{"gene":"RFX3","run_date":"2026-06-10T06:43:36","timeline":{"discoveries":[{"year":2004,"finding":"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.","method":"Rfx3 knockout mouse, RT-PCR for D2lic expression, electron microscopy of nodal cilia","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean KO with defined cellular phenotype, replicated across multiple readouts (LR asymmetry, cilia morphology, target gene expression), published in peer-reviewed journal","pmids":["15121860"],"is_preprint":false},{"year":2006,"finding":"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.","method":"Rfx3 knockout mouse, ultrastructural analysis (electron microscopy), immunohistochemistry, RT-PCR","journal":"The European journal of neuroscience","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean KO with defined cellular and molecular phenotypes, multiple orthogonal methods, peer-reviewed","pmids":["16930429"],"is_preprint":false},{"year":2007,"finding":"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.","method":"Rfx3 knockout mouse, immunofluorescence, glucose tolerance tests, electron microscopy of primary cilia","journal":"Diabetes","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean KO with defined cellular phenotype across multiple endocrine lineages, multiple orthogonal methods","pmids":["17229940"],"is_preprint":false},{"year":2009,"finding":"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.","method":"Primary multiciliated cell culture from Rfx3-/- mouse brain, cilia motility assays, ChIP (direct promoter binding of dynein genes), RT-PCR for FOXJ1","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — direct promoter binding demonstrated by ChIP, clean KO cell system, multiple orthogonal readouts including motility assays","pmids":["19671664"],"is_preprint":false},{"year":2010,"finding":"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.","method":"Rfx3 knockout and pancreas-specific conditional knockout mice, quantitative ChIP, ChIP sequencing, bandshift assay, RNA interference in Min6 cells, immunofluorescence, RT-PCR","journal":"Diabetes","confidence":"High","confidence_rationale":"Tier 1 / Strong — direct DNA binding shown by ChIP-seq, bandshift assay, and ChIP; replicated in multiple genetic models and cell lines","pmids":["20413507"],"is_preprint":false},{"year":2012,"finding":"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.","method":"Rfx3 knockout mouse, conditional genetic inactivation, brain explant cultures with ectopic FGF8, transplantation assays, immunohistochemistry, in situ hybridization","journal":"PLoS genetics","confidence":"High","confidence_rationale":"Tier 2 / Strong — epistasis and transplantation experiments, conditional inactivation, multiple orthogonal methods including explant rescue","pmids":["22479201"],"is_preprint":false},{"year":2013,"finding":"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.","method":"Plasmid-mediated gene transfer into human airway basal cells, co-immunoprecipitation, promoter-reporter assays, TaqMan RT-PCR","journal":"Respiratory research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP demonstrated interaction; functional synergy shown in reporter assays; single lab, primary human cells","pmids":["23822649"],"is_preprint":false},{"year":2015,"finding":"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.","method":"Rfx3 knockout mouse, DiI axon tracing, immunohistochemistry, comparison with Inpp5e mutant mice","journal":"Human molecular genetics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean KO with defined axon-tracing phenotype, corroborated by independent ciliary mutant, single lab","pmids":["25631876"],"is_preprint":false},{"year":2018,"finding":"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.","method":"Chemical reporters of protein fatty acylation, mass spectrometry, site-directed mutagenesis of the conserved cysteine, ciliogenesis assays, Hedgehog signaling reporter assays","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro acylation assay with mass spectrometry, mutagenesis with functional readouts (dimerization, ciliogenesis, Hh signaling), multiple orthogonal methods","pmids":["30127002"],"is_preprint":false},{"year":2018,"finding":"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.","method":"Electromobility shift assay (EMSA), supershift experiments with RFX1/RFX3 antibodies, chromatin immunoprecipitation of AAV genomes from HEK-293 cells","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — EMSA with supershift and ChIP of viral genome; single lab, two orthogonal methods","pmids":["29317724"],"is_preprint":false},{"year":2025,"finding":"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.","method":"iPSC-derived neurons and forebrain organoids with CRISPR RFX3 loss-of-function, transcriptomics, ChIP-seq for RFX3 and CREB binding, neuronal depolarization assays","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ChIP-seq and transcriptomics in human neurons, multiple orthogonal genomic methods; preprint, single lab","pmids":["40060598"],"is_preprint":true},{"year":2025,"finding":"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.","method":"Proximity labeling (BioID) in rodent forebrain neurons, ChIP-seq for CREB1 and CRTC1 before and after glutamatergic stimulation, motif enrichment analysis","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — proximity labeling plus ChIP-seq; preprint, single lab, two orthogonal methods","pmids":["40631264"],"is_preprint":true},{"year":2025,"finding":"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.","method":"CRISPR/Cas9 RFX3 KO iPSC lines differentiated into pancreatic islet organoids, scRNA-seq, bulk RNA-seq, glucose-stimulated insulin secretion assays, RFX3 overexpression rescue","journal":"Diabetologia","confidence":"High","confidence_rationale":"Tier 2 / Strong — CRISPR KO in human stem cell model, rescue experiment, multiple orthogonal methods including single-cell and bulk transcriptomics and functional secretion assays; peer-reviewed","pmids":["40263183"],"is_preprint":false},{"year":2024,"finding":"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.","method":"Single-cell transcriptomics, ChIP-seq, ATAC-seq in cochlear hair cells; conditional Rfx3 knockout mouse (inner ear)","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ChIP-seq showing direct binding, ATAC-seq, and scRNA-seq; preprint, single lab, multiple orthogonal genomic methods","pmids":[],"is_preprint":true},{"year":2024,"finding":"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.","method":"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","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct protein interaction confirmed by co-IP and mutagenesis, chromatin binding by CUT&RUN; preprint, single lab","pmids":[],"is_preprint":true}],"current_model":"RFX3 is a transcription factor that directly binds X-box-like DNA motifs in promoters and enhancers of ciliogenesis genes (including intraflagellar transport components, axonemal dyneins, and hair bundle genes such as Triobp) and endocrine differentiation genes (including glucokinase/Gck via Pal-1/Pal-2 elements), acting as either an activator or repressor; its transcriptional activity requires homodimerization that is regulated by enzyme-independent S-fatty acylation at a conserved cysteine in the dimerization domain, and it forms functional heterodimers and co-activator complexes with FOXJ1 and cooperates with CREB1 at activity-dependent enhancers in neurons, collectively controlling ciliogenesis, pancreatic beta-cell differentiation, neuronal synaptic gene expression, and left-right axis specification."},"narrative":{"mechanistic_narrative":"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].","teleology":[{"year":2004,"claim":"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","pmids":["15121860"],"confidence":"High","gaps":["Did not identify the full set of direct ciliary target genes","Did not resolve whether RFX3 acts alone or in complex at target promoters"]},{"year":2006,"claim":"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","pmids":["16930429"],"confidence":"High","gaps":["Did not establish direct vs. indirect regulation of SCO-spondin","Mechanism linking cilia loss to choroid plexus disorganization not defined"]},{"year":2007,"claim":"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","pmids":["17229940"],"confidence":"High","gaps":["Direct target genes in endocrine progenitors not yet identified","Did not separate cilia-dependent from cilia-independent transcriptional effects"]},{"year":2009,"claim":"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","pmids":["19671664"],"confidence":"High","gaps":["Whether RFX3 regulates FOXJ1 directly or indirectly not resolved","Cofactor requirements at dynein promoters not defined"]},{"year":2010,"claim":"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","pmids":["20413507"],"confidence":"High","gaps":["Genome-wide target catalog beyond Gck/Glut-2 not fully characterized","Did not address dimerization or cofactor dependence of binding"]},{"year":2012,"claim":"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","pmids":["22479201"],"confidence":"High","gaps":["Direct transcriptional targets driving FGF8/GLI3 changes not identified","Cilia-dependence of the patterning defect not fully dissected here"]},{"year":2013,"claim":"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","pmids":["23822649"],"confidence":"Medium","gaps":["Single lab, primary cells; interaction interface not mapped at this stage","Whether synergy requires direct DNA co-occupancy not shown"]},{"year":2015,"claim":"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","pmids":["25631876"],"confidence":"Medium","gaps":["Direct transcriptional targets controlling Slit1/Netrin1 heterotopias not defined","Causal cilia mechanism inferred from phenocopy, not directly tested"]},{"year":2018,"claim":"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","pmids":["30127002"],"confidence":"High","gaps":["Physiological signals regulating acylation status unknown","Whether acylation modulates partner interactions not tested"]},{"year":2018,"claim":"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","pmids":["29317724"],"confidence":"Medium","gaps":["Functional consequence for AAV transduction efficiency not fully established","Single lab; relationship to RFX3's endogenous program unclear"]},{"year":2025,"claim":"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)","pmids":["40060598"],"confidence":"Medium","gaps":["Preprint, single lab","Whether RFX3 directly recruits CREB or alters chromatin accessibility not resolved"]},{"year":2025,"claim":"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)","pmids":["40631264"],"confidence":"Medium","gaps":["Preprint, single lab","Direct vs. proximity-based interactions not distinguished"]},{"year":2025,"claim":"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","pmids":["40263183"],"confidence":"High","gaps":["Direct human islet target genes not enumerated","Mechanism of increased enterochromaffin specification not defined"]},{"year":2024,"claim":"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)","pmids":[],"confidence":"Medium","gaps":["Preprint, single lab","Determinants of activator vs. repressor switching not defined","Functional role of cytoplasmic relocalization unknown"]},{"year":2024,"claim":"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)","pmids":[],"confidence":"Medium","gaps":["Preprint, single lab; interaction not validated structurally in vitro","Competition between activating and repressing forkhead partners not quantified"]},{"year":null,"claim":"How RFX3 selects between activator and repressor states and between tissue-specific partner sets across cilia, endocrine, and neuronal contexts remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["Signals controlling fatty acylation and partner choice unknown","No unified model linking dimerization state to activator/repressor output across tissues"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0003677","term_label":"DNA binding","supporting_discovery_ids":[3,4,9,14]},{"term_id":"GO:0140110","term_label":"transcription regulator activity","supporting_discovery_ids":[0,4,6,14]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[9,14]}],"pathway":[{"term_id":"R-HSA-74160","term_label":"Gene expression (Transcription)","supporting_discovery_ids":[4,6,10]},{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[0,2,5,12]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[8,10]}],"complexes":[],"partners":["FOXJ1","FOXN3","FOXN4","CREB1","CRTC1","MEF2C","RFX1"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"P48380","full_name":"Transcription factor RFX3","aliases":["Regulatory factor X 3"],"length_aa":749,"mass_kda":83.5,"function":"Transcription factor required for ciliogenesis and islet cell differentiation during endocrine pancreas development. Essential for the differentiation of nodal monocilia and left-right asymmetry specification during embryogenesis. Required for the biogenesis of motile cilia by governing growth and beating efficiency of motile cells. Also required for ciliated ependymal cell differentiation. Regulates the expression of genes involved in ciliary assembly (DYNC2LI1, FOXJ1 and BBS4) and genes involved in ciliary motility (DNAH11, DNAH9 and DNAH5) (By similarity). Together with RFX6, participates in the differentiation of 4 of the 5 islet cell types during endocrine pancreas development, with the exception of pancreatic PP (polypeptide-producing) cells. Regulates transcription by forming a heterodimer with another RFX protein and binding to the X-box in the promoter of target genes (PubMed:20148032). Represses transcription of MAP1A in non-neuronal cells but not in neuronal cells (PubMed:12411430)","subcellular_location":"Nucleus","url":"https://www.uniprot.org/uniprotkb/P48380/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/RFX3","classification":"Not Classified","n_dependent_lines":0,"n_total_lines":1208,"dependency_fraction":0.0},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/RFX3","total_profiled":1310},"omim":[{"mim_id":"612659","title":"REGULATORY FACTOR X, 6; RFX6","url":"https://www.omim.org/entry/612659"},{"mim_id":"604110","title":"ADHESION G PROTEIN-COUPLED RECEPTOR G1; ADGRG1","url":"https://www.omim.org/entry/604110"},{"mim_id":"601337","title":"REGULATORY FACTOR X, 3; RFX3","url":"https://www.omim.org/entry/601337"},{"mim_id":"600006","title":"REGULATORY FACTOR X, 1; RFX1","url":"https://www.omim.org/entry/600006"},{"mim_id":"165240","title":"GLI-KRUPPEL FAMILY MEMBER 3; GLI3","url":"https://www.omim.org/entry/165240"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Nucleoplasm","reliability":"Supported"},{"location":"Golgi apparatus","reliability":"Additional"},{"location":"Vesicles","reliability":"Additional"},{"location":"Cytosol","reliability":"Additional"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in all","driving_tissues":[{"tissue":"testis","ntpm":19.8}],"url":"https://www.proteinatlas.org/search/RFX3"},"hgnc":{"alias_symbol":[],"prev_symbol":[]},"alphafold":{"accession":"P48380","domains":[{"cath_id":"1.10.10.10","chopping":"158-266","consensus_level":"high","plddt":80.9528,"start":158,"end":266},{"cath_id":"-","chopping":"323-388_403-475","consensus_level":"high","plddt":91.1322,"start":323,"end":475},{"cath_id":"1.10.490","chopping":"516-662","consensus_level":"high","plddt":91.9803,"start":516,"end":662}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/P48380","model_url":"https://alphafold.ebi.ac.uk/files/AF-P48380-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-P48380-F1-predicted_aligned_error_v6.png","plddt_mean":66.62},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=RFX3","jax_strain_url":"https://www.jax.org/strain/search?query=RFX3"},"sequence":{"accession":"P48380","fasta_url":"https://rest.uniprot.org/uniprotkb/P48380.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/P48380/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/P48380"}},"corpus_meta":[{"pmid":"15121860","id":"PMC_15121860","title":"The transcription factor RFX3 directs nodal cilium development and left-right asymmetry specification.","date":"2004","source":"Molecular and cellular biology","url":"https://pubmed.ncbi.nlm.nih.gov/15121860","citation_count":175,"is_preprint":false},{"pmid":"19671664","id":"PMC_19671664","title":"RFX3 governs growth and beating efficiency of motile cilia in mouse and controls the expression of genes involved in human ciliopathies.","date":"2009","source":"Journal of cell science","url":"https://pubmed.ncbi.nlm.nih.gov/19671664","citation_count":101,"is_preprint":false},{"pmid":"16930429","id":"PMC_16930429","title":"A deficiency in RFX3 causes hydrocephalus associated with abnormal differentiation of ependymal cells.","date":"2006","source":"The European journal of neuroscience","url":"https://pubmed.ncbi.nlm.nih.gov/16930429","citation_count":92,"is_preprint":false},{"pmid":"23822649","id":"PMC_23822649","title":"RFX3 modulation of FOXJ1 regulation of cilia genes in the human airway epithelium.","date":"2013","source":"Respiratory research","url":"https://pubmed.ncbi.nlm.nih.gov/23822649","citation_count":80,"is_preprint":false},{"pmid":"17229940","id":"PMC_17229940","title":"Novel function of the ciliogenic transcription factor RFX3 in development of the endocrine pancreas.","date":"2007","source":"Diabetes","url":"https://pubmed.ncbi.nlm.nih.gov/17229940","citation_count":79,"is_preprint":false},{"pmid":"22479201","id":"PMC_22479201","title":"The ciliogenic transcription factor RFX3 regulates early midline distribution of guidepost neurons required for corpus callosum development.","date":"2012","source":"PLoS genetics","url":"https://pubmed.ncbi.nlm.nih.gov/22479201","citation_count":62,"is_preprint":false},{"pmid":"20413507","id":"PMC_20413507","title":"The transcription factor Rfx3 regulates beta-cell differentiation, function, and glucokinase 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thalamocortical tract by regulating the patterning of prethalamus and ventral telencephalon.","date":"2015","source":"Human molecular genetics","url":"https://pubmed.ncbi.nlm.nih.gov/25631876","citation_count":23,"is_preprint":false},{"pmid":"31700260","id":"PMC_31700260","title":"Ginsenoside Rg3 and Korean Red Ginseng extract epigenetically regulate the tumor-related long noncoding RNAs RFX3-AS1 and STXBP5-AS1.","date":"2019","source":"Journal of ginseng research","url":"https://pubmed.ncbi.nlm.nih.gov/31700260","citation_count":22,"is_preprint":false},{"pmid":"29317724","id":"PMC_29317724","title":"RFX1 and RFX3 Transcription Factors Interact with the D Sequence of Adeno-Associated Virus Inverted Terminal Repeat and Regulate AAV Transduction.","date":"2018","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/29317724","citation_count":9,"is_preprint":false},{"pmid":"35416616","id":"PMC_35416616","title":"CircRFX3 Up-regulates Its Host Gene RFX3 to Facilitate 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formation by regulating the expression of  <i>Insm1, Ikzf2</i>  , and  <i>Triobp</i>  genes","date":"2024-09-26","source":"bioRxiv","url":"https://doi.org/10.1101/2024.09.24.614849","citation_count":0,"is_preprint":true},{"pmid":null,"id":"bio_10.1101_2025.07.23.25332035","title":"A Rare Variant in <i>TRIOBP</i> Linked to Occupational Noise Exposure in Meniere Disease","date":"2025-07-24","source":"bioRxiv","url":"https://doi.org/10.1101/2025.07.23.25332035","citation_count":0,"is_preprint":true},{"pmid":null,"id":"bio_10.1101_2025.03.28.25324850","title":"Whole-Genome Sequencing Reveals Individual and Cohort Level Insights into Chromosome 9p Syndromes","date":"2025-03-30","source":"bioRxiv","url":"https://doi.org/10.1101/2025.03.28.25324850","citation_count":0,"is_preprint":true},{"pmid":null,"id":"bio_10.1101_2024.07.30.602578","title":"Ciliary biology intersects autism and congenital heart 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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.\",\n      \"method\": \"Rfx3 knockout mouse, RT-PCR for D2lic expression, electron microscopy of nodal cilia\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean KO with defined cellular phenotype, replicated across multiple readouts (LR asymmetry, cilia morphology, target gene expression), published in peer-reviewed journal\",\n      \"pmids\": [\"15121860\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"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.\",\n      \"method\": \"Rfx3 knockout mouse, ultrastructural analysis (electron microscopy), immunohistochemistry, RT-PCR\",\n      \"journal\": \"The European journal of neuroscience\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean KO with defined cellular and molecular phenotypes, multiple orthogonal methods, peer-reviewed\",\n      \"pmids\": [\"16930429\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"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.\",\n      \"method\": \"Rfx3 knockout mouse, immunofluorescence, glucose tolerance tests, electron microscopy of primary cilia\",\n      \"journal\": \"Diabetes\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean KO with defined cellular phenotype across multiple endocrine lineages, multiple orthogonal methods\",\n      \"pmids\": [\"17229940\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"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.\",\n      \"method\": \"Primary multiciliated cell culture from Rfx3-/- mouse brain, cilia motility assays, ChIP (direct promoter binding of dynein genes), RT-PCR for FOXJ1\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — direct promoter binding demonstrated by ChIP, clean KO cell system, multiple orthogonal readouts including motility assays\",\n      \"pmids\": [\"19671664\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"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.\",\n      \"method\": \"Rfx3 knockout and pancreas-specific conditional knockout mice, quantitative ChIP, ChIP sequencing, bandshift assay, RNA interference in Min6 cells, immunofluorescence, RT-PCR\",\n      \"journal\": \"Diabetes\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — direct DNA binding shown by ChIP-seq, bandshift assay, and ChIP; replicated in multiple genetic models and cell lines\",\n      \"pmids\": [\"20413507\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"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.\",\n      \"method\": \"Rfx3 knockout mouse, conditional genetic inactivation, brain explant cultures with ectopic FGF8, transplantation assays, immunohistochemistry, in situ hybridization\",\n      \"journal\": \"PLoS genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — epistasis and transplantation experiments, conditional inactivation, multiple orthogonal methods including explant rescue\",\n      \"pmids\": [\"22479201\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"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.\",\n      \"method\": \"Plasmid-mediated gene transfer into human airway basal cells, co-immunoprecipitation, promoter-reporter assays, TaqMan RT-PCR\",\n      \"journal\": \"Respiratory research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP demonstrated interaction; functional synergy shown in reporter assays; single lab, primary human cells\",\n      \"pmids\": [\"23822649\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"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.\",\n      \"method\": \"Rfx3 knockout mouse, DiI axon tracing, immunohistochemistry, comparison with Inpp5e mutant mice\",\n      \"journal\": \"Human molecular genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean KO with defined axon-tracing phenotype, corroborated by independent ciliary mutant, single lab\",\n      \"pmids\": [\"25631876\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"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.\",\n      \"method\": \"Chemical reporters of protein fatty acylation, mass spectrometry, site-directed mutagenesis of the conserved cysteine, ciliogenesis assays, Hedgehog signaling reporter assays\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro acylation assay with mass spectrometry, mutagenesis with functional readouts (dimerization, ciliogenesis, Hh signaling), multiple orthogonal methods\",\n      \"pmids\": [\"30127002\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"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.\",\n      \"method\": \"Electromobility shift assay (EMSA), supershift experiments with RFX1/RFX3 antibodies, chromatin immunoprecipitation of AAV genomes from HEK-293 cells\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — EMSA with supershift and ChIP of viral genome; single lab, two orthogonal methods\",\n      \"pmids\": [\"29317724\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"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.\",\n      \"method\": \"iPSC-derived neurons and forebrain organoids with CRISPR RFX3 loss-of-function, transcriptomics, ChIP-seq for RFX3 and CREB binding, neuronal depolarization assays\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP-seq and transcriptomics in human neurons, multiple orthogonal genomic methods; preprint, single lab\",\n      \"pmids\": [\"40060598\"],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"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.\",\n      \"method\": \"Proximity labeling (BioID) in rodent forebrain neurons, ChIP-seq for CREB1 and CRTC1 before and after glutamatergic stimulation, motif enrichment analysis\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — proximity labeling plus ChIP-seq; preprint, single lab, two orthogonal methods\",\n      \"pmids\": [\"40631264\"],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"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.\",\n      \"method\": \"CRISPR/Cas9 RFX3 KO iPSC lines differentiated into pancreatic islet organoids, scRNA-seq, bulk RNA-seq, glucose-stimulated insulin secretion assays, RFX3 overexpression rescue\",\n      \"journal\": \"Diabetologia\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — CRISPR KO in human stem cell model, rescue experiment, multiple orthogonal methods including single-cell and bulk transcriptomics and functional secretion assays; peer-reviewed\",\n      \"pmids\": [\"40263183\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"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.\",\n      \"method\": \"Single-cell transcriptomics, ChIP-seq, ATAC-seq in cochlear hair cells; conditional Rfx3 knockout mouse (inner ear)\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP-seq showing direct binding, ATAC-seq, and scRNA-seq; preprint, single lab, multiple orthogonal genomic methods\",\n      \"pmids\": [],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"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.\",\n      \"method\": \"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\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct protein interaction confirmed by co-IP and mutagenesis, chromatin binding by CUT&RUN; preprint, single lab\",\n      \"pmids\": [],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"RFX3 is a transcription factor that directly binds X-box-like DNA motifs in promoters and enhancers of ciliogenesis genes (including intraflagellar transport components, axonemal dyneins, and hair bundle genes such as Triobp) and endocrine differentiation genes (including glucokinase/Gck via Pal-1/Pal-2 elements), acting as either an activator or repressor; its transcriptional activity requires homodimerization that is regulated by enzyme-independent S-fatty acylation at a conserved cysteine in the dimerization domain, and it forms functional heterodimers and co-activator complexes with FOXJ1 and cooperates with CREB1 at activity-dependent enhancers in neurons, collectively controlling ciliogenesis, pancreatic beta-cell differentiation, neuronal synaptic gene expression, and left-right axis specification.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"RFX3 is a DNA-binding transcription factor that serves as a master regulator of ciliogenesis and of endocrine and neuronal differentiation programs [#0, #2, #3]. 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 [#0, #1, #3]. 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 [#2, #4, #12]. 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 [#8]. 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 [#6, #14]. In neurons, RFX3 cooperates with CREB1/CRTC1 at activity-dependent enhancers to facilitate depolarization-induced transcription [#10, #11]. 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 [#5, #7, #14].\",\n  \"teleology\": [\n    {\n      \"year\": 2004,\n      \"claim\": \"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.\",\n      \"evidence\": \"Rfx3 knockout mouse with EM of nodal cilia and RT-PCR of D2lic\",\n      \"pmids\": [\"15121860\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not identify the full set of direct ciliary target genes\", \"Did not resolve whether RFX3 acts alone or in complex at target promoters\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"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.\",\n      \"evidence\": \"Rfx3 knockout mouse with EM, immunohistochemistry, RT-PCR\",\n      \"pmids\": [\"16930429\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not establish direct vs. indirect regulation of SCO-spondin\", \"Mechanism linking cilia loss to choroid plexus disorganization not defined\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Demonstrated a non-neural role in pancreatic endocrine cell differentiation and islet cilia formation, showing RFX3 governs the balance of hormone-producing lineages.\",\n      \"evidence\": \"Rfx3 knockout mouse, immunofluorescence, glucose tolerance tests, EM\",\n      \"pmids\": [\"17229940\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct target genes in endocrine progenitors not yet identified\", \"Did not separate cilia-dependent from cilia-independent transcriptional effects\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Provided direct binding evidence that RFX3 occupies axonemal dynein promoters and promotes FOXJ1 expression, mechanistically linking it to ciliary motility programs.\",\n      \"evidence\": \"Rfx3-/- primary multiciliated cells, ChIP, motility assays, RT-PCR\",\n      \"pmids\": [\"19671664\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether RFX3 regulates FOXJ1 directly or indirectly not resolved\", \"Cofactor requirements at dynein promoters not defined\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"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.\",\n      \"evidence\": \"Rfx3 KO and conditional KO mice, ChIP-seq, qChIP, bandshift, RNAi in Min6 cells\",\n      \"pmids\": [\"20413507\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Genome-wide target catalog beyond Gck/Glut-2 not fully characterized\", \"Did not address dimerization or cofactor dependence of binding\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"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.\",\n      \"evidence\": \"Rfx3 KO, conditional inactivation, explant ectopic FGF8 and transplantation assays\",\n      \"pmids\": [\"22479201\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct transcriptional targets driving FGF8/GLI3 changes not identified\", \"Cilia-dependence of the patterning defect not fully dissected here\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"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.\",\n      \"evidence\": \"Co-IP, promoter-reporter and RT-PCR in human airway basal cells\",\n      \"pmids\": [\"23822649\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab, primary cells; interaction interface not mapped at this stage\", \"Whether synergy requires direct DNA co-occupancy not shown\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Implicated RFX3 in thalamocortical axon guidance through prethalamic/telencephalic patterning, with phenocopy by a ciliary mutant supporting cilia-dependent signaling.\",\n      \"evidence\": \"Rfx3 KO with DiI tracing, IHC, comparison to Inpp5e mutant\",\n      \"pmids\": [\"25631876\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct transcriptional targets controlling Slit1/Netrin1 heterotopias not defined\", \"Causal cilia mechanism inferred from phenocopy, not directly tested\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"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.\",\n      \"evidence\": \"Chemical acylation reporters, mass spectrometry, cysteine mutagenesis, ciliogenesis and Hh reporter assays\",\n      \"pmids\": [\"30127002\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Physiological signals regulating acylation status unknown\", \"Whether acylation modulates partner interactions not tested\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"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.\",\n      \"evidence\": \"EMSA with supershift, ChIP of AAV genomes from HEK-293 cells\",\n      \"pmids\": [\"29317724\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Functional consequence for AAV transduction efficiency not fully established\", \"Single lab; relationship to RFX3's endogenous program unclear\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"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.\",\n      \"evidence\": \"iPSC-derived neurons/organoids with CRISPR RFX3 LOF, RFX3 and CREB ChIP-seq, transcriptomics, depolarization assays (preprint)\",\n      \"pmids\": [\"40060598\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Preprint, single lab\", \"Whether RFX3 directly recruits CREB or alters chromatin accessibility not resolved\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Defined activity-dependent protein partnerships of RFX3 with MEF2C and CREB1/CRTC1, supporting cooperative chromatin binding at synaptic-activity enhancers.\",\n      \"evidence\": \"BioID proximity labeling in rodent forebrain neurons, CREB1/CRTC1 ChIP-seq with glutamatergic stimulation, motif enrichment (preprint)\",\n      \"pmids\": [\"40631264\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Preprint, single lab\", \"Direct vs. proximity-based interactions not distinguished\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"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.\",\n      \"evidence\": \"CRISPR RFX3 KO iPSC islet organoids, scRNA-seq, bulk RNA-seq, GSIS assays, overexpression rescue\",\n      \"pmids\": [\"40263183\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct human islet target genes not enumerated\", \"Mechanism of increased enterochromaffin specification not defined\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"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.\",\n      \"evidence\": \"scRNA-seq, ChIP-seq, ATAC-seq, conditional inner-ear Rfx3 KO (preprint)\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Preprint, single lab\", \"Determinants of activator vs. repressor switching not defined\", \"Functional role of cytoplasmic relocalization unknown\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"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.\",\n      \"evidence\": \"CUT&RUN, co-IP/pulldown, dimerization-domain mutagenesis, AlphaFold3 prediction, reporter assays in Foxn3 KO retina (preprint)\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Preprint, single lab; interaction not validated structurally in vitro\", \"Competition between activating and repressing forkhead partners not quantified\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How RFX3 selects between activator and repressor states and between tissue-specific partner sets across cilia, endocrine, and neuronal contexts remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Signals controlling fatty acylation and partner choice unknown\", \"No unified model linking dimerization state to activator/repressor output across tissues\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0003677\", \"supporting_discovery_ids\": [3, 4, 9, 14]},\n      {\"term_id\": \"GO:0140110\", \"supporting_discovery_ids\": [0, 4, 6, 14]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [9, 14]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-74160\", \"supporting_discovery_ids\": [4, 6, 10]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [0, 2, 5, 12]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [8, 10]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"FOXJ1\", \"FOXN3\", \"FOXN4\", \"CREB1\", \"CRTC1\", \"MEF2C\", \"RFX1\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":7,"faith_pct":85.71428571428571}}