Affinage

RFX5

DNA-binding protein RFX5 · UniProt P48382

Length
616 aa
Mass
65.3 kDa
Annotated
2026-04-28
27 papers in source corpus 19 papers cited in narrative 19 extracted findings

Mechanistic narrative

Synthesis pass · prose summary of the discoveries below

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).

Mechanistic history

Synthesis pass · year-by-year structured walk · 16 steps
  1. 1995 High

    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

    PMID:7495736

    Open questions at the time
    • The molecular mechanism by which RFX5 acts on these promoters was unknown
    • Relationship to CIITA was undefined
  2. 1997 High

    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

    PMID:9177217

    Open questions at the time
    • How CIITA is recruited to the assembled RFX complex on chromatin was not resolved
    • Whether additional cofactors participate was unknown
  3. 1998 High

    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

    PMID:9491996

    Open questions at the time
    • 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
  4. 2000 High

    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

    PMID:10779326

    Open questions at the time
    • 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
  5. 2002 High

    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

    PMID:12101253

    Open questions at the time
    • Whether dimerization is constitutive or regulated was not determined
  6. 2003 High

    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

    PMID:12968017

    Open questions at the time
    • Mechanism by which RFX5 switches from activation to repression was not defined
    • Whether CIITA participates in collagen repression was not tested
  7. 2006 Medium

    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

    PMID:16730065

    Open questions at the time
    • 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
  8. 2008 Medium

    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

    PMID:18723135

    Open questions at the time
    • Structural mechanism of autoinhibition relief was not determined at atomic resolution
    • In vivo validation of autoinhibition model was lacking
  9. 2010 High

    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

    PMID:20637319 PMID:20732328

    Open questions at the time
    • Full-length trimer structure on DNA was not available
    • How autoinhibition is relieved structurally remained unresolved
  10. 2012 Medium

    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

    PMID:23079621

    Open questions at the time
    • Specific acetylated residues on RFX5 were not mapped
    • Whether SIRT1 regulation affects MHC-II as well as collagen targets was not tested
  11. 2013 Medium

    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

    PMID:23911394

    Open questions at the time
    • 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
  12. 2016 Medium

    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

    PMID:27840983

    Open questions at the time
    • Physiological significance of TPP1 regulation by RFX5 was not established
    • Whether the RFX trimer or RFX5 alone activates TPP1 was not tested
  13. 2020 Medium

    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

    PMID:32883983

    Open questions at the time
    • Whether RFX5 binds the KDM4A promoter as part of the full trimer was not assessed
    • Generality beyond hepatocellular carcinoma cell lines was not tested
  14. 2022 High

    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

    PMID:35739396

    Open questions at the time
    • Whether GLUD1 activation requires the RFX trimer or RFX5 alone was not determined
    • Upstream signaling from CCL18 to RFX5 induction was not fully mapped
  15. 2025 High

    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

    PMID:40744500

    Open questions at the time
    • Whether nucleosome destabilization occurs in the context of the full RFX trimer is unknown
    • Functional consequences for transcription at endogenous loci were not demonstrated
  16. 2025 Medium

    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

    PMID:40220043

    Open questions at the time
    • Whether the RFX trimer is involved was not tested
    • In vivo tumor relevance was not demonstrated

Open questions

Synthesis pass · forward-looking unresolved questions
  • 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.
  • 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

Synthesis pass · controlled-vocabulary classification · explore literature graph →
Molecular activity
GO:0140110 transcription regulator activity 8 GO:0003677 DNA binding 3 GO:0042393 histone binding 1
Localization
GO:0005634 nucleus 3 GO:0000228 nuclear chromosome 1
Pathway
R-HSA-168256 Immune System 6 R-HSA-74160 Gene expression (Transcription) 6 R-HSA-162582 Signal Transduction 1 R-HSA-4839726 Chromatin organization 1
Partners
CIITACREBPRMT6RFXANKRFXAPSIRT1
Complex memberships
MHC-II enhanceosome (RFX/NF-Y/CREB/CIITA)RFX complex (RFX5/RFXANK/RFXAP)

Evidence

Reading pass · 19 per-paper findings extracted from the source corpus
Year Finding Method Journal Conf PMIDs
1997 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. Yeast two-hybrid, far-Western blot, GAL4 fusion reporter assays, cotransfection in CIITA-deficient cells Proceedings of the National Academy of Sciences of the United States of America High 9177217
1995 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. Complementation of regulatory mutants by cDNA transfection; genetic epistasis in cell lines deficient for CIITA or RFX5 International immunology High 7495736
2000 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. Domain deletion/mutagenesis, in vitro and in vivo complex assembly assays, electrophoretic mobility shift assays (EMSA), reporter assays Molecular and cellular biology High 10779326
2000 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. Immunoblot for RFX5 degradation, lactacystin inhibition, cell fractionation, chlamydial protein synthesis inhibition The Journal of experimental medicine High 10790427
2002 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. Yeast two-hybrid, in vitro binding, site-directed mutagenesis, EMSA, reporter assays in cells Molecular and cellular biology High 12101253
1998 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. Conditional gene knockout (RFX5-/- mice), flow cytometry, immunohistochemistry, T cell selection assays Immunity High 9491996
2003 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. Reporter assays with dominant-negative mutants, chromatin immunoprecipitation (ChIP), immunofluorescence for nuclear translocation, overexpression in human lung fibroblasts 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 overcome this autoinhibition, and a single RFX trimeric complex binds the proximal regulatory region of the MHC-II promoter. Electrophoretic mobility shift assays (EMSA) with purified recombinant proteins and mutant constructs Biochimica et biophysica acta Medium 18723135
2010 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. Solution NMR (15N- and 13C-edited), structural determination of heterotrimeric complex Journal of molecular biology High 20732328
2010 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. Steady-state fluorescence quenching, circular dichroism, FRET, chemical cross-linking with tandem mass spectrometry Biochimica et biophysica acta Medium 20637319
2012 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. Co-immunoprecipitation, overexpression/knockdown of SIRT1, NAMPT, resveratrol/inhibitor treatment, reporter assays, nuclear/cytoplasmic fractionation Biochemical and biophysical research communications Medium 23079621
2013 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. Site-directed mutagenesis of AT-hook motif, PRMT6 overexpression/knockdown, reporter assays, ChIP Molecular immunology Medium 23911394
2006 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. Co-immunoprecipitation with CREB mutants, reporter assays, chromatin immunoprecipitation (ChIP) Molecular immunology Medium 16730065
2022 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. RFX5 knockdown/overexpression in primary macrophages, ChIP, metabolic assays (glutamate utilization), flow cytometry for HLA-DR and T cell activation Nature metabolism High 35739396
2016 RFX5 binds directly to the TPP1 promoter region and transcriptionally activates TPP1 expression in hepatocellular carcinoma cells, as demonstrated by ChIP and reporter assays. ChIP, luciferase reporter assay, RFX5 overexpression/knockdown in HepG2 cells Oncology reports Medium 27840983
2020 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. ChIP-seq (ENCODE), ChIP-PCR, luciferase reporter assay, RFX5 deletion/overexpression, cell cycle analysis Scientific reports Medium 32883983
2025 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. Cryo-EM structure determination of RFX5 eDBD-nucleosome complex Nucleic acids research High 40744500
2025 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. Comparative enrichment proteomics with promoter probes bearing symmetric or asymmetric hmC modifications in human and mouse nuclear lysates bioRxivpreprint Low bio_10.1101_2025.06.27.661915
2025 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. Chromatin immunoprecipitation, luciferase reporter assay, RFX5 knockdown/overexpression, JAG1 knockdown rescue Human cell Medium 40220043

Source papers

Stage 0 corpus · 27 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 152 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 90 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 68 10779326
2022 The transcription factor RFX5 coordinates antigen-presenting function and resistance to nutrient stress in synovial macrophages. Nature metabolism 63 35739396
1998 Residual MHC class II expression on mature dendritic cells and activated B cells in RFX5-deficient mice. Immunity 60 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 49 12101253
2012 SIRT1 deacetylates RFX5 and antagonizes repression of collagen type I (COL1A2) transcription in smooth muscle cells. Biochemical and biophysical research communications 33 23079621
2003 Interferon gamma repression of collagen (COL1A2) transcription is mediated by the RFX5 complex. The Journal of biological chemistry 33 12968017
2020 RFX5 promotes the progression of hepatocellular carcinoma through transcriptional activation of KDM4A. Scientific reports 29 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 29 31096145
2016 The transcription factor RFX5 is a transcriptional activator of the TPP1 gene in hepatocellular carcinoma. Oncology reports 29 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
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
2006 CREB and phospho-CREB interact with RFX5 and CIITA to regulate MHC class II genes. Molecular immunology 20 16730065
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 9 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 7 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 1 40240550
2025 RFX5 promotes the progression of triple-negative breast cancer through transcriptional activation of JAG1. Human cell 0 40220043
2025 Structural basis of nucleosome binding and destabilization by the extended DNA binding domain of RFX5. Nucleic acids research 0 40744500
2025 RFX5 promotes the growth, motility, and inhibits apoptosis of gastric adenocarcinoma cells through the SIRT1/AMPK axis. Open life sciences 0 41079601
2022 The transcription factor RFX5 positively regulates expression of MHCIa in the red-spotted grouper (Epinephelus akaara). Fish & shellfish immunology 0 35051562