Affinage

RFX3

Transcription factor RFX3 · UniProt P48380

Length
749 aa
Mass
83.5 kDa
Annotated
2026-06-10
18 papers in source corpus 13 papers cited in narrative 15 extracted findings
Cross-family judge vs UniProt: Affinage preferred faithfulness: 6/7 claims corpus-supported (86%)

Mechanistic narrative

Synthesis pass · prose summary of the discoveries below

RFX3 is a DNA-binding transcription factor that serves as a master regulator of ciliogenesis and of endocrine and neuronal differentiation programs (PMID:15121860, PMID:17229940, PMID:19671664). It functions as an upstream activator of intraflagellar transport and ciliary motility genes, directly binding promoters of axonemal dynein genes and promoting expression of the ciliary transcription factor FOXJ1, and its loss in mice causes stunted nodal monocilia with left-right asymmetry defects, hydrocephalus, and impaired motile cilia (PMID:15121860, PMID:16930429, PMID:19671664). In the pancreas, RFX3 controls differentiation of insulin-, glucagon-, and ghrelin-producing islet cells and directly binds the Pal-1/Pal-2 elements of the glucokinase (Gck) neuroendocrine promoter to regulate Gck and Glut-2 expression, a requirement confirmed in human iPSC-derived islet organoids where RFX3 loss reduces hormone-secreting cells and impairs insulin secretion (PMID:17229940, PMID:20413507, PMID:40263183). RFX3 transcriptional output depends on homodimerization, which is regulated by enzyme-independent S-fatty acylation at a conserved cysteine in its dimerization domain; an acylation-deficient mutant loses dimerization, fails to drive ciliary gene expression, and impairs Hedgehog signaling (PMID:30127002). Through this same dimerization domain RFX3 forms functional partnerships, acting as a co-activator with FOXJ1 and engaging the forkhead proteins FOXN3 and FOXN4 via a shared LXXLXWL motif, with FOXN3 acting as a cilia-gene repressor (PMID:23822649). In neurons, RFX3 cooperates with CREB1/CRTC1 at activity-dependent enhancers to facilitate depolarization-induced transcription (PMID:40060598, PMID:40631264). RFX3 also acts in tissue patterning, regulating cortical-septal boundary formation and thalamocortical axon guidance via cilia-dependent signaling, and functions as both activator and repressor in cochlear hair cells (PMID:22479201, PMID:25631876).

Mechanistic history

Synthesis pass · year-by-year structured walk · 15 steps
  1. 2004 High

    Established RFX3 as an upstream transcriptional regulator of ciliogenesis by showing it controls intraflagellar transport gene expression and is required for nodal cilia and left-right axis determination, defining its core developmental function.

    Evidence Rfx3 knockout mouse with EM of nodal cilia and RT-PCR of D2lic

    PMID:15121860

    Open questions at the time
    • Did not identify the full set of direct ciliary target genes
    • Did not resolve whether RFX3 acts alone or in complex at target promoters
  2. 2006 High

    Extended the ciliary requirement to ependymal differentiation, showing RFX3 loss causes hydrocephalus and subcommissural organ agenesis with reduced SCO-spondin, broadening RFX3's role across multiciliated cell types.

    Evidence Rfx3 knockout mouse with EM, immunohistochemistry, RT-PCR

    PMID:16930429

    Open questions at the time
    • Did not establish direct vs. indirect regulation of SCO-spondin
    • Mechanism linking cilia loss to choroid plexus disorganization not defined
  3. 2007 High

    Demonstrated a non-neural role in pancreatic endocrine cell differentiation and islet cilia formation, showing RFX3 governs the balance of hormone-producing lineages.

    Evidence Rfx3 knockout mouse, immunofluorescence, glucose tolerance tests, EM

    PMID:17229940

    Open questions at the time
    • Direct target genes in endocrine progenitors not yet identified
    • Did not separate cilia-dependent from cilia-independent transcriptional effects
  4. 2009 High

    Provided direct binding evidence that RFX3 occupies axonemal dynein promoters and promotes FOXJ1 expression, mechanistically linking it to ciliary motility programs.

    Evidence Rfx3-/- primary multiciliated cells, ChIP, motility assays, RT-PCR

    PMID:19671664

    Open questions at the time
    • Whether RFX3 regulates FOXJ1 directly or indirectly not resolved
    • Cofactor requirements at dynein promoters not defined
  5. 2010 High

    Resolved a direct DNA-binding mechanism in beta-cells by mapping RFX3 to the Pal-1/Pal-2 elements of the Gck neuroendocrine promoter, defining specific cis-targets for endocrine maturation.

    Evidence Rfx3 KO and conditional KO mice, ChIP-seq, qChIP, bandshift, RNAi in Min6 cells

    PMID:20413507

    Open questions at the time
    • Genome-wide target catalog beyond Gck/Glut-2 not fully characterized
    • Did not address dimerization or cofactor dependence of binding
  6. 2012 High

    Showed RFX3 indirectly patterns the cortical-septal boundary through control of FGF8 and the GLI3 repressor-activator balance, connecting RFX3 to midline guidepost neuron positioning.

    Evidence Rfx3 KO, conditional inactivation, explant ectopic FGF8 and transplantation assays

    PMID:22479201

    Open questions at the time
    • Direct transcriptional targets driving FGF8/GLI3 changes not identified
    • Cilia-dependence of the patterning defect not fully dissected here
  7. 2013 Medium

    Identified FOXJ1 as a direct physical partner and showed RFX3 acts as a co-activator that synergizes with FOXJ1 on cilia gene promoters, refining how RFX3 boosts ciliary transcription.

    Evidence Co-IP, promoter-reporter and RT-PCR in human airway basal cells

    PMID:23822649

    Open questions at the time
    • Single lab, primary cells; interaction interface not mapped at this stage
    • Whether synergy requires direct DNA co-occupancy not shown
  8. 2015 Medium

    Implicated RFX3 in thalamocortical axon guidance through prethalamic/telencephalic patterning, with phenocopy by a ciliary mutant supporting cilia-dependent signaling.

    Evidence Rfx3 KO with DiI tracing, IHC, comparison to Inpp5e mutant

    PMID:25631876

    Open questions at the time
    • Direct transcriptional targets controlling Slit1/Netrin1 heterotopias not defined
    • Causal cilia mechanism inferred from phenocopy, not directly tested
  9. 2018 High

    Uncovered a post-translational control of RFX3 activity, showing enzyme-independent S-fatty acylation of a dimerization-domain cysteine is required for homodimerization, ciliary gene expression, and Hedgehog signaling.

    Evidence Chemical acylation reporters, mass spectrometry, cysteine mutagenesis, ciliogenesis and Hh reporter assays

    PMID:30127002

    Open questions at the time
    • Physiological signals regulating acylation status unknown
    • Whether acylation modulates partner interactions not tested
  10. 2018 Medium

    Showed RFX1/RFX3 heterodimers bind the AAV inverted terminal repeat D sequence and associate with AAV genomes, revealing a role in regulating AAV-mediated transgene expression.

    Evidence EMSA with supershift, ChIP of AAV genomes from HEK-293 cells

    PMID:29317724

    Open questions at the time
    • Functional consequence for AAV transduction efficiency not fully established
    • Single lab; relationship to RFX3's endogenous program unclear
  11. 2025 Medium

    Placed RFX3 in neuronal activity-dependent transcription, showing its binding sites co-localize with CREB and that RFX3 loss reduces CREB chromatin occupancy and CREB-target induction upon depolarization.

    Evidence iPSC-derived neurons/organoids with CRISPR RFX3 LOF, RFX3 and CREB ChIP-seq, transcriptomics, depolarization assays (preprint)

    PMID:40060598

    Open questions at the time
    • Preprint, single lab
    • Whether RFX3 directly recruits CREB or alters chromatin accessibility not resolved
  12. 2025 Medium

    Defined activity-dependent protein partnerships of RFX3 with MEF2C and CREB1/CRTC1, supporting cooperative chromatin binding at synaptic-activity enhancers.

    Evidence BioID proximity labeling in rodent forebrain neurons, CREB1/CRTC1 ChIP-seq with glutamatergic stimulation, motif enrichment (preprint)

    PMID:40631264

    Open questions at the time
    • Preprint, single lab
    • Direct vs. proximity-based interactions not distinguished
  13. 2025 High

    Confirmed in a human iPSC system that RFX3 is required for islet cell differentiation and beta-cell function, with overexpression rescue establishing causality at progenitor stages.

    Evidence CRISPR RFX3 KO iPSC islet organoids, scRNA-seq, bulk RNA-seq, GSIS assays, overexpression rescue

    PMID:40263183

    Open questions at the time
    • Direct human islet target genes not enumerated
    • Mechanism of increased enterochromaffin specification not defined
  14. 2024 Medium

    Expanded RFX3's regulatory repertoire to cochlear hair cells, showing dual activator/repressor function at the Triobp enhancer and fate genes, plus dynamic nuclear-to-cytoplasmic relocalization.

    Evidence scRNA-seq, ChIP-seq, ATAC-seq, conditional inner-ear Rfx3 KO (preprint)

    Open questions at the time
    • Preprint, single lab
    • Determinants of activator vs. repressor switching not defined
    • Functional role of cytoplasmic relocalization unknown
  15. 2024 Medium

    Mapped a shared LXXLXWL motif by which forkhead proteins FOXJ1/FOXN3/FOXN4 bind the RFX3 dimerization domain, defining the structural basis of RFX3-forkhead partnerships and identifying FOXN3 as a repressive partner limiting ciliogenesis.

    Evidence CUT&RUN, co-IP/pulldown, dimerization-domain mutagenesis, AlphaFold3 prediction, reporter assays in Foxn3 KO retina (preprint)

    Open questions at the time
    • Preprint, single lab; interaction not validated structurally in vitro
    • Competition between activating and repressing forkhead partners not quantified

Open questions

Synthesis pass · forward-looking unresolved questions
  • How RFX3 selects between activator and repressor states and between tissue-specific partner sets across cilia, endocrine, and neuronal contexts remains unresolved.
  • Signals controlling fatty acylation and partner choice unknown
  • No unified model linking dimerization state to activator/repressor output across tissues

Mechanism profile

Synthesis pass · controlled-vocabulary classification · explore literature graph →
Molecular activity
GO:0003677 DNA binding 4 GO:0140110 transcription regulator activity 4
Localization
GO:0005634 nucleus 2
Pathway
R-HSA-1266738 Developmental Biology 4 R-HSA-74160 Gene expression (Transcription) 3 R-HSA-162582 Signal Transduction 2

Evidence

Reading pass · 15 per-paper findings extracted from the source corpus
Year Finding Method Journal Conf PMIDs
2004 RFX3 is required for the development of nodal monocilia and left-right body axis determination; Rfx3-deficient mice show stunted nodal cilia and left-right asymmetry defects. RFX3 regulates expression of D2lic (mouse orthologue of a C. elegans intraflagellar transport gene), establishing RFX3 as an upstream transcriptional regulator of intraflagellar transport-dependent ciliogenesis. Rfx3 knockout mouse, RT-PCR for D2lic expression, electron microscopy of nodal cilia Molecular and cellular biology High 15121860
2006 RFX3 is necessary for differentiation of ciliated ependymal cells in the mouse brain; Rfx3-deficient mice develop hydrocephalus associated with defects in choroid plexus epithelial organization and agenesis of the subcommissural organ (SCO), with downregulation of SCO-spondin expression as early as E14.5. Rfx3 knockout mouse, ultrastructural analysis (electron microscopy), immunohistochemistry, RT-PCR The European journal of neuroscience High 16930429
2007 RFX3 is expressed in pancreatic endocrine progenitors and all major islet lineages; Rfx3-deficient mice show reduced insulin-, glucagon-, and ghrelin-producing cells with increased pancreatic polypeptide-positive cells, and primary cilia on islet cells are severely stunted, indicating RFX3 controls endocrine cell differentiation and cilia formation in the pancreas. Rfx3 knockout mouse, immunofluorescence, glucose tolerance tests, electron microscopy of primary cilia Diabetes High 17229940
2009 RFX3 is required for growth and beating efficiency of motile cilia in multiciliated brain cells. RFX3 promotes optimal expression of the FOXJ1 transcription factor and directly binds promoters of axonemal dynein genes to regulate their expression, linking RFX3 to ciliary motility programs. Primary multiciliated cell culture from Rfx3-/- mouse brain, cilia motility assays, ChIP (direct promoter binding of dynein genes), RT-PCR for FOXJ1 Journal of cell science High 19671664
2010 RFX3 is required for differentiation and function of mature beta-cells; it directly binds the Pal-1 and Pal-2 regulatory sequences in the neuroendocrine promoter of the glucokinase (Gck) gene, regulating Glut-2 and Gck expression. Loss of Rfx3 leads to accumulation of incompletely differentiated beta-cell precursors and glucose intolerance. Rfx3 knockout and pancreas-specific conditional knockout mice, quantitative ChIP, ChIP sequencing, bandshift assay, RNA interference in Min6 cells, immunofluorescence, RT-PCR Diabetes High 20413507
2012 RFX3 indirectly regulates corpus callosum formation by controlling patterning of the cortical-septal boundary required for distribution of midline guidepost neurons. Rfx3 deficiency leads to ectopic FGF8 expression at the rostro-commissural plate associated with a reduced GLI3 repressor-to-activator ratio, and ectopic FGF8 reproduces guidepost neuronal defects. Rfx3 knockout mouse, conditional genetic inactivation, brain explant cultures with ectopic FGF8, transplantation assays, immunohistochemistry, in situ hybridization PLoS genetics High 22479201
2013 RFX3 physically interacts with FOXJ1 (demonstrated by co-immunoprecipitation) and acts as a transcriptional co-activator; combined FOXJ1 + RFX3 transfection enhances cilia gene promoter activity and mRNA expression beyond FOXJ1 alone. RFX3 alone does not induce FOXJ1 expression or cilia gene expression. Plasmid-mediated gene transfer into human airway basal cells, co-immunoprecipitation, promoter-reporter assays, TaqMan RT-PCR Respiratory research Medium 23822649
2015 Rfx3 is required for proper patterning of the prethalamus and ventral telencephalon necessary for thalamocortical tract formation; Rfx3-deficient mice show misguided thalamocortical axons associated with heterotopias expressing Slit1 and Netrin1 guidance molecules. Identical defects in Inpp5e mutants corroborate a role for primary cilia signaling in this process. Rfx3 knockout mouse, DiI axon tracing, immunohistochemistry, comparison with Inpp5e mutant mice Human molecular genetics Medium 25631876
2018 RFX3 transcriptional activity is regulated by S-fatty acylation at a conserved cysteine residue in its dimerization domain. RFX3 undergoes enzyme-independent auto-fatty acylation with preference for 18-carbon stearic and oleic acids. A fatty acylation-deficient mutant shows decreased homodimerization, fails to promote ciliary gene expression and ciliogenesis, and impairs Hedgehog signaling. Chemical reporters of protein fatty acylation, mass spectrometry, site-directed mutagenesis of the conserved cysteine, ciliogenesis assays, Hedgehog signaling reporter assays Proceedings of the National Academy of Sciences of the United States of America High 30127002
2018 RFX1 homodimers and RFX1/RFX3 heterodimers bind specifically to the double-stranded D sequence of AAV2 and AAV1 inverted terminal repeats; RFX3 antibodies can pull down AAV genomes from transduced HEK-293 cells, indicating RFX3 interacts with AAV genomes in the nucleus and acts as a regulator of AAV-mediated transgene expression. Electromobility shift assay (EMSA), supershift experiments with RFX1/RFX3 antibodies, chromatin immunoprecipitation of AAV genomes from HEK-293 cells Scientific reports Medium 29317724
2025 In human iPSC-derived neurons, RFX3 binding sites co-localize with CREB binding sites near activity-dependent genes. Monoallelic RFX3 loss reduces CREB binding at activity-dependent enhancers and impairs induction of CREB targets upon neuronal depolarization, placing RFX3 as a co-regulator that facilitates activity-dependent transcription by enhancing CREB chromatin binding. iPSC-derived neurons and forebrain organoids with CRISPR RFX3 loss-of-function, transcriptomics, ChIP-seq for RFX3 and CREB binding, neuronal depolarization assays bioRxivpreprint Medium 40060598
2025 RFX3 interacts with MEF2C and CREB1/CRTC1 in an activity-dependent manner in neurons. Upon glutamatergic stimulation, CRTC1 and CREB1 are recruited to activity-dependent enhancers enriched for RFX3 motifs, suggesting cooperative chromatin binding between CREB1 and RFX3 in response to synaptic activity. Proximity labeling (BioID) in rodent forebrain neurons, ChIP-seq for CREB1 and CRTC1 before and after glutamatergic stimulation, motif enrichment analysis bioRxivpreprint Medium 40631264
2025 RFX3 is required for human pancreatic islet cell differentiation from iPSCs; RFX3 KO disrupts endocrine gene regulation, reduces hormone-secreting islet cells, impairs beta-cell function and insulin secretion, increases enterochromaffin cell specification, and increases apoptosis. RFX3 overexpression rescues dysregulated gene expression at progenitor stages. CRISPR/Cas9 RFX3 KO iPSC lines differentiated into pancreatic islet organoids, scRNA-seq, bulk RNA-seq, glucose-stimulated insulin secretion assays, RFX3 overexpression rescue Diabetologia High 40263183
2024 Rfx3 functions as both a transcriptional activator and repressor in cochlear outer hair cells, binding to the intronic enhancer of the hair bundle gene Triobp to regulate its spatiotemporal expression, and binding to differentiation/fate determination genes Tbx2, Insm1, and Ikzf2. Rfx3 and Rfx7 show dynamic subcellular localization shifting from nuclear to cytoplasmic at later developmental stages. Single-cell transcriptomics, ChIP-seq, ATAC-seq in cochlear hair cells; conditional Rfx3 knockout mouse (inner ear) bioRxivpreprint Medium
2024 RFX3 physically interacts with FOXJ1, FOXN3, and FOXN4 via a short hydrophobic motif (LXXLXWL) shared by these forkhead proteins; this motif binds the RFX3 dimerization domain. Mutations in RFX3 at the predicted interaction site disrupt association. FOXN3 functions as a repressor of cilia genes and limits primary cilia formation through its interaction with RFX3. CUT&RUN (chromatin binding), co-IP/pulldown assays, site-directed mutagenesis of RFX3 dimerization domain, AlphaFold3 structural prediction, transcriptional reporter assays in Foxn3 knockout retina bioRxivpreprint Medium

Source papers

Stage 0 corpus · 18 papers · ranked by NIH iCite citations
Year Title Journal Citations PMID
2004 The transcription factor RFX3 directs nodal cilium development and left-right asymmetry specification. Molecular and cellular biology 175 15121860
2009 RFX3 governs growth and beating efficiency of motile cilia in mouse and controls the expression of genes involved in human ciliopathies. Journal of cell science 101 19671664
2006 A deficiency in RFX3 causes hydrocephalus associated with abnormal differentiation of ependymal cells. The European journal of neuroscience 92 16930429
2013 RFX3 modulation of FOXJ1 regulation of cilia genes in the human airway epithelium. Respiratory research 80 23822649
2007 Novel function of the ciliogenic transcription factor RFX3 in development of the endocrine pancreas. Diabetes 79 17229940
2012 The ciliogenic transcription factor RFX3 regulates early midline distribution of guidepost neurons required for corpus callosum development. PLoS genetics 62 22479201
2010 The transcription factor Rfx3 regulates beta-cell differentiation, function, and glucokinase expression. Diabetes 61 20413507
2018 Auto-fatty acylation of transcription factor RFX3 regulates ciliogenesis. Proceedings of the National Academy of Sciences of the United States of America 27 30127002
2018 ATOH1/RFX1/RFX3 transcription factors facilitate the differentiation and characterisation of inner ear hair cell-like cells from patient-specific induced pluripotent stem cells harbouring A8344G mutation of mitochondrial DNA. Cell death & disease 24 29740017
2015 The ciliogenic transcription factor Rfx3 is required for the formation of the thalamocortical tract by regulating the patterning of prethalamus and ventral telencephalon. Human molecular genetics 23 25631876
2019 Ginsenoside Rg3 and Korean Red Ginseng extract epigenetically regulate the tumor-related long noncoding RNAs RFX3-AS1 and STXBP5-AS1. Journal of ginseng research 22 31700260
2018 RFX1 and RFX3 Transcription Factors Interact with the D Sequence of Adeno-Associated Virus Inverted Terminal Repeat and Regulate AAV Transduction. Scientific reports 9 29317724
2022 CircRFX3 Up-regulates Its Host Gene RFX3 to Facilitate Tumorigenesis and Progression of Glioma. Journal of molecular neuroscience : MN 6 35416616
2022 Downregulation of microRNA-342-3p Eases Insulin Resistance and Liver Gluconeogenesis via Regulating Rfx3 in Gestational Diabetes Mellitus. Critical reviews in eukaryotic gene expression 6 35997120
2025 Multi-omic analysis of the ciliogenic transcription factor RFX3 reveals a role in promoting activity-dependent responses via enhancing CREB binding in human neurons. bioRxiv : the preprint server for biology 4 40060598
2025 RFX3 is essential for the generation of functional human pancreatic islets from stem cells. Diabetologia 3 40263183
2025 Cytoplasmic and nuclear protein interaction networks of the synapto-nuclear messenger CRTC1 in neurons reveal cooperative chromatin binding between CREB1 and CRTC1, MEF2C and RFX3. bioRxiv : the preprint server for biology 2 40631264
2026 Whole Genome Sequence Analysis of Weight Loss in 16 972 Participants With COPD Reveals Novel Risk Loci in DRAIC and RFX3. Journal of cachexia, sarcopenia and muscle 0 42026014

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