Affinage

RFX5

DNA-binding protein RFX5 · UniProt P48382

Length
616 aa
Mass
65.3 kDa
Annotated
2026-06-10
28 papers in source corpus 19 papers cited in narrative 20 extracted findings
Cross-family judge vs UniProt: Affinage preferred faithfulness: 6/6 claims corpus-supported (100%)

Mechanistic narrative

Synthesis pass · prose summary of the discoveries below

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).

Mechanistic history

Synthesis pass · year-by-year structured walk · 14 steps
  1. 1995 Medium

    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

    PMID:7495736

    Open questions at the time
    • Did not define the molecular interaction or DNA-binding mechanism
    • Single lab, restricted to DM isotype genes
  2. 1997 High

    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

    PMID:9177217

    Open questions at the time
    • Did not map the structural basis of the RFX5-CIITA interface
    • Did not address contribution of other complex subunits
  3. 1998 High

    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

    PMID:9491996

    Open questions at the time
    • Did not explain the residual MHC-II expression in medulla/dendritic cells mechanistically
    • Did not test non-immune RFX5 functions
  4. 2002 High

    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

    PMID:12101253

    Open questions at the time
    • Atomic structure of the dimer not yet resolved
    • Did not address how dimerization couples to RFXAP/RFXANK binding
  5. 2000 High

    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

    PMID:10779326

    Open questions at the time
    • Did not resolve atomic structure of the RFX5-NF-Y contact
    • Did not quantify cooperativity thermodynamically
  6. 2008 Medium

    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

    PMID:18723135

    Open questions at the time
    • Structural basis of autoinhibition not defined
    • In vitro only, single lab
  7. 2010 High

    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

    PMID:20637319 PMID:20732328

    Open questions at the time
    • No structure of the full trimer on DNA
    • DNA binding domain structure with DNA not resolved at this stage
  8. 2003 High

    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

    PMID:12968017

    Open questions at the time
    • Mechanism of repression versus activation at distinct promoters not resolved
    • Corepressor recruitment not identified
  9. 2012 Medium

    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

    PMID:23079621

    Open questions at the time
    • Acetyltransferase that adds the mark not identified
    • Specific lysine residues not mapped
    • Single lab
  10. 2013 Medium

    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

    PMID:23911394

    Open questions at the time
    • Methylated residue not directly mapped at structural level
    • Mechanism of DQ-versus-DR selectivity unclear
  11. 2006 Medium

    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

    PMID:16730065

    Open questions at the time
    • CREB binding region on RFX5 not mapped
    • Functional importance relative to CIITA/NF-Y unquantified
  12. 2020 Medium

    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

    PMID:27840983 PMID:32883983

    Open questions at the time
    • Whether RFX5 acts as the trimeric complex or independently at these promoters not resolved
    • Single cancer lineage tested per study
  13. 2022 Medium

    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

    PMID:35739396

    Open questions at the time
    • Direct RFX5 binding at the GLUD1 locus not structurally validated
    • In vivo relevance beyond primary cells untested
  14. 2025 High

    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

    PMID:40220043 PMID:40744500

    Open questions at the time
    • Cryo-EM used isolated eDBD, not the full trimer
    • Pioneer-factor-like chromatin opening not tested genome-wide

Open questions

Synthesis pass · forward-looking unresolved questions
  • 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.
  • 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

Synthesis pass · controlled-vocabulary classification · explore literature graph →
Molecular activity
GO:0003677 DNA binding 5 GO:0140110 transcription regulator activity 4 GO:0042393 histone binding 1
Localization
GO:0005634 nucleus 2 GO:0000228 nuclear chromosome 1
Pathway
R-HSA-1643685 Disease 3 R-HSA-168256 Immune System 3 R-HSA-74160 Gene expression (Transcription) 3
Partners
CIITACREBIER3NF-YPRMT6RFXANKRFXAPSIRT1
Complex memberships
RFX complex (RFX5/RFXANK/RFXAP)

Evidence

Reading pass · 20 per-paper findings extracted from the source corpus
Year Finding Method Journal Conf PMIDs
1997 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. Yeast two-hybrid, far-Western blot, cotransfection reporter assay in CIITA-deficient cells Proceedings of the National Academy of Sciences of the United States of America High 9177217
1995 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. Complementation assay using cDNA transfection into MHC class II regulatory mutant cells International immunology Medium 7495736
2000 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. Domain deletion/mutagenesis, in vitro and in vivo RFX complex assembly assays, MHC-II promoter reporter assays Molecular and cellular biology High 10779326
2000 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. Immunoblot of RFX5 protein levels in infected cells, pharmacological inhibition with lactacystin, cell fractionation, inhibition of chlamydial vs. host protein synthesis The Journal of experimental medicine Medium 10790427
2002 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. In vivo and in vitro dimerization assays, site-directed mutagenesis (L66), electrophoretic mobility shift assay (EMSA), MHC-II transcription assay Molecular and cellular biology High 12101253
2003 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. Promoter reporter assay, dominant-negative overexpression, IFN-gamma treatment, chromatin immunoprecipitation (ChIP), immunofluorescence for nuclear translocation The Journal of biological chemistry High 12968017
2008 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. Electrophoretic mobility shift assay (EMSA) with recombinant proteins and MHC-II promoter DNA fragments Biochimica et biophysica acta Medium 18723135
2010 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. Solution NMR structure determination (15N- and 13C-edited), in vitro binding assays Journal of molecular biology High 20732328
2010 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. Steady-state fluorescence quenching, circular dichroism, FRET, chemical cross-linking followed by tandem mass spectrometry Biochimica et biophysica acta Medium 20637319
2012 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. Co-immunoprecipitation, overexpression and knockdown of SIRT1/NAMPT, SIRT1 agonist/inhibitor treatment, acetylation assays, subcellular fractionation, promoter reporter assay, ChIP Biochemical and biophysical research communications Medium 23079621
2013 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. AT-hook motif mutagenesis, PRMT6 overexpression/knockdown, MHC-II isotype-specific expression assays Molecular immunology Medium 23911394
2006 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. Co-immunoprecipitation, MHC-II promoter reporter assay, chromatin immunoprecipitation (ChIP) Molecular immunology Medium 16730065
2016 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. ChIP-PCR, luciferase reporter assay, RFX5 overexpression/knockdown with qRT-PCR Oncology reports Medium 27840983
2020 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. ChIP-PCR, luciferase reporter assay, ChIP-seq (ENCODE), RFX5 knockout/rescue with KDM4A overexpression, cell cycle and apoptosis assays Scientific reports Medium 32883983
2022 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. RFX5 knockdown/overexpression, CCL18 stimulation, GLUD1 expression analysis, HLA-DR surface expression, metabolic assays in primary synovial macrophages Nature metabolism Medium 35739396
1998 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. RFX5 knockout mouse (conditional gene targeting), flow cytometry for MHC-II expression, lymphocyte subset analysis Immunity High 9491996
2025 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. Cryo-EM structure determination of RFX5 eDBD-nucleosome complex Nucleic acids research High 40744500
2025 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. Chromatin immunoprecipitation, luciferase reporter assay, JAG1 knockdown rescue experiment, RFX5 overexpression/knockdown Human cell Medium 40220043
2026 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. Co-immunoprecipitation, dual-luciferase reporter assay, siRNA knockdown, qPCR and western blot Discover oncology Low 42209919
2024 RFX5 exhibits methylation-sensitive DNA binding: it discriminates between specific CpG hydroxymethylation (hmC) symmetries in CpG dyads, as identified by meSMiLE-seq assay. meSMiLE-seq (microfluidic selective ligand enrichment followed by sequencing with methylated vs unmethylated DNA probes) bioRxivpreprint Low

Source papers

Stage 0 corpus · 28 papers · ranked by NIH iCite citations
Year Title Journal Citations PMID
2000 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. The Journal of experimental medicine 153 10790427
1997 Specific complex formation between the type II bare lymphocyte syndrome-associated transactivators CIITA and RFX5. Proceedings of the National Academy of Sciences of the United States of America 111 9177217
1995 The two novel MHC class II transactivators RFX5 and CIITA both control expression of HLA-DM genes. International immunology 91 7495736
2000 A functionally essential domain of RFX5 mediates activation of major histocompatibility complex class II promoters by promoting cooperative binding between RFX and NF-Y. Molecular and cellular biology 70 10779326
2022 The transcription factor RFX5 coordinates antigen-presenting function and resistance to nutrient stress in synovial macrophages. Nature metabolism 65 35739396
1998 Residual MHC class II expression on mature dendritic cells and activated B cells in RFX5-deficient mice. Immunity 61 9491996
2002 Major histocompatibility complex class II transcriptional platform: assembly of nuclear factor Y and regulatory factor X (RFX) on DNA requires RFX5 dimers. Molecular and cellular biology 50 12101253
2003 Interferon gamma repression of collagen (COL1A2) transcription is mediated by the RFX5 complex. The Journal of biological chemistry 35 12968017
2012 SIRT1 deacetylates RFX5 and antagonizes repression of collagen type I (COL1A2) transcription in smooth muscle cells. Biochemical and biophysical research communications 34 23079621
2020 RFX5 promotes the progression of hepatocellular carcinoma through transcriptional activation of KDM4A. Scientific reports 30 32883983
2019 MiR-4319 hinders YAP expression to restrain non-small cell lung cancer growth through regulation of LIN28-mediated RFX5 stability. Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie 30 31096145
2016 The transcription factor RFX5 is a transcriptional activator of the TPP1 gene in hepatocellular carcinoma. Oncology reports 30 27840983
1997 Analysis of mutations and chromosomal localisation of the gene encoding RFX5, a novel transcription factor affected in major histocompatibility complex class II deficiency. Human mutation 27 9401005
2006 CREB and phospho-CREB interact with RFX5 and CIITA to regulate MHC class II genes. Molecular immunology 21 16730065
2008 Assembly of the RFX complex on the MHCII promoter: role of RFXAP and RFXB in relieving autoinhibition of RFX5. Biochimica et biophysica acta 20 18723135
1999 Molecular analysis of an MHC class II deficiency patient reveals a novel mutation in the RFX5 gene. Immunogenetics 19 10079298
1999 Discoordinate expression of invariant chain and MHC class II genes in class II transactivator-transfected fibroblasts defective for RFX5. Journal of immunology (Baltimore, Md. : 1950) 19 10395672
2013 Differential regulation of MHCII genes by PRMT6, via an AT-hook motif of RFX5. Molecular immunology 10 23911394
2010 Solution structure of the heterotrimeric complex between the interaction domains of RFX5 and RFXAP from the RFX gene regulatory complex. Journal of molecular biology 8 20732328
2010 DNA binding domain of RFX5: interactions with X-box DNA and RFXANK. Biochimica et biophysica acta 6 20637319
2022 Case Report: Novel splicing mutations in RFX5 causing MHC class II deficiency. Frontiers in genetics 5 36276949
2010 Structure and conformational studies on dityrosine formation in the DNA binding domain of RFX5. Biophysical chemistry 5 20457484
2025 A patient with RFX5 variant causing an expression defect in both HLA ABC and HLA DR. Immunologic research 3 40240550
2025 Structural basis of nucleosome binding and destabilization by the extended DNA binding domain of RFX5. Nucleic acids research 1 40744500
2022 The transcription factor RFX5 positively regulates expression of MHCIa in the red-spotted grouper (Epinephelus akaara). Fish & shellfish immunology 1 35051562
2026 IER3 as an RFX5 transcriptional co-activator promotes hepatocellular carcinoma progression via AKR1B10-mediated p53 transcriptional regulation. Discover oncology 0 42209919
2025 RFX5 promotes the progression of triple-negative breast cancer through transcriptional activation of JAG1. Human cell 0 40220043
2025 RFX5 promotes the growth, motility, and inhibits apoptosis of gastric adenocarcinoma cells through the SIRT1/AMPK axis. Open life sciences 0 41079601

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