{"gene":"TOX4","run_date":"2026-06-10T10:51:55","timeline":{"discoveries":[{"year":2010,"finding":"TOX4 physically binds to DNA adducts generated by platinum anticancer drugs (cisplatin, oxaliplatin, satraplatin); this interaction was validated by surface plasmon resonance imaging (SPRi) after initial identification via ligand-fishing with damaged plasmids coupled to magnetic beads and nanoHPLC-MS/MS of nuclear extracts.","method":"Ligand-fishing pulldown from nuclear extracts + nanoHPLC-MS/MS + surface plasmon resonance imaging (SPRi) validation","journal":"Archives of biochemistry and biophysics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — orthogonal methods (MS pulldown + SPRi), single lab; SPRi provides direct binding validation","pmids":["21184731"],"is_preprint":false},{"year":2013,"finding":"TOX4 is a binding partner of the LEDGF/p75 PWWP domain; the interaction is specific for PWWP domains of the HDGF family, requires PWWP residues essential for chromatin interaction, and occurs in vitro in the presence of DNA. TOX4 co-localizes with LEDGF/p75 in HeLa cells. Overexpression of the TOX4 PWWP-interacting region (PIR) inhibits HIV-1 (but not MLV) integration.","method":"Yeast two-hybrid, protein complementation assay, co-immunoprecipitation, in vitro binding assay, co-localization by fluorescence microscopy, single-round VSV-G pseudotyped HIV-1 infection assay","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal methods (Y2H, co-IP, PCA, in vitro binding, functional HIV assay), single lab","pmids":["24312278"],"is_preprint":false},{"year":2019,"finding":"Tox4 is required early during iPSC reprogramming to generate reprogramming intermediates, enable proper exogenous reprogramming factor expression, and facilitate closing of somatic enhancers and opening of pluripotency enhancers. Tox4 also assembles into a high-molecular-weight protein complex. Tox4 is similarly required for efficient fibroblast-to-neuron conversion.","method":"siRNA-mediated knockdown during iPSC reprogramming, chromatin accessibility assays, molecular weight fractionation","journal":"Journal of cell science","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — clean KD with defined cellular and chromatin phenotype, single lab, single primary method","pmids":["31519808"],"is_preprint":false},{"year":2021,"finding":"TOX4 regulates hepatic glucose production (HGP) independently of the insulin receptor/AKT-FoxO1 pathway; cAMP and dexamethasone regulate TOX4 expression; TOX4 inhibition decreases glucose production in primary hepatocytes and in vivo; combined genetic ablation of TOX4 and FoxO1 in liver has additive effects on glucose tolerance and gluconeogenesis; TOX4 ablation fails to reverse metabolic derangement caused by insulin receptor knockout, confirming insulin receptor-independent action.","method":"Genetic knockout/knockdown in primary hepatocytes and mouse models (diet-induced obese, db/db mice), glucose tolerance tests, gluconeogenesis assays, epistasis with FoxO1 and insulin receptor KO","journal":"Cell metabolism","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple genetic models (KO, KD, double KO epistasis), in vitro and in vivo, replicated across model systems","pmids":["34914893"],"is_preprint":false},{"year":2021,"finding":"MBD2 directly binds CpG islands in the Tox4 promoter (shown by chromatin immunoprecipitation), preventing promoter methylation and thereby activating Tox4 expression. Tox4, in turn, mediates myoglobin-induced apoptosis in renal tubular cells, as siRNA knockdown of Tox4 attenuates this apoptosis.","method":"Chromatin immunoprecipitation (ChIP), siRNA knockdown, MBD2 knockout mice, in vitro myoglobin treatment of BUMPT cells, in vivo glycerol-induced AKI model","journal":"Journal of cellular and molecular medicine","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ChIP establishes direct promoter binding, functional epistasis confirmed by siRNA and KO, two orthogonal approaches, single lab","pmids":["33764669"],"is_preprint":false},{"year":2022,"finding":"TOX4 facilitates promoter-proximal pausing of RNA Pol II by promoting dephosphorylation of CTD serine 2 and DSIF (restricting pause release), and promotes Pol II recycling/reinitiation by facilitating dephosphorylation of CTD serines 2 and 5. TOX4 preferentially binds PP1α among PP1 phosphatases and is capable of facilitating Pol II CTD dephosphorylation in vitro. Loss of TOX4 increases levels of phosphorylated Pol II CTD, decreases Pol II occupancy on promoters, and reduces transcriptional output.","method":"ChIP-seq (chromatin occupancy), 4sUDRB-seq (elongation rate), TT-seq (transcriptional output), in vitro CTD dephosphorylation assay, co-immunoprecipitation (PP1α binding)","journal":"Communications biology","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — in vitro biochemical dephosphorylation assay plus multiple genome-wide orthogonal methods (ChIP-seq, 4sUDRB-seq, TT-seq) and co-IP, all in one study","pmids":["35365735"],"is_preprint":false},{"year":2023,"finding":"TOX4 binds the NTSR1 promoter and activates NTSR1 transcription, promoting lung cancer cell migration and invasion. The lncRNA SLCO4A1-AS1 acts as a decoy for TOX4, interrupting its interaction with the NTSR1 promoter and preventing NTSR1 transcription. This was established by ChIP assay showing TOX4 promoter occupancy, RNA pulldown showing direct SLCO4A1-AS1–TOX4 interaction, and RIP.","method":"Chromatin immunoprecipitation (ChIP), RNA pulldown, RNA immunoprecipitation (RIP), Transwell migration/invasion assays, RNA-seq, western blotting","journal":"Journal of biomedical science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ChIP establishes direct promoter binding, RNA pulldown and RIP confirm lncRNA–TOX4 interaction, functional rescue experiments, single lab","pmids":["37726723"],"is_preprint":false},{"year":2023,"finding":"In murine T cell development, Tox4 conditional knockout reduces thymic cellularity, partially blocks T cell development, decreases CD8:CD4 ratio by reducing CD8 cell proliferation and increasing apoptosis, and impairs proliferation of the double-positive (DP) blast population partly via downregulation of Cdk1. Mechanistically, Tox4 facilitates transcriptional reinitiation and restricts elongation in a dephosphorylation-dependent manner, conserved between mouse and human.","method":"Conditional knockout in mice, single-cell RNA-seq, flow cytometry, mechanistic analysis of Pol II phosphorylation states","journal":"Communications biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean conditional KO with defined developmental phenotype plus mechanistic dephosphorylation analysis, scRNA-seq, consistent with prior human cell study (PMID:35365735)","pmids":["37286708"],"is_preprint":false},{"year":2024,"finding":"TRIM65 E3 ubiquitin ligase directly binds the N-terminal domain of TOX4 through its coiled-coil and SPRY structural domains, mediates K48-linked ubiquitination and proteasomal degradation of TOX4 (dependent on TRIM65's E3 ligase activity), and thereby suppresses TOX4-mediated apoptosis in intestinal epithelial cells during hypoxia-reoxygenation injury.","method":"Yeast two-hybrid (substrate identification), co-immunoprecipitation, immunofluorescence confocal co-localization, ubiquitination assay (K48 linkage), E3 ligase activity mutant, siRNA/KO functional rescue","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Y2H plus co-IP plus ubiquitination assay with ligase-dead mutant, single lab, multiple orthogonal methods","pmids":["38212319"],"is_preprint":false},{"year":2025,"finding":"In Drosophila, Tox4 requires zinc for binding to the PNUTS TFIIS N-terminal domain (TND), and binds the TND on a surface distinct from established TND-interacting transcriptional regulators. Selective disruption of the PNUTS–Tox4 interaction or the PNUTS–PP1 interaction impairs normal gene expression and chromosomal dispersal during oogenesis. Tox4 is dispensable for viability but essential for fertility, with both PNUTS-dependent and PNUTS-independent roles in germline development.","method":"Biochemical binding assays, structural analysis, in vivo Drosophila genetics (selective interaction mutants), gene expression analysis, cytological analysis of chromosome dispersal","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — combined biochemical, structural, cellular, and in vivo genetic experiments in one study; zinc-dependence and distinct binding surface established structurally","pmids":["40347473"],"is_preprint":false}],"current_model":"TOX4 is a regulatory subunit of the nuclear PP1 holoenzyme (together with PNUTS and WDR82) that preferentially binds PP1α and facilitates dephosphorylation of RNA Pol II CTD serines 2 and 5 as well as DSIF, thereby promoting promoter-proximal pausing, restricting pause release, and enabling Pol II recycling/reinitiation; it also binds directly to platinated DNA and to the LEDGF PWWP domain, activates transcription of target genes (e.g., NTSR1) by occupying their promoters, is subject to K48-ubiquitination and proteasomal degradation by TRIM65, is transcriptionally activated by MBD2 via CpG island binding at its promoter, and acts as an insulin receptor-independent regulator of hepatic glucose production downstream of cAMP/glucocorticoid signaling."},"narrative":{"mechanistic_narrative":"TOX4 is a chromatin-associated regulatory subunit of a nuclear protein phosphatase 1 (PP1) holoenzyme that controls RNA polymerase II transcription dynamics [PMID:35365735, PMID:40347473]. It preferentially binds PP1α and facilitates dephosphorylation of the Pol II CTD on serines 2 and 5 as well as DSIF, thereby restricting pause release and promoting Pol II recycling/reinitiation; loss of TOX4 elevates phosphorylated CTD, reduces promoter Pol II occupancy, and lowers transcriptional output [PMID:35365735]. This activity is structurally anchored through a zinc-dependent interaction with the PNUTS TFIIS N-terminal domain on a surface distinct from other TND-binding regulators, and selective disruption of either the PNUTS–TOX4 or PNUTS–PP1 contact impairs gene expression in vivo [PMID:40347473]. The same dephosphorylation-dependent reinitiation/elongation control operates in mammalian T cell development, where conditional loss of TOX4 blocks thymocyte development partly through downregulation of Cdk1 [PMID:37286708]. TOX4 also acts as a sequence/locus-directed transcriptional activator: it occupies the NTSR1 promoter to drive transcription and promote lung cancer cell migration and invasion, an activity antagonized by the decoy lncRNA SLCO4A1-AS1 [PMID:37726723]. Beyond transcription, TOX4 binds platinum-drug-induced DNA adducts and the LEDGF/p75 PWWP domain, the latter contributing to HIV-1 integration [PMID:21184731, PMID:24312278], and it is required during cell-fate reprogramming for enhancer remodeling [PMID:31519808]. TOX4 abundance is set by competing inputs: MBD2 binding to CpG islands protects the TOX4 promoter from methylation to activate its expression [PMID:33764669], while the TRIM65 E3 ligase mediates K48-linked ubiquitination and proteasomal degradation of TOX4 [PMID:38212319]. Physiologically, TOX4 regulates hepatic glucose production independently of the insulin receptor/AKT-FoxO1 axis and downstream of cAMP/glucocorticoid signaling [PMID:34914893], and it mediates apoptosis in renal tubular and intestinal epithelial injury contexts [PMID:33764669, PMID:38212319].","teleology":[{"year":2010,"claim":"Established a physical activity for TOX4 by showing it directly recognizes platinum-drug DNA adducts, implicating it in the cellular response to DNA damage.","evidence":"Ligand-fishing pulldown from nuclear extracts with nanoHPLC-MS/MS and SPRi binding validation","pmids":["21184731"],"confidence":"Medium","gaps":["Functional consequence of adduct binding (repair vs. damage signaling) not defined","Domain mediating DNA-adduct recognition not mapped","No link to its later-defined transcriptional role"]},{"year":2013,"claim":"Placed TOX4 on chromatin via a specific protein interaction by identifying it as a LEDGF/p75 PWWP-domain partner and linking that interaction to HIV-1 integration.","evidence":"Yeast two-hybrid, co-IP, in vitro DNA-dependent binding, co-localization, and HIV-1 vs. MLV integration assays","pmids":["24312278"],"confidence":"Medium","gaps":["TOX4 region binding the PWWP domain not finely mapped","Whether TOX4 directs lentiviral integration site selection unresolved","Relationship to PP1/transcription functions unknown"]},{"year":2019,"claim":"Showed TOX4 is functionally required for cell-fate change, acting early in reprogramming to remodel enhancers and assembling into a high-molecular-weight complex.","evidence":"siRNA knockdown during iPSC reprogramming and fibroblast-to-neuron conversion with chromatin accessibility assays and size fractionation","pmids":["31519808"],"confidence":"Medium","gaps":["Identity of the high-molecular-weight complex not determined","Mechanism linking TOX4 to enhancer opening/closing unknown","Direct target loci not defined"]},{"year":2021,"claim":"Defined a physiological role in metabolism, placing TOX4 as an insulin-receptor-independent regulator of hepatic glucose production downstream of cAMP/glucocorticoid signaling.","evidence":"Liver knockout/knockdown in mouse models, glucose tolerance and gluconeogenesis assays, and epistasis with FoxO1 and insulin receptor KO","pmids":["34914893"],"confidence":"High","gaps":["Gluconeogenic target genes directly controlled by TOX4 not enumerated","How cAMP/dexamethasone signaling converges on TOX4 unclear","Connection to its transcriptional/PP1 mechanism not drawn"]},{"year":2021,"claim":"Identified an upstream transcriptional control of TOX4 itself and a context-specific pro-apoptotic role, showing MBD2 protects the TOX4 promoter from methylation to drive expression.","evidence":"ChIP for MBD2 promoter binding, siRNA knockdown, MBD2 KO mice, and a glycerol-induced AKI model","pmids":["33764669"],"confidence":"Medium","gaps":["Downstream apoptotic effectors of TOX4 not identified","Generality of MBD2 control beyond renal tubular cells unknown","Direct vs. indirect role of TOX4 in apoptosis not separated"]},{"year":2022,"claim":"Resolved the core molecular mechanism: TOX4 directs PP1α-dependent dephosphorylation of the Pol II CTD and DSIF to control pausing, pause release, and reinitiation.","evidence":"ChIP-seq, 4sUDRB-seq, TT-seq, in vitro CTD dephosphorylation assay, and co-IP demonstrating PP1α preference","pmids":["35365735"],"confidence":"High","gaps":["How TOX4 is targeted to specific promoters not defined","Stoichiometry/architecture of the TOX4-PNUTS-PP1-WDR82 holoenzyme not resolved here","Selectivity for CTD Ser2 vs Ser5 sites mechanistically unexplained"]},{"year":2023,"claim":"Showed the dephosphorylation-dependent reinitiation/elongation mechanism is physiologically required and conserved, governing mammalian T cell development.","evidence":"Conditional knockout in mice with single-cell RNA-seq, flow cytometry, and Pol II phosphorylation analysis","pmids":["37286708"],"confidence":"High","gaps":["Why CD8 lineage is preferentially affected not fully explained","Whether Cdk1 is a direct TOX4 transcriptional target unresolved","Cell-type specificity of the requirement not addressed"]},{"year":2023,"claim":"Demonstrated locus-specific transcriptional activation by TOX4 and its regulation by a decoy lncRNA, linking TOX4 to cancer cell invasion via NTSR1.","evidence":"ChIP for promoter occupancy, RNA pulldown and RIP for SLCO4A1-AS1 interaction, and Transwell migration/invasion assays","pmids":["37726723"],"confidence":"Medium","gaps":["DNA sequence/structural basis of TOX4 promoter binding not defined","Whether activation requires PP1/Pol II mechanism unclear","Breadth of TOX4 direct target genes not mapped"]},{"year":2024,"claim":"Identified the route to TOX4 protein turnover, showing TRIM65 ubiquitinates and degrades TOX4 to suppress its pro-apoptotic activity in epithelial injury.","evidence":"Yeast two-hybrid, co-IP, K48-linkage ubiquitination assay with a ligase-dead mutant, and functional rescue in hypoxia-reoxygenation injury","pmids":["38212319"],"confidence":"Medium","gaps":["Signals triggering TRIM65-mediated TOX4 degradation unknown","Whether degradation tunes the transcriptional/PP1 function not tested","Generality beyond intestinal epithelium unaddressed"]},{"year":2025,"claim":"Provided the structural basis of TOX4 incorporation into the phosphatase complex, showing a zinc-dependent, distinct-surface contact with the PNUTS TND essential for germline gene expression and fertility.","evidence":"Biochemical binding and structural analysis plus in vivo Drosophila genetics with selective interaction-disrupting mutants and cytological chromosome analysis","pmids":["40347473"],"confidence":"High","gaps":["PNUTS-independent roles of TOX4 not mechanistically defined","Conservation of the zinc-dependent binding mode in mammals not directly shown","How TOX4 contributes to chromosome dispersal mechanistically unclear"]},{"year":null,"claim":"How TOX4 is recruited to specific genomic loci to reconcile its global Pol II CTD-phosphatase function with locus-specific transcriptional activation (e.g., NTSR1) remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No defined DNA-binding specificity for TOX4 at activated promoters","Unclear how the same protein both pauses/recycles Pol II broadly and selectively activates target genes","Integration of TOX4 abundance control (MBD2, TRIM65) with its transcriptional output not established"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[5]},{"term_id":"GO:0140110","term_label":"transcription regulator activity","supporting_discovery_ids":[5,6]},{"term_id":"GO:0003677","term_label":"DNA binding","supporting_discovery_ids":[0]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[5,9]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[1,5]},{"term_id":"GO:0005694","term_label":"chromosome","supporting_discovery_ids":[5,9]}],"pathway":[{"term_id":"R-HSA-74160","term_label":"Gene expression (Transcription)","supporting_discovery_ids":[5,6,7]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[4,8]},{"term_id":"R-HSA-1430728","term_label":"Metabolism","supporting_discovery_ids":[3]}],"complexes":["TOX4-PNUTS-PP1 nuclear phosphatase holoenzyme"],"partners":["PP1Α (PPP1CA)","PNUTS (PPP1R10)","LEDGF/P75 (PSIP1)","TRIM65","SLCO4A1-AS1"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"O94842","full_name":"TOX high mobility group box family member 4","aliases":[],"length_aa":621,"mass_kda":66.2,"function":"Transcription factor that modulates cell fate reprogramming from the somatic state to the pluripotent and neuronal fate (By similarity). In liver, controls the expression of hormone-regulated gluconeogenic genes such as G6PC1 and PCK1 (By similarity). This regulation is independent of the insulin receptor activation (By similarity). Also acts as a regulatory component of protein phosphatase 1 (PP1) complexes (PubMed:39603239, PubMed:39603240). Component of the PNUTS-PP1 protein phosphatase complex, a PP1 complex that regulates RNA polymerase II transcription pause-release (PubMed:39603239, PubMed:39603240). PNUTS-PP1 also plays a role in the control of chromatin structure and cell cycle progression during the transition from mitosis into interphase (PubMed:20516061)","subcellular_location":"Nucleus; Chromosome","url":"https://www.uniprot.org/uniprotkb/O94842/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/TOX4","classification":"Not Classified","n_dependent_lines":346,"n_total_lines":1208,"dependency_fraction":0.28642384105960267},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/TOX4","total_profiled":1310},"omim":[{"mim_id":"614032","title":"TOX HIGH MOBILITY GROUP BOX FAMILY MEMBER 4; TOX4","url":"https://www.omim.org/entry/614032"},{"mim_id":"611059","title":"WD REPEAT-CONTAINING PROTEIN 82; WDR82","url":"https://www.omim.org/entry/611059"},{"mim_id":"603771","title":"PROTEIN PHOSPHATASE 1, REGULATORY SUBUNIT 10; PPP1R10","url":"https://www.omim.org/entry/603771"},{"mim_id":"176875","title":"PROTEIN PHOSPHATASE 1, CATALYTIC SUBUNIT, ALPHA ISOFORM; PPP1CA","url":"https://www.omim.org/entry/176875"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Nucleoplasm","reliability":"Approved"},{"location":"Vesicles","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/TOX4"},"hgnc":{"alias_symbol":["LCP1"],"prev_symbol":["C14orf92","KIAA0737"]},"alphafold":{"accession":"O94842","domains":[{"cath_id":"1.10.30.10","chopping":"221-299","consensus_level":"medium","plddt":90.7384,"start":221,"end":299},{"cath_id":"-","chopping":"580-612","consensus_level":"medium","plddt":92.8639,"start":580,"end":612}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/O94842","model_url":"https://alphafold.ebi.ac.uk/files/AF-O94842-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-O94842-F1-predicted_aligned_error_v6.png","plddt_mean":54.72},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=TOX4","jax_strain_url":"https://www.jax.org/strain/search?query=TOX4"},"sequence":{"accession":"O94842","fasta_url":"https://rest.uniprot.org/uniprotkb/O94842.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/O94842/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/O94842"}},"corpus_meta":[{"pmid":"21184731","id":"PMC_21184731","title":"TOX4 and its binding partners recognize DNA adducts generated by platinum anticancer drugs.","date":"2010","source":"Archives of biochemistry and biophysics","url":"https://pubmed.ncbi.nlm.nih.gov/21184731","citation_count":32,"is_preprint":false},{"pmid":"34914893","id":"PMC_34914893","title":"TOX4, an insulin receptor-independent regulator of hepatic glucose production, is activated in diabetic liver.","date":"2021","source":"Cell metabolism","url":"https://pubmed.ncbi.nlm.nih.gov/34914893","citation_count":26,"is_preprint":false},{"pmid":"24312278","id":"PMC_24312278","title":"TOX4 and NOVA1 proteins are partners of the LEDGF PWWP domain and affect HIV-1 replication.","date":"2013","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/24312278","citation_count":20,"is_preprint":false},{"pmid":"33764669","id":"PMC_33764669","title":"MBD2 mediates renal cell apoptosis via activation of Tox4 during rhabdomyolysis-induced acute kidney injury.","date":"2021","source":"Journal of cellular and molecular medicine","url":"https://pubmed.ncbi.nlm.nih.gov/33764669","citation_count":20,"is_preprint":false},{"pmid":"37726723","id":"PMC_37726723","title":"LncRNA SLCO4A1-AS1 suppresses lung cancer progression by sequestering the TOX4-NTSR1 signaling axis.","date":"2023","source":"Journal of biomedical science","url":"https://pubmed.ncbi.nlm.nih.gov/37726723","citation_count":19,"is_preprint":false},{"pmid":"38212319","id":"PMC_38212319","title":"E3 ligase TRIM65 alleviates intestinal ischemia/reperfusion injury through inhibition of TOX4-mediated apoptosis.","date":"2024","source":"Cell death & disease","url":"https://pubmed.ncbi.nlm.nih.gov/38212319","citation_count":15,"is_preprint":false},{"pmid":"35365735","id":"PMC_35365735","title":"TOX4 facilitates promoter-proximal pausing and C-terminal domain dephosphorylation of RNA polymerase II in human cells.","date":"2022","source":"Communications biology","url":"https://pubmed.ncbi.nlm.nih.gov/35365735","citation_count":13,"is_preprint":false},{"pmid":"31519808","id":"PMC_31519808","title":"Tox4 modulates cell fate reprogramming.","date":"2019","source":"Journal of cell science","url":"https://pubmed.ncbi.nlm.nih.gov/31519808","citation_count":12,"is_preprint":false},{"pmid":"37286708","id":"PMC_37286708","title":"Tox4 regulates transcriptional elongation and reinitiation during murine T cell development.","date":"2023","source":"Communications biology","url":"https://pubmed.ncbi.nlm.nih.gov/37286708","citation_count":8,"is_preprint":false},{"pmid":"40347473","id":"PMC_40347473","title":"PNUTS:PP1 recruitment to Tox4 regulates chromosomal dispersal in Drosophila germline development.","date":"2025","source":"Cell reports","url":"https://pubmed.ncbi.nlm.nih.gov/40347473","citation_count":0,"is_preprint":false},{"pmid":"41029921","id":"PMC_41029921","title":"TOX4 Inhibition in Chronic Hyperglycemia: Effects on Glycation Stress, Hepatic Protection, Epigenetic Mechanisms, Signaling Pathways, and Beta Cell Dynamics.","date":"2025","source":"Current drug metabolism","url":"https://pubmed.ncbi.nlm.nih.gov/41029921","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":7375,"output_tokens":3018,"usd":0.033697,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":10460,"output_tokens":4140,"usd":0.0779,"stage2_stop_reason":"end_turn"},"total_usd":0.111597,"stage1_batch_id":"msgbatch_017qqVz33Ga63SCDwzb35XPb","stage2_batch_id":"msgbatch_01Scr2Ei9x4HJMiRGxwrmvLK","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2010,\n      \"finding\": \"TOX4 physically binds to DNA adducts generated by platinum anticancer drugs (cisplatin, oxaliplatin, satraplatin); this interaction was validated by surface plasmon resonance imaging (SPRi) after initial identification via ligand-fishing with damaged plasmids coupled to magnetic beads and nanoHPLC-MS/MS of nuclear extracts.\",\n      \"method\": \"Ligand-fishing pulldown from nuclear extracts + nanoHPLC-MS/MS + surface plasmon resonance imaging (SPRi) validation\",\n      \"journal\": \"Archives of biochemistry and biophysics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — orthogonal methods (MS pulldown + SPRi), single lab; SPRi provides direct binding validation\",\n      \"pmids\": [\"21184731\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"TOX4 is a binding partner of the LEDGF/p75 PWWP domain; the interaction is specific for PWWP domains of the HDGF family, requires PWWP residues essential for chromatin interaction, and occurs in vitro in the presence of DNA. TOX4 co-localizes with LEDGF/p75 in HeLa cells. Overexpression of the TOX4 PWWP-interacting region (PIR) inhibits HIV-1 (but not MLV) integration.\",\n      \"method\": \"Yeast two-hybrid, protein complementation assay, co-immunoprecipitation, in vitro binding assay, co-localization by fluorescence microscopy, single-round VSV-G pseudotyped HIV-1 infection assay\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal methods (Y2H, co-IP, PCA, in vitro binding, functional HIV assay), single lab\",\n      \"pmids\": [\"24312278\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Tox4 is required early during iPSC reprogramming to generate reprogramming intermediates, enable proper exogenous reprogramming factor expression, and facilitate closing of somatic enhancers and opening of pluripotency enhancers. Tox4 also assembles into a high-molecular-weight protein complex. Tox4 is similarly required for efficient fibroblast-to-neuron conversion.\",\n      \"method\": \"siRNA-mediated knockdown during iPSC reprogramming, chromatin accessibility assays, molecular weight fractionation\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — clean KD with defined cellular and chromatin phenotype, single lab, single primary method\",\n      \"pmids\": [\"31519808\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"TOX4 regulates hepatic glucose production (HGP) independently of the insulin receptor/AKT-FoxO1 pathway; cAMP and dexamethasone regulate TOX4 expression; TOX4 inhibition decreases glucose production in primary hepatocytes and in vivo; combined genetic ablation of TOX4 and FoxO1 in liver has additive effects on glucose tolerance and gluconeogenesis; TOX4 ablation fails to reverse metabolic derangement caused by insulin receptor knockout, confirming insulin receptor-independent action.\",\n      \"method\": \"Genetic knockout/knockdown in primary hepatocytes and mouse models (diet-induced obese, db/db mice), glucose tolerance tests, gluconeogenesis assays, epistasis with FoxO1 and insulin receptor KO\",\n      \"journal\": \"Cell metabolism\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple genetic models (KO, KD, double KO epistasis), in vitro and in vivo, replicated across model systems\",\n      \"pmids\": [\"34914893\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"MBD2 directly binds CpG islands in the Tox4 promoter (shown by chromatin immunoprecipitation), preventing promoter methylation and thereby activating Tox4 expression. Tox4, in turn, mediates myoglobin-induced apoptosis in renal tubular cells, as siRNA knockdown of Tox4 attenuates this apoptosis.\",\n      \"method\": \"Chromatin immunoprecipitation (ChIP), siRNA knockdown, MBD2 knockout mice, in vitro myoglobin treatment of BUMPT cells, in vivo glycerol-induced AKI model\",\n      \"journal\": \"Journal of cellular and molecular medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP establishes direct promoter binding, functional epistasis confirmed by siRNA and KO, two orthogonal approaches, single lab\",\n      \"pmids\": [\"33764669\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"TOX4 facilitates promoter-proximal pausing of RNA Pol II by promoting dephosphorylation of CTD serine 2 and DSIF (restricting pause release), and promotes Pol II recycling/reinitiation by facilitating dephosphorylation of CTD serines 2 and 5. TOX4 preferentially binds PP1α among PP1 phosphatases and is capable of facilitating Pol II CTD dephosphorylation in vitro. Loss of TOX4 increases levels of phosphorylated Pol II CTD, decreases Pol II occupancy on promoters, and reduces transcriptional output.\",\n      \"method\": \"ChIP-seq (chromatin occupancy), 4sUDRB-seq (elongation rate), TT-seq (transcriptional output), in vitro CTD dephosphorylation assay, co-immunoprecipitation (PP1α binding)\",\n      \"journal\": \"Communications biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — in vitro biochemical dephosphorylation assay plus multiple genome-wide orthogonal methods (ChIP-seq, 4sUDRB-seq, TT-seq) and co-IP, all in one study\",\n      \"pmids\": [\"35365735\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"TOX4 binds the NTSR1 promoter and activates NTSR1 transcription, promoting lung cancer cell migration and invasion. The lncRNA SLCO4A1-AS1 acts as a decoy for TOX4, interrupting its interaction with the NTSR1 promoter and preventing NTSR1 transcription. This was established by ChIP assay showing TOX4 promoter occupancy, RNA pulldown showing direct SLCO4A1-AS1–TOX4 interaction, and RIP.\",\n      \"method\": \"Chromatin immunoprecipitation (ChIP), RNA pulldown, RNA immunoprecipitation (RIP), Transwell migration/invasion assays, RNA-seq, western blotting\",\n      \"journal\": \"Journal of biomedical science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP establishes direct promoter binding, RNA pulldown and RIP confirm lncRNA–TOX4 interaction, functional rescue experiments, single lab\",\n      \"pmids\": [\"37726723\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"In murine T cell development, Tox4 conditional knockout reduces thymic cellularity, partially blocks T cell development, decreases CD8:CD4 ratio by reducing CD8 cell proliferation and increasing apoptosis, and impairs proliferation of the double-positive (DP) blast population partly via downregulation of Cdk1. Mechanistically, Tox4 facilitates transcriptional reinitiation and restricts elongation in a dephosphorylation-dependent manner, conserved between mouse and human.\",\n      \"method\": \"Conditional knockout in mice, single-cell RNA-seq, flow cytometry, mechanistic analysis of Pol II phosphorylation states\",\n      \"journal\": \"Communications biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean conditional KO with defined developmental phenotype plus mechanistic dephosphorylation analysis, scRNA-seq, consistent with prior human cell study (PMID:35365735)\",\n      \"pmids\": [\"37286708\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"TRIM65 E3 ubiquitin ligase directly binds the N-terminal domain of TOX4 through its coiled-coil and SPRY structural domains, mediates K48-linked ubiquitination and proteasomal degradation of TOX4 (dependent on TRIM65's E3 ligase activity), and thereby suppresses TOX4-mediated apoptosis in intestinal epithelial cells during hypoxia-reoxygenation injury.\",\n      \"method\": \"Yeast two-hybrid (substrate identification), co-immunoprecipitation, immunofluorescence confocal co-localization, ubiquitination assay (K48 linkage), E3 ligase activity mutant, siRNA/KO functional rescue\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Y2H plus co-IP plus ubiquitination assay with ligase-dead mutant, single lab, multiple orthogonal methods\",\n      \"pmids\": [\"38212319\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"In Drosophila, Tox4 requires zinc for binding to the PNUTS TFIIS N-terminal domain (TND), and binds the TND on a surface distinct from established TND-interacting transcriptional regulators. Selective disruption of the PNUTS–Tox4 interaction or the PNUTS–PP1 interaction impairs normal gene expression and chromosomal dispersal during oogenesis. Tox4 is dispensable for viability but essential for fertility, with both PNUTS-dependent and PNUTS-independent roles in germline development.\",\n      \"method\": \"Biochemical binding assays, structural analysis, in vivo Drosophila genetics (selective interaction mutants), gene expression analysis, cytological analysis of chromosome dispersal\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — combined biochemical, structural, cellular, and in vivo genetic experiments in one study; zinc-dependence and distinct binding surface established structurally\",\n      \"pmids\": [\"40347473\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"TOX4 is a regulatory subunit of the nuclear PP1 holoenzyme (together with PNUTS and WDR82) that preferentially binds PP1α and facilitates dephosphorylation of RNA Pol II CTD serines 2 and 5 as well as DSIF, thereby promoting promoter-proximal pausing, restricting pause release, and enabling Pol II recycling/reinitiation; it also binds directly to platinated DNA and to the LEDGF PWWP domain, activates transcription of target genes (e.g., NTSR1) by occupying their promoters, is subject to K48-ubiquitination and proteasomal degradation by TRIM65, is transcriptionally activated by MBD2 via CpG island binding at its promoter, and acts as an insulin receptor-independent regulator of hepatic glucose production downstream of cAMP/glucocorticoid signaling.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"TOX4 is a chromatin-associated regulatory subunit of a nuclear protein phosphatase 1 (PP1) holoenzyme that controls RNA polymerase II transcription dynamics [#5, #9]. It preferentially binds PP1\\u03b1 and facilitates dephosphorylation of the Pol II CTD on serines 2 and 5 as well as DSIF, thereby restricting pause release and promoting Pol II recycling/reinitiation; loss of TOX4 elevates phosphorylated CTD, reduces promoter Pol II occupancy, and lowers transcriptional output [#5]. This activity is structurally anchored through a zinc-dependent interaction with the PNUTS TFIIS N-terminal domain on a surface distinct from other TND-binding regulators, and selective disruption of either the PNUTS\\u2013TOX4 or PNUTS\\u2013PP1 contact impairs gene expression in vivo [#9]. The same dephosphorylation-dependent reinitiation/elongation control operates in mammalian T cell development, where conditional loss of TOX4 blocks thymocyte development partly through downregulation of Cdk1 [#7]. TOX4 also acts as a sequence/locus-directed transcriptional activator: it occupies the NTSR1 promoter to drive transcription and promote lung cancer cell migration and invasion, an activity antagonized by the decoy lncRNA SLCO4A1-AS1 [#6]. Beyond transcription, TOX4 binds platinum-drug-induced DNA adducts and the LEDGF/p75 PWWP domain, the latter contributing to HIV-1 integration [#0, #1], and it is required during cell-fate reprogramming for enhancer remodeling [#2]. TOX4 abundance is set by competing inputs: MBD2 binding to CpG islands protects the TOX4 promoter from methylation to activate its expression [#4], while the TRIM65 E3 ligase mediates K48-linked ubiquitination and proteasomal degradation of TOX4 [#8]. Physiologically, TOX4 regulates hepatic glucose production independently of the insulin receptor/AKT-FoxO1 axis and downstream of cAMP/glucocorticoid signaling [#3], and it mediates apoptosis in renal tubular and intestinal epithelial injury contexts [#4, #8].\",\n  \"teleology\": [\n    {\n      \"year\": 2010,\n      \"claim\": \"Established a physical activity for TOX4 by showing it directly recognizes platinum-drug DNA adducts, implicating it in the cellular response to DNA damage.\",\n      \"evidence\": \"Ligand-fishing pulldown from nuclear extracts with nanoHPLC-MS/MS and SPRi binding validation\",\n      \"pmids\": [\"21184731\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Functional consequence of adduct binding (repair vs. damage signaling) not defined\", \"Domain mediating DNA-adduct recognition not mapped\", \"No link to its later-defined transcriptional role\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Placed TOX4 on chromatin via a specific protein interaction by identifying it as a LEDGF/p75 PWWP-domain partner and linking that interaction to HIV-1 integration.\",\n      \"evidence\": \"Yeast two-hybrid, co-IP, in vitro DNA-dependent binding, co-localization, and HIV-1 vs. MLV integration assays\",\n      \"pmids\": [\"24312278\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"TOX4 region binding the PWWP domain not finely mapped\", \"Whether TOX4 directs lentiviral integration site selection unresolved\", \"Relationship to PP1/transcription functions unknown\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Showed TOX4 is functionally required for cell-fate change, acting early in reprogramming to remodel enhancers and assembling into a high-molecular-weight complex.\",\n      \"evidence\": \"siRNA knockdown during iPSC reprogramming and fibroblast-to-neuron conversion with chromatin accessibility assays and size fractionation\",\n      \"pmids\": [\"31519808\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Identity of the high-molecular-weight complex not determined\", \"Mechanism linking TOX4 to enhancer opening/closing unknown\", \"Direct target loci not defined\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Defined a physiological role in metabolism, placing TOX4 as an insulin-receptor-independent regulator of hepatic glucose production downstream of cAMP/glucocorticoid signaling.\",\n      \"evidence\": \"Liver knockout/knockdown in mouse models, glucose tolerance and gluconeogenesis assays, and epistasis with FoxO1 and insulin receptor KO\",\n      \"pmids\": [\"34914893\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Gluconeogenic target genes directly controlled by TOX4 not enumerated\", \"How cAMP/dexamethasone signaling converges on TOX4 unclear\", \"Connection to its transcriptional/PP1 mechanism not drawn\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Identified an upstream transcriptional control of TOX4 itself and a context-specific pro-apoptotic role, showing MBD2 protects the TOX4 promoter from methylation to drive expression.\",\n      \"evidence\": \"ChIP for MBD2 promoter binding, siRNA knockdown, MBD2 KO mice, and a glycerol-induced AKI model\",\n      \"pmids\": [\"33764669\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Downstream apoptotic effectors of TOX4 not identified\", \"Generality of MBD2 control beyond renal tubular cells unknown\", \"Direct vs. indirect role of TOX4 in apoptosis not separated\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Resolved the core molecular mechanism: TOX4 directs PP1\\u03b1-dependent dephosphorylation of the Pol II CTD and DSIF to control pausing, pause release, and reinitiation.\",\n      \"evidence\": \"ChIP-seq, 4sUDRB-seq, TT-seq, in vitro CTD dephosphorylation assay, and co-IP demonstrating PP1\\u03b1 preference\",\n      \"pmids\": [\"35365735\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How TOX4 is targeted to specific promoters not defined\", \"Stoichiometry/architecture of the TOX4-PNUTS-PP1-WDR82 holoenzyme not resolved here\", \"Selectivity for CTD Ser2 vs Ser5 sites mechanistically unexplained\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Showed the dephosphorylation-dependent reinitiation/elongation mechanism is physiologically required and conserved, governing mammalian T cell development.\",\n      \"evidence\": \"Conditional knockout in mice with single-cell RNA-seq, flow cytometry, and Pol II phosphorylation analysis\",\n      \"pmids\": [\"37286708\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Why CD8 lineage is preferentially affected not fully explained\", \"Whether Cdk1 is a direct TOX4 transcriptional target unresolved\", \"Cell-type specificity of the requirement not addressed\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Demonstrated locus-specific transcriptional activation by TOX4 and its regulation by a decoy lncRNA, linking TOX4 to cancer cell invasion via NTSR1.\",\n      \"evidence\": \"ChIP for promoter occupancy, RNA pulldown and RIP for SLCO4A1-AS1 interaction, and Transwell migration/invasion assays\",\n      \"pmids\": [\"37726723\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"DNA sequence/structural basis of TOX4 promoter binding not defined\", \"Whether activation requires PP1/Pol II mechanism unclear\", \"Breadth of TOX4 direct target genes not mapped\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Identified the route to TOX4 protein turnover, showing TRIM65 ubiquitinates and degrades TOX4 to suppress its pro-apoptotic activity in epithelial injury.\",\n      \"evidence\": \"Yeast two-hybrid, co-IP, K48-linkage ubiquitination assay with a ligase-dead mutant, and functional rescue in hypoxia-reoxygenation injury\",\n      \"pmids\": [\"38212319\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Signals triggering TRIM65-mediated TOX4 degradation unknown\", \"Whether degradation tunes the transcriptional/PP1 function not tested\", \"Generality beyond intestinal epithelium unaddressed\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Provided the structural basis of TOX4 incorporation into the phosphatase complex, showing a zinc-dependent, distinct-surface contact with the PNUTS TND essential for germline gene expression and fertility.\",\n      \"evidence\": \"Biochemical binding and structural analysis plus in vivo Drosophila genetics with selective interaction-disrupting mutants and cytological chromosome analysis\",\n      \"pmids\": [\"40347473\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"PNUTS-independent roles of TOX4 not mechanistically defined\", \"Conservation of the zinc-dependent binding mode in mammals not directly shown\", \"How TOX4 contributes to chromosome dispersal mechanistically unclear\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How TOX4 is recruited to specific genomic loci to reconcile its global Pol II CTD-phosphatase function with locus-specific transcriptional activation (e.g., NTSR1) remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No defined DNA-binding specificity for TOX4 at activated promoters\", \"Unclear how the same protein both pauses/recycles Pol II broadly and selectively activates target genes\", \"Integration of TOX4 abundance control (MBD2, TRIM65) with its transcriptional output not established\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [5]},\n      {\"term_id\": \"GO:0140110\", \"supporting_discovery_ids\": [5, 6]},\n      {\"term_id\": \"GO:0003677\", \"supporting_discovery_ids\": [0]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [5, 9]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [1, 5]},\n      {\"term_id\": \"GO:0005694\", \"supporting_discovery_ids\": [5, 9]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-74160\", \"supporting_discovery_ids\": [5, 6, 7]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [4, 8]},\n      {\"term_id\": \"R-HSA-1430728\", \"supporting_discovery_ids\": [3]}\n    ],\n    \"complexes\": [\n      \"TOX4-PNUTS-PP1 nuclear phosphatase holoenzyme\"\n    ],\n    \"partners\": [\n      \"PP1\\u03b1 (PPP1CA)\",\n      \"PNUTS (PPP1R10)\",\n      \"LEDGF/p75 (PSIP1)\",\n      \"TRIM65\",\n      \"SLCO4A1-AS1\"\n    ],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":8,"faith_total":8,"faith_pct":100.0}}