{"gene":"PHRF1","run_date":"2026-06-10T06:43:35","timeline":{"discoveries":[{"year":2013,"finding":"PHRF1 functions as an E3 ubiquitin ligase that ubiquitinates TGIF (TG-interacting factor) at lysine 130, driving its proteasomal degradation. This TGIF degradation releases cPML to redistribute into the cytoplasm, where cPML associates with SARA and coordinates Smad2 phosphorylation/activation by the TGF-β receptor, thereby promoting TGF-β cytostatic signaling.","method":"Ubiquitination assay, immunoprecipitation, site-directed mutagenesis (K130 of TGIF), cell fractionation, tumor xenograft reconstitution","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — multiple orthogonal methods (ubiquitination assay, mutagenesis identifying K130, cPML redistribution, in vivo reconstitution) in a single rigorous study","pmids":["23911286"],"is_preprint":false},{"year":2015,"finding":"PHRF1 localizes rapidly to DNA damage lesions upon genotoxic insults. Its PHD domain binds constitutively to di- and trimethylated histone H3 lysine 36 (H3K36me2/me3). The SDTE motif (S915DT917E) is required for interaction with NBS1. Both the PHD domain and SDTE motif are required for PHRF1's ability to promote non-homologous end-joining (NHEJ). PHRF1 also mediates PARP1 polyubiquitination leading to its proteasomal degradation.","method":"Immunoprecipitation, peptide pull-down assay, plasmid-based NHEJ reporter assay, site-directed mutagenesis (SDTEADAE), overexpression/ablation in H1299 reporter cells, live-cell localization after DNA damage","journal":"Cell death & disease","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — multiple orthogonal methods (pull-down for H3K36me2/3 binding, mutagenesis of SDTE motif, functional NHEJ reporter, PARP1 ubiquitination assay) in a single study","pmids":["25855964"],"is_preprint":false},{"year":2015,"finding":"PML-RARα disrupts the PHRF1 tumor suppressor network by competing with PHRF1 for binding to TGIF, thereby blocking PHRF1-mediated TGIF ubiquitination and degradation. This results in cPML sequestration and inactivation, suppressing TGF-β cytostatic signaling and promoting acute promyelocytic leukemia (APL). Enforcing PHRF1 activity restores TGF-β signaling in human blasts and suppresses APL formation in a mouse model.","method":"Co-immunoprecipitation (competition binding), TGIF ubiquitination assay, TGF-β reporter assay, mouse APL model with PHRF1 reconstitution","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — Co-IP competition, functional ubiquitination assay, in vivo mouse APL model with rescue experiment","pmids":["25683711"],"is_preprint":false},{"year":2020,"finding":"PHRF1 promotes lung cancer cell migration and invasion by modulating ZEB1 transcription. PHRF1 associates with the phosphorylated C-terminal repeat domain (CTD) of Rpb1 (large subunit of RNA Pol II) via its C-terminal SRI domain. Chromatin immunoprecipitation showed PHRF1 binds the proximal region adjacent to the ZEB1 transcription start site. SRI domain deletion abolishes both Rpb1 association and ZEB1 upregulation.","method":"Co-immunoprecipitation, chromatin immunoprecipitation (ChIP), SRI domain deletion mutagenesis, transwell invasion/migration assay","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — reciprocal Co-IP plus ChIP plus domain mutagenesis, single lab","pmids":["32730336"],"is_preprint":false},{"year":2023,"finding":"In colorectal cancer HCT116-p53-/- cells, PHRF1 promotes invasion via its C-terminal SRI domain by modulating SOX4 expression. PHRF1 knockout reduces SOX4 levels and impairs invasion; reintroduction of SOX4 partially restores invasive capability. ZEB1 expression was not affected (negative finding for ZEB1 in this context).","method":"CRISPR-Cas9 PHRF1 knockout, SRI domain deletion, transwell invasion assay, SOX4 rescue experiment, expression profiling","journal":"Anticancer research","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — CRISPR KO, domain mutagenesis, and rescue experiment in a single lab","pmids":["38030167"],"is_preprint":false},{"year":2023,"finding":"PHRF1 promotes IgA class switch recombination in CH12F3-2A B cells. PHRF1 knockout reduces IgA production and decreases levels of PARP1, NELF-A, and NELF-D. Reintroduction of PARP1 partially restores IgA switching in PHRF1 knockout cells. However, IgA and other Ig class switches were not significantly decreased in PHRF1-deficient primary splenic B cells from CD19-Cre mice (negative finding in primary cells).","method":"CRISPR-Cas9 knockout, shRNA silencing, flow cytometry for IgA, PARP1 rescue, CD19-Cre conditional knockout mice","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — CRISPR KO and rescue in cell line, with discordant in vivo result reducing overall confidence","pmids":["37540725"],"is_preprint":false},{"year":2025,"finding":"PHRF1 mono-ubiquitinates TopBP1 at lysine 73, which enhances the TopBP1-ATR interaction and promotes ATR activation during replication stress. PHRF1 is recruited to DNA lesions in a manner dependent on its PHD domain and histone methylation. PHRF1 depletion disrupts ATR activation and sensitizes cells to replication stress agents. Conditional knockout of Phrf1 in mice causes early lethality with impaired ATR-Chk1 axis signaling.","method":"Co-immunoprecipitation, in vitro ubiquitination assay, site-directed mutagenesis (K73 of TopBP1), ATR/Chk1 activation assays, conditional mouse knockout, replication stress sensitivity assays","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — in vitro ubiquitination assay with K73 mutagenesis, Co-IP, functional ATR activation, and in vivo mouse KO in a single rigorous study","pmids":["40052822"],"is_preprint":false},{"year":2025,"finding":"The PHD finger of PHRF1 robustly binds the N-terminal region of histone H3. A cancer-associated P221L mutation in the PHD finger abolishes histone H3 interaction and fails to rescue defective DNA damage response (DDR) in PHRF1 knockout cells, demonstrating that H3 binding by the PHD finger is required for proper DDR. PHRF1 also regulates transcription and RNA splicing as shown by RNA-seq and proteomics.","method":"Biochemical binding assays, site-directed mutagenesis (P221L), PHRF1 knockout cells, DDR functional rescue assay, RNA-seq, proteomics","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — biochemical H3 binding, P221L mutagenesis abolishing interaction, functional DDR rescue in KO cells, orthogonal RNA-seq/proteomics","pmids":["40671529"],"is_preprint":false},{"year":2025,"finding":"PHRF1 acts as an E3 ubiquitin ligase for p53, targeting it for ubiquitin-proteasome-mediated degradation. In XPC-deficient bladder cancer cells, KDM4A overactivation suppresses PHRF1 expression, leading to nuclear p53 accumulation and autophagy induction in response to cisplatin. Under cisplatin-induced DNA damage where MDM2 function is impaired, PHRF1 retains its E3 ligase activity toward p53.","method":"Western blot (p53 accumulation), ubiquitination assay, KDM4A inhibitor treatment, mouse xenograft model, XPC knockdown/knockout","journal":"Advanced science","confidence":"Medium","confidence_rationale":"Tier 2–3 / Weak — ubiquitination assay and xenograft model, single lab, limited mechanistic detail in abstract","pmids":["41215711"],"is_preprint":false},{"year":2016,"finding":"Overexpression of PHRF1 in H1299 non-small cell lung cancer cells inhibits proliferation and tumorigenicity, arrests cell cycle in G1 phase, decreases TGIF and c-Myc protein levels, and increases p21 protein levels, consistent with its role in TGF-β cytostatic signaling.","method":"Overexpression in H1299 cells, flow cytometry cell cycle analysis, soft agar assay, tumor xenograft, Western blot","journal":"Oncotarget","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — multiple cellular assays (cell cycle, xenograft, protein expression) in a single lab without deep mechanistic dissection","pmids":["27608840"],"is_preprint":false}],"current_model":"PHRF1 is a PHD and RING finger domain-containing E3 ubiquitin ligase that: (1) ubiquitinates TGIF at K130 to drive its degradation and release cPML for TGF-β/Smad2 signaling; (2) mono-ubiquitinates TopBP1 at K73 to enhance TopBP1-ATR interaction and ATR activation during replication stress; (3) binds H3K36me2/3 via its PHD domain and NBS1 via its SDTE motif to promote NHEJ at DNA damage sites; (4) associates with RNA Pol II CTD via its SRI domain to regulate ZEB1 and SOX4 transcription and cell invasion; (5) ubiquitinates PARP1 for proteasomal degradation; and (6) ubiquitinates p53 for degradation in a context where MDM2 is impaired—with the PHD finger's binding to histone H3 N-terminus being essential for proper DNA damage response."},"narrative":{"mechanistic_narrative":"PHRF1 is a chromatin-associated PHD- and RING-finger E3 ubiquitin ligase that couples histone-mark recognition to ubiquitin-dependent control of TGF-β signaling, the DNA damage and replication-stress response, and tumor cell invasion [PMID:23911286, PMID:25855964, PMID:40052822]. As a tumor suppressor in the TGF-β axis, PHRF1 ubiquitinates TGIF at K130 to drive its proteasomal degradation, releasing cytoplasmic PML to coordinate SARA-dependent Smad2 activation and cytostatic signaling; PML-RARα subverts this network by competing for TGIF binding, blocking degradation and promoting acute promyelocytic leukemia, and enforced PHRF1 activity restores TGF-β signaling and suppresses APL [PMID:23911286, PMID:25683711]. PHRF1 is recruited to DNA lesions through its PHD finger, which binds di-/trimethylated H3K36 and the N-terminal region of histone H3, and through its SDTE motif, which binds NBS1; this engagement supports non-homologous end-joining, and a cancer-associated P221L PHD mutation that abolishes H3 binding fails to rescue the DNA damage response [PMID:25855964, PMID:40671529]. During replication stress PHRF1 mono-ubiquitinates TopBP1 at K73 to enhance the TopBP1-ATR interaction and ATR-Chk1 activation, with conditional Phrf1 loss causing early embryonic lethality and impaired ATR-Chk1 signaling [PMID:40052822]. PHRF1 additionally ubiquitinates PARP1 and p53 for proteasomal degradation, and through its SRI domain associates with the phosphorylated RNA Pol II CTD to regulate ZEB1 and SOX4 transcription and drive cancer cell migration and invasion [PMID:25855964, PMID:41215711, PMID:32730336, PMID:38030167].","teleology":[{"year":2013,"claim":"Established PHRF1's first defined molecular activity—that it is an E3 ligase whose substrate TGIF links it to TGF-β cytostatic signaling—answering what biological pathway PHRF1 controls.","evidence":"Ubiquitination assay, K130 site-directed mutagenesis, cPML redistribution and tumor xenograft reconstitution","pmids":["23911286"],"confidence":"High","gaps":["Did not define how PHRF1 is regulated or recruited","E2 enzyme partner not identified"]},{"year":2015,"claim":"Defined the chromatin-recruitment logic of PHRF1—PHD-domain reading of H3K36me2/3 and SDTE-mediated NBS1 binding—linking it to DNA repair via NHEJ, and identified PARP1 as an additional ubiquitination substrate.","evidence":"Peptide pull-down, SDTE mutagenesis, NHEJ reporter assay and PARP1 ubiquitination assay in H1299 cells","pmids":["25855964"],"confidence":"High","gaps":["Did not connect repair role to a specific ubiquitination substrate at the lesion","Functional consequence of PARP1 degradation not detailed"]},{"year":2015,"claim":"Showed the TGIF/PHRF1 tumor-suppressor axis is a target of oncogenic disruption, with PML-RARα competing for TGIF to drive APL—establishing disease relevance and a rescue model.","evidence":"Competition Co-IP, TGIF ubiquitination and TGF-β reporter assays, mouse APL model with PHRF1 reconstitution","pmids":["25683711"],"confidence":"High","gaps":["Structural basis of the TGIF-binding competition not resolved","Generalizability beyond APL not tested"]},{"year":2016,"claim":"Connected PHRF1 expression to a concrete cell-cycle/tumor-suppressor phenotype, showing overexpression reduces TGIF and c-Myc, raises p21, and arrests cells in G1.","evidence":"Overexpression in H1299 cells, cell-cycle flow cytometry, soft agar and xenograft assays","pmids":["27608840"],"confidence":"Medium","gaps":["Did not dissect direct vs indirect effects on c-Myc and p21","Single cell line"]},{"year":2020,"claim":"Revealed a transcriptional, ligase-independent arm of PHRF1—SRI-domain association with the phosphorylated Pol II CTD—driving ZEB1 expression and lung cancer invasion.","evidence":"Reciprocal Co-IP, ChIP at the ZEB1 TSS, SRI deletion mutagenesis and transwell invasion assays","pmids":["32730336"],"confidence":"Medium","gaps":["Single lab","Direct vs indirect transcriptional control not separated from ligase function"]},{"year":2023,"claim":"Extended the SRI-dependent invasion role to colorectal cancer through SOX4 rather than ZEB1, indicating context-specific transcriptional targets.","evidence":"CRISPR PHRF1 knockout, SRI deletion, SOX4 rescue and transwell invasion in HCT116-p53-/- cells","pmids":["38030167"],"confidence":"Medium","gaps":["Mechanism of target selection (SOX4 vs ZEB1) unexplained","Single lab"]},{"year":2023,"claim":"Tested whether PHRF1 contributes to antibody class switching, finding it promotes IgA switching and PARP1/NELF levels in a B-cell line but with a discordant negative result in primary B cells.","evidence":"CRISPR knockout, shRNA, flow cytometry for IgA, PARP1 rescue and CD19-Cre conditional mice","pmids":["37540725"],"confidence":"Medium","gaps":["Cell-line vs primary B-cell discordance unresolved","Mechanistic link between PHRF1, PARP1/NELF and CSR not defined"]},{"year":2025,"claim":"Defined PHRF1's role in the replication-stress response by identifying TopBP1 K73 mono-ubiquitination as the event that enhances TopBP1-ATR interaction and ATR-Chk1 activation, with mouse KO confirming essentiality.","evidence":"In vitro ubiquitination with K73 mutagenesis, Co-IP, ATR/Chk1 activation assays and conditional mouse knockout","pmids":["40052822"],"confidence":"High","gaps":["E2 partner and regulation of mono- vs poly-ubiquitination not defined","Relationship to NHEJ role not integrated"]},{"year":2025,"claim":"Demonstrated that PHD-finger binding to the histone H3 N-terminus is functionally required for the DNA damage response, using a cancer P221L mutation that abolishes binding and rescue.","evidence":"Biochemical H3 binding, P221L mutagenesis, DDR rescue in knockout cells, RNA-seq and proteomics","pmids":["40671529"],"confidence":"High","gaps":["Splicing/transcription regulatory mechanism not mechanistically resolved","Relative contribution of H3 vs H3K36me binding not separated"]},{"year":2025,"claim":"Identified p53 as a PHRF1 ubiquitination substrate operating when MDM2 is impaired, placing PHRF1 in a cisplatin-response circuit downstream of KDM4A/XPC in bladder cancer.","evidence":"p53 ubiquitination assay, KDM4A inhibition, XPC knockdown and mouse xenograft model","pmids":["41215711"],"confidence":"Medium","gaps":["Direct vs indirect p53 ubiquitination not fully resolved","Single lab, limited mechanistic detail"]},{"year":null,"claim":"How PHRF1's distinct activities—substrate ubiquitination (TGIF, TopBP1, PARP1, p53), chromatin reading via the PHD finger, and SRI-dependent transcriptional control—are coordinated and selected in a given cellular context remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unifying model for substrate/target selection across pathways","E2 partners unidentified","No structural model of the multidomain protein on chromatin"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0016874","term_label":"ligase activity","supporting_discovery_ids":[0,1,6,8]},{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,1,6,8]},{"term_id":"GO:0042393","term_label":"histone binding","supporting_discovery_ids":[1,7]},{"term_id":"GO:0140110","term_label":"transcription regulator activity","supporting_discovery_ids":[3,4]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[1,7,8]},{"term_id":"GO:0000228","term_label":"nuclear chromosome","supporting_discovery_ids":[1,6]}],"pathway":[{"term_id":"R-HSA-73894","term_label":"DNA Repair","supporting_discovery_ids":[1,7]},{"term_id":"R-HSA-8953897","term_label":"Cellular responses to stimuli","supporting_discovery_ids":[6]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0,2]},{"term_id":"R-HSA-74160","term_label":"Gene expression (Transcription)","supporting_discovery_ids":[3,4]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[0,1,6,8]}],"complexes":[],"partners":["TGIF1","NBS1","TOPBP1","PARP1","TP53","POLR2A","PML"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q9P1Y6","full_name":"PHD and RING finger domain-containing protein 1","aliases":[],"length_aa":1649,"mass_kda":178.7,"function":"","subcellular_location":"","url":"https://www.uniprot.org/uniprotkb/Q9P1Y6/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/PHRF1","classification":"Not Classified","n_dependent_lines":2,"n_total_lines":1208,"dependency_fraction":0.0016556291390728477},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/PHRF1","total_profiled":1310},"omim":[{"mim_id":"611780","title":"PHD AND RING FINGER DOMAIN-CONTAINING PROTEIN 1; PHRF1","url":"https://www.omim.org/entry/611780"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"","locations":[],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in many","driving_tissues":[],"url":"https://www.proteinatlas.org/search/PHRF1"},"hgnc":{"alias_symbol":["KIAA1542","PPP1R125"],"prev_symbol":["RNF221"]},"alphafold":{"accession":"Q9P1Y6","domains":[{"cath_id":"3.30.40,3.30.40","chopping":"108-175","consensus_level":"medium","plddt":87.2369,"start":108,"end":175},{"cath_id":"3.30.40.10","chopping":"184-227","consensus_level":"medium","plddt":84.9395,"start":184,"end":227},{"cath_id":"1.10.8","chopping":"1562-1631","consensus_level":"medium","plddt":92.1039,"start":1562,"end":1631}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9P1Y6","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9P1Y6-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9P1Y6-F1-predicted_aligned_error_v6.png","plddt_mean":45.72},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=PHRF1","jax_strain_url":"https://www.jax.org/strain/search?query=PHRF1"},"sequence":{"accession":"Q9P1Y6","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9P1Y6.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9P1Y6/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9P1Y6"}},"corpus_meta":[{"pmid":"18204446","id":"PMC_18204446","title":"Genome-wide association scan in women with systemic lupus erythematosus identifies susceptibility variants in ITGAM, PXK, KIAA1542 and other loci.","date":"2008","source":"Nature genetics","url":"https://pubmed.ncbi.nlm.nih.gov/18204446","citation_count":1075,"is_preprint":false},{"pmid":"20112359","id":"PMC_20112359","title":"Genetic variation at the IRF7/PHRF1 locus is associated with autoantibody profile and serum interferon-alpha activity in lupus patients.","date":"2010","source":"Arthritis and rheumatism","url":"https://pubmed.ncbi.nlm.nih.gov/20112359","citation_count":125,"is_preprint":false},{"pmid":"25855964","id":"PMC_25855964","title":"PHRF1 promotes genome integrity by modulating non-homologous end-joining.","date":"2015","source":"Cell death & disease","url":"https://pubmed.ncbi.nlm.nih.gov/25855964","citation_count":33,"is_preprint":false},{"pmid":"23911286","id":"PMC_23911286","title":"Identification of PHRF1 as a tumor suppressor that promotes the TGF-β cytostatic program through selective release of TGIF-driven PML inactivation.","date":"2013","source":"Cell reports","url":"https://pubmed.ncbi.nlm.nih.gov/23911286","citation_count":31,"is_preprint":false},{"pmid":"21167895","id":"PMC_21167895","title":"Association of genetic variations in the STAT4 and IRF7/KIAA1542 regions with systemic lupus erythematosus in a Northern Han Chinese population.","date":"2010","source":"Human immunology","url":"https://pubmed.ncbi.nlm.nih.gov/21167895","citation_count":28,"is_preprint":false},{"pmid":"25683711","id":"PMC_25683711","title":"Disruption of the PHRF1 Tumor Suppressor Network by PML-RARα Drives Acute Promyelocytic Leukemia Pathogenesis.","date":"2015","source":"Cell reports","url":"https://pubmed.ncbi.nlm.nih.gov/25683711","citation_count":21,"is_preprint":false},{"pmid":"27608840","id":"PMC_27608840","title":"Overexpression of PHRF1 attenuates the proliferation and tumorigenicity of non-small cell lung cancer cells.","date":"2016","source":"Oncotarget","url":"https://pubmed.ncbi.nlm.nih.gov/27608840","citation_count":11,"is_preprint":false},{"pmid":"32730336","id":"PMC_32730336","title":"PHRF1 promotes migration and invasion by modulating ZEB1 expression.","date":"2020","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/32730336","citation_count":5,"is_preprint":false},{"pmid":"38669818","id":"PMC_38669818","title":"Research Note: Chicken breast muscle satellite cell function: effect of expression of CNN1 and PHRF1.","date":"2024","source":"Poultry science","url":"https://pubmed.ncbi.nlm.nih.gov/38669818","citation_count":4,"is_preprint":false},{"pmid":"38030167","id":"PMC_38030167","title":"PHRF1 Promotes Cell Invasion by Modulating SOX4 Expression in Colorectal Cancer HCT116-p53-/- Cells.","date":"2023","source":"Anticancer research","url":"https://pubmed.ncbi.nlm.nih.gov/38030167","citation_count":3,"is_preprint":false},{"pmid":"41215711","id":"PMC_41215711","title":"XPC Deficiency Activate Cisplatin-Mediated Autophagy in Bladder Cancer by Limiting Novel PHRF1-Mediated Ubiquitination of the p53 Protein.","date":"2025","source":"Advanced science (Weinheim, Baden-Wurttemberg, Germany)","url":"https://pubmed.ncbi.nlm.nih.gov/41215711","citation_count":3,"is_preprint":false},{"pmid":"37540725","id":"PMC_37540725","title":"PHRF1 promotes the class switch recombination of IgA in CH12F3-2A cells.","date":"2023","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/37540725","citation_count":2,"is_preprint":false},{"pmid":"40671529","id":"PMC_40671529","title":"Histone H3 N-terminal recognition by the PHD finger of PHRF1 is required for proper DNA damage response.","date":"2025","source":"Nucleic acids research","url":"https://pubmed.ncbi.nlm.nih.gov/40671529","citation_count":2,"is_preprint":false},{"pmid":"40052822","id":"PMC_40052822","title":"Mono-ubiquitination of TopBP1 by PHRF1 enhances ATR activation and genomic stability.","date":"2025","source":"Nucleic acids research","url":"https://pubmed.ncbi.nlm.nih.gov/40052822","citation_count":1,"is_preprint":false},{"pmid":"39605374","id":"PMC_39605374","title":"Histone H3 N-terminal recognition by the PHD finger of PHRF1 is required for proper DNA damage response.","date":"2024","source":"bioRxiv : the preprint server for biology","url":"https://pubmed.ncbi.nlm.nih.gov/39605374","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":9236,"output_tokens":3061,"usd":0.036811,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":10398,"output_tokens":3718,"usd":0.07247,"stage2_stop_reason":"end_turn"},"total_usd":0.109281,"stage1_batch_id":"msgbatch_01CNoDbkChHEkgrJnL7BwtGJ","stage2_batch_id":"msgbatch_016Y4x9E9Wg5VQDANj9quSQD","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2013,\n      \"finding\": \"PHRF1 functions as an E3 ubiquitin ligase that ubiquitinates TGIF (TG-interacting factor) at lysine 130, driving its proteasomal degradation. This TGIF degradation releases cPML to redistribute into the cytoplasm, where cPML associates with SARA and coordinates Smad2 phosphorylation/activation by the TGF-β receptor, thereby promoting TGF-β cytostatic signaling.\",\n      \"method\": \"Ubiquitination assay, immunoprecipitation, site-directed mutagenesis (K130 of TGIF), cell fractionation, tumor xenograft reconstitution\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — multiple orthogonal methods (ubiquitination assay, mutagenesis identifying K130, cPML redistribution, in vivo reconstitution) in a single rigorous study\",\n      \"pmids\": [\"23911286\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"PHRF1 localizes rapidly to DNA damage lesions upon genotoxic insults. Its PHD domain binds constitutively to di- and trimethylated histone H3 lysine 36 (H3K36me2/me3). The SDTE motif (S915DT917E) is required for interaction with NBS1. Both the PHD domain and SDTE motif are required for PHRF1's ability to promote non-homologous end-joining (NHEJ). PHRF1 also mediates PARP1 polyubiquitination leading to its proteasomal degradation.\",\n      \"method\": \"Immunoprecipitation, peptide pull-down assay, plasmid-based NHEJ reporter assay, site-directed mutagenesis (SDTEADAE), overexpression/ablation in H1299 reporter cells, live-cell localization after DNA damage\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — multiple orthogonal methods (pull-down for H3K36me2/3 binding, mutagenesis of SDTE motif, functional NHEJ reporter, PARP1 ubiquitination assay) in a single study\",\n      \"pmids\": [\"25855964\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"PML-RARα disrupts the PHRF1 tumor suppressor network by competing with PHRF1 for binding to TGIF, thereby blocking PHRF1-mediated TGIF ubiquitination and degradation. This results in cPML sequestration and inactivation, suppressing TGF-β cytostatic signaling and promoting acute promyelocytic leukemia (APL). Enforcing PHRF1 activity restores TGF-β signaling in human blasts and suppresses APL formation in a mouse model.\",\n      \"method\": \"Co-immunoprecipitation (competition binding), TGIF ubiquitination assay, TGF-β reporter assay, mouse APL model with PHRF1 reconstitution\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — Co-IP competition, functional ubiquitination assay, in vivo mouse APL model with rescue experiment\",\n      \"pmids\": [\"25683711\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"PHRF1 promotes lung cancer cell migration and invasion by modulating ZEB1 transcription. PHRF1 associates with the phosphorylated C-terminal repeat domain (CTD) of Rpb1 (large subunit of RNA Pol II) via its C-terminal SRI domain. Chromatin immunoprecipitation showed PHRF1 binds the proximal region adjacent to the ZEB1 transcription start site. SRI domain deletion abolishes both Rpb1 association and ZEB1 upregulation.\",\n      \"method\": \"Co-immunoprecipitation, chromatin immunoprecipitation (ChIP), SRI domain deletion mutagenesis, transwell invasion/migration assay\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — reciprocal Co-IP plus ChIP plus domain mutagenesis, single lab\",\n      \"pmids\": [\"32730336\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"In colorectal cancer HCT116-p53-/- cells, PHRF1 promotes invasion via its C-terminal SRI domain by modulating SOX4 expression. PHRF1 knockout reduces SOX4 levels and impairs invasion; reintroduction of SOX4 partially restores invasive capability. ZEB1 expression was not affected (negative finding for ZEB1 in this context).\",\n      \"method\": \"CRISPR-Cas9 PHRF1 knockout, SRI domain deletion, transwell invasion assay, SOX4 rescue experiment, expression profiling\",\n      \"journal\": \"Anticancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — CRISPR KO, domain mutagenesis, and rescue experiment in a single lab\",\n      \"pmids\": [\"38030167\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"PHRF1 promotes IgA class switch recombination in CH12F3-2A B cells. PHRF1 knockout reduces IgA production and decreases levels of PARP1, NELF-A, and NELF-D. Reintroduction of PARP1 partially restores IgA switching in PHRF1 knockout cells. However, IgA and other Ig class switches were not significantly decreased in PHRF1-deficient primary splenic B cells from CD19-Cre mice (negative finding in primary cells).\",\n      \"method\": \"CRISPR-Cas9 knockout, shRNA silencing, flow cytometry for IgA, PARP1 rescue, CD19-Cre conditional knockout mice\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — CRISPR KO and rescue in cell line, with discordant in vivo result reducing overall confidence\",\n      \"pmids\": [\"37540725\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"PHRF1 mono-ubiquitinates TopBP1 at lysine 73, which enhances the TopBP1-ATR interaction and promotes ATR activation during replication stress. PHRF1 is recruited to DNA lesions in a manner dependent on its PHD domain and histone methylation. PHRF1 depletion disrupts ATR activation and sensitizes cells to replication stress agents. Conditional knockout of Phrf1 in mice causes early lethality with impaired ATR-Chk1 axis signaling.\",\n      \"method\": \"Co-immunoprecipitation, in vitro ubiquitination assay, site-directed mutagenesis (K73 of TopBP1), ATR/Chk1 activation assays, conditional mouse knockout, replication stress sensitivity assays\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — in vitro ubiquitination assay with K73 mutagenesis, Co-IP, functional ATR activation, and in vivo mouse KO in a single rigorous study\",\n      \"pmids\": [\"40052822\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"The PHD finger of PHRF1 robustly binds the N-terminal region of histone H3. A cancer-associated P221L mutation in the PHD finger abolishes histone H3 interaction and fails to rescue defective DNA damage response (DDR) in PHRF1 knockout cells, demonstrating that H3 binding by the PHD finger is required for proper DDR. PHRF1 also regulates transcription and RNA splicing as shown by RNA-seq and proteomics.\",\n      \"method\": \"Biochemical binding assays, site-directed mutagenesis (P221L), PHRF1 knockout cells, DDR functional rescue assay, RNA-seq, proteomics\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — biochemical H3 binding, P221L mutagenesis abolishing interaction, functional DDR rescue in KO cells, orthogonal RNA-seq/proteomics\",\n      \"pmids\": [\"40671529\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"PHRF1 acts as an E3 ubiquitin ligase for p53, targeting it for ubiquitin-proteasome-mediated degradation. In XPC-deficient bladder cancer cells, KDM4A overactivation suppresses PHRF1 expression, leading to nuclear p53 accumulation and autophagy induction in response to cisplatin. Under cisplatin-induced DNA damage where MDM2 function is impaired, PHRF1 retains its E3 ligase activity toward p53.\",\n      \"method\": \"Western blot (p53 accumulation), ubiquitination assay, KDM4A inhibitor treatment, mouse xenograft model, XPC knockdown/knockout\",\n      \"journal\": \"Advanced science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Weak — ubiquitination assay and xenograft model, single lab, limited mechanistic detail in abstract\",\n      \"pmids\": [\"41215711\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Overexpression of PHRF1 in H1299 non-small cell lung cancer cells inhibits proliferation and tumorigenicity, arrests cell cycle in G1 phase, decreases TGIF and c-Myc protein levels, and increases p21 protein levels, consistent with its role in TGF-β cytostatic signaling.\",\n      \"method\": \"Overexpression in H1299 cells, flow cytometry cell cycle analysis, soft agar assay, tumor xenograft, Western blot\",\n      \"journal\": \"Oncotarget\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — multiple cellular assays (cell cycle, xenograft, protein expression) in a single lab without deep mechanistic dissection\",\n      \"pmids\": [\"27608840\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"PHRF1 is a PHD and RING finger domain-containing E3 ubiquitin ligase that: (1) ubiquitinates TGIF at K130 to drive its degradation and release cPML for TGF-β/Smad2 signaling; (2) mono-ubiquitinates TopBP1 at K73 to enhance TopBP1-ATR interaction and ATR activation during replication stress; (3) binds H3K36me2/3 via its PHD domain and NBS1 via its SDTE motif to promote NHEJ at DNA damage sites; (4) associates with RNA Pol II CTD via its SRI domain to regulate ZEB1 and SOX4 transcription and cell invasion; (5) ubiquitinates PARP1 for proteasomal degradation; and (6) ubiquitinates p53 for degradation in a context where MDM2 is impaired—with the PHD finger's binding to histone H3 N-terminus being essential for proper DNA damage response.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"PHRF1 is a chromatin-associated PHD- and RING-finger E3 ubiquitin ligase that couples histone-mark recognition to ubiquitin-dependent control of TGF-\\u03b2 signaling, the DNA damage and replication-stress response, and tumor cell invasion [#0, #1, #6]. As a tumor suppressor in the TGF-\\u03b2 axis, PHRF1 ubiquitinates TGIF at K130 to drive its proteasomal degradation, releasing cytoplasmic PML to coordinate SARA-dependent Smad2 activation and cytostatic signaling; PML-RAR\\u03b1 subverts this network by competing for TGIF binding, blocking degradation and promoting acute promyelocytic leukemia, and enforced PHRF1 activity restores TGF-\\u03b2 signaling and suppresses APL [#0, #2]. PHRF1 is recruited to DNA lesions through its PHD finger, which binds di-/trimethylated H3K36 and the N-terminal region of histone H3, and through its SDTE motif, which binds NBS1; this engagement supports non-homologous end-joining, and a cancer-associated P221L PHD mutation that abolishes H3 binding fails to rescue the DNA damage response [#1, #7]. During replication stress PHRF1 mono-ubiquitinates TopBP1 at K73 to enhance the TopBP1-ATR interaction and ATR-Chk1 activation, with conditional Phrf1 loss causing early embryonic lethality and impaired ATR-Chk1 signaling [#6]. PHRF1 additionally ubiquitinates PARP1 and p53 for proteasomal degradation, and through its SRI domain associates with the phosphorylated RNA Pol II CTD to regulate ZEB1 and SOX4 transcription and drive cancer cell migration and invasion [#1, #8, #3, #4].\",\n  \"teleology\": [\n    {\n      \"year\": 2013,\n      \"claim\": \"Established PHRF1's first defined molecular activity\\u2014that it is an E3 ligase whose substrate TGIF links it to TGF-\\u03b2 cytostatic signaling\\u2014answering what biological pathway PHRF1 controls.\",\n      \"evidence\": \"Ubiquitination assay, K130 site-directed mutagenesis, cPML redistribution and tumor xenograft reconstitution\",\n      \"pmids\": [\"23911286\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not define how PHRF1 is regulated or recruited\", \"E2 enzyme partner not identified\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Defined the chromatin-recruitment logic of PHRF1\\u2014PHD-domain reading of H3K36me2/3 and SDTE-mediated NBS1 binding\\u2014linking it to DNA repair via NHEJ, and identified PARP1 as an additional ubiquitination substrate.\",\n      \"evidence\": \"Peptide pull-down, SDTE mutagenesis, NHEJ reporter assay and PARP1 ubiquitination assay in H1299 cells\",\n      \"pmids\": [\"25855964\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not connect repair role to a specific ubiquitination substrate at the lesion\", \"Functional consequence of PARP1 degradation not detailed\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Showed the TGIF/PHRF1 tumor-suppressor axis is a target of oncogenic disruption, with PML-RAR\\u03b1 competing for TGIF to drive APL\\u2014establishing disease relevance and a rescue model.\",\n      \"evidence\": \"Competition Co-IP, TGIF ubiquitination and TGF-\\u03b2 reporter assays, mouse APL model with PHRF1 reconstitution\",\n      \"pmids\": [\"25683711\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of the TGIF-binding competition not resolved\", \"Generalizability beyond APL not tested\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Connected PHRF1 expression to a concrete cell-cycle/tumor-suppressor phenotype, showing overexpression reduces TGIF and c-Myc, raises p21, and arrests cells in G1.\",\n      \"evidence\": \"Overexpression in H1299 cells, cell-cycle flow cytometry, soft agar and xenograft assays\",\n      \"pmids\": [\"27608840\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Did not dissect direct vs indirect effects on c-Myc and p21\", \"Single cell line\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Revealed a transcriptional, ligase-independent arm of PHRF1\\u2014SRI-domain association with the phosphorylated Pol II CTD\\u2014driving ZEB1 expression and lung cancer invasion.\",\n      \"evidence\": \"Reciprocal Co-IP, ChIP at the ZEB1 TSS, SRI deletion mutagenesis and transwell invasion assays\",\n      \"pmids\": [\"32730336\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab\", \"Direct vs indirect transcriptional control not separated from ligase function\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Extended the SRI-dependent invasion role to colorectal cancer through SOX4 rather than ZEB1, indicating context-specific transcriptional targets.\",\n      \"evidence\": \"CRISPR PHRF1 knockout, SRI deletion, SOX4 rescue and transwell invasion in HCT116-p53-/- cells\",\n      \"pmids\": [\"38030167\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism of target selection (SOX4 vs ZEB1) unexplained\", \"Single lab\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Tested whether PHRF1 contributes to antibody class switching, finding it promotes IgA switching and PARP1/NELF levels in a B-cell line but with a discordant negative result in primary B cells.\",\n      \"evidence\": \"CRISPR knockout, shRNA, flow cytometry for IgA, PARP1 rescue and CD19-Cre conditional mice\",\n      \"pmids\": [\"37540725\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Cell-line vs primary B-cell discordance unresolved\", \"Mechanistic link between PHRF1, PARP1/NELF and CSR not defined\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Defined PHRF1's role in the replication-stress response by identifying TopBP1 K73 mono-ubiquitination as the event that enhances TopBP1-ATR interaction and ATR-Chk1 activation, with mouse KO confirming essentiality.\",\n      \"evidence\": \"In vitro ubiquitination with K73 mutagenesis, Co-IP, ATR/Chk1 activation assays and conditional mouse knockout\",\n      \"pmids\": [\"40052822\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"E2 partner and regulation of mono- vs poly-ubiquitination not defined\", \"Relationship to NHEJ role not integrated\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Demonstrated that PHD-finger binding to the histone H3 N-terminus is functionally required for the DNA damage response, using a cancer P221L mutation that abolishes binding and rescue.\",\n      \"evidence\": \"Biochemical H3 binding, P221L mutagenesis, DDR rescue in knockout cells, RNA-seq and proteomics\",\n      \"pmids\": [\"40671529\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Splicing/transcription regulatory mechanism not mechanistically resolved\", \"Relative contribution of H3 vs H3K36me binding not separated\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Identified p53 as a PHRF1 ubiquitination substrate operating when MDM2 is impaired, placing PHRF1 in a cisplatin-response circuit downstream of KDM4A/XPC in bladder cancer.\",\n      \"evidence\": \"p53 ubiquitination assay, KDM4A inhibition, XPC knockdown and mouse xenograft model\",\n      \"pmids\": [\"41215711\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct vs indirect p53 ubiquitination not fully resolved\", \"Single lab, limited mechanistic detail\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How PHRF1's distinct activities\\u2014substrate ubiquitination (TGIF, TopBP1, PARP1, p53), chromatin reading via the PHD finger, and SRI-dependent transcriptional control\\u2014are coordinated and selected in a given cellular context remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No unifying model for substrate/target selection across pathways\", \"E2 partners unidentified\", \"No structural model of the multidomain protein on chromatin\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0016874\", \"supporting_discovery_ids\": [0, 1, 6, 8]},\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 1, 6, 8]},\n      {\"term_id\": \"GO:0042393\", \"supporting_discovery_ids\": [1, 7]},\n      {\"term_id\": \"GO:0140110\", \"supporting_discovery_ids\": [3, 4]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [1, 7, 8]},\n      {\"term_id\": \"GO:0000228\", \"supporting_discovery_ids\": [1, 6]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-73894\", \"supporting_discovery_ids\": [1, 7]},\n      {\"term_id\": \"R-HSA-8953897\", \"supporting_discovery_ids\": [6]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 2]},\n      {\"term_id\": \"R-HSA-74160\", \"supporting_discovery_ids\": [3, 4]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [0, 1, 6, 8]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"TGIF1\", \"NBS1\", \"TopBP1\", \"PARP1\", \"TP53\", \"POLR2A\", \"PML\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"faith_supported":5,"faith_total":5,"faith_pct":100.0}}