{"gene":"ZFP64","run_date":"2026-06-11T09:02:06","timeline":{"discoveries":[{"year":2008,"finding":"ZFP64 acts as a coactivator of Notch1: it physically associates with the intracellular domain of Notch1, is recruited to the promoters of Notch target genes Hes1 and Hey1, and transactivates them. ZFP64 expression is directly transactivated by Runx2. In C2C12 mesenchymal cells, ZFP64 suppresses myogenic differentiation and promotes osteoblastic differentiation.","method":"Co-immunoprecipitation (ZFP64–Notch1 ICD association), ChIP (promoter recruitment to Hes1/Hey1), promoter transactivation assays, Runx2 knockdown/overexpression, C2C12 differentiation gain-of-function/loss-of-function assays","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP, ChIP, promoter assays, and functional differentiation readout all in one focused study; multiple orthogonal methods establishing the same mechanism","pmids":["18430783"],"is_preprint":false},{"year":2022,"finding":"PKCα directly phosphorylates ZFP64 at serine 226, causing its nuclear translocation. Nuclear ZFP64 then transcriptionally activates CSF1 (macrophage colony-stimulating factor), driving M2 macrophage polarization and immunosuppression in hepatocellular carcinoma.","method":"Mass spectrometry (phosphorylation site identification), ChIP-seq (ZFP64 binding to CSF1 promoter), nuclear fractionation/immunofluorescence (translocation), in vitro kinase assay, orthotopic xenograft and transgenic mouse models, coculture system, mass cytometry","journal":"Journal of hepatology","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — phosphorylation site mapped by MS, ChIP-seq for direct promoter binding, direct kinase assay, multiple orthogonal in vitro and in vivo methods in one study","pmids":["35219791"],"is_preprint":false},{"year":2022,"finding":"ZFP64 acts as a transcription factor that directly binds the GAL-1 (Galectin-1) promoter and activates GAL-1 transcription, thereby inducing stem-cell-like phenotypes and an immunosuppressive tumor microenvironment in gastric cancer cells.","method":"ChIP-seq (ZFP64 binding to GAL-1 promoter), dual-luciferase reporter gene assay, ZFP64 knockdown/overexpression, subcutaneous xenograft and humanized mouse models","journal":"Journal of experimental & clinical cancer research : CR","confidence":"High","confidence_rationale":"Tier 2 / Moderate — ChIP-seq plus luciferase reporter (two orthogonal methods) in a single focused study with in vivo validation","pmids":["34996504"],"is_preprint":false},{"year":2022,"finding":"ZFP64 transcriptionally activates PD-1 and CTLA-4 by binding to their promoters in esophageal cancer cells, and its overexpression promotes tumor growth in orthotopic xenograft models.","method":"Dual-luciferase reporter assay and immunohistochemistry for PD-1/CTLA-4 expression, gain-of-function assays, orthotopic xenograft mouse model","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2-3 / Weak — luciferase reporter for promoter binding, single lab, limited orthogonal mechanistic validation","pmids":["35843097"],"is_preprint":false},{"year":2023,"finding":"ZFP64 recruits HDAC1 to the NUMB gene promoter, leading to histone deacetylation and repression of NUMB expression. Loss of NUMB activates the Notch1 signaling pathway to promote gallbladder cancer progression.","method":"Co-immunoprecipitation (ZFP64–HDAC1 interaction), ChIP (ZFP64/HDAC1 at NUMB promoter), ZFP64 knockdown/overexpression, in vitro and in vivo tumor models","journal":"Cancers","confidence":"Medium","confidence_rationale":"Tier 2-3 / Weak — Co-IP and ChIP performed, single lab, limited orthogonal methods","pmids":["37760477"],"is_preprint":false},{"year":2024,"finding":"ZFP64 directly binds the promoters of glycolysis-related genes (ALDOC, ENO2, HK2, SPAG4) and activates their transcription, promoting glycolysis-dependent stem-cell-like properties and tumorigenesis in breast cancer cells.","method":"ChIP assay (ZFP64 binding to glycolytic gene promoters), dual-luciferase reporter assay, mRNA-seq, ZFP64 knockdown/overexpression, ENO2/HK2 inhibitor rescue experiments, subcutaneous xenograft models","journal":"Biology direct","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ChIP and dual-luciferase reporter are two orthogonal methods for the same promoter binding claim, with functional rescue, single lab","pmids":["39294751"],"is_preprint":false},{"year":2024,"finding":"KDM1A (LSD1) demethylase activates ZFP64 expression by removing the repressive H3K9me2 histone mark from the ZFP64 promoter. ZFP64 in turn transcriptionally activates CENPL, promoting epithelial ovarian cancer cell proliferation and invasion.","method":"ChIP (KDM1A occupancy and H3K9me2 levels at ZFP64 promoter), ZFP64 and CENPL knockdown/overexpression, KDM1A knockdown rescue with ZFP64 restoration, in vivo subcutaneous tumor models","journal":"Cytotechnology","confidence":"Medium","confidence_rationale":"Tier 2-3 / Weak — ChIP for epigenetic mark plus functional rescue, single lab, limited replication","pmids":["39628712"],"is_preprint":false},{"year":2025,"finding":"ZFP64 in cerebellar Purkinje cells promotes activity-dependent climbing fiber (CF) synapse elimination during postnatal development, acting presumably downstream of P/Q-type voltage-dependent Ca2+ channels (P/Q-VDCCs). PC-specific knockdown of ZFP64 delayed CF synapse pruning and dendritic extension of CF innervation. ZFP64 regulates semaphorin 3A (Sema3A) expression, and Sema3A knockdown partially restored the CF elimination defect caused by ZFP64 or P/Q-VDCC knockdown.","method":"Purkinje cell-specific AAV-mediated ZFP64 knockdown, electrophysiological recording of CF synapse number, morphological analysis of CF innervation, genetic epistasis with Sema3A knockdown and P/Q-VDCC knockdown","journal":"iScience","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — cell-type-specific knockdown with defined electrophysiological and morphological phenotype, epistasis experiment, single lab","pmids":["40546958"],"is_preprint":false},{"year":2025,"finding":"CAFs-derived lactate increases histone lactylation (H3K18la) at the ZFP64 locus, elevating ZFP64 expression in TNBC cells. Elevated ZFP64 then directly binds the promoters of GCH1 and FTH1 and activates their transcription, suppressing ferroptosis and conferring doxorubicin resistance.","method":"ChIP (ZFP64 binding to GCH1 and FTH1 promoters), dual-luciferase reporter, histone lactylation detection (pan-Kla, H3K18la), CAF co-culture system, ZFP64 knockdown with GCH1/FTH1 overexpression rescue, in vivo xenograft","journal":"Journal of translational medicine","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — ChIP and luciferase reporter for promoter binding, rescue experiments, single lab, multiple orthogonal methods","pmids":["40022222"],"is_preprint":false},{"year":2025,"finding":"ZFP64 directly binds the TUBB3 promoter to activate its transcription in gastric cancer cells; the lncRNA FIRRE promotes this activity by binding to ZFP64, and ZFP64 knockdown abrogates FIRRE-driven TUBB3 upregulation both in vitro and in vivo.","method":"ChIP (ZFP64 at TUBB3 promoter), dual-luciferase reporter assay, RNA immunoprecipitation (RIP, FIRRE–ZFP64 interaction), ZFP64 knockdown rescue experiments, in vivo tumor models","journal":"Cancer letters","confidence":"Medium","confidence_rationale":"Tier 2-3 / Weak — ChIP, luciferase, and RIP for the interaction/binding claims, single lab","pmids":["39706253"],"is_preprint":false},{"year":2025,"finding":"CRISPR-Cas9 knockout of ZFP64 in K562 and HUDEP-2 cells increases HBG (γ-globin) mRNA expression by 1.5–2.5-fold, identifying ZFP64 as a repressor of fetal hemoglobin gene expression.","method":"CRISPR-Cas9 knockout of ZFP64 in K562 and HUDEP-2 cell lines, RT-qPCR for HBG mRNA levels","journal":"Cell biochemistry and biophysics","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single loss-of-function assay with mRNA readout only, no promoter binding or mechanistic pathway placement, single lab","pmids":["40392482"],"is_preprint":false},{"year":1997,"finding":"Mouse Zfp64 encodes a 614-amino-acid protein with ten C2H2 zinc finger motifs and multiple predicted phosphorylation sites for PKC, CK II, tyrosine kinase, and cAMP/cGMP-dependent protein kinases; it is expressed in all developing and mature mouse tissues examined.","method":"cDNA cloning, sequence analysis, in situ hybridization/Northern blot for expression pattern, chromosomal mapping","journal":"Gene","confidence":"Low","confidence_rationale":"Tier 3 / Weak — cloning and sequence-based prediction only, no functional assays performed","pmids":["9034307"],"is_preprint":false}],"current_model":"ZFP64 is a C2H2 zinc finger transcription factor that drives target gene expression by directly binding promoters (CSF1, GAL-1, Hes1/Hey1, GCH1, FTH1, ALDOC/ENO2/HK2, TUBB3, CENPL, PD-1/CTLA-4) and, in some contexts, by recruiting co-regulators (acting as a Notch1 coactivator or recruiting HDAC1 to repress NUMB); its activity and nuclear localization are regulated post-translationally by PKCα-mediated phosphorylation at S226, and its expression is epigenetically controlled by KDM1A-dependent H3K9me2 removal and by CAF-derived lactate-induced histone lactylation, with established roles in mesenchymal cell differentiation, cerebellar synapse elimination (downstream of P/Q-VDCCs via Sema3A), and transcriptional repression of fetal hemoglobin genes."},"narrative":{"mechanistic_narrative":"ZFP64 is a C2H2 zinc finger transcription factor that controls cell-fate and differentiation programs by directly binding target gene promoters and, in selected contexts, recruiting co-regulators [PMID:18430783, PMID:35219791]. It first emerged as a Notch1 coactivator that associates with the Notch1 intracellular domain, is recruited to the Hes1 and Hey1 promoters, and—itself transactivated by Runx2—suppresses myogenic and promotes osteoblastic differentiation in mesenchymal cells [PMID:18430783]. Beyond direct transcriptional activation, ZFP64 can also repress transcription by recruiting HDAC1 to deacetylate and silence the NUMB promoter [PMID:37760477]. Its activity is gated post-translationally: PKCα directly phosphorylates ZFP64 at serine 226 to drive nuclear translocation and subsequent transcriptional activation of CSF1 [PMID:35219791]. Across multiple tumor settings ZFP64 activates a broad repertoire of direct targets—GAL-1 [PMID:34996504], glycolytic genes including ALDOC, ENO2 and HK2 [PMID:39294751], the antioxidant/ferroptosis genes GCH1 and FTH1 [PMID:40022222], TUBB3 [PMID:39706253], CENPL [PMID:39628712], and the immune checkpoint genes PD-1 and CTLA-4 [PMID:35843097]—thereby supporting glycolysis, stemness, immunosuppression and therapy resistance. ZFP64 expression is itself epigenetically regulated, being activated by KDM1A-dependent removal of H3K9me2 [PMID:39628712] and by CAF-derived lactate driving H3K18 histone lactylation at its locus [PMID:40022222], and its target output can be modulated by the lncRNA FIRRE binding ZFP64 to enhance TUBB3 induction [PMID:39706253]. In the nervous system, ZFP64 in Purkinje cells promotes activity-dependent climbing-fiber synapse elimination downstream of P/Q-type voltage-dependent Ca2+ channels by regulating Sema3A expression [PMID:40546958].","teleology":[{"year":2008,"claim":"Established ZFP64's first mechanistic identity—as a Notch1 coactivator and Runx2 effector—linking it to a differentiation decision between myogenic and osteoblastic fates.","evidence":"Co-IP of ZFP64 with Notch1 ICD, ChIP at Hes1/Hey1 promoters, promoter transactivation, and C2C12 gain/loss-of-function differentiation assays","pmids":["18430783"],"confidence":"High","gaps":["Whether the same Notch-coactivator mechanism operates in the later tumor contexts is untested","Direct DNA-binding sequence specificity of ZFP64 not defined"]},{"year":2022,"claim":"Identified the post-translational switch governing ZFP64 nuclear entry, showing PKCα phosphorylation at S226 converts cytoplasmic ZFP64 into a nuclear CSF1-activating transcription factor that reshapes the tumor immune microenvironment.","evidence":"MS phosphosite mapping, in vitro kinase assay, nuclear fractionation/IF, ChIP-seq at CSF1 promoter, and orthotopic/transgenic HCC mouse models","pmids":["35219791"],"confidence":"High","gaps":["Upstream signals activating PKCα toward ZFP64 not defined","Whether S226 phosphorylation regulates targets beyond CSF1 is untested"]},{"year":2022,"claim":"Extended ZFP64 to direct activation of immunosuppression and stemness programs in gastric and esophageal cancers via GAL-1 and the checkpoint genes PD-1/CTLA-4.","evidence":"ChIP-seq and dual-luciferase reporter at GAL-1 promoter (gastric); luciferase reporter and IHC for PD-1/CTLA-4 with orthotopic xenografts (esophageal)","pmids":["34996504","35843097"],"confidence":"Medium","gaps":["PD-1/CTLA-4 binding rests on reporter assays without ChIP confirmation","Cofactor requirements for these activation events unknown"]},{"year":2023,"claim":"Revealed ZFP64 also functions as a transcriptional repressor by recruiting HDAC1 to deacetylate the NUMB promoter, indirectly amplifying Notch1 signaling.","evidence":"Co-IP of ZFP64–HDAC1, ChIP at NUMB promoter, and knockdown/overexpression in gallbladder cancer models","pmids":["37760477"],"confidence":"Medium","gaps":["What dictates activator versus repressor mode at a given promoter is unknown","Single-lab, limited orthogonal validation of the HDAC1 interaction"]},{"year":2024,"claim":"Defined ZFP64 as a direct driver of glycolytic metabolism and as an epigenetically controlled node, activating glycolysis genes and CENPL while being switched on by KDM1A-mediated H3K9me2 removal.","evidence":"ChIP and dual-luciferase at glycolytic gene promoters with inhibitor rescue (breast); ChIP of KDM1A/H3K9me2 at ZFP64 promoter and rescue (ovarian)","pmids":["39294751","39628712"],"confidence":"Medium","gaps":["Direct DNA-binding motif shared across glycolytic targets not defined","CENPL regulation supported by a single low-replication study"]},{"year":2025,"claim":"Connected ZFP64 to therapy resistance and lncRNA-modulated transcription, showing CAF-lactate-induced histone lactylation elevates ZFP64 to suppress ferroptosis (GCH1/FTH1), and that FIRRE binds ZFP64 to enhance TUBB3 induction.","evidence":"ChIP/luciferase at GCH1, FTH1 and TUBB3 promoters; H3K18la detection and CAF co-culture; RIP for FIRRE–ZFP64; rescue and xenograft experiments","pmids":["40022222","39706253"],"confidence":"Medium","gaps":["Generality of histone-lactylation control of ZFP64 beyond TNBC untested","Whether FIRRE alters ZFP64 DNA binding or stability not resolved"]},{"year":2025,"claim":"Placed ZFP64 in neuronal circuit refinement, demonstrating it drives activity-dependent climbing-fiber synapse elimination downstream of P/Q-VDCCs through Sema3A.","evidence":"Purkinje-cell-specific AAV knockdown with electrophysiology, CF morphology, and genetic epistasis with Sema3A and P/Q-VDCC knockdown","pmids":["40546958"],"confidence":"Medium","gaps":["Direct binding of ZFP64 to the Sema3A locus not shown","Single-lab knockdown rather than genetic knockout"]},{"year":2025,"claim":"Implicated ZFP64 as a repressor of fetal hemoglobin, showing its knockout de-represses γ-globin in erythroid cell lines.","evidence":"CRISPR-Cas9 knockout in K562 and HUDEP-2 with RT-qPCR for HBG mRNA","pmids":["40392482"],"confidence":"Low","gaps":["No promoter binding or mechanistic pathway placement; mRNA readout only","Not independently confirmed in primary erythroid cells"]},{"year":null,"claim":"The intrinsic DNA-binding sequence specificity of ZFP64 and the rules determining when it activates versus represses a given promoter remain undefined.","evidence":"","pmids":[],"confidence":"Low","gaps":["No consensus binding motif established across its many reported targets","No structural model of ZFP64 zinc fingers on DNA","Cofactor composition that distinguishes activator from HDAC1-repressor mode unresolved"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140110","term_label":"transcription regulator activity","supporting_discovery_ids":[0,1,2,4,5,8,9]},{"term_id":"GO:0003677","term_label":"DNA binding","supporting_discovery_ids":[1,2,5,8,9]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[1]}],"pathway":[{"term_id":"R-HSA-74160","term_label":"Gene expression (Transcription)","supporting_discovery_ids":[0,1,2,5,8,9]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0,4]},{"term_id":"R-HSA-4839726","term_label":"Chromatin organization","supporting_discovery_ids":[4,6,8]}],"complexes":[],"partners":["NOTCH1","HDAC1","PRKCA","RUNX2","FIRRE"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q9NTW7","full_name":"Zinc finger protein 64","aliases":["Zinc finger protein 338"],"length_aa":645,"mass_kda":72.2,"function":"May be involved in the regulation of mesenchymal cell differentiation through transactivation of NOTCH1 target genes","subcellular_location":"Nucleus","url":"https://www.uniprot.org/uniprotkb/Q9NTW7/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/ZFP64","classification":"Not Classified","n_dependent_lines":8,"n_total_lines":1208,"dependency_fraction":0.006622516556291391},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"PARP1","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/ZFP64","total_profiled":1310},"omim":[{"mim_id":"618111","title":"ZINC FINGER PROTEIN 64; ZFP64","url":"https://www.omim.org/entry/618111"},{"mim_id":"618081","title":"IMMUNOGLOBULIN-LIKE DOMAIN-CONTAINING RECEPTOR 2; ILDR2","url":"https://www.omim.org/entry/618081"},{"mim_id":"600211","title":"RUNT-RELATED TRANSCRIPTION FACTOR 2; RUNX2","url":"https://www.omim.org/entry/600211"},{"mim_id":"194544","title":"ZINC FINGER PROTEIN 70; ZNF70","url":"https://www.omim.org/entry/194544"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Nucleoplasm","reliability":"Approved"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/ZFP64"},"hgnc":{"alias_symbol":["FLJ10734","dJ831D17.1","FLJ12628","dJ548G19.1"],"prev_symbol":["ZNF338"]},"alphafold":{"accession":"Q9NTW7","domains":[{"cath_id":"3.30.160.60","chopping":"153-226","consensus_level":"medium","plddt":80.0018,"start":153,"end":226}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9NTW7","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9NTW7-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9NTW7-F1-predicted_aligned_error_v6.png","plddt_mean":63.91},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=ZFP64","jax_strain_url":"https://www.jax.org/strain/search?query=ZFP64"},"sequence":{"accession":"Q9NTW7","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9NTW7.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9NTW7/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9NTW7"}},"corpus_meta":[{"pmid":"35219791","id":"PMC_35219791","title":"PKCα/ZFP64/CSF1 axis resets the tumor microenvironment and fuels anti-PD1 resistance in hepatocellular carcinoma.","date":"2022","source":"Journal of hepatology","url":"https://pubmed.ncbi.nlm.nih.gov/35219791","citation_count":139,"is_preprint":false},{"pmid":"40022222","id":"PMC_40022222","title":"Cancer-associated fibroblasts promote doxorubicin resistance in triple-negative breast cancer through enhancing ZFP64 histone lactylation to regulate ferroptosis.","date":"2025","source":"Journal of translational medicine","url":"https://pubmed.ncbi.nlm.nih.gov/40022222","citation_count":46,"is_preprint":false},{"pmid":"34996504","id":"PMC_34996504","title":"Targeting ZFP64/GAL-1 axis promotes therapeutic effect of nab-paclitaxel and reverses immunosuppressive microenvironment in gastric cancer.","date":"2022","source":"Journal of experimental & clinical cancer research : CR","url":"https://pubmed.ncbi.nlm.nih.gov/34996504","citation_count":44,"is_preprint":false},{"pmid":"18430783","id":"PMC_18430783","title":"Zfp64 participates in Notch signaling and regulates differentiation in mesenchymal cells.","date":"2008","source":"Journal of cell science","url":"https://pubmed.ncbi.nlm.nih.gov/18430783","citation_count":41,"is_preprint":false},{"pmid":"35521817","id":"PMC_35521817","title":"ZFP64::NCOA3 gene fusion defines a novel subset of spindle cell rhabdomyosarcoma.","date":"2022","source":"Genes, chromosomes & cancer","url":"https://pubmed.ncbi.nlm.nih.gov/35521817","citation_count":11,"is_preprint":false},{"pmid":"9034307","id":"PMC_9034307","title":"A search for a mammalian homologue of the Drosophila photoreceptor development gene glass yields Zfp64, a zinc finger encoding gene which maps to the distal end of mouse chromosome 2.","date":"1997","source":"Gene","url":"https://pubmed.ncbi.nlm.nih.gov/9034307","citation_count":11,"is_preprint":false},{"pmid":"35843097","id":"PMC_35843097","title":"ZFP64 transcriptionally activates PD-1 and CTLA-4 and plays an oncogenic role in esophageal cancer.","date":"2022","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/35843097","citation_count":9,"is_preprint":false},{"pmid":"37760477","id":"PMC_37760477","title":"ZFP64 Promotes Gallbladder Cancer Progression through Recruiting HDAC1 to Activate NOTCH1 Signaling Pathway.","date":"2023","source":"Cancers","url":"https://pubmed.ncbi.nlm.nih.gov/37760477","citation_count":8,"is_preprint":false},{"pmid":"39294751","id":"PMC_39294751","title":"ZFP64 drives glycolysis-mediated stem cell-like properties and tumorigenesis in breast cancer.","date":"2024","source":"Biology direct","url":"https://pubmed.ncbi.nlm.nih.gov/39294751","citation_count":5,"is_preprint":false},{"pmid":"41075874","id":"PMC_41075874","title":"Clinicopathologic and Molecular Genetic Features of Spindle Cell Rhabdomyosarcoma Harboring ZFP64::NCOA2/3 Fusions: A Series of 14 Cases.","date":"2025","source":"Modern pathology : an official journal of the United States and Canadian Academy of Pathology, Inc","url":"https://pubmed.ncbi.nlm.nih.gov/41075874","citation_count":4,"is_preprint":false},{"pmid":"39706253","id":"PMC_39706253","title":"LncRNA FIRRE drives gastric cancer progression via ZFP64-mediated TUBB3 promoter activation.","date":"2024","source":"Cancer letters","url":"https://pubmed.ncbi.nlm.nih.gov/39706253","citation_count":3,"is_preprint":false},{"pmid":"40546958","id":"PMC_40546958","title":"The transcription factor ZFP64 promotes activity-dependent synapse elimination during postnatal cerebellar development.","date":"2025","source":"iScience","url":"https://pubmed.ncbi.nlm.nih.gov/40546958","citation_count":2,"is_preprint":false},{"pmid":"40392482","id":"PMC_40392482","title":"Investigate the Effect of ZFP64 on mRNA Expression of HBG Based on Bioinformatics and Experimental Validation.","date":"2025","source":"Cell biochemistry and biophysics","url":"https://pubmed.ncbi.nlm.nih.gov/40392482","citation_count":1,"is_preprint":false},{"pmid":"39628712","id":"PMC_39628712","title":"KDM1A-mediated ZFP64 demethylation activates CENPL to promote epithelial ovarian cancer progression.","date":"2024","source":"Cytotechnology","url":"https://pubmed.ncbi.nlm.nih.gov/39628712","citation_count":1,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":9714,"output_tokens":3318,"usd":0.039456,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":10756,"output_tokens":3342,"usd":0.068665,"stage2_stop_reason":"end_turn"},"total_usd":0.108121,"stage1_batch_id":"msgbatch_01UT1AahFPCAyn2uVweS7A9z","stage2_batch_id":"msgbatch_01HuKWtKNT4Pqj1bc8eEP5RV","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2008,\n      \"finding\": \"ZFP64 acts as a coactivator of Notch1: it physically associates with the intracellular domain of Notch1, is recruited to the promoters of Notch target genes Hes1 and Hey1, and transactivates them. ZFP64 expression is directly transactivated by Runx2. In C2C12 mesenchymal cells, ZFP64 suppresses myogenic differentiation and promotes osteoblastic differentiation.\",\n      \"method\": \"Co-immunoprecipitation (ZFP64–Notch1 ICD association), ChIP (promoter recruitment to Hes1/Hey1), promoter transactivation assays, Runx2 knockdown/overexpression, C2C12 differentiation gain-of-function/loss-of-function assays\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP, ChIP, promoter assays, and functional differentiation readout all in one focused study; multiple orthogonal methods establishing the same mechanism\",\n      \"pmids\": [\"18430783\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"PKCα directly phosphorylates ZFP64 at serine 226, causing its nuclear translocation. Nuclear ZFP64 then transcriptionally activates CSF1 (macrophage colony-stimulating factor), driving M2 macrophage polarization and immunosuppression in hepatocellular carcinoma.\",\n      \"method\": \"Mass spectrometry (phosphorylation site identification), ChIP-seq (ZFP64 binding to CSF1 promoter), nuclear fractionation/immunofluorescence (translocation), in vitro kinase assay, orthotopic xenograft and transgenic mouse models, coculture system, mass cytometry\",\n      \"journal\": \"Journal of hepatology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — phosphorylation site mapped by MS, ChIP-seq for direct promoter binding, direct kinase assay, multiple orthogonal in vitro and in vivo methods in one study\",\n      \"pmids\": [\"35219791\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"ZFP64 acts as a transcription factor that directly binds the GAL-1 (Galectin-1) promoter and activates GAL-1 transcription, thereby inducing stem-cell-like phenotypes and an immunosuppressive tumor microenvironment in gastric cancer cells.\",\n      \"method\": \"ChIP-seq (ZFP64 binding to GAL-1 promoter), dual-luciferase reporter gene assay, ZFP64 knockdown/overexpression, subcutaneous xenograft and humanized mouse models\",\n      \"journal\": \"Journal of experimental & clinical cancer research : CR\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP-seq plus luciferase reporter (two orthogonal methods) in a single focused study with in vivo validation\",\n      \"pmids\": [\"34996504\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"ZFP64 transcriptionally activates PD-1 and CTLA-4 by binding to their promoters in esophageal cancer cells, and its overexpression promotes tumor growth in orthotopic xenograft models.\",\n      \"method\": \"Dual-luciferase reporter assay and immunohistochemistry for PD-1/CTLA-4 expression, gain-of-function assays, orthotopic xenograft mouse model\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Weak — luciferase reporter for promoter binding, single lab, limited orthogonal mechanistic validation\",\n      \"pmids\": [\"35843097\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"ZFP64 recruits HDAC1 to the NUMB gene promoter, leading to histone deacetylation and repression of NUMB expression. Loss of NUMB activates the Notch1 signaling pathway to promote gallbladder cancer progression.\",\n      \"method\": \"Co-immunoprecipitation (ZFP64–HDAC1 interaction), ChIP (ZFP64/HDAC1 at NUMB promoter), ZFP64 knockdown/overexpression, in vitro and in vivo tumor models\",\n      \"journal\": \"Cancers\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Weak — Co-IP and ChIP performed, single lab, limited orthogonal methods\",\n      \"pmids\": [\"37760477\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"ZFP64 directly binds the promoters of glycolysis-related genes (ALDOC, ENO2, HK2, SPAG4) and activates their transcription, promoting glycolysis-dependent stem-cell-like properties and tumorigenesis in breast cancer cells.\",\n      \"method\": \"ChIP assay (ZFP64 binding to glycolytic gene promoters), dual-luciferase reporter assay, mRNA-seq, ZFP64 knockdown/overexpression, ENO2/HK2 inhibitor rescue experiments, subcutaneous xenograft models\",\n      \"journal\": \"Biology direct\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP and dual-luciferase reporter are two orthogonal methods for the same promoter binding claim, with functional rescue, single lab\",\n      \"pmids\": [\"39294751\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"KDM1A (LSD1) demethylase activates ZFP64 expression by removing the repressive H3K9me2 histone mark from the ZFP64 promoter. ZFP64 in turn transcriptionally activates CENPL, promoting epithelial ovarian cancer cell proliferation and invasion.\",\n      \"method\": \"ChIP (KDM1A occupancy and H3K9me2 levels at ZFP64 promoter), ZFP64 and CENPL knockdown/overexpression, KDM1A knockdown rescue with ZFP64 restoration, in vivo subcutaneous tumor models\",\n      \"journal\": \"Cytotechnology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Weak — ChIP for epigenetic mark plus functional rescue, single lab, limited replication\",\n      \"pmids\": [\"39628712\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"ZFP64 in cerebellar Purkinje cells promotes activity-dependent climbing fiber (CF) synapse elimination during postnatal development, acting presumably downstream of P/Q-type voltage-dependent Ca2+ channels (P/Q-VDCCs). PC-specific knockdown of ZFP64 delayed CF synapse pruning and dendritic extension of CF innervation. ZFP64 regulates semaphorin 3A (Sema3A) expression, and Sema3A knockdown partially restored the CF elimination defect caused by ZFP64 or P/Q-VDCC knockdown.\",\n      \"method\": \"Purkinje cell-specific AAV-mediated ZFP64 knockdown, electrophysiological recording of CF synapse number, morphological analysis of CF innervation, genetic epistasis with Sema3A knockdown and P/Q-VDCC knockdown\",\n      \"journal\": \"iScience\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — cell-type-specific knockdown with defined electrophysiological and morphological phenotype, epistasis experiment, single lab\",\n      \"pmids\": [\"40546958\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"CAFs-derived lactate increases histone lactylation (H3K18la) at the ZFP64 locus, elevating ZFP64 expression in TNBC cells. Elevated ZFP64 then directly binds the promoters of GCH1 and FTH1 and activates their transcription, suppressing ferroptosis and conferring doxorubicin resistance.\",\n      \"method\": \"ChIP (ZFP64 binding to GCH1 and FTH1 promoters), dual-luciferase reporter, histone lactylation detection (pan-Kla, H3K18la), CAF co-culture system, ZFP64 knockdown with GCH1/FTH1 overexpression rescue, in vivo xenograft\",\n      \"journal\": \"Journal of translational medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — ChIP and luciferase reporter for promoter binding, rescue experiments, single lab, multiple orthogonal methods\",\n      \"pmids\": [\"40022222\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"ZFP64 directly binds the TUBB3 promoter to activate its transcription in gastric cancer cells; the lncRNA FIRRE promotes this activity by binding to ZFP64, and ZFP64 knockdown abrogates FIRRE-driven TUBB3 upregulation both in vitro and in vivo.\",\n      \"method\": \"ChIP (ZFP64 at TUBB3 promoter), dual-luciferase reporter assay, RNA immunoprecipitation (RIP, FIRRE–ZFP64 interaction), ZFP64 knockdown rescue experiments, in vivo tumor models\",\n      \"journal\": \"Cancer letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Weak — ChIP, luciferase, and RIP for the interaction/binding claims, single lab\",\n      \"pmids\": [\"39706253\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"CRISPR-Cas9 knockout of ZFP64 in K562 and HUDEP-2 cells increases HBG (γ-globin) mRNA expression by 1.5–2.5-fold, identifying ZFP64 as a repressor of fetal hemoglobin gene expression.\",\n      \"method\": \"CRISPR-Cas9 knockout of ZFP64 in K562 and HUDEP-2 cell lines, RT-qPCR for HBG mRNA levels\",\n      \"journal\": \"Cell biochemistry and biophysics\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single loss-of-function assay with mRNA readout only, no promoter binding or mechanistic pathway placement, single lab\",\n      \"pmids\": [\"40392482\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1997,\n      \"finding\": \"Mouse Zfp64 encodes a 614-amino-acid protein with ten C2H2 zinc finger motifs and multiple predicted phosphorylation sites for PKC, CK II, tyrosine kinase, and cAMP/cGMP-dependent protein kinases; it is expressed in all developing and mature mouse tissues examined.\",\n      \"method\": \"cDNA cloning, sequence analysis, in situ hybridization/Northern blot for expression pattern, chromosomal mapping\",\n      \"journal\": \"Gene\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — cloning and sequence-based prediction only, no functional assays performed\",\n      \"pmids\": [\"9034307\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"ZFP64 is a C2H2 zinc finger transcription factor that drives target gene expression by directly binding promoters (CSF1, GAL-1, Hes1/Hey1, GCH1, FTH1, ALDOC/ENO2/HK2, TUBB3, CENPL, PD-1/CTLA-4) and, in some contexts, by recruiting co-regulators (acting as a Notch1 coactivator or recruiting HDAC1 to repress NUMB); its activity and nuclear localization are regulated post-translationally by PKCα-mediated phosphorylation at S226, and its expression is epigenetically controlled by KDM1A-dependent H3K9me2 removal and by CAF-derived lactate-induced histone lactylation, with established roles in mesenchymal cell differentiation, cerebellar synapse elimination (downstream of P/Q-VDCCs via Sema3A), and transcriptional repression of fetal hemoglobin genes.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"ZFP64 is a C2H2 zinc finger transcription factor that controls cell-fate and differentiation programs by directly binding target gene promoters and, in selected contexts, recruiting co-regulators [#0, #1]. It first emerged as a Notch1 coactivator that associates with the Notch1 intracellular domain, is recruited to the Hes1 and Hey1 promoters, and—itself transactivated by Runx2—suppresses myogenic and promotes osteoblastic differentiation in mesenchymal cells [#0]. Beyond direct transcriptional activation, ZFP64 can also repress transcription by recruiting HDAC1 to deacetylate and silence the NUMB promoter [#4]. Its activity is gated post-translationally: PKCα directly phosphorylates ZFP64 at serine 226 to drive nuclear translocation and subsequent transcriptional activation of CSF1 [#1]. Across multiple tumor settings ZFP64 activates a broad repertoire of direct targets—GAL-1 [#2], glycolytic genes including ALDOC, ENO2 and HK2 [#5], the antioxidant/ferroptosis genes GCH1 and FTH1 [#8], TUBB3 [#9], CENPL [#6], and the immune checkpoint genes PD-1 and CTLA-4 [#3]—thereby supporting glycolysis, stemness, immunosuppression and therapy resistance. ZFP64 expression is itself epigenetically regulated, being activated by KDM1A-dependent removal of H3K9me2 [#6] and by CAF-derived lactate driving H3K18 histone lactylation at its locus [#8], and its target output can be modulated by the lncRNA FIRRE binding ZFP64 to enhance TUBB3 induction [#9]. In the nervous system, ZFP64 in Purkinje cells promotes activity-dependent climbing-fiber synapse elimination downstream of P/Q-type voltage-dependent Ca2+ channels by regulating Sema3A expression [#7].\",\n  \"teleology\": [\n    {\n      \"year\": 2008,\n      \"claim\": \"Established ZFP64's first mechanistic identity—as a Notch1 coactivator and Runx2 effector—linking it to a differentiation decision between myogenic and osteoblastic fates.\",\n      \"evidence\": \"Co-IP of ZFP64 with Notch1 ICD, ChIP at Hes1/Hey1 promoters, promoter transactivation, and C2C12 gain/loss-of-function differentiation assays\",\n      \"pmids\": [\"18430783\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether the same Notch-coactivator mechanism operates in the later tumor contexts is untested\", \"Direct DNA-binding sequence specificity of ZFP64 not defined\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Identified the post-translational switch governing ZFP64 nuclear entry, showing PKCα phosphorylation at S226 converts cytoplasmic ZFP64 into a nuclear CSF1-activating transcription factor that reshapes the tumor immune microenvironment.\",\n      \"evidence\": \"MS phosphosite mapping, in vitro kinase assay, nuclear fractionation/IF, ChIP-seq at CSF1 promoter, and orthotopic/transgenic HCC mouse models\",\n      \"pmids\": [\"35219791\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Upstream signals activating PKCα toward ZFP64 not defined\", \"Whether S226 phosphorylation regulates targets beyond CSF1 is untested\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Extended ZFP64 to direct activation of immunosuppression and stemness programs in gastric and esophageal cancers via GAL-1 and the checkpoint genes PD-1/CTLA-4.\",\n      \"evidence\": \"ChIP-seq and dual-luciferase reporter at GAL-1 promoter (gastric); luciferase reporter and IHC for PD-1/CTLA-4 with orthotopic xenografts (esophageal)\",\n      \"pmids\": [\"34996504\", \"35843097\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"PD-1/CTLA-4 binding rests on reporter assays without ChIP confirmation\", \"Cofactor requirements for these activation events unknown\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Revealed ZFP64 also functions as a transcriptional repressor by recruiting HDAC1 to deacetylate the NUMB promoter, indirectly amplifying Notch1 signaling.\",\n      \"evidence\": \"Co-IP of ZFP64–HDAC1, ChIP at NUMB promoter, and knockdown/overexpression in gallbladder cancer models\",\n      \"pmids\": [\"37760477\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"What dictates activator versus repressor mode at a given promoter is unknown\", \"Single-lab, limited orthogonal validation of the HDAC1 interaction\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Defined ZFP64 as a direct driver of glycolytic metabolism and as an epigenetically controlled node, activating glycolysis genes and CENPL while being switched on by KDM1A-mediated H3K9me2 removal.\",\n      \"evidence\": \"ChIP and dual-luciferase at glycolytic gene promoters with inhibitor rescue (breast); ChIP of KDM1A/H3K9me2 at ZFP64 promoter and rescue (ovarian)\",\n      \"pmids\": [\"39294751\", \"39628712\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct DNA-binding motif shared across glycolytic targets not defined\", \"CENPL regulation supported by a single low-replication study\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Connected ZFP64 to therapy resistance and lncRNA-modulated transcription, showing CAF-lactate-induced histone lactylation elevates ZFP64 to suppress ferroptosis (GCH1/FTH1), and that FIRRE binds ZFP64 to enhance TUBB3 induction.\",\n      \"evidence\": \"ChIP/luciferase at GCH1, FTH1 and TUBB3 promoters; H3K18la detection and CAF co-culture; RIP for FIRRE–ZFP64; rescue and xenograft experiments\",\n      \"pmids\": [\"40022222\", \"39706253\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Generality of histone-lactylation control of ZFP64 beyond TNBC untested\", \"Whether FIRRE alters ZFP64 DNA binding or stability not resolved\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Placed ZFP64 in neuronal circuit refinement, demonstrating it drives activity-dependent climbing-fiber synapse elimination downstream of P/Q-VDCCs through Sema3A.\",\n      \"evidence\": \"Purkinje-cell-specific AAV knockdown with electrophysiology, CF morphology, and genetic epistasis with Sema3A and P/Q-VDCC knockdown\",\n      \"pmids\": [\"40546958\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct binding of ZFP64 to the Sema3A locus not shown\", \"Single-lab knockdown rather than genetic knockout\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Implicated ZFP64 as a repressor of fetal hemoglobin, showing its knockout de-represses γ-globin in erythroid cell lines.\",\n      \"evidence\": \"CRISPR-Cas9 knockout in K562 and HUDEP-2 with RT-qPCR for HBG mRNA\",\n      \"pmids\": [\"40392482\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No promoter binding or mechanistic pathway placement; mRNA readout only\", \"Not independently confirmed in primary erythroid cells\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The intrinsic DNA-binding sequence specificity of ZFP64 and the rules determining when it activates versus represses a given promoter remain undefined.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No consensus binding motif established across its many reported targets\", \"No structural model of ZFP64 zinc fingers on DNA\", \"Cofactor composition that distinguishes activator from HDAC1-repressor mode unresolved\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140110\", \"supporting_discovery_ids\": [0, 1, 2, 4, 5, 8, 9]},\n      {\"term_id\": \"GO:0003677\", \"supporting_discovery_ids\": [1, 2, 5, 8, 9]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [1]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-74160\", \"supporting_discovery_ids\": [0, 1, 2, 5, 8, 9]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 4]},\n      {\"term_id\": \"R-HSA-4839726\", \"supporting_discovery_ids\": [4, 6, 8]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"NOTCH1\", \"HDAC1\", \"PRKCA\", \"RUNX2\", \"FIRRE\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}