Affinage

RFX3

Transcription factor RFX3 · UniProt P48380

Length
749 aa
Mass
83.5 kDa
Annotated
2026-04-28
18 papers in source corpus 15 papers cited in narrative 15 extracted findings

Mechanistic narrative

Synthesis pass · prose summary of the discoveries below

RFX3 is a transcription factor that binds X-box motifs to orchestrate ciliogenesis, neuroendocrine cell differentiation, and activity-dependent neuronal gene expression. It directly activates intraflagellar transport and axonemal dynein genes required for both primary and motile cilia assembly and motility, and it physically interacts with FOXJ1 as a co-activator and with FOXN3/FOXN4 via a conserved hydrophobic motif in its dimerization domain to modulate ciliary gene programs (PMID:15121860, PMID:19671664, PMID:23822649). RFX3 undergoes auto-S-fatty acylation at a conserved dimerization-domain cysteine, which is required for homodimerization, transcriptional activity, ciliogenesis, and Hedgehog signaling (PMID:30127002). Beyond ciliogenesis, RFX3 directly regulates glucokinase and Glut-2 in pancreatic beta-cells to control glucose-stimulated insulin secretion, is essential for pancreatic islet cell differentiation, and co-localizes with CREB at activity-dependent enhancers in neurons to facilitate stimulus-dependent transcription (PMID:20413507, PMID:40263183, PMID:40060598).

Mechanistic history

Synthesis pass · year-by-year structured walk · 11 steps
  1. 2004 High

    Establishing RFX3 as a ciliogenesis transcription factor: Rfx3 knockout mice revealed that RFX3 is essential for nodal monocilia elongation and regulates the intraflagellar transport gene D2lic, placing RFX3 upstream of the cilia assembly machinery and linking it to left-right asymmetry determination.

    Evidence Rfx3 KO mouse with ultrastructural cilia analysis and RT-PCR for D2lic

    PMID:15121860

    Open questions at the time
    • Whether RFX3 directly binds D2lic promoter was not tested
    • Role in motile versus primary cilia was unresolved
    • Downstream ciliogenesis targets beyond D2lic were unknown
  2. 2006 High

    Extending the ciliogenesis role to brain ependymal cells: Rfx3 deficiency caused failure of ciliated ependymal cell differentiation and hydrocephalus due to subcommissural organ agenesis, broadening RFX3's requirement beyond nodal cilia to multiciliated brain epithelia.

    Evidence Rfx3 KO mouse with ultrastructural, immunohistochemical, and RT-PCR analysis of brain ependyma

    PMID:16930429

    Open questions at the time
    • Direct transcriptional targets in ependymal cells not identified
    • Whether hydrocephalus results from ciliary motility defects versus secretory defects was unclear
  3. 2007 High

    Revealing a pancreatic endocrine role: Rfx3 deficiency reduced insulin-, glucagon-, and ghrelin-producing islet cells, stunted primary cilia on islet cells, and impaired glucose tolerance, establishing RFX3 as a regulator of pancreatic endocrine differentiation.

    Evidence Rfx3 KO mouse with immunofluorescence, electron microscopy, and glucose tolerance testing

    PMID:17229940

    Open questions at the time
    • Direct transcriptional targets in beta-cells were not identified
    • Whether the metabolic phenotype was cilia-dependent or cilia-independent was unresolved
  4. 2009 High

    Demonstrating direct promoter binding at motile cilia genes: ChIP showed RFX3 directly binds axonemal dynein gene promoters in brain multiciliated cells and regulates FOXJ1 expression, establishing a direct transcriptional mechanism for ciliary motility control.

    Evidence ChIP in primary Rfx3 KO mouse brain cell cultures with cilia beat frequency measurements

    PMID:19671664

    Open questions at the time
    • Whether RFX3 and FOXJ1 function in a shared complex was unknown
    • Genome-wide target repertoire was not mapped
  5. 2010 High

    Identifying direct beta-cell targets: RFX3 was shown to directly bind the glucokinase neuroendocrine promoter (Pal-1/Pal-2 sites) and regulate glucokinase and Glut-2 expression, mechanistically linking RFX3 to glucose-stimulated insulin secretion independently of cilia.

    Evidence ChIP-seq, EMSA, RNAi in Min6 cells, and pancreas-specific conditional KO

    PMID:20413507

    Open questions at the time
    • Full set of RFX3 beta-cell targets beyond glucokinase/Glut-2 was not defined
    • Whether cilia-dependent signaling also contributes to the beta-cell phenotype was unresolved
  6. 2012 High

    Uncovering an indirect role in brain axon guidance: RFX3 deficiency disrupted corpus callosum formation by altering GLI3 repressor/activator balance and causing ectopic FGF8 expression at the cortical-septal boundary, demonstrating a cilia-signaling-dependent mechanism for axon tract patterning.

    Evidence Rfx3 conditional KO, transplantation assays, brain explant FGF8 phenocopy, GLI3 isoform analysis

    PMID:22479201

    Open questions at the time
    • How RFX3/cilia control GLI3 processing was not resolved at the molecular level
    • Whether other Hedgehog-dependent brain structures are similarly affected was not tested
  7. 2013 High

    Establishing physical interaction with FOXJ1: Co-immunoprecipitation and promoter assays showed RFX3 physically associates with FOXJ1 and acts as a transcriptional co-activator of ciliary genes, explaining how RFX3 enhances but cannot independently drive cilia gene expression.

    Evidence Reciprocal co-IP and promoter-reporter assays in primary human airway basal cells

    PMID:23822649

    Open questions at the time
    • The structural basis for the RFX3-FOXJ1 interaction was unknown
    • Whether the interaction is required in vivo was not tested
  8. 2018 High

    Discovering auto-S-fatty acylation as a regulatory mechanism: RFX3 was shown to undergo enzyme-independent auto-acylation at a conserved dimerization-domain cysteine, and this modification is required for homodimerization, ciliary gene expression, ciliogenesis, and Hedgehog signaling, revealing a novel post-translational activation mechanism for an RFX family member.

    Evidence Chemical reporters, mass spectrometry, active-site mutagenesis, ciliogenesis and Hedgehog reporter assays

    PMID:30127002

    Open questions at the time
    • Whether fatty acylation is dynamically regulated in vivo is unknown
    • Structural consequences of acylation on the dimerization domain were not resolved
    • Whether other RFX family members undergo similar auto-acylation was not determined
  9. 2024 Medium

    Defining a shared FOX-family interaction motif: A conserved LXXLXWL motif in FOXJ1, FOXN3, and FOXN4 was shown to mediate binding to the RFX3 dimerization domain, enabling transcriptional activation (FOXN4) or repression (FOXN3) of cilia genes, expanding the combinatorial logic of RFX3-mediated transcription.

    Evidence Co-IP with domain mutagenesis, CUT&RUN, transcriptional reporter assays, AlphaFold3 structural prediction (preprint)

    PMID:bio_10.1101_2024.10.28.620684

    Open questions at the time
    • Awaits peer review
    • In vivo functional validation of the motif mutants is lacking
    • Whether the interaction interface overlaps with the acylation site was not tested
  10. 2025 High

    Extending RFX3's role to human pancreatic islet differentiation: CRISPR KO in iPSC-derived islet organoids confirmed RFX3 is required for human beta-cell differentiation and insulin secretion, with overexpression rescuing the phenotype, validating the mouse findings in a human model.

    Evidence CRISPR/Cas9 RFX3 KO in human iPSCs, islet organoid differentiation, scRNA-seq, glucose-stimulated insulin secretion, overexpression rescue

    PMID:40263183

    Open questions at the time
    • Direct RFX3 targets in human islet cells were not mapped genome-wide
    • Whether the enterochromaffin cell fate switch is cilia-dependent is unknown
  11. 2025 Medium

    Revealing a role in activity-dependent neuronal transcription: RFX3 loss in human iPSC-derived neurons impaired activity-dependent gene expression; RFX3 and CREB co-occupy activity-dependent enhancers, and RFX3 deficiency reduces CREB binding, suggesting RFX3 facilitates CREB-dependent chromatin access at stimulus-responsive loci.

    Evidence RFX3 KO/het iPSC-derived neurons, ChIP-seq, transcriptomics, depolarization assays; proximity labeling (BioID) and ChIP-seq in rodent neurons (preprints)

    PMID:40060598 PMID:40631264

    Open questions at the time
    • Both studies are preprints awaiting peer review
    • Whether RFX3 directly contacts CREB/CRTC1 or acts through chromatin remodeling is unresolved
    • Causal contribution of RFX3 to neuronal activity phenotypes in vivo is not established

Open questions

Synthesis pass · forward-looking unresolved questions
  • Key unresolved questions include the structural basis for RFX3 auto-acylation and its dynamic regulation in vivo, the full genome-wide target repertoire across cell types, and whether RFX3's cilia-independent functions (beta-cell metabolism, neuronal activity-dependent transcription) represent a broader non-ciliary transcriptional program.
  • No crystal or cryo-EM structure of RFX3 dimerization domain with acylation
  • Genome-wide target comparison across ciliated vs non-ciliated cell types is lacking
  • In vivo relevance of RFX3-CREB interaction for neuronal function is untested

Mechanism profile

Synthesis pass · controlled-vocabulary classification · explore literature graph →
Molecular activity
GO:0140110 transcription regulator activity 7 GO:0003677 DNA binding 4
Localization
GO:0005634 nucleus 4
Pathway
R-HSA-74160 Gene expression (Transcription) 8 R-HSA-1266738 Developmental Biology 5 R-HSA-1852241 Organelle biogenesis and maintenance 4 R-HSA-162582 Signal Transduction 2
Partners
CREB1CRTC1FOXJ1FOXN3FOXN4RFX1

Evidence

Reading pass · 15 per-paper findings extracted from the source corpus
Year Finding Method Journal Conf PMIDs
2004 RFX3 is essential for the elongation of nodal monocilia during embryonic development; Rfx3-deficient mice exhibit stunted nodal cilia, leading to left-right asymmetry defects. RFX3 regulates expression of D2lic (mouse orthologue of a C. elegans intraflagellar transport gene), placing RFX3 upstream of intraflagellar transport in ciliogenesis. Rfx3 knockout mouse (loss-of-function), ultrastructural analysis of nodal cilia, RT-PCR for D2lic expression Molecular and cellular biology High 15121860
2006 RFX3 is required for differentiation of ciliated ependymal cells (including subcommissural organ and choroid plexus cells) in the mouse brain; Rfx3 deficiency causes hydrocephalus associated with agenesis of the subcommissural organ and downregulation of SCO-spondin expression. Rfx3 knockout mouse, ultrastructural analysis, immunohistochemistry, RT-PCR for SCO-spondin The European journal of neuroscience High 16930429
2007 RFX3 is expressed in pancreatic endocrine progenitors and all major islet cell lineages; Rfx3-deficient mice exhibit reduced insulin-, glucagon-, and ghrelin-producing cells, increased pancreatic polypeptide cells, stunted primary cilia on islet cells, and impaired glucose tolerance, demonstrating a role for RFX3 in pancreatic endocrine cell differentiation. Rfx3 knockout mouse, immunofluorescence, electron microscopy of primary cilia, glucose tolerance testing Diabetes High 17229940
2009 RFX3 is required for the growth and beating efficiency of motile cilia in multiciliated brain cells; RFX3 regulates FOXJ1 transcription factor expression and directly binds the promoters of axonemal dynein genes required for ciliary motility. Rfx3 knockout primary mouse brain cell cultures, ChIP (direct promoter binding), cilia beat frequency measurements Journal of cell science High 19671664
2010 RFX3 is required for the 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 gene, regulating glucokinase and Glut-2 expression and glucose-stimulated insulin secretion. Rfx3 knockout mouse, pancreas-specific conditional KO, RNA interference in Min6 cells, quantitative ChIP, ChIP sequencing, bandshift (EMSA) assays Diabetes High 20413507
2012 RFX3 controls corpus callosum formation indirectly by regulating patterning of the cortical-septal boundary before E12.5, which leads to proper distribution of guidepost neurons; RFX3 deficiency results in ectopic FGF8 expression associated with a reduced GLI3 repressor-to-activator ratio, and ectopic FGF8 reproduces the guidepost neuron defects. Rfx3 knockout mouse, conditional genetic inactivation, transplantation assays, brain explant cultures with FGF8, GLI3 isoform analysis PLoS genetics High 22479201
2013 RFX3 physically interacts with FOXJ1 and acts as a transcriptional co-activator; combined FOXJ1+RFX3 transfection enhances ciliated gene promoter activity and mRNA expression beyond FOXJ1 alone in human airway basal cells, while RFX3 alone cannot induce cilia-related gene expression. Co-immunoprecipitation of FOXJ1 and RFX3, plasmid-mediated gene transfer in primary human airway basal cells, promoter-reporter assays, TaqMan PCR Respiratory research High 23822649
2015 RFX3 is required for proper formation of the thalamocortical tract by establishing the correct cellular environment in the ventral telencephalon; Rfx3 deficiency causes heterotopias expressing Slit1 and Netrin1 guidance cues, leading to aberrant thalamocortical axon projections; identical defects occur in Inpp5e mutants, indicating primary cilia signaling underlies tract formation. Rfx3 knockout mouse, DiI axon tracing, immunohistochemistry, genetic epistasis with Inpp5e mutants Human molecular genetics High 25631876
2018 RFX3 undergoes enzyme-independent auto-S-fatty acylation (preferentially by stearic and oleic acid) at a conserved cysteine in its dimerization domain; this modification promotes homodimerization, ciliary gene expression, ciliogenesis, cilia elongation, and Hedgehog signaling. A fatty acylation-deficient mutant fails in all these functions. Chemical reporters of protein fatty acylation, mass spectrometry, active-site mutagenesis, dimerization assays, ciliogenesis assays, Hedgehog signaling reporters 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 AAV inverted terminal repeats, and RFX proteins can be pulled down with the AAV genome in transduced HEK-293 cells, indicating RFX3 acts as a regulator of AAV-mediated transgene expression. Electromobility shift assay (EMSA), supershift experiments, co-immunoprecipitation/pulldown with AAV genome from transduced cells Scientific reports Medium 29317724
2025 In human iPSC-derived neurons, monoallelic RFX3 loss diminishes neuronal activity-dependent gene expression; RFX3 binding sites co-localize with CREB binding sites near activity-dependent genes, and RFX3 deficiency leads to decreased CREB binding and impaired induction of CREB targets upon neuronal depolarization. iPSC-derived neurons and forebrain organoids with biallelic or monoallelic RFX3 loss, transcriptomics, ChIP-seq/DNA binding analysis, neuronal depolarization assays bioRxivpreprint Medium 40060598
2025 CRTC1 and CREB1 interact with RFX3 in an activity-dependent manner in rodent forebrain neurons; glutamatergic stimulation recruits CRTC1 and CREB1 to activity-dependent enhancers enriched in RFX3 motifs, indicating cooperative chromatin binding between CREB1 and RFX3 at activity-dependent loci. Proximity labeling (BioID) in rodent forebrain neurons, ChIP-seq, neuronal activity stimulation assays bioRxivpreprint Medium 40631264
2024 A short hydrophobic motif (LXXLXWL) shared by FOXJ1, FOXN4, and FOXN3 is required for physical association with the RFX3 dimerization domain; mutations in RFX3 at the predicted interaction site disrupted association with FOXN3 or FOXN4, and this interaction mediates both transcriptional repression (by FOXN3) and activation (by FOXN4) of cilia genes. CUT&RUN chromatin profiling, co-immunoprecipitation, domain mutagenesis, AlphaFold3 structural prediction, in vitro transcriptional reporter assays bioRxivpreprint Medium bio_10.1101_2024.10.28.620684
2025 RFX3 is required for human pancreatic islet cell differentiation; RFX3 knockout in iPSC-derived islet organoids reduces hormone-secreting cells, impairs beta-cell function and insulin secretion, and leads to increased enterochromaffin cell specification; RFX3 overexpression rescues dysregulated gene expression. CRISPR/Cas9 RFX3 KO in iPSCs, pancreatic islet organoid differentiation, single-cell RNA-seq, bulk RNA-seq, glucose-stimulated insulin secretion assay, RFX3 overexpression rescue Diabetologia High 40263183
2024 In cochlear outer hair cells, Rfx3 binds intronic enhancers of Triobp and regulatory regions of Insm1, Ikzf2, and Tbx2, functioning as either a transcriptional activator or repressor to regulate hair bundle formation and outer hair cell differentiation and maintenance. ChIP-seq, ATAC-seq, single-cell transcriptomics integration, Rfx3 conditional knockout mouse bioRxivpreprint Medium bio_10.1101_2024.09.24.614849

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
2007 Novel function of the ciliogenic transcription factor RFX3 in development of the endocrine pancreas. Diabetes 79 17229940
2013 RFX3 modulation of FOXJ1 regulation of cilia genes in the human airway epithelium. Respiratory research 78 23822649
2012 The ciliogenic transcription factor RFX3 regulates early midline distribution of guidepost neurons required for corpus callosum development. PLoS genetics 61 22479201
2010 The transcription factor Rfx3 regulates beta-cell differentiation, function, and glucokinase expression. Diabetes 60 20413507
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
2018 Auto-fatty acylation of transcription factor RFX3 regulates ciliogenesis. Proceedings of the National Academy of Sciences of the United States of America 24 30127002
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 8 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 3 40060598
2025 RFX3 is essential for the generation of functional human pancreatic islets from stem cells. Diabetologia 2 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 1 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