{"gene":"USF2","run_date":"2026-04-28T21:43:01","timeline":{"discoveries":[{"year":1996,"finding":"USF2 contains two distinct nuclear localization signals: the basic region and the highly conserved USF-specific region (USR), both of which are required for nuclear localization. Two transcriptional activation domains were identified: the USR (context-dependent, requires the adenovirus major late minimal promoter or an initiator element) and the exon 5-encoded region (active in multiple contexts). Activity of the exon 5 domain varies with conformation induced by alternatively spliced exon 4.","method":"Indirect immunofluorescence of transiently transfected cells, cotransfection assays with deletion mutants fused to DNA-binding domains of USF2 or GAL4, reporter assays with multiple promoter constructs","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 1-2 — multiple orthogonal methods (immunofluorescence, deletion mutagenesis, reporter assays) in a single rigorous study","pmids":["8657110"],"is_preprint":false},{"year":1997,"finding":"USF2 (via its bHLH domain) forms heterodimers with the bZip transcription factor c-Maf through the USF2 basic region and HLH motif; the leucine zipper is required for USF2 homodimerization but not for c-Maf heterodimerization. USF2 binding to c-Maf markedly reduces c-Maf DNA-binding activity, suggesting a mechanism of transcriptional repression.","method":"Yeast two-hybrid, in vitro binding with USF2 deletion/mutation mutants, DNA binding assays","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2-3 — single lab, multiple binding and functional assays but no in vivo confirmation","pmids":["9070273"],"is_preprint":false},{"year":1997,"finding":"USF2 activity is required for glucose-stimulated L-pyruvate kinase (L-PK) promoter activity in single living islet beta-cells; microinjection of anti-USF2 antibodies inhibited L-PK promoter activity by 71–87% at high glucose, and this effect depended on the intact upstream L4 box (two E-boxes) in the L-PK promoter.","method":"Photon-counting digital imaging of luciferase in single living cells, microinjection of antibodies, promoter deletion constructs","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1-2 — novel single-cell imaging method with antibody microinjection and promoter deletion controls in a single rigorous study","pmids":["9252379"],"is_preprint":false},{"year":1998,"finding":"USF2 undergoes nuclear translocation in mast cells upon activation by IL-3 or IgE-antigen stimulation. Prevention of this translocation using a peptide derived from the USF-specific NLS region significantly inhibited IL-3-mediated mast cell survival, directly linking USF2 nuclear translocation to cell viability.","method":"Subcellular fractionation/localization, peptide inhibition of nuclear translocation, cell viability assays","journal":"Journal of immunology","confidence":"Medium","confidence_rationale":"Tier 2-3 — direct localization experiment with functional consequence via peptide inhibition, single lab","pmids":["9743349"],"is_preprint":false},{"year":1999,"finding":"The coactivator p300 interacts functionally with USF2 to potentiate USF2-dependent transcriptional activation of the F1F0 ATP synthase alpha-subunit (ATPA) initiator element. Wild-type adenovirus E1A (but not p300-binding-deficient E1A mutants) inhibited this activation; overexpression of p300 reversed the E1A inhibitory effect.","method":"Transfection/reporter assays, E1A mutant expression, p300 overexpression rescue experiments","journal":"Biochimica et biophysica acta","confidence":"Medium","confidence_rationale":"Tier 2-3 — functional interaction demonstrated with mutant controls, single lab","pmids":["10434034"],"is_preprint":false},{"year":2000,"finding":"USF1, USF2, and alpha-Pal/NRF-1 are the major transcription factors binding the FMR1 promoter in brain and testis extracts. Methylation abolishes alpha-Pal/NRF-1 binding and affects USF1/USF2 binding to a lesser degree, suggesting methylation inhibits FMR1 transcription partly by blocking these factor binding events.","method":"EMSA with brain and testis extracts, methylation-dependent binding assays, transcriptional activity assays in neuronally derived cells with mutation analysis","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods (EMSA, footprinting, transfection) with physiologically relevant extracts; replicated across multiple experiments","pmids":["11058604"],"is_preprint":false},{"year":2001,"finding":"USF1 and USF2 bind to E-box motifs (especially E-box B) in the APC tumor suppressor gene promoter and are required for its basal transcriptional activity; mutation of E-box B abolished basal promoter activity in HCT-116 cells.","method":"EMSA, transient transfection with E-box deletion/mutation constructs, cotransfection with USF expression vectors","journal":"Journal of cellular biochemistry","confidence":"Medium","confidence_rationale":"Tier 2-3 — EMSA plus functional reporter assays with mutagenesis, single lab","pmids":["11241666"],"is_preprint":false},{"year":2001,"finding":"A USF1/USF2 heterodimer activates human FcεRI alpha chain gene expression by binding to a CAGCTG (E-box) element in the first intron. Overexpression of USF2 antisense repressed the FcεRI alpha chain promoter and decreased alpha chain mRNA in mast cell lines.","method":"EMSA with antibodies and in vitro translation products, antisense overexpression, promoter-reporter assays","journal":"European journal of immunology","confidence":"Medium","confidence_rationale":"Tier 2-3 — EMSA with antibody supershift, antisense knockdown with mRNA readout, single lab","pmids":["11180124"],"is_preprint":false},{"year":2001,"finding":"USF1 and USF2 bind the E-box in the proximal promoter of the HOXB4 gene and activate its transcription in hematopoietic stem cells (K562 and CD34+ cells) via activation of the MAPK pathway; this was shown by yeast one-hybrid screening, EMSA, and cotransfection assays.","method":"Yeast one-hybrid screen, EMSA, cotransfection with MAPK pathway analysis, reporter assays in K562 and CD34+ cells","journal":"The Journal of experimental medicine","confidence":"Medium","confidence_rationale":"Tier 2 — multiple methods including yeast one-hybrid, EMSA, and functional cotransfection; single lab","pmids":["11085749"],"is_preprint":false},{"year":2002,"finding":"ChREBP, not USF2, mediates glucose-stimulated endogenous L-PK expression in INS-1 insulin-secreting cells. Inducible overexpression of USF-1 and USF-2 did not alter basal or glucose-responsive L-PK mRNA, and dominant-negative USF (which abolished USF binding to the L-PK promoter) also had no effect on L-PK expression in INS-1 cells.","method":"Tet-on inducible expression of USF1, USF2, and dominant-negative mutants; Northern blot; EMSA; nuclear run-on experiments; Western blot of nuclear fractions","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1-2 — rigorous inducible system with dominant negatives, EMSA, and nuclear run-on; multiple orthogonal approaches","pmids":["12087089"],"is_preprint":false},{"year":2002,"finding":"USF-1 and USF-2 bind an E-box at -893/-888 in the iNOS promoter in vivo and act as transcriptional repressors of IL-1β-induced iNOS expression in murine mesangial cells. Mutation of the E-box augmented iNOS response, and cotransfection of dominant-negative USF-2 or cis-element decoys enhanced IL-1β stimulation.","method":"Site-directed mutagenesis of iNOS promoter, EMSA with supershift, cotransfection with USF expression vectors and dominant-negative mutants, cis-decoy experiments","journal":"American journal of physiology. Cell physiology","confidence":"High","confidence_rationale":"Tier 1-2 — multiple orthogonal methods including mutagenesis, EMSA, dominant-negative cotransfection, and decoy experiments","pmids":["12225970"],"is_preprint":false},{"year":2003,"finding":"USF2, but not USF1, specifically transactivates the IGF2R promoter in nontumorigenic mammary epithelial cells (MCF-10A); USF1 and USF2 both bind IGF2R E-boxes in vitro and in chromatin, but a USF-specific dominant-negative mutant markedly decreased IGF2R mRNA. IGF2R promoter activity was USF-independent in breast cancer cell lines, suggesting loss of USF function contributes to IGF2R downregulation in cancer.","method":"In vitro DNA binding, chromatin immunoprecipitation (ChIP), transactivation assays with overexpressed USF2 vs USF1, dominant-negative USF expression, endogenous mRNA measurement","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods including ChIP, in vitro binding, and dominant-negative KD across multiple cell lines","pmids":["12857727"],"is_preprint":false},{"year":2004,"finding":"USF1 and USF2 constitutively bind the class B E-box in the proximal promoter of the human HO-1 gene in vivo, are required for high-level HO-1 expression, and enhance HO-1 transcription in response to heme and cadmium in human renal proximal tubular epithelial cells. A single guanine contact point in the E-box is essential for USF binding.","method":"DMS in vivo footprinting, ChIP, EMSA, overexpression of USF1/USF2, dominant-negative USF expression, reporter assays","journal":"The Biochemical journal","confidence":"High","confidence_rationale":"Tier 1-2 — in vivo footprinting, ChIP, and functional assays with dominant negatives; multiple orthogonal methods","pmids":["15242350"],"is_preprint":false},{"year":2004,"finding":"A novel USF2 isoform (USF2c), generated by alternative splicing using a cryptic acceptor site within exon 6 (missing exons 4, 5, and part of exon 6), encodes a ~29 kDa protein that binds as a homodimer to the cathepsin B promoter E-box and acts as a transcriptional repressor of cathepsin B expression.","method":"RT-PCR cloning, in vitro transcription/translation, EMSA, cotransfection reporter assays","journal":"Gene","confidence":"Medium","confidence_rationale":"Tier 2-3 — in vitro binding and functional reporter assays, single lab","pmids":["15276216"],"is_preprint":false},{"year":2005,"finding":"USF1 and USF2 bind to a repressor E-box site at -114 to -119 of the human MCT1 promoter and act as repressors of MCT1 transcription in Caco-2 colonic cells, as shown by EMSA, site-directed mutagenesis, and cotransfection of USF expression vectors decreasing endogenous MCT1 expression.","method":"EMSA, site-directed mutagenesis, cotransfection with USF expression vectors, endogenous MCT1 mRNA measurement","journal":"American journal of physiology. Gastrointestinal and liver physiology","confidence":"Medium","confidence_rationale":"Tier 2-3 — EMSA plus mutagenesis plus overexpression effect on endogenous gene; single lab","pmids":["15691871"],"is_preprint":false},{"year":2005,"finding":"Transcriptionally active USF2 suppresses the tumorigenicity and anchorage-independent growth of c-Myc-transformed fibroblasts; this required transcriptionally active (not inactive) USF2, and cotransfection assays indicated active USF2 inhibited cellular transformation by preventing c-Myc-mediated transcriptional repression.","method":"Stable cell line generation, soft agar colony assay, nude mice xenograft, cotransfection with USF and Myc dominant-negative mutants","journal":"Experimental cell research","confidence":"Medium","confidence_rationale":"Tier 2 — in vivo xenograft plus in vitro epistasis with dominant-negative mutants; single lab","pmids":["15541720"],"is_preprint":false},{"year":2005,"finding":"USF2 (LysRS partner) is regulated by Ap4A in mast cells: LysRS associates with USF2 and negatively regulates its activity; Ap4A produced by LysRS acts as a positive regulator of USF2 transcriptional activity, analogous to the mechanism described for MITF. The Hint protein negatively regulates USF2 in this pathway.","method":"Co-immunoprecipitation (Co-IP), functional transcriptional assays in mast cells, biochemical assays of Ap4A synthesis","journal":"Molecular and cellular biology","confidence":"Medium","confidence_rationale":"Tier 2-3 — Co-IP plus functional transcriptional assays; single lab","pmids":["16199869"],"is_preprint":false},{"year":2006,"finding":"USF2 functions as a tumor suppressor in prostate carcinogenesis: ectopic USF2 expression in androgen-independent PC-3 cells inhibited anchorage-independent growth (90-98%), invasion (80%), and in vivo tumorigenicity (80-90%) in nude mice. USF2-null mice exhibited marked prostate hyperplasia, confirming an in vivo role in normal prostate growth and differentiation.","method":"Western blot of cancer cell lines, ectopic overexpression, soft agar assay, Matrigel invasion assay, nude mouse xenograft, USF2 knockout mice generation and phenotyping, IHC of human prostate cancer tissues","journal":"Oncogene","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods including KO mouse with phenotype, xenograft, and in vitro functional assays; replicated across in vitro and in vivo systems","pmids":["16186802"],"is_preprint":false},{"year":2008,"finding":"Whole-genome ChIP-chip analysis revealed that USF1 and USF2 bind predominantly near transcription start sites of protein-coding genes in a liver cell line, their binding positively correlates with gene expression levels, and they frequently occupy bidirectional promoters. USF1 and USF2 co-occupy promoters of nuclear-mitochondrial genes and lipid/cholesterol metabolism genes, often in collaboration with GABPA/NRF-2.","method":"ChIP-chip (genome-wide, 35 bp resolution) in HepG2 liver cell line, correlation with expression data","journal":"Genome research","confidence":"High","confidence_rationale":"Tier 2 — genome-wide ChIP-chip is a strong method; single lab but large-scale systematic analysis","pmids":["18230803"],"is_preprint":false},{"year":2008,"finding":"USF-2 interacts with Fra-1 (an AP-1 factor) via protein-protein interaction, and this USF-2/Fra-1 cooperation regulates HO-1 promoter activity in opposing ways in primary versus tumor cells. Mutation of either the E-box or AP-1 site in the HO-1 promoter only partially affected USF-dependent regulation; double mutation abolished it. USF-2 lacking its USF-specific region (USR) or exon 4-encoded transactivation domain lost regulatory capacity.","method":"Protein-protein interaction studies, promoter mutagenesis (E-box and AP-1 site), USF-2 domain deletion analysis, reporter assays in primary hepatocytes vs tumor cell lines","journal":"Antioxidants & redox signaling","confidence":"Medium","confidence_rationale":"Tier 2-3 — protein interaction plus mutagenesis in two cell types; single lab","pmids":["18331200"],"is_preprint":false},{"year":2009,"finding":"HINT1 co-immunoprecipitates with USF2 in HepG2 hepatoma cells and inhibits USF2 transcriptional activity, as well as inhibiting endogenous cyclin D1 and TGFβ2 expression controlled by USF2.","method":"Co-immunoprecipitation, reporter assays for USF2 transcriptional activity, endogenous gene expression analysis after HINT1 overexpression","journal":"International journal of cancer","confidence":"Medium","confidence_rationale":"Tier 2-3 — Co-IP plus functional transcriptional assays; single lab","pmids":["19089909"],"is_preprint":false},{"year":2009,"finding":"The rs1867277 A allele in the FOXE1 5' UTR specifically recruits the USF1/USF2 transcription factor complex (but not the G allele), as demonstrated by DNA-binding assays, and this allele-specific recruitment leads to differential transcriptional regulation of FOXE1.","method":"DNA-binding assays (EMSA with USF1/USF2 antibodies), transfection/reporter assays with allele-specific constructs","journal":"PLoS genetics","confidence":"Medium","confidence_rationale":"Tier 2-3 — EMSA with antibody supershift plus reporter assay; single lab but validated in two independent cohorts at the GWAS level","pmids":["19730683"],"is_preprint":false},{"year":2011,"finding":"In TGF-β signaling, USF2 operates in a SMAD/p53/USF2→PAI-1 transcriptional axis: SMAD2/3, pp60c-src, EGFR, and p53 activation are each required for TGF-β1-induced PAI-1 expression in the context of renal fibrosis and ureteral obstruction.","method":"Pathway inhibitor studies, reporter assays, in vivo UUO mouse model with pathway component activation measured","journal":"Cell and tissue research","confidence":"Medium","confidence_rationale":"Tier 2-3 — in vivo model plus multiple pathway inhibitors; review-style synthesis with experimental data cited from multiple studies","pmids":["21638209"],"is_preprint":false},{"year":2011,"finding":"ID1 inhibits USF2 transcriptional activity in mesangial cells, thereby blocking TGF-β-induced apoptosis. TGF-β upregulates USF2, which increases BAX expression and apoptosis; BMPs induce ID1 which counteracts USF2 activity to exert an anti-apoptotic effect.","method":"Expression manipulation of ID1 and USF2, apoptosis assays, BAX expression measurement, reporter assays in human mesangial cells","journal":"American journal of physiology. Renal physiology","confidence":"Medium","confidence_rationale":"Tier 2-3 — functional assays with overexpression/knockdown and apoptosis readout; single lab","pmids":["21921026"],"is_preprint":false},{"year":2013,"finding":"HIF2α physically interacts with USF2 in a manner dependent on HIF2α's N-TAD domain, and this interaction is required for selective activation of HIF2 target genes. USF2 exhibits specific binding to the promoters of HIF2 (but not HIF1) target genes even when overexpressed, and RNA Pol II association with HIF2 target genes is USF2-dependent.","method":"siRNA knockdown, inhibitor studies, reporter assays, chromatin immunoprecipitation (ChIP), co-immunoprecipitation with domain deletion mutants, RNA Pol II ChIP","journal":"PloS one","confidence":"High","confidence_rationale":"Tier 2 — Co-IP with domain mapping, ChIP, and reporter assays; multiple orthogonal methods establishing USF2 as a HIF2-specific transcriptional partner","pmids":["23991099"],"is_preprint":false},{"year":2018,"finding":"USF2 binds to E-box motifs in the Smurf1 and Smurf2 promoters (in vitro and in vivo by ChIP) and inhibits their transcriptional activity, resulting in decreased Smurf mRNA and enhanced TGF-β pathway activity in breast cancer cells.","method":"Luciferase reporter assays with Smurf promoters, ChIP, overexpression of USF2, endogenous mRNA measurement","journal":"Cellular signalling","confidence":"Medium","confidence_rationale":"Tier 2-3 — ChIP plus reporter and mRNA assays; single lab","pmids":["30244169"],"is_preprint":false},{"year":2018,"finding":"USF2 and PLAG1 co-regulate MSI2 (Musashi-2) transcription in human hematopoietic stem and progenitor cells; both factors bind the MSI2 promoter (confirmed by ChIP-seq), are required for efficient transactivation of endogenous MSI2, and their coincident overexpression expands CD34+ cells in vitro.","method":"Luciferase reporter assays, ChIP-seq (global co-binding analysis), shRNA knockdown of USF2 and PLAG1, overexpression in cord blood cells with CD34+ expansion assay","journal":"Stem cell reports","confidence":"High","confidence_rationale":"Tier 2 — genome-wide ChIP-seq co-binding, reporter assays, and functional overexpression with defined phenotypic readout; multiple orthogonal approaches","pmids":["29641991"],"is_preprint":false},{"year":2019,"finding":"CDK5 phosphorylates USF2 at two serine residues, S155 and S222; phosphorylation at these sites stabilizes the USF2 protein and regulates cellular growth and migration in cancer cells.","method":"In vitro kinase assays, site-directed mutagenesis (S155A and S222A), protein stability assays, functional cell growth and migration assays","journal":"Cancers","confidence":"High","confidence_rationale":"Tier 1-2 — in vitro kinase assay plus mutagenesis plus functional readouts; multiple orthogonal methods","pmids":["31013770"],"is_preprint":false},{"year":2019,"finding":"USF2 directly binds the ATF4 transcriptional initiation region and regulates its transcriptional activity in periodontal ligament cells; USF2 overexpression promotes osteogenic differentiation (osteoblast-specific gene expression and mineralization), and this effect is reversed by simultaneous ATF4 silencing.","method":"Luciferase reporter assays, overexpression and silencing systems, Alizarin red staining, qRT-PCR, ELISA, microarray","journal":"Journal of periodontal research","confidence":"Medium","confidence_rationale":"Tier 2-3 — reporter assays with epistasis (USF2 OE + ATF4 KD rescue), single lab","pmids":["31448831"],"is_preprint":false},{"year":2020,"finding":"USF2 directly regulates proinflammatory cytokine production (IL-17A, IFN-γ, IL-22, GM-CSF) and T-bet expression in pathogenic Th17 cells; shRNA targeting USF2 in these cells reduced expression of these cytokines, identifying USF2 as an upstream regulator of the Th17 pathogenic transcriptional program.","method":"shRNA knockdown of USF2 in isolated Th17 cells, gene expression analysis, gene set enrichment analysis identifying USF2 as upstream regulator","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"Medium","confidence_rationale":"Tier 2-3 — shRNA KD with defined cytokine readouts; single study but in primary human cells","pmids":["33203678"],"is_preprint":false},{"year":2021,"finding":"USF2 transcriptionally activates THBS1 by binding to its promoter, promoting oxidative stress and activating the TGF-β/Smad3/NLRP3/Caspase-1 pathway to stimulate pyroptosis in sepsis-induced acute kidney injury. USF2 knockdown reduced THBS1, inhibited this pathway, and ameliorated kidney injury.","method":"ChIP/reporter assays for USF2→THBS1 transcriptional activation, USF2 knockdown (shRNA/siRNA), in vivo mouse AKI model, pyroptosis assays (Caspase-1, GSDMD-N, LDH), pathway protein measurements","journal":"Pharmacological research","confidence":"Medium","confidence_rationale":"Tier 2-3 — ChIP and reporter assays for direct binding, plus in vivo knockdown with phenotypic readout; single lab","pmids":["34756923"],"is_preprint":false},{"year":2021,"finding":"TGF-β upregulates USF2, which transcriptionally activates S100A8 by directly binding its promoter (confirmed by luciferase reporter and ChIP assays), thereby promoting EMT and metastasis in colorectal cancer cells. Extracellular S100A8 conversely suppresses the USF2/S100A8 axis.","method":"Luciferase reporter assays, ChIP, Western blot, migration/invasion assays, mouse metastasis models, siRNA knockdown","journal":"Cancer communications","confidence":"Medium","confidence_rationale":"Tier 2-3 — ChIP and reporter assays confirming direct binding plus in vivo mouse model; single lab","pmids":["33389821"],"is_preprint":false},{"year":2022,"finding":"USF2 transcriptionally represses TXNRD1 by directly binding to two E-box sites in the TXNRD1 promoter in hepatocellular carcinoma cells, functioning as a tumor suppressor through downstream repression of TXNRD1-Akt/mTOR signaling.","method":"ChIP, reporter assays, USF2 overexpression/knockdown, TXNRD1 functional assays, in vitro and in vivo experiments","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2-3 — ChIP confirming direct E-box binding plus functional mechanistic assays; single lab","pmids":["36319631"],"is_preprint":false},{"year":2023,"finding":"USF2 transcriptionally activates YTHDF1 (an m6A reader) by binding to its promoter; YTHDF1 then suppresses autophagy by increasing m6A-mediated stability of Cdc25A (an autophagy inhibitor), thereby exacerbating ischemia-reperfusion injury in neurons. Usf2 interference improved autophagy and reduced neuronal apoptosis in vitro and in vivo.","method":"Promoter binding assays, lentiviral shRNA knockdown, MCAO in vivo model, m6A stability assays, autophagy and apoptosis readouts","journal":"Molecular neurobiology","confidence":"Medium","confidence_rationale":"Tier 2-3 — promoter binding plus epistasis (USF2→YTHDF1→Cdc25A) with in vivo and in vitro validation; single lab","pmids":["37914905"],"is_preprint":false},{"year":2023,"finding":"USF2 negatively regulates STX6 transcription in hepatocellular carcinoma; under USF2 control, STX6 promotes autophagosome-lysosome fusion and accelerates LC3B degradation, promoting HCC progression.","method":"Reporter assays, USF2 and STX6 overexpression/knockdown, autophagy flux assays, in vitro and in vivo HCC functional experiments","journal":"International journal of biological sciences","confidence":"Medium","confidence_rationale":"Tier 2-3 — reporter assays plus functional autophagy flux experiments; single lab","pmids":["37564208"],"is_preprint":false},{"year":2024,"finding":"USF2 acts as a transcriptional repressor of lysosomal and autophagy genes under nutrient-rich conditions by binding CLEAR motifs together with HDAC1, reducing histone H3K27 acetylation and chromatin accessibility. Under starvation, USF2 competes with TFEB for CLEAR motif binding in a phosphorylation-dependent manner; GSK3β-mediated phosphorylation of USF2 S155 governs its DNA-binding activity and lysosomal gene repression.","method":"ChIP, chromatin accessibility assays, histone acetylation profiling, reporter assays, co-IP with HDAC1, USF2-TFEB competition assays, GSK3β kinase assays with S155 mutagenesis, USF2 knockout cells/mice","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1-2 — multiple orthogonal methods (ChIP, chromatin accessibility, histone modification, in vivo KO, kinase assay with mutagenesis) in a single rigorous study","pmids":["39333072"],"is_preprint":false}],"current_model":"USF2 is a bHLH-leucine zipper transcription factor that binds E-box/CLEAR motifs at target gene promoters as homodimers or USF1/USF2 heterodimers; it can activate or repress transcription depending on context (promoter architecture, binding partners, and phosphorylation state), interacts with coactivators (p300) and corepressors (HDAC1, HINT1, ID1), is regulated by CDK5-mediated phosphorylation (S155, S222) that controls its stability and DNA-binding activity, undergoes signal-dependent nuclear translocation in mast cells, competes with TFEB at CLEAR motifs to suppress lysosomal/autophagy genes under nutrient-rich conditions in a GSK3β-phosphorylation-dependent manner, and functions as a context-dependent transcriptional regulator of target genes involved in metabolism, fibrosis (PAI-1, THBS1), immunity (FcεRI, Th17 cytokines), proliferation, and tumor suppression (IGF2R, prostate)."},"narrative":{"teleology":[{"year":1996,"claim":"Mapping USF2's functional architecture resolved how a bHLH-zip protein achieves nuclear entry and context-dependent transactivation, identifying dual NLS elements and two activation domains modulated by alternative splicing.","evidence":"Deletion mutagenesis fused to GAL4/USF2 DNA-binding domains, immunofluorescence of transfected cells, reporter assays with multiple promoter constructs","pmids":["8657110"],"confidence":"High","gaps":["Crystal structure of USF2 activation domains not determined","Mechanism by which exon 4 splicing alters exon 5 domain conformation unknown"]},{"year":1997,"claim":"Discovery of USF2/c-Maf heterodimerization via the bHLH domain established that USF2 can sequester non-canonical partners to repress their DNA binding, expanding its regulatory repertoire beyond E-box-dependent mechanisms.","evidence":"Yeast two-hybrid, in vitro binding with deletion/mutation mutants, DNA binding assays","pmids":["9070273"],"confidence":"Medium","gaps":["No in vivo confirmation of USF2–c-Maf interaction","Physiological target genes of this heterodimer not identified"]},{"year":1997,"claim":"Single-cell antibody microinjection demonstrated that USF2 is required for glucose-stimulated L-PK promoter activity in pancreatic β-cells, providing the first evidence that USF2 mediates metabolic gene regulation in a stimulus-dependent manner.","evidence":"Photon-counting imaging of luciferase in living islet β-cells, anti-USF2 antibody microinjection, promoter deletion constructs","pmids":["9252379"],"confidence":"High","gaps":["Later work in INS-1 cells attributed glucose-responsive L-PK expression to ChREBP rather than USF2, indicating cell-type specificity (PMID:12087089)"]},{"year":1998,"claim":"Demonstration that USF2 undergoes signal-dependent nuclear translocation in mast cells upon IL-3 or IgE stimulation, with NLS-derived peptide blockade impairing cell survival, established USF2 as a signal-regulated transcription factor controlling immune cell viability.","evidence":"Subcellular fractionation, NLS-peptide inhibition of nuclear translocation, cell viability assays in mast cells","pmids":["9743349"],"confidence":"Medium","gaps":["Upstream kinase/signaling pathway triggering nuclear import not identified","Peptide specificity for USF2 versus other NLS-containing proteins not fully addressed"]},{"year":1999,"claim":"Identification of p300 as a functional coactivator of USF2 at the ATP synthase α-subunit initiator element revealed a coactivator-dependent mechanism for USF2-mediated transcriptional activation, linking USF2 to the HAT-dependent chromatin remodeling machinery.","evidence":"Reporter assays with E1A mutants (p300-binding-deficient), p300 overexpression rescue","pmids":["10434034"],"confidence":"Medium","gaps":["Direct physical interaction between USF2 and p300 not demonstrated by co-IP","Whether p300 recruitment is required at all USF2 target promoters is unknown"]},{"year":2001,"claim":"Multiple studies established USF2 as a versatile E-box-dependent transcriptional regulator of diverse target genes including APC, FcεRI, and HOXB4, demonstrating that USF1/USF2 homo- and heterodimers can either activate or repress transcription depending on the target promoter.","evidence":"EMSA with supershift, reporter assays with E-box mutations, antisense knockdown of USF2, cotransfection in multiple cell types (HCT-116, mast cells, K562/CD34+ cells)","pmids":["11241666","11180124","11085749"],"confidence":"Medium","gaps":["Determinants of whether USF2 activates or represses at a given E-box not resolved","Genome-wide target repertoire not yet known at this stage"]},{"year":2002,"claim":"USF2 was shown to repress iNOS transcription by binding a distal E-box in the iNOS promoter, directly demonstrating a gene-repressive function distinct from its activator role, and was confirmed to be dispensable for glucose-responsive L-PK expression in INS-1 cells.","evidence":"Dominant-negative USF2, cis-decoy experiments, site-directed mutagenesis (iNOS); inducible USF expression, nuclear run-on (L-PK)","pmids":["12225970","12087089"],"confidence":"High","gaps":["Chromatin-level mechanism of USF2-mediated repression at iNOS not elucidated","Cell-type determinants of USF2 dispensability at metabolic promoters unclear"]},{"year":2003,"claim":"ChIP and dominant-negative experiments revealed USF2 specifically transactivates IGF2R in nontumorigenic mammary cells but not in breast cancer lines, providing the first evidence that loss of USF2 function could contribute to oncogenesis through deregulation of a specific target gene.","evidence":"ChIP, in vitro binding, reporter assays, dominant-negative USF, endogenous mRNA measurement across MCF-10A and cancer cell lines","pmids":["12857727"],"confidence":"High","gaps":["Mechanism of USF2 inactivation in cancer cells not determined","Whether IGF2R loss is the primary mediator of the tumorigenic phenotype was not tested"]},{"year":2005,"claim":"Functional evidence from multiple systems established USF2 as a regulator of LysRS/Ap4A signaling in mast cells and a suppressor of c-Myc-driven transformation, with transcriptionally active USF2 required to inhibit anchorage-independent growth and in vivo tumorigenesis.","evidence":"Soft agar and nude mouse xenograft with active/inactive USF2 mutants; co-IP of LysRS–USF2 complex, Ap4A functional assays in mast cells","pmids":["15541720","16199869"],"confidence":"Medium","gaps":["Whether USF2 tumor suppression is c-Myc-specific or general not resolved","HINT1 regulation of USF2 in vivo not validated"]},{"year":2006,"claim":"In vivo knockout and xenograft studies definitively established USF2 as a prostate tumor suppressor: USF2 overexpression suppressed PC-3 tumorigenicity by 80–90%, while USF2-null mice developed marked prostate hyperplasia.","evidence":"USF2 KO mice with prostate phenotyping, ectopic expression in PC-3 cells, soft agar/invasion/xenograft assays, IHC of human prostate cancer","pmids":["16186802"],"confidence":"High","gaps":["USF2 target genes mediating prostate tumor suppression not identified","Whether USF2 loss is sufficient for frank malignancy versus hyperplasia not resolved"]},{"year":2008,"claim":"Genome-wide ChIP-chip in liver cells revealed that USF1/USF2 predominantly bind near transcription start sites, co-occupy bidirectional promoters, and positively correlate with gene expression, establishing USF2 as a widespread promoter-proximal activator with a preference for metabolic and nuclear-mitochondrial genes.","evidence":"ChIP-chip at 35 bp resolution in HepG2 cells, correlation with expression data, co-occupancy analysis with GABPA/NRF-2","pmids":["18230803"],"confidence":"High","gaps":["ChIP-chip performed in a single cell type (HepG2); tissue-specific binding not addressed","Causal role of USF binding at individual loci not established"]},{"year":2009,"claim":"Identification of HINT1 as a USF2-interacting repressor that inhibits cyclin D1 and TGFβ2 expression, and of Fra-1 as a cooperative partner at the HO-1 promoter, revealed that USF2 output depends on its protein interaction network at individual target genes.","evidence":"Co-IP of HINT1–USF2, reporter and endogenous gene assays; USF2–Fra-1 interaction studies with E-box/AP-1 double mutagenesis","pmids":["19089909","18331200"],"confidence":"Medium","gaps":["Structural basis of HINT1–USF2 interaction not determined","Genome-wide extent of USF2–Fra-1 co-regulation unknown"]},{"year":2013,"claim":"Discovery that HIF2α physically interacts with USF2 through its N-TAD domain, and that USF2 is specifically required for RNA Pol II recruitment to HIF2 (but not HIF1) target genes, resolved how hypoxia signaling achieves HIF paralog specificity at select promoters.","evidence":"Co-IP with domain deletion mutants, ChIP for USF2 and Pol II at HIF2 vs HIF1 targets, siRNA knockdown, reporter assays","pmids":["23991099"],"confidence":"High","gaps":["Whether USF2 DNA-binding activity is required for HIF2 cooperation or just protein scaffolding is unclear","Genome-wide scope of USF2-dependent HIF2 targets not mapped"]},{"year":2018,"claim":"ChIP-seq co-binding analysis in hematopoietic progenitors showed USF2 and PLAG1 co-regulate MSI2 transcription and that their combined activity expands CD34+ cells, positioning USF2 as a regulator of stem cell self-renewal.","evidence":"ChIP-seq, shRNA knockdown, reporter assays, overexpression in cord blood CD34+ cells with expansion assay","pmids":["29641991"],"confidence":"High","gaps":["Broader in vivo hematopoietic phenotype of USF2 loss in this context not characterized","Whether USF2–PLAG1 cooperation extends beyond the MSI2 locus unknown"]},{"year":2019,"claim":"Identification of CDK5 as a kinase that phosphorylates USF2 at S155 and S222 to stabilize the protein established a post-translational regulatory mechanism controlling USF2 abundance and its effects on cell growth and migration.","evidence":"In vitro kinase assays, phospho-dead mutagenesis (S155A, S222A), protein stability and functional cell assays","pmids":["31013770"],"confidence":"High","gaps":["In vivo confirmation of CDK5-dependent USF2 phosphorylation not provided","Downstream targets specifically affected by phosphorylation-stabilized USF2 not identified"]},{"year":2020,"claim":"shRNA knockdown in primary human Th17 cells identified USF2 as an upstream regulator of the pathogenic Th17 cytokine program (IL-17A, IFN-γ, IL-22, GM-CSF, T-bet), extending USF2's functional scope to adaptive immunity.","evidence":"shRNA knockdown in isolated human Th17 cells, gene expression and gene set enrichment analysis","pmids":["33203678"],"confidence":"Medium","gaps":["Direct promoter binding of USF2 at Th17 cytokine loci not demonstrated by ChIP","In vivo Th17 phenotype of USF2 deficiency not tested"]},{"year":2021,"claim":"USF2 was shown to transcriptionally activate pro-fibrotic (THBS1) and pro-metastatic (S100A8) target genes through direct promoter binding, linking USF2 to TGF-β-driven pathology in acute kidney injury and colorectal cancer EMT.","evidence":"ChIP/reporter assays for USF2→THBS1 and USF2→S100A8, shRNA knockdown, in vivo AKI and metastasis models","pmids":["34756923","33389821"],"confidence":"Medium","gaps":["Whether USF2 is a general mediator of TGF-β transcriptional output or acts at selected targets not resolved","Relative contribution of USF2 versus other E-box factors at these loci not quantified"]},{"year":2024,"claim":"A comprehensive mechanistic study resolved how USF2 represses lysosomal/autophagy genes under nutrient-rich conditions: USF2 recruits HDAC1 to CLEAR motifs, reduces H3K27 acetylation, and competes with TFEB; GSK3β phosphorylation of S155 governs USF2 DNA-binding activity and this repression, establishing USF2 as a nutrient-sensing transcriptional switch at CLEAR elements.","evidence":"ChIP, chromatin accessibility, histone acetylation profiling, co-IP with HDAC1, USF2–TFEB competition assays, GSK3β kinase assay with S155 mutagenesis, USF2 KO cells and mice","pmids":["39333072"],"confidence":"High","gaps":["Whether mTORC1 or other nutrient sensors act upstream of GSK3β→USF2 not fully delineated","Relative importance of CDK5 vs GSK3β phosphorylation at S155 in different cellular contexts not resolved"]},{"year":null,"claim":"Key unresolved questions include: (1) how USF2 switches between activator and repressor modes at different E-box-containing promoters, (2) the full phosphorylation-dependent interactome that dictates cofactor choice, and (3) whether USF2 tumor suppressor function in prostate and liver operates through CLEAR-motif lysosomal gene repression, TXNRD1 repression, or additional pathways.","evidence":"","pmids":[],"confidence":"Low","gaps":["Structural basis of activator/repressor switch undetermined","No integrative model unifying CDK5 and GSK3β phosphorylation with cofactor selection","Causal target genes for prostate tumor suppression remain unidentified"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0003677","term_label":"DNA binding","supporting_discovery_ids":[0,2,5,6,7,8,10,11,12,13,14,18,25,26,35]},{"term_id":"GO:0140110","term_label":"transcription regulator activity","supporting_discovery_ids":[0,2,4,10,11,12,15,17,24,25,26,29,30,31,32,35]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[1,15,23,35]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[0,3,18,35]}],"pathway":[{"term_id":"R-HSA-74160","term_label":"Gene expression (Transcription)","supporting_discovery_ids":[0,2,4,10,11,12,18,24,26,29,35]},{"term_id":"R-HSA-9612973","term_label":"Autophagy","supporting_discovery_ids":[33,34,35]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[3,7,29]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[22,25,30]},{"term_id":"R-HSA-4839726","term_label":"Chromatin organization","supporting_discovery_ids":[35]}],"complexes":["USF1/USF2 heterodimer"],"partners":["USF1","HDAC1","HINT1","HIF2A","PLAG1","FOSL1","MAF","ID1"],"other_free_text":[]},"mechanistic_narrative":"USF2 is a bHLH-leucine zipper transcription factor that binds E-box and CLEAR motifs as a homodimer or USF1/USF2 heterodimer to activate or repress a broad spectrum of target genes governing metabolism, immunity, fibrosis, autophagy, and tumor suppression. USF2 contains two nuclear localization signals (the basic region and the USF-specific region) and two transactivation domains whose activity depends on alternative splicing and promoter context; it cooperates with coactivators such as p300 and HIF2α, and is inhibited by corepressors including HDAC1, HINT1, and ID1 [PMID:8657110, PMID:10434034, PMID:23991099, PMID:39333072]. Under nutrient-rich conditions, USF2 recruits HDAC1 to CLEAR motifs to repress lysosomal and autophagy genes by reducing H3K27 acetylation, and competes with TFEB upon starvation in a manner regulated by GSK3β-mediated phosphorylation of S155, while CDK5 phosphorylation of S155 and S222 independently stabilizes USF2 protein to control cell growth [PMID:39333072, PMID:31013770]. USF2 functions as a tumor suppressor in prostate and liver cancers—USF2-null mice develop prostate hyperplasia, and ectopic USF2 suppresses tumorigenicity in prostate cancer xenografts—while also activating proinflammatory cytokine programs in pathogenic Th17 cells and driving fibrotic gene expression (PAI-1, THBS1) through TGF-β signaling [PMID:16186802, PMID:33203678, PMID:34756923]."},"prefetch_data":{"uniprot":{"accession":"Q15853","full_name":"Upstream stimulatory factor 2","aliases":["Class B basic helix-loop-helix protein 12","bHLHb12","FOS-interacting protein","FIP","Major late transcription factor 2","Upstream transcription factor 2"],"length_aa":346,"mass_kda":37.0,"function":"Transcription factor that binds to a symmetrical DNA sequence (E-boxes) (5'-CACGTG-3') that is found in a variety of viral and cellular promoters","subcellular_location":"Nucleus","url":"https://www.uniprot.org/uniprotkb/Q15853/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/USF2","classification":"Not Classified","n_dependent_lines":323,"n_total_lines":1208,"dependency_fraction":0.2673841059602649},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/USF2","total_profiled":1310},"omim":[{"mim_id":"612822","title":"UTP20 SMALL SUBUNIT PROCESSOME COMPONENT; UTP20","url":"https://www.omim.org/entry/612822"},{"mim_id":"606464","title":"HEPCIDIN ANTIMICROBIAL PEPTIDE; HAMP","url":"https://www.omim.org/entry/606464"},{"mim_id":"602868","title":"CELL DIVISION CYCLE 5-LIKE; CDC5L","url":"https://www.omim.org/entry/602868"},{"mim_id":"601728","title":"PHOSPHATASE AND TENSIN HOMOLOG; PTEN","url":"https://www.omim.org/entry/601728"},{"mim_id":"600390","title":"UPSTREAM TRANSCRIPTION FACTOR 2, FOS-INTERACTING; USF2","url":"https://www.omim.org/entry/600390"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Nucleoplasm","reliability":"Supported"},{"location":"Vesicles","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/USF2"},"hgnc":{"alias_symbol":["FIP","bHLHb12"],"prev_symbol":[]},"alphafold":{"accession":"Q15853","domains":[],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q15853","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q15853-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q15853-F1-predicted_aligned_error_v6.png","plddt_mean":58.47},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=USF2","jax_strain_url":"https://www.jax.org/strain/search?query=USF2"},"sequence":{"accession":"Q15853","fasta_url":"https://rest.uniprot.org/uniprotkb/Q15853.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q15853/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q15853"}},"corpus_meta":[{"pmid":"6384177","id":"PMC_6384177","title":"Replacement of the fip gene of Escherichia coli by an inactive gene cloned on a plasmid.","date":"1984","source":"Journal of 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USF2 transcriptional activity in FcepsilonRI-activated mast cells.","date":"2005","source":"Molecular and cellular biology","url":"https://pubmed.ncbi.nlm.nih.gov/16199869","citation_count":62,"is_preprint":false},{"pmid":"12837692","id":"PMC_12837692","title":"82-FIP, a novel FMRP (fragile X mental retardation protein) interacting protein, shows a cell cycle-dependent intracellular localization.","date":"2003","source":"Human molecular genetics","url":"https://pubmed.ncbi.nlm.nih.gov/12837692","citation_count":60,"is_preprint":false},{"pmid":"3881414","id":"PMC_3881414","title":"Thioredoxin is the bacterial protein encoded by fip that is required for filamentous bacteriophage f1 assembly.","date":"1985","source":"Journal of bacteriology","url":"https://pubmed.ncbi.nlm.nih.gov/3881414","citation_count":58,"is_preprint":false},{"pmid":"11578135","id":"PMC_11578135","title":"A comparison of lymphatic tissues from cats with spontaneous feline infectious peritonitis (FIP), cats with FIP 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Two transcriptional activation domains were identified: the USR (context-dependent, requires the adenovirus major late minimal promoter or an initiator element) and the exon 5-encoded region (active in multiple contexts). Activity of the exon 5 domain varies with conformation induced by alternatively spliced exon 4.\",\n      \"method\": \"Indirect immunofluorescence of transiently transfected cells, cotransfection assays with deletion mutants fused to DNA-binding domains of USF2 or GAL4, reporter assays with multiple promoter constructs\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — multiple orthogonal methods (immunofluorescence, deletion mutagenesis, reporter assays) in a single rigorous study\",\n      \"pmids\": [\"8657110\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1997,\n      \"finding\": \"USF2 (via its bHLH domain) forms heterodimers with the bZip transcription factor c-Maf through the USF2 basic region and HLH motif; the leucine zipper is required for USF2 homodimerization but not for c-Maf heterodimerization. USF2 binding to c-Maf markedly reduces c-Maf DNA-binding activity, suggesting a mechanism of transcriptional repression.\",\n      \"method\": \"Yeast two-hybrid, in vitro binding with USF2 deletion/mutation mutants, DNA binding assays\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — single lab, multiple binding and functional assays but no in vivo confirmation\",\n      \"pmids\": [\"9070273\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1997,\n      \"finding\": \"USF2 activity is required for glucose-stimulated L-pyruvate kinase (L-PK) promoter activity in single living islet beta-cells; microinjection of anti-USF2 antibodies inhibited L-PK promoter activity by 71–87% at high glucose, and this effect depended on the intact upstream L4 box (two E-boxes) in the L-PK promoter.\",\n      \"method\": \"Photon-counting digital imaging of luciferase in single living cells, microinjection of antibodies, promoter deletion constructs\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — novel single-cell imaging method with antibody microinjection and promoter deletion controls in a single rigorous study\",\n      \"pmids\": [\"9252379\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"USF2 undergoes nuclear translocation in mast cells upon activation by IL-3 or IgE-antigen stimulation. Prevention of this translocation using a peptide derived from the USF-specific NLS region significantly inhibited IL-3-mediated mast cell survival, directly linking USF2 nuclear translocation to cell viability.\",\n      \"method\": \"Subcellular fractionation/localization, peptide inhibition of nuclear translocation, cell viability assays\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — direct localization experiment with functional consequence via peptide inhibition, single lab\",\n      \"pmids\": [\"9743349\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"The coactivator p300 interacts functionally with USF2 to potentiate USF2-dependent transcriptional activation of the F1F0 ATP synthase alpha-subunit (ATPA) initiator element. Wild-type adenovirus E1A (but not p300-binding-deficient E1A mutants) inhibited this activation; overexpression of p300 reversed the E1A inhibitory effect.\",\n      \"method\": \"Transfection/reporter assays, E1A mutant expression, p300 overexpression rescue experiments\",\n      \"journal\": \"Biochimica et biophysica acta\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — functional interaction demonstrated with mutant controls, single lab\",\n      \"pmids\": [\"10434034\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"USF1, USF2, and alpha-Pal/NRF-1 are the major transcription factors binding the FMR1 promoter in brain and testis extracts. Methylation abolishes alpha-Pal/NRF-1 binding and affects USF1/USF2 binding to a lesser degree, suggesting methylation inhibits FMR1 transcription partly by blocking these factor binding events.\",\n      \"method\": \"EMSA with brain and testis extracts, methylation-dependent binding assays, transcriptional activity assays in neuronally derived cells with mutation analysis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods (EMSA, footprinting, transfection) with physiologically relevant extracts; replicated across multiple experiments\",\n      \"pmids\": [\"11058604\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"USF1 and USF2 bind to E-box motifs (especially E-box B) in the APC tumor suppressor gene promoter and are required for its basal transcriptional activity; mutation of E-box B abolished basal promoter activity in HCT-116 cells.\",\n      \"method\": \"EMSA, transient transfection with E-box deletion/mutation constructs, cotransfection with USF expression vectors\",\n      \"journal\": \"Journal of cellular biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — EMSA plus functional reporter assays with mutagenesis, single lab\",\n      \"pmids\": [\"11241666\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"A USF1/USF2 heterodimer activates human FcεRI alpha chain gene expression by binding to a CAGCTG (E-box) element in the first intron. Overexpression of USF2 antisense repressed the FcεRI alpha chain promoter and decreased alpha chain mRNA in mast cell lines.\",\n      \"method\": \"EMSA with antibodies and in vitro translation products, antisense overexpression, promoter-reporter assays\",\n      \"journal\": \"European journal of immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — EMSA with antibody supershift, antisense knockdown with mRNA readout, single lab\",\n      \"pmids\": [\"11180124\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"USF1 and USF2 bind the E-box in the proximal promoter of the HOXB4 gene and activate its transcription in hematopoietic stem cells (K562 and CD34+ cells) via activation of the MAPK pathway; this was shown by yeast one-hybrid screening, EMSA, and cotransfection assays.\",\n      \"method\": \"Yeast one-hybrid screen, EMSA, cotransfection with MAPK pathway analysis, reporter assays in K562 and CD34+ cells\",\n      \"journal\": \"The Journal of experimental medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple methods including yeast one-hybrid, EMSA, and functional cotransfection; single lab\",\n      \"pmids\": [\"11085749\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"ChREBP, not USF2, mediates glucose-stimulated endogenous L-PK expression in INS-1 insulin-secreting cells. Inducible overexpression of USF-1 and USF-2 did not alter basal or glucose-responsive L-PK mRNA, and dominant-negative USF (which abolished USF binding to the L-PK promoter) also had no effect on L-PK expression in INS-1 cells.\",\n      \"method\": \"Tet-on inducible expression of USF1, USF2, and dominant-negative mutants; Northern blot; EMSA; nuclear run-on experiments; Western blot of nuclear fractions\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — rigorous inducible system with dominant negatives, EMSA, and nuclear run-on; multiple orthogonal approaches\",\n      \"pmids\": [\"12087089\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"USF-1 and USF-2 bind an E-box at -893/-888 in the iNOS promoter in vivo and act as transcriptional repressors of IL-1β-induced iNOS expression in murine mesangial cells. Mutation of the E-box augmented iNOS response, and cotransfection of dominant-negative USF-2 or cis-element decoys enhanced IL-1β stimulation.\",\n      \"method\": \"Site-directed mutagenesis of iNOS promoter, EMSA with supershift, cotransfection with USF expression vectors and dominant-negative mutants, cis-decoy experiments\",\n      \"journal\": \"American journal of physiology. Cell physiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — multiple orthogonal methods including mutagenesis, EMSA, dominant-negative cotransfection, and decoy experiments\",\n      \"pmids\": [\"12225970\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"USF2, but not USF1, specifically transactivates the IGF2R promoter in nontumorigenic mammary epithelial cells (MCF-10A); USF1 and USF2 both bind IGF2R E-boxes in vitro and in chromatin, but a USF-specific dominant-negative mutant markedly decreased IGF2R mRNA. IGF2R promoter activity was USF-independent in breast cancer cell lines, suggesting loss of USF function contributes to IGF2R downregulation in cancer.\",\n      \"method\": \"In vitro DNA binding, chromatin immunoprecipitation (ChIP), transactivation assays with overexpressed USF2 vs USF1, dominant-negative USF expression, endogenous mRNA measurement\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods including ChIP, in vitro binding, and dominant-negative KD across multiple cell lines\",\n      \"pmids\": [\"12857727\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"USF1 and USF2 constitutively bind the class B E-box in the proximal promoter of the human HO-1 gene in vivo, are required for high-level HO-1 expression, and enhance HO-1 transcription in response to heme and cadmium in human renal proximal tubular epithelial cells. A single guanine contact point in the E-box is essential for USF binding.\",\n      \"method\": \"DMS in vivo footprinting, ChIP, EMSA, overexpression of USF1/USF2, dominant-negative USF expression, reporter assays\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — in vivo footprinting, ChIP, and functional assays with dominant negatives; multiple orthogonal methods\",\n      \"pmids\": [\"15242350\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"A novel USF2 isoform (USF2c), generated by alternative splicing using a cryptic acceptor site within exon 6 (missing exons 4, 5, and part of exon 6), encodes a ~29 kDa protein that binds as a homodimer to the cathepsin B promoter E-box and acts as a transcriptional repressor of cathepsin B expression.\",\n      \"method\": \"RT-PCR cloning, in vitro transcription/translation, EMSA, cotransfection reporter assays\",\n      \"journal\": \"Gene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — in vitro binding and functional reporter assays, single lab\",\n      \"pmids\": [\"15276216\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"USF1 and USF2 bind to a repressor E-box site at -114 to -119 of the human MCT1 promoter and act as repressors of MCT1 transcription in Caco-2 colonic cells, as shown by EMSA, site-directed mutagenesis, and cotransfection of USF expression vectors decreasing endogenous MCT1 expression.\",\n      \"method\": \"EMSA, site-directed mutagenesis, cotransfection with USF expression vectors, endogenous MCT1 mRNA measurement\",\n      \"journal\": \"American journal of physiology. Gastrointestinal and liver physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — EMSA plus mutagenesis plus overexpression effect on endogenous gene; single lab\",\n      \"pmids\": [\"15691871\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Transcriptionally active USF2 suppresses the tumorigenicity and anchorage-independent growth of c-Myc-transformed fibroblasts; this required transcriptionally active (not inactive) USF2, and cotransfection assays indicated active USF2 inhibited cellular transformation by preventing c-Myc-mediated transcriptional repression.\",\n      \"method\": \"Stable cell line generation, soft agar colony assay, nude mice xenograft, cotransfection with USF and Myc dominant-negative mutants\",\n      \"journal\": \"Experimental cell research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — in vivo xenograft plus in vitro epistasis with dominant-negative mutants; single lab\",\n      \"pmids\": [\"15541720\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"USF2 (LysRS partner) is regulated by Ap4A in mast cells: LysRS associates with USF2 and negatively regulates its activity; Ap4A produced by LysRS acts as a positive regulator of USF2 transcriptional activity, analogous to the mechanism described for MITF. The Hint protein negatively regulates USF2 in this pathway.\",\n      \"method\": \"Co-immunoprecipitation (Co-IP), functional transcriptional assays in mast cells, biochemical assays of Ap4A synthesis\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — Co-IP plus functional transcriptional assays; single lab\",\n      \"pmids\": [\"16199869\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"USF2 functions as a tumor suppressor in prostate carcinogenesis: ectopic USF2 expression in androgen-independent PC-3 cells inhibited anchorage-independent growth (90-98%), invasion (80%), and in vivo tumorigenicity (80-90%) in nude mice. USF2-null mice exhibited marked prostate hyperplasia, confirming an in vivo role in normal prostate growth and differentiation.\",\n      \"method\": \"Western blot of cancer cell lines, ectopic overexpression, soft agar assay, Matrigel invasion assay, nude mouse xenograft, USF2 knockout mice generation and phenotyping, IHC of human prostate cancer tissues\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods including KO mouse with phenotype, xenograft, and in vitro functional assays; replicated across in vitro and in vivo systems\",\n      \"pmids\": [\"16186802\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Whole-genome ChIP-chip analysis revealed that USF1 and USF2 bind predominantly near transcription start sites of protein-coding genes in a liver cell line, their binding positively correlates with gene expression levels, and they frequently occupy bidirectional promoters. USF1 and USF2 co-occupy promoters of nuclear-mitochondrial genes and lipid/cholesterol metabolism genes, often in collaboration with GABPA/NRF-2.\",\n      \"method\": \"ChIP-chip (genome-wide, 35 bp resolution) in HepG2 liver cell line, correlation with expression data\",\n      \"journal\": \"Genome research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genome-wide ChIP-chip is a strong method; single lab but large-scale systematic analysis\",\n      \"pmids\": [\"18230803\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"USF-2 interacts with Fra-1 (an AP-1 factor) via protein-protein interaction, and this USF-2/Fra-1 cooperation regulates HO-1 promoter activity in opposing ways in primary versus tumor cells. Mutation of either the E-box or AP-1 site in the HO-1 promoter only partially affected USF-dependent regulation; double mutation abolished it. USF-2 lacking its USF-specific region (USR) or exon 4-encoded transactivation domain lost regulatory capacity.\",\n      \"method\": \"Protein-protein interaction studies, promoter mutagenesis (E-box and AP-1 site), USF-2 domain deletion analysis, reporter assays in primary hepatocytes vs tumor cell lines\",\n      \"journal\": \"Antioxidants & redox signaling\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — protein interaction plus mutagenesis in two cell types; single lab\",\n      \"pmids\": [\"18331200\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"HINT1 co-immunoprecipitates with USF2 in HepG2 hepatoma cells and inhibits USF2 transcriptional activity, as well as inhibiting endogenous cyclin D1 and TGFβ2 expression controlled by USF2.\",\n      \"method\": \"Co-immunoprecipitation, reporter assays for USF2 transcriptional activity, endogenous gene expression analysis after HINT1 overexpression\",\n      \"journal\": \"International journal of cancer\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — Co-IP plus functional transcriptional assays; single lab\",\n      \"pmids\": [\"19089909\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"The rs1867277 A allele in the FOXE1 5' UTR specifically recruits the USF1/USF2 transcription factor complex (but not the G allele), as demonstrated by DNA-binding assays, and this allele-specific recruitment leads to differential transcriptional regulation of FOXE1.\",\n      \"method\": \"DNA-binding assays (EMSA with USF1/USF2 antibodies), transfection/reporter assays with allele-specific constructs\",\n      \"journal\": \"PLoS genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — EMSA with antibody supershift plus reporter assay; single lab but validated in two independent cohorts at the GWAS level\",\n      \"pmids\": [\"19730683\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"In TGF-β signaling, USF2 operates in a SMAD/p53/USF2→PAI-1 transcriptional axis: SMAD2/3, pp60c-src, EGFR, and p53 activation are each required for TGF-β1-induced PAI-1 expression in the context of renal fibrosis and ureteral obstruction.\",\n      \"method\": \"Pathway inhibitor studies, reporter assays, in vivo UUO mouse model with pathway component activation measured\",\n      \"journal\": \"Cell and tissue research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — in vivo model plus multiple pathway inhibitors; review-style synthesis with experimental data cited from multiple studies\",\n      \"pmids\": [\"21638209\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"ID1 inhibits USF2 transcriptional activity in mesangial cells, thereby blocking TGF-β-induced apoptosis. TGF-β upregulates USF2, which increases BAX expression and apoptosis; BMPs induce ID1 which counteracts USF2 activity to exert an anti-apoptotic effect.\",\n      \"method\": \"Expression manipulation of ID1 and USF2, apoptosis assays, BAX expression measurement, reporter assays in human mesangial cells\",\n      \"journal\": \"American journal of physiology. Renal physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — functional assays with overexpression/knockdown and apoptosis readout; single lab\",\n      \"pmids\": [\"21921026\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"HIF2α physically interacts with USF2 in a manner dependent on HIF2α's N-TAD domain, and this interaction is required for selective activation of HIF2 target genes. USF2 exhibits specific binding to the promoters of HIF2 (but not HIF1) target genes even when overexpressed, and RNA Pol II association with HIF2 target genes is USF2-dependent.\",\n      \"method\": \"siRNA knockdown, inhibitor studies, reporter assays, chromatin immunoprecipitation (ChIP), co-immunoprecipitation with domain deletion mutants, RNA Pol II ChIP\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — Co-IP with domain mapping, ChIP, and reporter assays; multiple orthogonal methods establishing USF2 as a HIF2-specific transcriptional partner\",\n      \"pmids\": [\"23991099\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"USF2 binds to E-box motifs in the Smurf1 and Smurf2 promoters (in vitro and in vivo by ChIP) and inhibits their transcriptional activity, resulting in decreased Smurf mRNA and enhanced TGF-β pathway activity in breast cancer cells.\",\n      \"method\": \"Luciferase reporter assays with Smurf promoters, ChIP, overexpression of USF2, endogenous mRNA measurement\",\n      \"journal\": \"Cellular signalling\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — ChIP plus reporter and mRNA assays; single lab\",\n      \"pmids\": [\"30244169\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"USF2 and PLAG1 co-regulate MSI2 (Musashi-2) transcription in human hematopoietic stem and progenitor cells; both factors bind the MSI2 promoter (confirmed by ChIP-seq), are required for efficient transactivation of endogenous MSI2, and their coincident overexpression expands CD34+ cells in vitro.\",\n      \"method\": \"Luciferase reporter assays, ChIP-seq (global co-binding analysis), shRNA knockdown of USF2 and PLAG1, overexpression in cord blood cells with CD34+ expansion assay\",\n      \"journal\": \"Stem cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genome-wide ChIP-seq co-binding, reporter assays, and functional overexpression with defined phenotypic readout; multiple orthogonal approaches\",\n      \"pmids\": [\"29641991\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"CDK5 phosphorylates USF2 at two serine residues, S155 and S222; phosphorylation at these sites stabilizes the USF2 protein and regulates cellular growth and migration in cancer cells.\",\n      \"method\": \"In vitro kinase assays, site-directed mutagenesis (S155A and S222A), protein stability assays, functional cell growth and migration assays\",\n      \"journal\": \"Cancers\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — in vitro kinase assay plus mutagenesis plus functional readouts; multiple orthogonal methods\",\n      \"pmids\": [\"31013770\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"USF2 directly binds the ATF4 transcriptional initiation region and regulates its transcriptional activity in periodontal ligament cells; USF2 overexpression promotes osteogenic differentiation (osteoblast-specific gene expression and mineralization), and this effect is reversed by simultaneous ATF4 silencing.\",\n      \"method\": \"Luciferase reporter assays, overexpression and silencing systems, Alizarin red staining, qRT-PCR, ELISA, microarray\",\n      \"journal\": \"Journal of periodontal research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — reporter assays with epistasis (USF2 OE + ATF4 KD rescue), single lab\",\n      \"pmids\": [\"31448831\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"USF2 directly regulates proinflammatory cytokine production (IL-17A, IFN-γ, IL-22, GM-CSF) and T-bet expression in pathogenic Th17 cells; shRNA targeting USF2 in these cells reduced expression of these cytokines, identifying USF2 as an upstream regulator of the Th17 pathogenic transcriptional program.\",\n      \"method\": \"shRNA knockdown of USF2 in isolated Th17 cells, gene expression analysis, gene set enrichment analysis identifying USF2 as upstream regulator\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — shRNA KD with defined cytokine readouts; single study but in primary human cells\",\n      \"pmids\": [\"33203678\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"USF2 transcriptionally activates THBS1 by binding to its promoter, promoting oxidative stress and activating the TGF-β/Smad3/NLRP3/Caspase-1 pathway to stimulate pyroptosis in sepsis-induced acute kidney injury. USF2 knockdown reduced THBS1, inhibited this pathway, and ameliorated kidney injury.\",\n      \"method\": \"ChIP/reporter assays for USF2→THBS1 transcriptional activation, USF2 knockdown (shRNA/siRNA), in vivo mouse AKI model, pyroptosis assays (Caspase-1, GSDMD-N, LDH), pathway protein measurements\",\n      \"journal\": \"Pharmacological research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — ChIP and reporter assays for direct binding, plus in vivo knockdown with phenotypic readout; single lab\",\n      \"pmids\": [\"34756923\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"TGF-β upregulates USF2, which transcriptionally activates S100A8 by directly binding its promoter (confirmed by luciferase reporter and ChIP assays), thereby promoting EMT and metastasis in colorectal cancer cells. Extracellular S100A8 conversely suppresses the USF2/S100A8 axis.\",\n      \"method\": \"Luciferase reporter assays, ChIP, Western blot, migration/invasion assays, mouse metastasis models, siRNA knockdown\",\n      \"journal\": \"Cancer communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — ChIP and reporter assays confirming direct binding plus in vivo mouse model; single lab\",\n      \"pmids\": [\"33389821\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"USF2 transcriptionally represses TXNRD1 by directly binding to two E-box sites in the TXNRD1 promoter in hepatocellular carcinoma cells, functioning as a tumor suppressor through downstream repression of TXNRD1-Akt/mTOR signaling.\",\n      \"method\": \"ChIP, reporter assays, USF2 overexpression/knockdown, TXNRD1 functional assays, in vitro and in vivo experiments\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — ChIP confirming direct E-box binding plus functional mechanistic assays; single lab\",\n      \"pmids\": [\"36319631\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"USF2 transcriptionally activates YTHDF1 (an m6A reader) by binding to its promoter; YTHDF1 then suppresses autophagy by increasing m6A-mediated stability of Cdc25A (an autophagy inhibitor), thereby exacerbating ischemia-reperfusion injury in neurons. Usf2 interference improved autophagy and reduced neuronal apoptosis in vitro and in vivo.\",\n      \"method\": \"Promoter binding assays, lentiviral shRNA knockdown, MCAO in vivo model, m6A stability assays, autophagy and apoptosis readouts\",\n      \"journal\": \"Molecular neurobiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — promoter binding plus epistasis (USF2→YTHDF1→Cdc25A) with in vivo and in vitro validation; single lab\",\n      \"pmids\": [\"37914905\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"USF2 negatively regulates STX6 transcription in hepatocellular carcinoma; under USF2 control, STX6 promotes autophagosome-lysosome fusion and accelerates LC3B degradation, promoting HCC progression.\",\n      \"method\": \"Reporter assays, USF2 and STX6 overexpression/knockdown, autophagy flux assays, in vitro and in vivo HCC functional experiments\",\n      \"journal\": \"International journal of biological sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — reporter assays plus functional autophagy flux experiments; single lab\",\n      \"pmids\": [\"37564208\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"USF2 acts as a transcriptional repressor of lysosomal and autophagy genes under nutrient-rich conditions by binding CLEAR motifs together with HDAC1, reducing histone H3K27 acetylation and chromatin accessibility. Under starvation, USF2 competes with TFEB for CLEAR motif binding in a phosphorylation-dependent manner; GSK3β-mediated phosphorylation of USF2 S155 governs its DNA-binding activity and lysosomal gene repression.\",\n      \"method\": \"ChIP, chromatin accessibility assays, histone acetylation profiling, reporter assays, co-IP with HDAC1, USF2-TFEB competition assays, GSK3β kinase assays with S155 mutagenesis, USF2 knockout cells/mice\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — multiple orthogonal methods (ChIP, chromatin accessibility, histone modification, in vivo KO, kinase assay with mutagenesis) in a single rigorous study\",\n      \"pmids\": [\"39333072\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"USF2 is a bHLH-leucine zipper transcription factor that binds E-box/CLEAR motifs at target gene promoters as homodimers or USF1/USF2 heterodimers; it can activate or repress transcription depending on context (promoter architecture, binding partners, and phosphorylation state), interacts with coactivators (p300) and corepressors (HDAC1, HINT1, ID1), is regulated by CDK5-mediated phosphorylation (S155, S222) that controls its stability and DNA-binding activity, undergoes signal-dependent nuclear translocation in mast cells, competes with TFEB at CLEAR motifs to suppress lysosomal/autophagy genes under nutrient-rich conditions in a GSK3β-phosphorylation-dependent manner, and functions as a context-dependent transcriptional regulator of target genes involved in metabolism, fibrosis (PAI-1, THBS1), immunity (FcεRI, Th17 cytokines), proliferation, and tumor suppression (IGF2R, prostate).\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"USF2 is a bHLH-leucine zipper transcription factor that binds E-box and CLEAR motifs as a homodimer or USF1/USF2 heterodimer to activate or repress a broad spectrum of target genes governing metabolism, immunity, fibrosis, autophagy, and tumor suppression. USF2 contains two nuclear localization signals (the basic region and the USF-specific region) and two transactivation domains whose activity depends on alternative splicing and promoter context; it cooperates with coactivators such as p300 and HIF2α, and is inhibited by corepressors including HDAC1, HINT1, and ID1 [PMID:8657110, PMID:10434034, PMID:23991099, PMID:39333072]. Under nutrient-rich conditions, USF2 recruits HDAC1 to CLEAR motifs to repress lysosomal and autophagy genes by reducing H3K27 acetylation, and competes with TFEB upon starvation in a manner regulated by GSK3β-mediated phosphorylation of S155, while CDK5 phosphorylation of S155 and S222 independently stabilizes USF2 protein to control cell growth [PMID:39333072, PMID:31013770]. USF2 functions as a tumor suppressor in prostate and liver cancers—USF2-null mice develop prostate hyperplasia, and ectopic USF2 suppresses tumorigenicity in prostate cancer xenografts—while also activating proinflammatory cytokine programs in pathogenic Th17 cells and driving fibrotic gene expression (PAI-1, THBS1) through TGF-β signaling [PMID:16186802, PMID:33203678, PMID:34756923].\",\n  \"teleology\": [\n    {\n      \"year\": 1996,\n      \"claim\": \"Mapping USF2's functional architecture resolved how a bHLH-zip protein achieves nuclear entry and context-dependent transactivation, identifying dual NLS elements and two activation domains modulated by alternative splicing.\",\n      \"evidence\": \"Deletion mutagenesis fused to GAL4/USF2 DNA-binding domains, immunofluorescence of transfected cells, reporter assays with multiple promoter constructs\",\n      \"pmids\": [\"8657110\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Crystal structure of USF2 activation domains not determined\", \"Mechanism by which exon 4 splicing alters exon 5 domain conformation unknown\"]\n    },\n    {\n      \"year\": 1997,\n      \"claim\": \"Discovery of USF2/c-Maf heterodimerization via the bHLH domain established that USF2 can sequester non-canonical partners to repress their DNA binding, expanding its regulatory repertoire beyond E-box-dependent mechanisms.\",\n      \"evidence\": \"Yeast two-hybrid, in vitro binding with deletion/mutation mutants, DNA binding assays\",\n      \"pmids\": [\"9070273\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No in vivo confirmation of USF2–c-Maf interaction\", \"Physiological target genes of this heterodimer not identified\"]\n    },\n    {\n      \"year\": 1997,\n      \"claim\": \"Single-cell antibody microinjection demonstrated that USF2 is required for glucose-stimulated L-PK promoter activity in pancreatic β-cells, providing the first evidence that USF2 mediates metabolic gene regulation in a stimulus-dependent manner.\",\n      \"evidence\": \"Photon-counting imaging of luciferase in living islet β-cells, anti-USF2 antibody microinjection, promoter deletion constructs\",\n      \"pmids\": [\"9252379\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Later work in INS-1 cells attributed glucose-responsive L-PK expression to ChREBP rather than USF2, indicating cell-type specificity (PMID:12087089)\"]\n    },\n    {\n      \"year\": 1998,\n      \"claim\": \"Demonstration that USF2 undergoes signal-dependent nuclear translocation in mast cells upon IL-3 or IgE stimulation, with NLS-derived peptide blockade impairing cell survival, established USF2 as a signal-regulated transcription factor controlling immune cell viability.\",\n      \"evidence\": \"Subcellular fractionation, NLS-peptide inhibition of nuclear translocation, cell viability assays in mast cells\",\n      \"pmids\": [\"9743349\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Upstream kinase/signaling pathway triggering nuclear import not identified\", \"Peptide specificity for USF2 versus other NLS-containing proteins not fully addressed\"]\n    },\n    {\n      \"year\": 1999,\n      \"claim\": \"Identification of p300 as a functional coactivator of USF2 at the ATP synthase α-subunit initiator element revealed a coactivator-dependent mechanism for USF2-mediated transcriptional activation, linking USF2 to the HAT-dependent chromatin remodeling machinery.\",\n      \"evidence\": \"Reporter assays with E1A mutants (p300-binding-deficient), p300 overexpression rescue\",\n      \"pmids\": [\"10434034\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct physical interaction between USF2 and p300 not demonstrated by co-IP\", \"Whether p300 recruitment is required at all USF2 target promoters is unknown\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Multiple studies established USF2 as a versatile E-box-dependent transcriptional regulator of diverse target genes including APC, FcεRI, and HOXB4, demonstrating that USF1/USF2 homo- and heterodimers can either activate or repress transcription depending on the target promoter.\",\n      \"evidence\": \"EMSA with supershift, reporter assays with E-box mutations, antisense knockdown of USF2, cotransfection in multiple cell types (HCT-116, mast cells, K562/CD34+ cells)\",\n      \"pmids\": [\"11241666\", \"11180124\", \"11085749\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Determinants of whether USF2 activates or represses at a given E-box not resolved\", \"Genome-wide target repertoire not yet known at this stage\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"USF2 was shown to repress iNOS transcription by binding a distal E-box in the iNOS promoter, directly demonstrating a gene-repressive function distinct from its activator role, and was confirmed to be dispensable for glucose-responsive L-PK expression in INS-1 cells.\",\n      \"evidence\": \"Dominant-negative USF2, cis-decoy experiments, site-directed mutagenesis (iNOS); inducible USF expression, nuclear run-on (L-PK)\",\n      \"pmids\": [\"12225970\", \"12087089\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Chromatin-level mechanism of USF2-mediated repression at iNOS not elucidated\", \"Cell-type determinants of USF2 dispensability at metabolic promoters unclear\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"ChIP and dominant-negative experiments revealed USF2 specifically transactivates IGF2R in nontumorigenic mammary cells but not in breast cancer lines, providing the first evidence that loss of USF2 function could contribute to oncogenesis through deregulation of a specific target gene.\",\n      \"evidence\": \"ChIP, in vitro binding, reporter assays, dominant-negative USF, endogenous mRNA measurement across MCF-10A and cancer cell lines\",\n      \"pmids\": [\"12857727\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism of USF2 inactivation in cancer cells not determined\", \"Whether IGF2R loss is the primary mediator of the tumorigenic phenotype was not tested\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Functional evidence from multiple systems established USF2 as a regulator of LysRS/Ap4A signaling in mast cells and a suppressor of c-Myc-driven transformation, with transcriptionally active USF2 required to inhibit anchorage-independent growth and in vivo tumorigenesis.\",\n      \"evidence\": \"Soft agar and nude mouse xenograft with active/inactive USF2 mutants; co-IP of LysRS–USF2 complex, Ap4A functional assays in mast cells\",\n      \"pmids\": [\"15541720\", \"16199869\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether USF2 tumor suppression is c-Myc-specific or general not resolved\", \"HINT1 regulation of USF2 in vivo not validated\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"In vivo knockout and xenograft studies definitively established USF2 as a prostate tumor suppressor: USF2 overexpression suppressed PC-3 tumorigenicity by 80–90%, while USF2-null mice developed marked prostate hyperplasia.\",\n      \"evidence\": \"USF2 KO mice with prostate phenotyping, ectopic expression in PC-3 cells, soft agar/invasion/xenograft assays, IHC of human prostate cancer\",\n      \"pmids\": [\"16186802\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"USF2 target genes mediating prostate tumor suppression not identified\", \"Whether USF2 loss is sufficient for frank malignancy versus hyperplasia not resolved\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Genome-wide ChIP-chip in liver cells revealed that USF1/USF2 predominantly bind near transcription start sites, co-occupy bidirectional promoters, and positively correlate with gene expression, establishing USF2 as a widespread promoter-proximal activator with a preference for metabolic and nuclear-mitochondrial genes.\",\n      \"evidence\": \"ChIP-chip at 35 bp resolution in HepG2 cells, correlation with expression data, co-occupancy analysis with GABPA/NRF-2\",\n      \"pmids\": [\"18230803\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"ChIP-chip performed in a single cell type (HepG2); tissue-specific binding not addressed\", \"Causal role of USF binding at individual loci not established\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Identification of HINT1 as a USF2-interacting repressor that inhibits cyclin D1 and TGFβ2 expression, and of Fra-1 as a cooperative partner at the HO-1 promoter, revealed that USF2 output depends on its protein interaction network at individual target genes.\",\n      \"evidence\": \"Co-IP of HINT1–USF2, reporter and endogenous gene assays; USF2–Fra-1 interaction studies with E-box/AP-1 double mutagenesis\",\n      \"pmids\": [\"19089909\", \"18331200\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Structural basis of HINT1–USF2 interaction not determined\", \"Genome-wide extent of USF2–Fra-1 co-regulation unknown\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Discovery that HIF2α physically interacts with USF2 through its N-TAD domain, and that USF2 is specifically required for RNA Pol II recruitment to HIF2 (but not HIF1) target genes, resolved how hypoxia signaling achieves HIF paralog specificity at select promoters.\",\n      \"evidence\": \"Co-IP with domain deletion mutants, ChIP for USF2 and Pol II at HIF2 vs HIF1 targets, siRNA knockdown, reporter assays\",\n      \"pmids\": [\"23991099\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether USF2 DNA-binding activity is required for HIF2 cooperation or just protein scaffolding is unclear\", \"Genome-wide scope of USF2-dependent HIF2 targets not mapped\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"ChIP-seq co-binding analysis in hematopoietic progenitors showed USF2 and PLAG1 co-regulate MSI2 transcription and that their combined activity expands CD34+ cells, positioning USF2 as a regulator of stem cell self-renewal.\",\n      \"evidence\": \"ChIP-seq, shRNA knockdown, reporter assays, overexpression in cord blood CD34+ cells with expansion assay\",\n      \"pmids\": [\"29641991\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Broader in vivo hematopoietic phenotype of USF2 loss in this context not characterized\", \"Whether USF2–PLAG1 cooperation extends beyond the MSI2 locus unknown\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Identification of CDK5 as a kinase that phosphorylates USF2 at S155 and S222 to stabilize the protein established a post-translational regulatory mechanism controlling USF2 abundance and its effects on cell growth and migration.\",\n      \"evidence\": \"In vitro kinase assays, phospho-dead mutagenesis (S155A, S222A), protein stability and functional cell assays\",\n      \"pmids\": [\"31013770\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo confirmation of CDK5-dependent USF2 phosphorylation not provided\", \"Downstream targets specifically affected by phosphorylation-stabilized USF2 not identified\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"shRNA knockdown in primary human Th17 cells identified USF2 as an upstream regulator of the pathogenic Th17 cytokine program (IL-17A, IFN-γ, IL-22, GM-CSF, T-bet), extending USF2's functional scope to adaptive immunity.\",\n      \"evidence\": \"shRNA knockdown in isolated human Th17 cells, gene expression and gene set enrichment analysis\",\n      \"pmids\": [\"33203678\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct promoter binding of USF2 at Th17 cytokine loci not demonstrated by ChIP\", \"In vivo Th17 phenotype of USF2 deficiency not tested\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"USF2 was shown to transcriptionally activate pro-fibrotic (THBS1) and pro-metastatic (S100A8) target genes through direct promoter binding, linking USF2 to TGF-β-driven pathology in acute kidney injury and colorectal cancer EMT.\",\n      \"evidence\": \"ChIP/reporter assays for USF2→THBS1 and USF2→S100A8, shRNA knockdown, in vivo AKI and metastasis models\",\n      \"pmids\": [\"34756923\", \"33389821\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether USF2 is a general mediator of TGF-β transcriptional output or acts at selected targets not resolved\", \"Relative contribution of USF2 versus other E-box factors at these loci not quantified\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"A comprehensive mechanistic study resolved how USF2 represses lysosomal/autophagy genes under nutrient-rich conditions: USF2 recruits HDAC1 to CLEAR motifs, reduces H3K27 acetylation, and competes with TFEB; GSK3β phosphorylation of S155 governs USF2 DNA-binding activity and this repression, establishing USF2 as a nutrient-sensing transcriptional switch at CLEAR elements.\",\n      \"evidence\": \"ChIP, chromatin accessibility, histone acetylation profiling, co-IP with HDAC1, USF2–TFEB competition assays, GSK3β kinase assay with S155 mutagenesis, USF2 KO cells and mice\",\n      \"pmids\": [\"39333072\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether mTORC1 or other nutrient sensors act upstream of GSK3β→USF2 not fully delineated\", \"Relative importance of CDK5 vs GSK3β phosphorylation at S155 in different cellular contexts not resolved\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Key unresolved questions include: (1) how USF2 switches between activator and repressor modes at different E-box-containing promoters, (2) the full phosphorylation-dependent interactome that dictates cofactor choice, and (3) whether USF2 tumor suppressor function in prostate and liver operates through CLEAR-motif lysosomal gene repression, TXNRD1 repression, or additional pathways.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Structural basis of activator/repressor switch undetermined\", \"No integrative model unifying CDK5 and GSK3β phosphorylation with cofactor selection\", \"Causal target genes for prostate tumor suppression remain unidentified\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0003677\", \"supporting_discovery_ids\": [0, 2, 5, 6, 7, 8, 10, 11, 12, 13, 14, 18, 25, 26, 35]},\n      {\"term_id\": \"GO:0140110\", \"supporting_discovery_ids\": [0, 2, 4, 10, 11, 12, 15, 17, 24, 25, 26, 29, 30, 31, 32, 35]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [1, 15, 23, 35]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [0, 3, 18, 35]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-74160\", \"supporting_discovery_ids\": [0, 2, 4, 10, 11, 12, 18, 24, 26, 29, 35]},\n      {\"term_id\": \"R-HSA-9612973\", \"supporting_discovery_ids\": [33, 34, 35]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [3, 7, 29]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [22, 25, 30]},\n      {\"term_id\": \"R-HSA-4839726\", \"supporting_discovery_ids\": [35]}\n    ],\n    \"complexes\": [\n      \"USF1/USF2 heterodimer\"\n    ],\n    \"partners\": [\n      \"USF1\",\n      \"HDAC1\",\n      \"HINT1\",\n      \"HIF2A\",\n      \"PLAG1\",\n      \"FOSL1\",\n      \"MAF\",\n      \"ID1\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}