"claim":"Establishing RFX5 as an essential regulator of MHC class II and non-classical HLA-DM genes resolved a key missing link in how MHC-II expression is controlled in human cells.","evidence":"Genetic complementation of RFX5-deficient regulatory mutant cell lines restored HLA-DMA/DMB expression","pmids":["7495736"],"confidence":"High","gaps":["The molecular mechanism by which RFX5 acts on these promoters was unknown","Relationship to CIITA was undefined"]},{"year":1997,"claim":"Demonstrating that RFX5 directly interacts with CIITA established a division of labor: RFX5 provides promoter specificity via DNA binding while CIITA supplies the transcriptional activation domain.","evidence":"Yeast two-hybrid, far-Western blot, and GAL4 fusion reporter assays in CIITA-deficient cells","pmids":["9177217"],"confidence":"High","gaps":["How CIITA is recruited to the assembled RFX complex on chromatin was not resolved","Whether additional cofactors participate was unknown"]},{"year":1998,"claim":"RFX5 knockout mice revealed that RFX5 is required for MHC-II expression in most antigen-presenting cells but dispensable in activated dendritic cells and B cells, establishing cell-type specificity of the requirement.","evidence":"RFX5−/− mice analyzed by flow cytometry, immunohistochemistry, and T cell selection assays","pmids":["9491996"],"confidence":"High","gaps":["Identity of the compensatory factor(s) enabling residual MHC-II in dendritic cells was not determined","Impact on non-MHC-II target genes in vivo was unexplored"]},{"year":2000,"claim":"Domain mapping of RFX5 separated its N-terminal complex-assembly function from a C-terminal domain that mediates cooperative NF-Y binding at the Y box, defining the architecture underlying enhanceosome formation on MHC-II promoters.","evidence":"Deletion/point mutagenesis with EMSA, co-assembly assays, and reporter assays","pmids":["10779326"],"confidence":"High","gaps":["Structural basis of the C-terminal domain–NF-Y interaction was not resolved","Whether Y-box cooperation occurs at non-MHC-II promoters was unknown"]},{"year":2002,"claim":"Showing that RFX5 homodimerizes via a leucine-rich N-terminal stretch and that dimerization is essential for higher-order DNA–protein complex formation explained how a single gene product nucleates the multisubunit enhanceosome.","evidence":"Yeast two-hybrid, in vitro binding, Leu66 point mutagenesis, EMSA, and reporter assays","pmids":["12101253"],"confidence":"High","gaps":["Whether dimerization is constitutive or regulated was not determined"]},{"year":2003,"claim":"Discovery that the intact RFX trimer represses COL1A2 transcription under IFN-γ stimulation expanded RFX5 function beyond immune gene activation to transcriptional repression of extracellular matrix genes.","evidence":"ChIP at collagen promoter, dominant-negative mutants, immunofluorescence for nuclear translocation in lung fibroblasts","pmids":["12968017"],"confidence":"High","gaps":["Mechanism by which RFX5 switches from activation to repression was not defined","Whether CIITA participates in collagen repression was not tested"]},{"year":2006,"claim":"Identification of phospho-CREB as a direct interactor of both RFX5 and CIITA at MHC-II promoters added a signal-responsive component to the enhanceosome model.","evidence":"Co-immunoprecipitation with CREB mutants, ChIP at HLA-DRA promoter, reporter assays","pmids":["16730065"],"confidence":"Medium","gaps":["Whether CREB interaction is required for all MHC-II loci or only a subset was not tested","Stoichiometry of CREB within the enhanceosome was unknown"]},{"year":2008,"claim":"Showing that RFX5 DNA binding is autoinhibited and requires both RFXAP and RFXANK for relief explained why isolated RFX5 cannot bind X-box DNA efficiently and why all three subunits are genetically required.","evidence":"EMSA with purified recombinant RFX5 and truncation mutants reconstituted with RFXAP/RFXANK","pmids":["18723135"],"confidence":"Medium","gaps":["Structural mechanism of autoinhibition relief was not determined at atomic resolution","In vivo validation of autoinhibition model was lacking"]},{"year":2010,"claim":"NMR structure of the RFX5(N)₂–RFXAP(C) complex and biophysical characterization of the DBD–X-box interaction provided the first atomic-level view of how the trimer assembles and engages DNA.","evidence":"Solution NMR of the heterotrimeric complex; fluorescence quenching, FRET, and CD for DBD–DNA interaction","pmids":["20732328","20637319"],"confidence":"High","gaps":["Full-length trimer structure on DNA was not available","How autoinhibition is relieved structurally remained unresolved"]},{"year":2012,"claim":"Discovery that SIRT1 deacetylates RFX5 and promotes its nuclear expulsion and proteasomal degradation established the first post-translational regulatory axis controlling RFX5 activity, linking NAD⁺ metabolism to MHC-II and collagen regulation.","evidence":"Co-immunoprecipitation, SIRT1 overexpression/knockdown, pharmacological modulation, nuclear/cytoplasmic fractionation in smooth muscle cells","pmids":["23079621"],"confidence":"Medium","gaps":["Specific acetylated residues on RFX5 were not mapped","Whether SIRT1 regulation affects MHC-II as well as collagen targets was not tested"]},{"year":2013,"claim":"PRMT6-mediated arginine methylation of the RFX5 AT-hook motif selectively downregulates HLA-DQ but not HLA-DR, revealing an isotype-specific epigenetic control mechanism within the MHC-II locus.","evidence":"AT-hook mutagenesis, PRMT6 overexpression/knockdown, ChIP and reporter assays","pmids":["23911394"],"confidence":"Medium","gaps":["Whether methylation disrupts DNA binding or protein–protein interaction at the AT-hook was not distinguished","In vivo relevance in primary antigen-presenting cells was not shown"]},{"year":2016,"claim":"Identification of TPP1 as a direct transcriptional target of RFX5 in hepatocellular carcinoma cells extended the non-immune target repertoire of RFX5.","evidence":"ChIP and luciferase reporter assay with RFX5 overexpression/knockdown in HepG2 cells","pmids":["27840983"],"confidence":"Medium","gaps":["Physiological significance of TPP1 regulation by RFX5 was not established","Whether the RFX trimer or RFX5 alone activates TPP1 was not tested"]},{"year":2020,"claim":"RFX5 transactivation of KDM4A linked the RFX5 transcription factor to epigenetic regulation and cell cycle control via p53 in hepatocellular carcinoma.","evidence":"ChIP-PCR, luciferase reporter, RFX5 deletion/overexpression, cell cycle analysis","pmids":["32883983"],"confidence":"Medium","gaps":["Whether RFX5 binds the KDM4A promoter as part of the full trimer was not assessed","Generality beyond hepatocellular carcinoma cell lines was not tested"]},{"year":2022,"claim":"RFX5 induction by CCL18 in synovial macrophages activates GLUD1 to reprogram glutamate metabolism and simultaneously upregulates HLA-DR, linking RFX5 to metabolic adaptation in inflammatory tissue.","evidence":"RFX5 knockdown/overexpression in primary macrophages, ChIP, glutamate utilization assays, flow cytometry for HLA-DR and T cell activation","pmids":["35739396"],"confidence":"High","gaps":["Whether GLUD1 activation requires the RFX trimer or RFX5 alone was not determined","Upstream signaling from CCL18 to RFX5 induction was not fully mapped"]},{"year":2025,"claim":"Cryo-EM structure of the RFX5 extended DNA binding domain on a nucleosome revealed that RFX5 directly contacts histones and distorts nucleosomal DNA to increase accessibility, establishing RFX5 as a factor capable of chromatin remodeling without ATP hydrolysis.","evidence":"Cryo-EM structure determination of RFX5 eDBD–nucleosome complex","pmids":["40744500"],"confidence":"High","gaps":["Whether nucleosome destabilization occurs in the context of the full RFX trimer is unknown","Functional consequences for transcription at endogenous loci were not demonstrated"]},{"year":2025,"claim":"RFX5 transcriptional activation of JAG1 and consequent Notch signaling in breast cancer expanded its oncogenic target repertoire.","evidence":"ChIP, luciferase reporter, epistasis rescue with JAG1 knockdown in triple-negative breast cancer cells","pmids":["40220043"],"confidence":"Medium","gaps":["Whether the RFX trimer is involved was not tested","In vivo tumor relevance was not demonstrated"]},{"year":null,"claim":"A complete structural model of the full RFX trimer bound to an MHC-II promoter in chromatin context, the precise mechanism by which RFX5 switches between transcriptional activation and repression, and the physiological relevance of RFX5 nucleosome destabilization at endogenous loci remain to be determined.","evidence":"","pmids":[],"confidence":"High","gaps":["No full-length trimer-on-DNA structure exists","Activation-vs-repression switch mechanism is undefined","In vivo nucleosome remodeling activity not validated at endogenous promoters"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0003677","term_label":"DNA binding","supporting_discovery_ids":[7,9,16]},{"term_id":"GO:0140110","term_label":"transcription regulator activity","supporting_discovery_ids":[0,1,2,6,13,14,15,18]},{"term_id":"GO:0042393","term_label":"histone binding","supporting_discovery_ids":[16]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[6,10,11]},{"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":[0,1,2,5,11,13]},{"term_id":"R-HSA-74160","term_label":"Gene expression (Transcription)","supporting_discovery_ids":[0,2,6,14,15,18]},{"term_id":"R-HSA-4839726","term_label":"Chromatin organization","supporting_discovery_ids":[16]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[18]}],"complexes":["RFX complex (RFX5/RFXANK/RFXAP)","MHC-II enhanceosome (RFX/NF-Y/CREB/CIITA)"],"partners":["RFXANK","RFXAP","CIITA","CREB","SIRT1","PRMT6"],"other_free_text":[]},"mechanistic_narrative":"RFX5 is the DNA-binding subunit of the trimeric RFX complex (with RFXANK and RFXAP) that serves as a master organizer of MHC class II gene transcription and regulates diverse non-immune target genes in a context-dependent manner. RFX5 homodimerizes through an N-terminal leucine-rich coiled-coil that also recruits RFXAP, and its DNA binding domain is autoinhibited until relieved by assembly with both RFXANK and RFXAP; the assembled trimer binds X-box motifs in MHC-II promoters, cooperates with NF-Y at the Y box via the RFX5 C-terminal domain, and recruits the coactivator CIITA to drive transcription [PMID:12101253, PMID:10779326, PMID:18723135, PMID:9177217]. RFX5 stability and nuclear localization are regulated by SIRT1-mediated deacetylation and PRMT6-mediated arginine methylation of its AT-hook motif, the latter conferring MHC-II isotype-specific control [PMID:23079621, PMID:23911394]. Beyond MHC-II, RFX5 represses COL1A2 transcription, activates GLUD1 to enable glutamate-dependent metabolism in macrophages, transactivates KDM4A and JAG1 promoters in cancer cells, and its extended DNA binding domain directly engages and destabilizes nucleosomes to increase chromatin accessibility [PMID:12968017, PMID:35739396, PMID:32883983, PMID:40220043, PMID:40744500]."},"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":152,"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":90,"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":68,"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":63,"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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biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/12968017","citation_count":33,"is_preprint":false},{"pmid":"31096145","id":"PMC_31096145","title":"MiR-4319 hinders YAP expression to restrain non-small cell lung cancer growth through regulation of LIN28-mediated RFX5 stability.","date":"2019","source":"Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie","url":"https://pubmed.ncbi.nlm.nih.gov/31096145","citation_count":29,"is_preprint":false},{"pmid":"27840983","id":"PMC_27840983","title":"The transcription factor RFX5 is a transcriptional activator of the TPP1 gene in hepatocellular carcinoma.","date":"2016","source":"Oncology reports","url":"https://pubmed.ncbi.nlm.nih.gov/27840983","citation_count":29,"is_preprint":false},{"pmid":"32883983","id":"PMC_32883983","title":"RFX5 promotes the progression of hepatocellular carcinoma through transcriptional activation of KDM4A.","date":"2020","source":"Scientific 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cotransfection 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 — reciprocal interaction assays (Y2H + far-Western) plus functional reporter rescue, replicated context\",\n      \"pmids\": [\"9177217\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1995,\n      \"finding\": \"RFX5 and CIITA are both essential regulators of HLA-DMA and HLA-DMB gene expression, controlling both constitutive and IFN-γ-inducible expression of DM genes in addition to classical MHC class II genes.\",\n      \"method\": \"Complementation of regulatory mutants by cDNA transfection; genetic epistasis in cell lines deficient for CIITA or RFX5\",\n      \"journal\": \"International immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — clean genetic complementation with defined molecular readout, foundational paper\",\n      \"pmids\": [\"7495736\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"The N-terminal region of RFX5 is required for association with RFXANK and RFXAP and for assembly of the trimeric RFX complex; a separate C-terminal domain of RFX5 mediates cooperative binding between the RFX complex and NF-Y at the Y box of MHC-II promoters, and this cooperative binding is essential for transcriptional activation.\",\n      \"method\": \"Domain deletion/mutagenesis, in vitro and in vivo complex assembly assays, electrophoretic mobility shift assays (EMSA), reporter assays\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — mutagenesis combined with in vitro binding assays and functional reporter assays in multiple cell lines\",\n      \"pmids\": [\"10779326\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"Chlamydia infection degrades RFX5 via a lactacystin-sensitive, chlamydia-dependent proteasome-like activity present in the cytosolic fraction of infected cells, leading to suppression of both constitutive and IFN-γ-inducible MHC class I expression.\",\n      \"method\": \"Immunoblot for RFX5 degradation, lactacystin inhibition, cell fractionation, chlamydial protein synthesis inhibition\",\n      \"journal\": \"The Journal of experimental medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods (pharmacological inhibition, fractionation, protein synthesis block) in a single study\",\n      \"pmids\": [\"10790427\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"RFX5 forms homodimers in vivo and in vitro through a leucine-rich stretch (residues 62-68) N-terminal to its DNA binding domain; leucine at position 66 is critical for self-association, and dimerization-deficient RFX5 mutants fail to support higher-order DNA-protein complex formation on MHC-II conserved upstream sequences or MHC-II transcription in vivo.\",\n      \"method\": \"Yeast two-hybrid, in vitro binding, site-directed mutagenesis, EMSA, reporter assays in cells\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — mutagenesis combined with in vitro and in vivo functional validation\",\n      \"pmids\": [\"12101253\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"RFX5 knockout mice lack MHC-II expression in thymic cortex, resting B cells, and macrophages, causing failure of positive selection of CD4+ T cells; however, some residual MHC-II expression persists in mature dendritic cells and activated B cells, demonstrating a cell-type-specific requirement for RFX5.\",\n      \"method\": \"Conditional gene knockout (RFX5-/- mice), flow cytometry, immunohistochemistry, T cell selection assays\",\n      \"journal\": \"Immunity\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — clean knockout mouse with defined cellular and immunological phenotypes\",\n      \"pmids\": [\"9491996\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"The intact RFX5 trimeric complex (RFX5, RFXB/RFXANK, RFXAP) is required for maximum repression of collagen COL1A2 transcription; IFN-γ increases nuclear translocation of all three RFX complex subunits and their occupancy at the collagen transcription start site, while dominant-negative RFX5 mutants reverse IFN-γ-mediated collagen repression.\",\n      \"method\": \"Reporter assays with dominant-negative mutants, chromatin immunoprecipitation (ChIP), immunofluorescence for nuclear translocation, overexpression in human lung fibroblasts\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — ChIP plus dominant-negative rescue and reporter assays, multiple orthogonal methods\",\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 overcome this autoinhibition, and a single RFX trimeric complex binds the proximal regulatory region of the MHC-II promoter.\",\n      \"method\": \"Electrophoretic mobility shift assays (EMSA) with purified recombinant proteins and mutant constructs\",\n      \"journal\": \"Biochimica et biophysica acta\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 — in vitro reconstitution with purified proteins, single lab\",\n      \"pmids\": [\"18723135\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"The NMR solution structure of the RFX5(N)2-RFXAP(C) heterotrimeric complex reveals that two RFX5 N-terminal domains form an antiparallel coiled-coil 'staple' structure, with RFXAP(C) adopting a V-shaped helical structure that packs within the RFX5 dimer; leucine residues in the leucine-rich region (62-LYLYLQL-68) contribute to both the RFX5 dimer interface and the RFX5-RFXAP interface.\",\n      \"method\": \"Solution NMR (15N- and 13C-edited), structural determination of heterotrimeric complex\",\n      \"journal\": \"Journal of molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — atomic-resolution structure with residue-level functional mapping, validated against prior mutagenesis data\",\n      \"pmids\": [\"20732328\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"The DNA binding domain (DBD) of RFX5 directly interacts with X-box DNA in an entropy-driven, enthalpy-favorable manner, and directly binds RFXANK in vitro (Kd ~128 nM) even in the absence of RFXAP, as measured by fluorescence and FRET methods.\",\n      \"method\": \"Steady-state fluorescence quenching, circular dichroism, FRET, chemical cross-linking with tandem mass spectrometry\",\n      \"journal\": \"Biochimica et biophysica acta\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1-2 — multiple biophysical methods in vitro, single lab\",\n      \"pmids\": [\"20637319\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"SIRT1 forms a complex with RFX5, deacetylates it, promotes its nuclear expulsion and proteasomal degradation, thereby antagonizing RFX5-mediated repression of COL1A2 transcription in smooth muscle cells; IFN-γ represses COL1A2 by downregulating SIRT1, increasing RFX5 acetylation.\",\n      \"method\": \"Co-immunoprecipitation, overexpression/knockdown of SIRT1, NAMPT, resveratrol/inhibitor treatment, reporter assays, nuclear/cytoplasmic fractionation\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — co-IP plus pharmacological and genetic manipulation, single lab\",\n      \"pmids\": [\"23079621\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"An AT-hook motif in RFX5 is involved in regulating transcription of HLA-DQ but not HLA-DR MHC-II genes; PRMT6, an arginine methyltransferase, methylates this AT-hook motif and selectively downregulates HLA-DQ expression in an AT-hook-dependent manner, providing isotype-specific regulation of MHC-II.\",\n      \"method\": \"Site-directed mutagenesis of AT-hook motif, PRMT6 overexpression/knockdown, reporter assays, ChIP\",\n      \"journal\": \"Molecular immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — mutagenesis combined with functional reporter assays and ChIP, single lab\",\n      \"pmids\": [\"23911394\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"CREB and phospho-CREB interact directly with RFX5 (through the C-terminal portion of CREB) and with CIITA to form part of the MHC-II transcriptional regulatory complex; phosphorylation of CREB enhances transcription from MHC-II promoters and phospho-CREB is found at the HLA-DRA promoter by ChIP.\",\n      \"method\": \"Co-immunoprecipitation with CREB mutants, reporter assays, chromatin immunoprecipitation (ChIP)\",\n      \"journal\": \"Molecular immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal co-IP with multiple mutants plus ChIP, single lab\",\n      \"pmids\": [\"16730065\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"CCL18 induces RFX5 expression in synovial macrophages; RFX5 selectively upregulates GLUD1 transcription to enable glutamate utilization for energy under glucose withdrawal, and simultaneously enhances surface HLA-DR expression to promote antigen-specific T cell expansion.\",\n      \"method\": \"RFX5 knockdown/overexpression in primary macrophages, ChIP, metabolic assays (glutamate utilization), flow cytometry for HLA-DR and T cell activation\",\n      \"journal\": \"Nature metabolism\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods (ChIP, metabolic assays, flow cytometry) with genetic loss-of-function in primary cells\",\n      \"pmids\": [\"35739396\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"RFX5 binds directly to the TPP1 promoter region and transcriptionally activates TPP1 expression in hepatocellular carcinoma cells, as demonstrated by ChIP and reporter assays.\",\n      \"method\": \"ChIP, luciferase reporter assay, RFX5 overexpression/knockdown in HepG2 cells\",\n      \"journal\": \"Oncology reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — ChIP plus reporter assay, single lab\",\n      \"pmids\": [\"27840983\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"RFX5 directly binds the KDM4A promoter and transcriptionally activates KDM4A expression in hepatocellular carcinoma; the RFX5-KDM4A pathway promotes cell cycle progression from G0/G1 to S phase and inhibits apoptosis through regulation of p53 and downstream genes.\",\n      \"method\": \"ChIP-seq (ENCODE), ChIP-PCR, luciferase reporter assay, RFX5 deletion/overexpression, cell cycle analysis\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — ChIP-PCR plus luciferase reporter and functional rescue, single lab\",\n      \"pmids\": [\"32883983\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Cryo-EM structure of the RFX5 extended DNA binding domain (eDBD) bound to a nucleosome reveals that eDBD engages nucleosomal DNA at superhelical location +2 and contacts histones; RFX5 eDBD induces localized distortion of the bound DNA gyre and detachment of the adjacent DNA gyre, increasing DNA accessibility.\",\n      \"method\": \"Cryo-EM structure determination of RFX5 eDBD-nucleosome complex\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — atomic-resolution cryo-EM structure with mechanistic characterization of nucleosome distortion\",\n      \"pmids\": [\"40744500\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"RFX5 discriminates between strand-symmetric and strand-asymmetric 5-hydroxymethylcytosine (hmC) modifications in CpG dyads, identified as a reader of specific hmC symmetries in the nuclear proteome.\",\n      \"method\": \"Comparative enrichment proteomics with promoter probes bearing symmetric or asymmetric hmC modifications in human and mouse nuclear lysates\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — single pulldown/enrichment proteomics method, preprint, no functional validation of hmC reading\",\n      \"pmids\": [\"bio_10.1101_2025.06.27.661915\"],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"RFX5 transcriptionally activates JAG1 by binding to its promoter (−1890/+15 or −1359/+15 region), thereby activating Notch signaling (Notch1, NICD, Hes1) in triple-negative breast cancer cells.\",\n      \"method\": \"Chromatin immunoprecipitation, luciferase reporter assay, RFX5 knockdown/overexpression, JAG1 knockdown rescue\",\n      \"journal\": \"Human cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — ChIP plus luciferase reporter and epistasis rescue, single lab\",\n      \"pmids\": [\"40220043\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"RFX5 is the largest subunit of the trimeric RFX transcription factor complex (with RFXANK and RFXAP), which binds X-box sequences in MHC class II promoters through an autoinhibited DNA binding domain that is activated upon complex assembly; RFX5 homodimerizes via a leucine-rich N-terminal coiled-coil that also recruits RFXAP (as resolved by NMR and cryo-EM), its C-terminal domain mediates cooperative binding with NF-Y to activate MHC-II transcription, it directly interacts with CIITA (which provides the transcriptional activation function), and it is subject to post-translational regulation by SIRT1-mediated deacetylation and PRMT6-mediated arginine methylation that control its stability, localization, and target-gene selectivity; beyond MHC-II regulation, RFX5 represses collagen (COL1A2) and activates non-immune targets including GLUD1, TPP1, KDM4A, and JAG1 in a context-dependent manner, and its extended DNA binding domain can directly bind and destabilize nucleosomes to increase chromatin accessibility.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"RFX5 is the DNA-binding subunit of the trimeric RFX complex (with RFXANK and RFXAP) that serves as a master organizer of MHC class II gene transcription and regulates diverse non-immune target genes in a context-dependent manner. RFX5 homodimerizes through an N-terminal leucine-rich coiled-coil that also recruits RFXAP, and its DNA binding domain is autoinhibited until relieved by assembly with both RFXANK and RFXAP; the assembled trimer binds X-box motifs in MHC-II promoters, cooperates with NF-Y at the Y box via the RFX5 C-terminal domain, and recruits the coactivator CIITA to drive transcription [PMID:12101253, PMID:10779326, PMID:18723135, PMID:9177217]. RFX5 stability and nuclear localization are regulated by SIRT1-mediated deacetylation and PRMT6-mediated arginine methylation of its AT-hook motif, the latter conferring MHC-II isotype-specific control [PMID:23079621, PMID:23911394]. Beyond MHC-II, RFX5 represses COL1A2 transcription, activates GLUD1 to enable glutamate-dependent metabolism in macrophages, transactivates KDM4A and JAG1 promoters in cancer cells, and its extended DNA binding domain directly engages and destabilizes nucleosomes to increase chromatin accessibility [PMID:12968017, PMID:35739396, PMID:32883983, PMID:40220043, PMID:40744500].\",\n  \"teleology\": [\n    {\n      \"year\": 1995,\n      \"claim\": \"Establishing RFX5 as an essential regulator of MHC class II and non-classical HLA-DM genes resolved a key missing link in how MHC-II expression is controlled in human cells.\",\n      \"evidence\": \"Genetic complementation of RFX5-deficient regulatory mutant cell lines restored HLA-DMA/DMB expression\",\n      \"pmids\": [\"7495736\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"The molecular mechanism by which RFX5 acts on these promoters was unknown\", \"Relationship to CIITA was undefined\"]\n    },\n    {\n      \"year\": 1997,\n      \"claim\": \"Demonstrating that RFX5 directly interacts with CIITA established a division of labor: RFX5 provides promoter specificity via DNA binding while CIITA supplies the transcriptional activation domain.\",\n      \"evidence\": \"Yeast two-hybrid, far-Western blot, and GAL4 fusion reporter assays in CIITA-deficient cells\",\n      \"pmids\": [\"9177217\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How CIITA is recruited to the assembled RFX complex on chromatin was not resolved\", \"Whether additional cofactors participate was unknown\"]\n    },\n    {\n      \"year\": 1998,\n      \"claim\": \"RFX5 knockout mice revealed that RFX5 is required for MHC-II expression in most antigen-presenting cells but dispensable in activated dendritic cells and B cells, establishing cell-type specificity of the requirement.\",\n      \"evidence\": \"RFX5−/− mice analyzed by flow cytometry, immunohistochemistry, and T cell selection assays\",\n      \"pmids\": [\"9491996\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Identity of the compensatory factor(s) enabling residual MHC-II in dendritic cells was not determined\", \"Impact on non-MHC-II target genes in vivo was unexplored\"]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"Domain mapping of RFX5 separated its N-terminal complex-assembly function from a C-terminal domain that mediates cooperative NF-Y binding at the Y box, defining the architecture underlying enhanceosome formation on MHC-II promoters.\",\n      \"evidence\": \"Deletion/point mutagenesis with EMSA, co-assembly assays, and reporter assays\",\n      \"pmids\": [\"10779326\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of the C-terminal domain–NF-Y interaction was not resolved\", \"Whether Y-box cooperation occurs at non-MHC-II promoters was unknown\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Showing that RFX5 homodimerizes via a leucine-rich N-terminal stretch and that dimerization is essential for higher-order DNA–protein complex formation explained how a single gene product nucleates the multisubunit enhanceosome.\",\n      \"evidence\": \"Yeast two-hybrid, in vitro binding, Leu66 point mutagenesis, EMSA, and reporter assays\",\n      \"pmids\": [\"12101253\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether dimerization is constitutive or regulated was not determined\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Discovery that the intact RFX trimer represses COL1A2 transcription under IFN-γ stimulation expanded RFX5 function beyond immune gene activation to transcriptional repression of extracellular matrix genes.\",\n      \"evidence\": \"ChIP at collagen promoter, dominant-negative mutants, immunofluorescence for nuclear translocation in lung fibroblasts\",\n      \"pmids\": [\"12968017\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism by which RFX5 switches from activation to repression was not defined\", \"Whether CIITA participates in collagen repression was not tested\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Identification of phospho-CREB as a direct interactor of both RFX5 and CIITA at MHC-II promoters added a signal-responsive component to the enhanceosome model.\",\n      \"evidence\": \"Co-immunoprecipitation with CREB mutants, ChIP at HLA-DRA promoter, reporter assays\",\n      \"pmids\": [\"16730065\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether CREB interaction is required for all MHC-II loci or only a subset was not tested\", \"Stoichiometry of CREB within the enhanceosome was unknown\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Showing that RFX5 DNA binding is autoinhibited and requires both RFXAP and RFXANK for relief explained why isolated RFX5 cannot bind X-box DNA efficiently and why all three subunits are genetically required.\",\n      \"evidence\": \"EMSA with purified recombinant RFX5 and truncation mutants reconstituted with RFXAP/RFXANK\",\n      \"pmids\": [\"18723135\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Structural mechanism of autoinhibition relief was not determined at atomic resolution\", \"In vivo validation of autoinhibition model was lacking\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"NMR structure of the RFX5(N)₂–RFXAP(C) complex and biophysical characterization of the DBD–X-box interaction provided the first atomic-level view of how the trimer assembles and engages DNA.\",\n      \"evidence\": \"Solution NMR of the heterotrimeric complex; fluorescence quenching, FRET, and CD for DBD–DNA interaction\",\n      \"pmids\": [\"20732328\", \"20637319\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Full-length trimer structure on DNA was not available\", \"How autoinhibition is relieved structurally remained unresolved\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Discovery that SIRT1 deacetylates RFX5 and promotes its nuclear expulsion and proteasomal degradation established the first post-translational regulatory axis controlling RFX5 activity, linking NAD⁺ metabolism to MHC-II and collagen regulation.\",\n      \"evidence\": \"Co-immunoprecipitation, SIRT1 overexpression/knockdown, pharmacological modulation, nuclear/cytoplasmic fractionation in smooth muscle cells\",\n      \"pmids\": [\"23079621\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Specific acetylated residues on RFX5 were not mapped\", \"Whether SIRT1 regulation affects MHC-II as well as collagen targets was not tested\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"PRMT6-mediated arginine methylation of the RFX5 AT-hook motif selectively downregulates HLA-DQ but not HLA-DR, revealing an isotype-specific epigenetic control mechanism within the MHC-II locus.\",\n      \"evidence\": \"AT-hook mutagenesis, PRMT6 overexpression/knockdown, ChIP and reporter assays\",\n      \"pmids\": [\"23911394\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether methylation disrupts DNA binding or protein–protein interaction at the AT-hook was not distinguished\", \"In vivo relevance in primary antigen-presenting cells was not shown\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Identification of TPP1 as a direct transcriptional target of RFX5 in hepatocellular carcinoma cells extended the non-immune target repertoire of RFX5.\",\n      \"evidence\": \"ChIP and luciferase reporter assay with RFX5 overexpression/knockdown in HepG2 cells\",\n      \"pmids\": [\"27840983\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Physiological significance of TPP1 regulation by RFX5 was not established\", \"Whether the RFX trimer or RFX5 alone activates TPP1 was not tested\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"RFX5 transactivation of KDM4A linked the RFX5 transcription factor to epigenetic regulation and cell cycle control via p53 in hepatocellular carcinoma.\",\n      \"evidence\": \"ChIP-PCR, luciferase reporter, RFX5 deletion/overexpression, cell cycle analysis\",\n      \"pmids\": [\"32883983\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether RFX5 binds the KDM4A promoter as part of the full trimer was not assessed\", \"Generality beyond hepatocellular carcinoma cell lines was not tested\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"RFX5 induction by CCL18 in synovial macrophages activates GLUD1 to reprogram glutamate metabolism and simultaneously upregulates HLA-DR, linking RFX5 to metabolic adaptation in inflammatory tissue.\",\n      \"evidence\": \"RFX5 knockdown/overexpression in primary macrophages, ChIP, glutamate utilization assays, flow cytometry for HLA-DR and T cell activation\",\n      \"pmids\": [\"35739396\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether GLUD1 activation requires the RFX trimer or RFX5 alone was not determined\", \"Upstream signaling from CCL18 to RFX5 induction was not fully mapped\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Cryo-EM structure of the RFX5 extended DNA binding domain on a nucleosome revealed that RFX5 directly contacts histones and distorts nucleosomal DNA to increase accessibility, establishing RFX5 as a factor capable of chromatin remodeling without ATP hydrolysis.\",\n      \"evidence\": \"Cryo-EM structure determination of RFX5 eDBD–nucleosome complex\",\n      \"pmids\": [\"40744500\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether nucleosome destabilization occurs in the context of the full RFX trimer is unknown\", \"Functional consequences for transcription at endogenous loci were not demonstrated\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"RFX5 transcriptional activation of JAG1 and consequent Notch signaling in breast cancer expanded its oncogenic target repertoire.\",\n      \"evidence\": \"ChIP, luciferase reporter, epistasis rescue with JAG1 knockdown in triple-negative breast cancer cells\",\n      \"pmids\": [\"40220043\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether the RFX trimer is involved was not tested\", \"In vivo tumor relevance was not demonstrated\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"A complete structural model of the full RFX trimer bound to an MHC-II promoter in chromatin context, the precise mechanism by which RFX5 switches between transcriptional activation and repression, and the physiological relevance of RFX5 nucleosome destabilization at endogenous loci remain to be determined.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No full-length trimer-on-DNA structure exists\", \"Activation-vs-repression switch mechanism is undefined\", \"In vivo nucleosome remodeling activity not validated at endogenous promoters\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0003677\", \"supporting_discovery_ids\": [7, 9, 16]},\n      {\"term_id\": \"GO:0140110\", \"supporting_discovery_ids\": [0, 1, 2, 6, 13, 14, 15, 18]},\n      {\"term_id\": \"GO:0042393\", \"supporting_discovery_ids\": [16]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [6, 10, 11]},\n      {\"term_id\": \"GO:0000228\", \"supporting_discovery_ids\": [16]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [0, 1, 2, 5, 11, 13]},\n      {\"term_id\": \"R-HSA-74160\", \"supporting_discovery_ids\": [0, 2, 6, 14, 15, 18]},\n      {\"term_id\": \"R-HSA-4839726\", \"supporting_discovery_ids\": [16]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [18]}\n    ],\n    \"complexes\": [\n      \"RFX complex (RFX5/RFXANK/RFXAP)\",\n      \"MHC-II enhanceosome (RFX/NF-Y/CREB/CIITA)\"\n    ],\n    \"partners\": [\n      \"RFXANK\",\n      \"RFXAP\",\n      \"CIITA\",\n      \"CREB\",\n      \"SIRT1\",\n      \"PRMT6\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}