{"gene":"CHFR","run_date":"2026-06-09T22:57:18","timeline":{"discoveries":[{"year":2000,"finding":"CHFR defines a mitotic stress checkpoint that delays entry into metaphase (prophase checkpoint). Cells expressing wild-type CHFR delay chromosome condensation in response to microtubule poisons (nocodazole, taxol); cells lacking CHFR expression enter metaphase without delay. Ectopic expression of wild-type CHFR restored the cell cycle delay and increased cell survival under mitotic stress.","method":"Ectopic expression of wild-type vs. mutant CHFR in human cancer cell lines; cell cycle analysis after microtubule poison treatment","journal":"Nature","confidence":"High","confidence_rationale":"Tier 2 / Strong — founding study with clear loss-of-function and rescue experiments; replicated extensively by subsequent labs","pmids":["10935642"],"is_preprint":false},{"year":2002,"finding":"CHFR is an E3 ubiquitin ligase; its RING finger domain is required for ubiquitin ligase activity. Chfr auto-ubiquitinates in vitro and in vivo, and mutations of conserved RING-finger residues abolish ligase activity. Chfr delays mitotic entry by negatively regulating Cdc2 kinase activation at the G2-M transition by prolonging phosphorylation of Cdc2 tyrosine 15. The direct target of the Chfr pathway is Polo-like kinase 1 (Plk1): Chfr ubiquitinates Plk1, delaying activation of Cdc25C and inactivation of Wee1, thereby delaying Cdc2 activation. The Chfr pathway requires ubiquitin-dependent protein degradation (inactivating RING-domain mutations, poly-ubiquitination interference, or proteasome inhibition all abolish the delay).","method":"In vitro ubiquitination assays; in vivo overexpression in HEK293T; Xenopus cell-free extracts; RING-domain mutagenesis; proteasome inhibitor experiments","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution, mutagenesis, cell-free system, multiple orthogonal methods in one study; Plk1 as substrate replicated by subsequent labs","pmids":["11807090"],"is_preprint":false},{"year":2002,"finding":"CHFR RING finger domain is required for ubiquitin ligase activity (auto-ubiquitination in vitro with E1, Ubc4/Ubc5, and ubiquitin). RING domain deletion mutant is defective in inhibiting chromosome condensation after taxol treatment. CHFR expression is cell cycle regulated and dependent on its ubiquitin ligase activity.","method":"In vitro ubiquitination assay with purified components; stable cell lines expressing wild-type or RING-deleted CHFR; taxol treatment cell cycle assay","journal":"Cancer research","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution and cell-based mutagenesis; independent confirmation of RING-domain requirement","pmids":["11912157"],"is_preprint":false},{"year":2002,"finding":"Crystal structure of the CHFR FHA domain (native and in complex with tungstate, a phosphate analog) revealed a beta-sandwich fold similar to Rad53 FHA domains but crystallizing as a segment-swapped dimer. The ability to recognize tungstate suggests the CHFR FHA domain shares the ability of other FHA domains to bind phosphoproteins, though sequence/structure differences indicate distinct binding properties across FHA domain families.","method":"X-ray crystallography (structure determination of FHA domain native and tungstate-bound forms)","journal":"Structure (London, England : 1993)","confidence":"High","confidence_rationale":"Tier 1 / Moderate — crystal structure with ligand complex; single lab but structural method is definitive","pmids":["12121644"],"is_preprint":false},{"year":2003,"finding":"CHFR catalyzes the formation of non-canonical Lys63-linked polyubiquitin chains using Ubc13-Mms2 as the ubiquitin-conjugating enzyme. Ubc13-Mms2-dependent Lys63 chains are not associated with proteasomal targeting but with stress signaling. In mitosis, Chfr undergoes a phosphorylation-dependent electrophoretic mobility shift with no change in overall protein levels (contrary to a prior report suggesting Chfr undergoes proteasome-dependent degradation in mitosis).","method":"In vitro ubiquitination assay with Ubc13-Mms2; polyubiquitin chain linkage analysis; cell cycle protein level analysis","journal":"Oncogene","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution identifying Lys63-linkage; mechanistically distinct from Lys48 chains; single lab but biochemically rigorous","pmids":["14562038"],"is_preprint":false},{"year":2004,"finding":"In mammalian cells, the antephase checkpoint (blocking mitotic entry in response to microtubule poisons) is primarily mediated by the p38 stress kinases and requires the Chfr protein. The checkpoint requires ubiquitylation but not proteasome activity, consistent with Chfr forming Lys63-linked (non-degradative) ubiquitin chains.","method":"Genetic/pharmacological inhibition of p38 kinases; checkpoint assays in Chfr-deficient vs. Chfr-expressing cell lines; proteasome inhibitor experiments","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — epistasis experiments placing Chfr downstream of p38; proteasome independence confirmed with inhibitors; published in high-quality journal","pmids":["15302856"],"is_preprint":false},{"year":2004,"finding":"PML bodies control the nuclear dynamics and function of CHFR. CHFR is a dynamic component of PML bodies; FRET analysis identified a fraction of CHFR that interacts with PML in living cells. A trans-dominant CHFR mutant that inhibits checkpoint function also prevents colocalization/interaction with PML. In PML−/− cells, CHFR distribution and mobility are perturbed, accompanied by aberrations in mitotic entry and response to spindle depolymerization.","method":"Live-cell FRET; fluorescence recovery analysis; PML-knockout cells; checkpoint assays","journal":"Nature structural & molecular biology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — FRET in living cells, PML-KO rescue experiments, direct localization tied to checkpoint function","pmids":["15467728"],"is_preprint":false},{"year":2005,"finding":"CHFR physically interacts with Aurora A and ubiquitinates Aurora A both in vitro and in vivo, controlling Aurora A expression levels. Chfr-deficient mice are cancer-prone with chromosomal instability in embryonic fibroblasts, establishing Chfr as a tumor suppressor that maintains chromosomal stability partly through control of Aurora A.","method":"Co-immunoprecipitation; in vitro ubiquitination assay; Chfr knockout mouse model; MEF chromosome analysis","journal":"Nature genetics","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — in vitro ubiquitination + co-IP + KO mouse model; replicated by other labs","pmids":["15793587"],"is_preprint":false},{"year":2005,"finding":"CHFR delays chromosome condensation in prophase by excluding Cyclin B1 from the nucleus. In CHFR-expressing cells arrested by microtubule poisons, Cyclin A/Cdc2 is activated but Aurora-A, Aurora-B, and Cyclin B1/Cdc2 are inactive; Cyclin B1 is excluded from the nucleus. Ectopic expression of Cyclin B1 with a mutant nuclear export sequence induces chromosome condensation and overrides the CHFR checkpoint.","method":"Stable cell lines expressing wild-type CHFR; immunofluorescence and cell cycle marker analysis after microtubule poison treatment; ectopic Cyclin B1 nuclear export mutant expression","journal":"Oncogene","confidence":"High","confidence_rationale":"Tier 2 / Moderate — mechanistic rescue experiment with Cyclin B1 NES mutant; clear functional phenotype readout; single lab but multiple orthogonal approaches","pmids":["15674323"],"is_preprint":false},{"year":2007,"finding":"USP7/HAUSP is a deubiquitinase that interacts with Chfr and removes ubiquitin moieties from auto-ubiquitinated Chfr both in vivo and in vitro, resulting in Chfr accumulation. USP7-mediated deubiquitination of Chfr leads to its stabilization and may be a key regulatory step for Chfr activation.","method":"Immunoaffinity purification and mass spectrometry; co-immunoprecipitation; in vitro deubiquitination assay","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP and in vitro deubiquitination assay; single lab with two orthogonal methods","pmids":["17442268"],"is_preprint":false},{"year":2008,"finding":"Endogenous CHFR localizes to the cytoplasm and to the mitotic spindle during mitosis. CHFR interacts with TCTP and beta-tubulin (confirmed by biochemical analysis of endogenous proteins). The Chfr-TCTP interaction is stable throughout the cell cycle but is diminished by complete depolymerization of microtubules, suggesting Chfr may sense microtubule disruption through this interaction to activate the prophase checkpoint. High-level ectopic Chfr overexpression shifts localization to the nucleus/PML bodies and causes protein degradation in an autoubiquitination/proteasome-dependent manner.","method":"Two-hybrid screen; co-immunoprecipitation of endogenous proteins; immunofluorescence with three different anti-Chfr antibodies; nocodazole microtubule depolymerization assay","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — endogenous Co-IP and direct localization experiments; single lab; localization tied to functional interpretation","pmids":["18504434"],"is_preprint":false},{"year":2008,"finding":"The FHA domain of CHFR is critical for its anti-proliferative and G2/M checkpoint functions; the RING finger and cysteine-rich domains are required for E3 ligase activity but not for growth suppression. Domain deletion analysis established that checkpoint function (FHA domain) and ubiquitin ligase activity (RING + Cys domains) are separable activities.","method":"Domain deletion mutagenesis; cell proliferation assays; cell cycle checkpoint assays; in vitro/in vivo ubiquitination assays","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — mutagenesis combined with functional cell cycle assays; single lab","pmids":["18335050"],"is_preprint":false},{"year":2008,"finding":"CHFR interacts with alpha-tubulin and can regulate its ubiquitination in response to nocodazole and control the amount of acetylated alpha-tubulin (a spindle component). CHFR also interacts with the spindle checkpoint protein MAD2; decreased CHFR expression results in mislocalization of MAD2 and BUBR1 during mitosis and impaired MAD2/CDC20 complex formation, leading to multipolar spindles and genomic instability.","method":"siRNA knockdown of CHFR in MCF10A; co-immunoprecipitation (CHFR with Aurora A, alpha-tubulin, MAD2); ubiquitination assays; fluorescence microscopy of mitotic spindle; chromosome counting","journal":"Neoplasia (New York, N.Y.)","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — Co-IP plus functional KD phenotype; single lab; multiple interacting partners identified","pmids":["18592005"],"is_preprint":false},{"year":2009,"finding":"CHFR binds and ubiquitinates HDAC1 (histone deacetylase 1) both in vitro and in vivo, targeting it for degradation. Ectopic expression of CHFR in cancer cells leads to downregulation of HDAC1, upregulation of p21(CIP1/WAF1), KAI1, and E-cadherin, G1 arrest, and reduced invasiveness. Identified via affinity purification/mass spectrometry.","method":"Affinity purification combined with mass spectrometry; co-immunoprecipitation; in vitro ubiquitination assay; invasion assays; xenograft model","journal":"Nature cell biology","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — AP-MS identification + in vitro ubiquitination + co-IP + functional cell and in vivo assays; published in high-quality journal","pmids":["19182791"],"is_preprint":false},{"year":2009,"finding":"CHFR interacts with Kif22 (a chromokinesin that binds DNA and microtubules) and ubiquitinates Kif22 as a novel substrate, leading to Kif22 downregulation. Chfr-mediated Kif22 down-regulation is critical for the maintenance of chromosome stability.","method":"Co-immunoprecipitation; in vitro ubiquitination assay; siRNA knockdown; chromosome stability assays","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus in vitro ubiquitination of substrate; single lab with two orthogonal methods","pmids":["19321445"],"is_preprint":false},{"year":2009,"finding":"Activated PKB/Akt phosphorylates CHFR in vitro and in vivo. Expression of a CHFR mutant that cannot be phosphorylated by PKB results in reduced Plk1 levels and inhibition of mitotic entry. Thus, PKB facilitates mitotic entry after DNA damage by phosphorylating and inhibiting CHFR checkpoint function.","method":"In vitro kinase assay (PKB phosphorylating CHFR); phospho-mutant CHFR expression; Plk1 level analysis; mitotic entry assay","journal":"Molecular cancer research : MCR","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro kinase assay plus phospho-mutant functional rescue; single lab","pmids":["14638868"],"is_preprint":false},{"year":2009,"finding":"Nuclear localization of CHFR is mediated by a nuclear localization signal (NLS); a lysine-rich stretch (KKK) at amino acids 257-259 is essential for nuclear localization. Nuclear localization is essential for CHFR checkpoint function but not for its stability.","method":"NLS mutagenesis (KKK→AAA); immunofluorescence localization; checkpoint functional assays","journal":"Molecules and cells","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — site-directed mutagenesis linked to localization and functional checkpoint readout; single lab","pmids":["19326084"],"is_preprint":false},{"year":2010,"finding":"Crystal structure of the cysteine-rich region of human CHFR (residues 425–664) at 1.9 Å resolution revealed a multizinc binding domain with elaborate topology containing the PAR-binding zinc finger (PBZ). Co-crystal structures with PAR-like ligands (ADP-ribose, AMP, diadenosine pyrophosphate) defined the PBZ-PAR interaction: the PBZ recognizes two adenine-containing subunits of PAR and the connecting phosphate backbone. Site-specific mutagenesis and surface plasmon resonance validated this interaction. The PBZ-PAR interaction is required for antephase checkpoint function.","method":"X-ray crystallography (1.9 Å); co-crystal structures with PAR analogs; site-specific mutagenesis; surface plasmon resonance","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure + co-crystal with ligands + mutagenesis + SPR binding validation in one study","pmids":["20880844"],"is_preprint":false},{"year":2010,"finding":"CHFR functions as a ubiquitin ligase for HLTF (helicase-like transcription factor), binding to and ubiquitinating HLTF to target it for proteasomal degradation. CHFR-mediated HLTF downregulation reduces PAI-1 expression, inhibiting cell migration.","method":"Co-immunoprecipitation; in vitro ubiquitination assay; cell migration assay","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — Co-IP and in vitro ubiquitination; functional migration readout; single lab","pmids":["20388495"],"is_preprint":false},{"year":2011,"finding":"STIL promotes Chfr auto-ubiquitination and reduces its protein stability. In cells lacking STIL (MEFs, E9.5 embryos), Chfr protein levels are elevated and Plk1 (a Chfr substrate) is reduced. Knockdown of Chfr or overexpression of Plk1 reverses the abnormal mitotic phenotypes of STIL-deficient fibroblasts. STIL is thus identified as the first negative regulator of the Chfr mitotic checkpoint.","method":"Genetic KO (Stil-/- mice); siRNA knockdown; protein stability assays; rescue by Chfr KD or Plk1 OE; flow cytometry","journal":"Journal of cell science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic KO + functional rescue experiments; single lab","pmids":["21245198"],"is_preprint":false},{"year":2011,"finding":"RNF8 and CHFR synergistically regulate histone ubiquitination to control histone H4 Lys16 acetylation through MRG15-dependent acetyltransferase complexes. This chromatin remodeling function is required for ATM activation after DNA damage. Double-knockout (RNF8/Chfr DKO) cells show suppressed DNA damage-induced ATM kinase activation, hypersensitivity to ionizing radiation, and DKO mice develop thymic lymphomas with clonal chromosome translocations.","method":"Double-knockout mouse model; chromatin fractionation; histone ubiquitination and acetylation assays; ATM activation assays; radiation sensitivity assays","journal":"Nature structural & molecular biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — KO mouse model + biochemical histone modification assays + ATM activation readout; published in high-quality journal","pmids":["21706008"],"is_preprint":false},{"year":2011,"finding":"Auto-ubiquitylation of Chfr at G2 phase is required for Plk1 accumulation and mitotic entry. Chfr auto-ubiquitylation mutants (Chfr-K2A) do not undergo cell cycle-dependent degradation; elevated Chfr-K2A levels at G2/M reduce Plk1 levels and delay mitotic entry by suppressing cyclin B1/Cdk1 kinase activity. Overexpression of Plk1 in Chfr-K2A cells restores kinase activity and promotes mitotic entry. Aurora A levels are not affected, identifying Plk1 as the major Chfr target in controlling mitotic entry timing.","method":"Site-directed mutagenesis of Chfr auto-ubiquitylation sites; cell cycle analysis; Plk1 and Aurora A level measurement; cyclin B1/Cdk1 kinase assay; Plk1 overexpression rescue","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Moderate — mutagenesis + functional rescue experiment + kinase assay; single lab with multiple orthogonal methods","pmids":["21768102"],"is_preprint":false},{"year":2011,"finding":"CHFR is recruited to DNA double-strand breaks (DSBs) via poly(ADP-ribose) (PAR). At DSBs, CHFR regulates the first wave of protein ubiquitination. CHFR ubiquitinates PARP1 and regulates chromatin-associated PARP1 in vivo, establishing CHFR as an important E3 ligase in the early DNA damage response that mediates crosstalk between ubiquitination and poly-ADP-ribosylation.","method":"Recruitment assay to laser-induced DSBs; PAR-binding experiments; in vivo ubiquitination assay; chromatin fractionation","journal":"Nucleic acids research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct DSB recruitment assay and in vivo ubiquitination; single lab","pmids":["23268447"],"is_preprint":false},{"year":2011,"finding":"CHFR binds MAD2 via its cysteine-rich domain; deletion of this domain abolishes the CHFR/MAD2 interaction. The cysteine-rich domain is required for proper MAD2 localization and for promoting the MAD2/CDC20 interaction, thus enabling spindle assembly checkpoint function.","method":"Domain deletion mutagenesis; co-immunoprecipitation; MAD2 localization by immunofluorescence; MAD2/CDC20 interaction assay","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — domain deletion + Co-IP + localization; single lab with multiple readouts","pmids":["21575600"],"is_preprint":false},{"year":2012,"finding":"CHFR interacts with PARP-1; mitotic stress induces PARP-1 auto-PARylation, which enhances the CHFR-PARP-1 interaction and increases polyubiquitination/degradation of PARP-1. Decreased PARP-1 protein levels promote cell cycle arrest at prophase. In CHFR-silenced cells, PARP-1 is not degraded after mitotic stress and cells progress into mitosis. Chfr knockout mice and CHFR-silenced primary gastric cancer tissues have higher PARP-1 protein levels.","method":"Affinity purification/mass spectrometry; co-immunoprecipitation; in vitro ubiquitination; Chfr KO mouse tissue analysis; cell cycle assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — AP-MS identification + in vitro ubiquitination + Co-IP + KO mouse validation; multiple orthogonal methods","pmids":["22337872"],"is_preprint":false},{"year":2012,"finding":"SRG3/mBAF155 blocks CHFR-mediated ubiquitination and degradation of core SWI/SNF-BAF complex components (BRG1, SNF5, BAF60a). CHFR interacts with BRG1, SNF5, and BAF60a and ubiquitinates them for proteasomal degradation; SRG3/mBAF155 stabilizes these components by blocking their interaction with CHFR.","method":"Co-immunoprecipitation; in vitro ubiquitination assay; protein stability assays","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — Co-IP and in vitro ubiquitination of multiple substrates; single lab","pmids":["22285184"],"is_preprint":false},{"year":2013,"finding":"CHFR ubiquitinates and regulates TOPK/PBK (a serine/threonine kinase) levels as part of its checkpoint function. TOPK in turn phosphorylates and inactivates PTEN, which activates Akt, leading to proper G2/M progression. TOPK and PTEN are identified as new players in CHFR-mediated mitotic checkpoint signaling.","method":"Co-immunoprecipitation; in vitro ubiquitination assay; kinase assay (TOPK phosphorylation of PTEN); CHFR knockdown/overexpression","journal":"Cellular signalling","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — Co-IP + in vitro ubiquitination + kinase assay; single lab; pathway placement by epistasis","pmids":["24012691"],"is_preprint":false},{"year":2015,"finding":"The CHFR PBZ domain mediates a protein interaction with poly-ADP-ribosylated PARP1, leading to stabilization of CHFR. Disruption of the CHFR-PARP1 interaction (via PARP1 knockdown or PBZ peptide overexpression) causes loss of CHFR protein expression. A small-molecule inhibitor ('A3') targeting the CHFR PBZ domain disrupts the CHFR-PARP1 interaction and inhibits mitotic checkpoint function, showing therapeutic synergy with docetaxel in vitro and in xenograft models.","method":"shRNA knockdown; PBZ domain peptide overexpression; computational docking screen; in vitro protein-protein interaction assay; cell viability and colony formation assays; xenograft mouse model","journal":"Oncotarget","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — functional domain disruption + in vivo xenograft; single lab; confirms PAR-binding mechanistic role","pmids":["26356822"],"is_preprint":false},{"year":2016,"finding":"CHFR functions as an E3 ubiquitin ligase for SIRT1, responsible for its proteasomal degradation under oxidative stress. CHFR interacts with SIRT1 and ubiquitinates it. CHFR-mediated SIRT1 degradation is enhanced when SIRT1 is phosphorylated by JNK1 upon oxidative stress, leading to increased p53 acetylation and apoptosis. JNK inhibition prevents SIRT1 phosphorylation and CHFR-mediated degradation.","method":"Co-immunoprecipitation; in vivo ubiquitination assay; protein stability assay; JNK inhibitor experiments; p53 acetylation assay","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — Co-IP plus ubiquitination and functional apoptosis readout; single lab","pmids":["27883020"],"is_preprint":false},{"year":2021,"finding":"CHFR associates with and ubiquitinates ZEB1 in a ubiquitination-dependent manner, leading to decreased ZEB1 expression in TNBC. CHFR represses FASN expression through ZEB1, causing cell death under chemotherapy. HDAC inhibitor TSA increases CHFR expression independent of histone acetylation, destabilizing ZEB1 and sensitizing TNBC cells to chemotherapy.","method":"Affinity purification/mass spectrometry; co-immunoprecipitation; in vivo ubiquitination assay; CHFR overexpression/knockdown; cell viability assays","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — AP-MS identification + Co-IP + ubiquitination assay; single lab","pmids":["34462429"],"is_preprint":false},{"year":2021,"finding":"PARP1 interacts with and poly(ADP-ribosyl)ates RNF126, which then recruits CHFR (via its PAR-binding PBZ domain) to ubiquitinate and degrade RNF126. RNF126 is required for ATR-Chk1 signaling activation by IR or PARP inhibitors; CHFR-mediated RNF126 degradation thus modulates DNA damage checkpoint signaling.","method":"Co-immunoprecipitation; in vitro and in vivo ubiquitination assay; PAR-binding assays; siRNA knockdown; cell viability assays","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — Co-IP and ubiquitination assay identifying new substrate; single lab","pmids":["34388456"],"is_preprint":false},{"year":2023,"finding":"CHFR is required for ubiquitylation-dependent degradation of VE-cadherin in endothelial cells. Endothelial-restricted Chfr knockout (ChfrΔEC) mice show augmented VE-cadherin expression at adherens junctions and abrogated LPS-induced VE-cadherin degradation. LPS-induced lung endothelial barrier breakdown, inflammatory neutrophil extravasation, and mortality are suppressed in ChfrΔEC mice. FoxO1 transcription factor is identified as an upstream regulator of CHFR expression.","method":"Endothelial-specific conditional Chfr knockout mice; immunofluorescence; VE-cadherin ubiquitination assay; LPS challenge model; endothelial permeability assay","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Moderate — conditional KO mouse with specific vascular phenotype + mechanistic ubiquitination assay; single lab but in vivo validation","pmids":["37852964"],"is_preprint":false},{"year":2007,"finding":"In S. cerevisiae Chfr homologs (Chf1/Chf2), Ubc4 is required for G1 cell cycle delay and Chf protein turnover (Lys48-type modification), while Ubc13/Mms2 is required for G2 delay and does not contribute to protein turnover (Lys63-type modification). Site-specific mutagenesis identified the Lys residues modified by each E2. This dual E2 specificity accounts for different cell cycle functions.","method":"Yeast genetic assays; in vitro reconstitution with purified components; tandem mass spectrometry linkage analysis; site-directed mutagenesis","journal":"Cell cycle (Georgetown, Tex.)","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution + MS linkage analysis + mutagenesis; functional dissection of two E2 specificities in ortholog","pmids":["18202552"],"is_preprint":false},{"year":2004,"finding":"S. cerevisiae Dma1 and Dma2 (budding yeast Chfr homologs) are involved in proper spindle positioning, likely by regulating septin ring deposition at the bud neck. DMA2 overexpression causes defects in septin ring disassembly and cytokinesis that can be rescued by eliminating the spindle position checkpoint protein Bub2 or overproducing Tem1. dma1Δdma2Δ cells fail to activate the spindle position checkpoint in response to dynein loss.","method":"Genetic deletion analysis; DMA2 overexpression; epistasis with Bub2/Tem1; spindle position checkpoint assays","journal":"Molecular biology of the cell","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic epistasis in yeast ortholog; functional checkpoint assays; single lab","pmids":["15146058"],"is_preprint":false},{"year":2012,"finding":"CHFR interacts with LRP1 in human vascular smooth muscle cells. Aggregated LDL (agLDL) downregulates CHFR expression and reduces the CHFR-LRP1 physical interaction, leading to reduced LRP1 ubiquitination and prolonged LRP1 half-life. CHFR silencing increases VSMC LRP1 expression specifically (not classical LDLR), indicating CHFR targets LRP1 for ubiquitin-mediated degradation.","method":"Co-immunoprecipitation; pulse-chase analysis; cycloheximide chase; CHFR siRNA silencing; 2D proteomics","journal":"Arteriosclerosis, thrombosis, and vascular biology","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — Co-IP + protein stability assay + KD phenotype; single lab; substrate identification without full in vitro reconstitution","pmids":["23241409"],"is_preprint":false}],"current_model":"CHFR is a multifunctional E3 ubiquitin ligase that (1) defines an antephase/prophase checkpoint by ubiquitinating Plk1 (via Lys48-linked chains with Ubc4/Ubc5 for proteasomal degradation) to suppress Cdc25C/Wee1 and thereby exclude Cyclin B1 from the nucleus, delaying chromosome condensation in response to mitotic stress through a pathway requiring p38 kinases; (2) forms non-degradative Lys63-linked polyubiquitin chains with Ubc13-Mms2 for stress signaling; (3) ubiquitinates additional mitotic substrates including Aurora A, HDAC1, PARP1, Kif22, TOPK, SIRT1, ZEB1, HLTF, BAF complex components (BRG1, SNF5, BAF60a), RNF126, and VE-cadherin to regulate diverse processes including chromosomal stability, transcription, DNA damage response, and endothelial barrier integrity; (4) is recruited to DNA double-strand breaks via PAR through its PBZ zinc-finger domain, where it mediates the first wave of damage-site ubiquitination and cooperates with RNF8 to regulate histone H4K16 acetylation and ATM activation; (5) localizes to the cytoplasm/mitotic spindle (interacting with TCTP and beta-tubulin) and to the nucleus via a KKK NLS (residues 257–259), with nuclear localization required for checkpoint function; (6) is stabilized by USP7/HAUSP-mediated deubiquitination and regulated by PKB/Akt phosphorylation (which inhibits checkpoint function); and (7) functions as a tumor suppressor whose loss is associated with chromosomal instability, cancer predisposition, and sensitivity to taxanes."},"narrative":{"mechanistic_narrative":"CHFR is a RING-domain E3 ubiquitin ligase and tumor suppressor that enforces a mitotic stress (antephase/prophase) checkpoint, delaying chromosome condensation in response to microtubule poisons [PMID:10935642]. Its checkpoint activity is genetically separable from its catalytic activity: the FHA domain mediates growth suppression and checkpoint function while the RING and cysteine-rich regions are required for ubiquitin ligase activity [PMID:18335050]. The principal effector of the checkpoint is Polo-like kinase 1 (Plk1), which CHFR ubiquitinates to prolong inhibitory Cdc2 tyrosine-15 phosphorylation by delaying Cdc25C activation and Wee1 inactivation, thereby excluding Cyclin B1 from the nucleus and blocking mitotic entry [PMID:11807090, PMID:15674323, PMID:21768102], a checkpoint that operates downstream of the p38 stress kinases [PMID:15302856]. CHFR builds distinct chain types with distinct E2s — Lys48-linked chains via Ubc4/Ubc5 that target substrates for proteasomal degradation, and non-degradative Lys63-linked chains via Ubc13-Mms2 for stress signaling [PMID:11912157, PMID:14562038, PMID:18202552]. Beyond Plk1, CHFR ubiquitinates a broad substrate set to maintain chromosomal stability and regulate transcription, migration, and apoptosis, including Aurora A [PMID:15793587], HDAC1 [PMID:19182791], Kif22 [PMID:19321445], PARP1 [PMID:23268447, PMID:22337872], HLTF [PMID:20388495], SIRT1 [PMID:27883020], ZEB1 [PMID:34462429], SWI/SNF-BAF components [PMID:22285184], RNF126 [PMID:34388456], and VE-cadherin in endothelial barrier control [PMID:37852964]. CHFR is recruited to DNA double-strand breaks through poly(ADP-ribose) binding by its cysteine-rich PAR-binding zinc finger (PBZ), whose structure and PAR-recognition mode are defined, where it directs the first wave of damage-site ubiquitination and cooperates with RNF8 to control histone H4K16 acetylation and ATM activation [PMID:20880844, PMID:21706008, PMID:23268447]. CHFR activity is set by counter-regulators: USP7/HAUSP deubiquitination stabilizes it [PMID:17442268], STIL promotes its auto-ubiquitination and turnover [PMID:21245198], and PKB/Akt phosphorylation inhibits its checkpoint function [PMID:14638868]. Loss of CHFR causes chromosomal instability and cancer predisposition in knockout mice [PMID:15793587, PMID:21706008].","teleology":[{"year":2000,"claim":"Established that CHFR exists and defines a previously unrecognized mitotic stress checkpoint controlling the timing of chromosome condensation.","evidence":"Ectopic wild-type vs. mutant CHFR expression and cell-cycle analysis after microtubule poisons in cancer cell lines","pmids":["10935642"],"confidence":"High","gaps":["Molecular activity of CHFR unknown at this stage","Direct substrates unidentified"]},{"year":2002,"claim":"Defined CHFR as a RING-domain E3 ubiquitin ligase and identified Plk1 as the checkpoint substrate, explaining how the checkpoint delays Cdc2 activation.","evidence":"In vitro ubiquitination, RING mutagenesis, Xenopus extracts, and proteasome inhibition; independent confirmation of RING requirement and cell-cycle-regulated expression","pmids":["11807090","11912157"],"confidence":"High","gaps":["Chain linkage type not yet resolved","Whether Plk1 degradation is the sole mechanism unclear"]},{"year":2002,"claim":"Determined the FHA domain structure, indicating a phosphopeptide-recognition module consistent with FHA family proteins.","evidence":"X-ray crystallography of FHA domain native and tungstate-bound forms","pmids":["12121644"],"confidence":"High","gaps":["Physiological phosphoprotein ligand of the FHA domain not identified","Crystallized as segment-swapped dimer of uncertain in vivo relevance"]},{"year":2003,"claim":"Showed CHFR can build non-degradative Lys63-linked chains via Ubc13-Mms2, distinguishing a signaling function from proteasomal targeting.","evidence":"In vitro ubiquitination with Ubc13-Mms2 and chain linkage analysis; mitotic mobility-shift analysis","pmids":["14562038"],"confidence":"High","gaps":["In vivo substrates of K63 chains not defined","Reconciliation with degradative K48 activity left open"]},{"year":2004,"claim":"Placed CHFR downstream of p38 stress kinases and confirmed the antephase checkpoint requires ubiquitylation but not the proteasome.","evidence":"p38 inhibition, checkpoint assays in Chfr-deficient cells, proteasome inhibitor experiments","pmids":["15302856"],"confidence":"High","gaps":["Direct link between p38 and CHFR activation not biochemically defined"]},{"year":2004,"claim":"Linked CHFR nuclear dynamics and checkpoint function to PML bodies.","evidence":"Live-cell FRET, fluorescence recovery, PML-knockout cells, checkpoint assays","pmids":["15467728"],"confidence":"High","gaps":["Mechanism by which PML bodies regulate CHFR activity unresolved"]},{"year":2005,"claim":"Identified Aurora A as a substrate and established CHFR as a chromosomal-stability tumor suppressor in vivo.","evidence":"Co-IP, in vitro ubiquitination, Chfr-knockout mice, MEF chromosome analysis","pmids":["15793587"],"confidence":"High","gaps":["Relative contribution of Aurora A vs. Plk1 to tumor suppression unclear"]},{"year":2005,"claim":"Defined the downstream output of the checkpoint as nuclear exclusion of Cyclin B1, preventing chromosome condensation.","evidence":"Wild-type CHFR cell lines, cell-cycle marker immunofluorescence, Cyclin B1 NES-mutant override experiment","pmids":["15674323"],"confidence":"High","gaps":["Mechanism connecting Plk1 ubiquitination to Cyclin B1 localization not fully traced"]},{"year":2008,"claim":"Separated CHFR's checkpoint function (FHA domain) from its catalytic activity (RING + cysteine-rich domains).","evidence":"Domain deletion mutagenesis with proliferation, checkpoint, and ubiquitination assays","pmids":["18335050"],"confidence":"Medium","gaps":["How FHA-dependent checkpoint function operates without requiring ligase activity not explained","Single lab"]},{"year":2008,"claim":"Localized endogenous CHFR to the cytoplasm and mitotic spindle and proposed microtubule sensing through TCTP/tubulin interactions.","evidence":"Two-hybrid screen, endogenous Co-IP, immunofluorescence with three antibodies, nocodazole depolymerization","pmids":["18504434"],"confidence":"Medium","gaps":["Causal role of TCTP/tubulin binding in checkpoint activation not demonstrated","Ectopic overexpression shifts localization, complicating interpretation"]},{"year":2008,"claim":"Extended CHFR's spindle role through alpha-tubulin and MAD2 regulation affecting spindle assembly checkpoint integrity.","evidence":"siRNA knockdown, Co-IP, ubiquitination assays, mitotic spindle microscopy, chromosome counting","pmids":["18592005"],"confidence":"Medium","gaps":["Direct vs. indirect effects on MAD2/BUBR1 localization not distinguished","Single lab"]},{"year":2009,"claim":"Expanded the substrate repertoire to transcriptional and motility regulators (HDAC1, Kif22, HLTF), linking CHFR to gene expression, invasion, and chromosome stability.","evidence":"AP-MS, Co-IP, in vitro ubiquitination, invasion/migration assays, xenograft model","pmids":["19182791","19321445","20388495"],"confidence":"Medium","gaps":["Physiological conditions selecting each substrate undefined","Some readouts rely on overexpression"]},{"year":2009,"claim":"Identified upstream regulation by PKB/Akt phosphorylation that inhibits checkpoint function and a KKK NLS required for nuclear checkpoint activity.","evidence":"In vitro kinase assay, phospho-mutant rescue, NLS mutagenesis, localization and checkpoint assays","pmids":["14638868","19326084"],"confidence":"Medium","gaps":["Phosphosite mapping limited","Single-lab findings"]},{"year":2010,"claim":"Defined the PBZ zinc finger structure and its PAR-recognition mode, establishing the molecular basis for PAR-dependent recruitment and antephase checkpoint function.","evidence":"1.9 Å crystal and co-crystal structures with PAR analogs, mutagenesis, SPR binding validation","pmids":["20880844"],"confidence":"High","gaps":["In vivo PAR sources driving CHFR recruitment in different contexts not fully mapped"]},{"year":2011,"claim":"Established CHFR's role in the early DNA damage response: PAR-dependent recruitment to DSBs, PARP1 ubiquitination, and RNF8-cooperative control of H4K16 acetylation and ATM activation.","evidence":"Laser-induced DSB recruitment, PAR binding, in vivo ubiquitination, RNF8/Chfr double-knockout mice, histone modification and ATM assays","pmids":["21706008","23268447"],"confidence":"High","gaps":["Order of events between CHFR and RNF8 at chromatin not fully resolved"]},{"year":2011,"claim":"Clarified the role of CHFR auto-ubiquitylation in normal cell-cycle timing, showing auto-ubiquitylation-driven turnover permits Plk1 accumulation for mitotic entry, with Plk1 the dominant target over Aurora A.","evidence":"Auto-ubiquitylation site mutants, cell-cycle analysis, Plk1/Aurora A measurement, Cyclin B1/Cdk1 kinase assay, Plk1 rescue","pmids":["21768102"],"confidence":"High","gaps":["Trigger controlling timing of auto-ubiquitylation not defined"]},{"year":2011,"claim":"Identified STIL as the first negative regulator promoting CHFR auto-ubiquitination and turnover.","evidence":"Stil-knockout mice/MEFs, siRNA, stability assays, rescue by Chfr KD or Plk1 OE","pmids":["21245198"],"confidence":"Medium","gaps":["Whether STIL acts directly on CHFR not established","Single lab"]},{"year":2012,"claim":"Showed PARP1 auto-PARylation under mitotic stress enhances CHFR-PARP1 interaction and PARP1 degradation, coupling PAR signaling to checkpoint arrest.","evidence":"AP-MS, Co-IP, in vitro ubiquitination, Chfr-KO mouse tissue, cell-cycle assays","pmids":["22337872"],"confidence":"High","gaps":["Quantitative contribution of PARP1 degradation to arrest vs. other substrates unclear"]},{"year":2012,"claim":"Connected CHFR to additional checkpoint and metabolic signaling via TOPK/PTEN/Akt and BAF complex stabilization by SRG3/mBAF155.","evidence":"Co-IP, in vitro ubiquitination, kinase assays, protein stability assays","pmids":["24012691","22285184"],"confidence":"Medium","gaps":["Pathway placements rest on epistasis in single labs","Physiological substrate selection conditions undefined"]},{"year":2015,"claim":"Demonstrated that PBZ-mediated CHFR-PARP1 interaction stabilizes CHFR and is a druggable node, with a PBZ-targeting compound synergizing with docetaxel.","evidence":"shRNA, PBZ peptide overexpression, docking screen, protein-interaction and viability assays, xenograft model","pmids":["26356822"],"confidence":"Medium","gaps":["Compound specificity and off-target effects not fully characterized","Single lab"]},{"year":2016,"claim":"Identified SIRT1 as an oxidative-stress substrate, linking CHFR to JNK1-dependent p53 acetylation and apoptosis.","evidence":"Co-IP, in vivo ubiquitination, stability assays, JNK inhibition, p53 acetylation assay","pmids":["27883020"],"confidence":"Medium","gaps":["Direct ubiquitination not reconstituted in vitro","Single lab"]},{"year":2021,"claim":"Extended CHFR substrate range to ZEB1 (chemo-sensitization in TNBC) and RNF126 (DNA damage checkpoint modulation), the latter via PAR-dependent recruitment.","evidence":"AP-MS, Co-IP, in vivo/in vitro ubiquitination, PAR-binding assays, viability assays","pmids":["34462429","34388456"],"confidence":"Medium","gaps":["Context-dependence of these substrate relationships not defined","Single-lab studies"]},{"year":2023,"claim":"Established a non-mitotic physiological role for CHFR in endothelial barrier integrity through VE-cadherin degradation, downstream of FoxO1.","evidence":"Endothelial-specific conditional Chfr knockout mice, immunofluorescence, VE-cadherin ubiquitination assay, LPS challenge, permeability assays","pmids":["37852964"],"confidence":"High","gaps":["Whether VE-cadherin is a direct ubiquitination substrate in vitro not shown","Link to mitotic functions unclear"]},{"year":null,"claim":"How CHFR integrates upstream stress signals (p38, JNK, Akt, PAR) to select among its many substrates and switch between K48 and K63 chain outputs in specific contexts remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unified model linking signal input to substrate/chain-type choice","FHA domain physiological phospholigand unidentified","Mechanism coupling cytoplasmic microtubule sensing to nuclear checkpoint output incomplete"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0016874","term_label":"ligase activity","supporting_discovery_ids":[1,2,7,13,22,24,32]},{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[1,7,13,14,22,24,28,31]},{"term_id":"GO:0003723","term_label":"RNA binding","supporting_discovery_ids":[17,22,27]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[6,16]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[10]},{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[10,12]},{"term_id":"GO:0000228","term_label":"nuclear chromosome","supporting_discovery_ids":[20,22]}],"pathway":[{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[0,1,8,21]},{"term_id":"R-HSA-73894","term_label":"DNA Repair","supporting_discovery_ids":[20,22,30]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[1,13,24]},{"term_id":"R-HSA-4839726","term_label":"Chromatin organization","supporting_discovery_ids":[20]}],"complexes":[],"partners":["PLK1","AURKA","HDAC1","PARP1","USP7","MAD2L1","TPT1","RNF8"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q96EP1","full_name":"E3 ubiquitin-protein ligase CHFR","aliases":["Checkpoint with forkhead and RING finger domains protein","RING finger protein 196","RING-type E3 ubiquitin transferase CHFR"],"length_aa":664,"mass_kda":73.4,"function":"E3 ubiquitin-protein ligase that functions in the antephase checkpoint by actively delaying passage into mitosis in response to microtubule poisons. Acts in early prophase before chromosome condensation, when the centrosome move apart from each other along the periphery of the nucleus. Probably involved in signaling the presence of mitotic stress caused by microtubule poisons by mediating the 'Lys-48'-linked ubiquitination of target proteins, leading to their degradation by the proteasome. Promotes the ubiquitination and subsequent degradation of AURKA and PLK1. Probably acts as a tumor suppressor, possibly by mediating the polyubiquitination of HDAC1, leading to its degradation. May also promote the formation of 'Lys-63'-linked polyubiquitin chains and functions with the specific ubiquitin-conjugating UBC13-MMS2 (UBE2N-UBE2V2) heterodimer. Substrates that are polyubiquitinated at 'Lys-63' are usually not targeted for degradation, but are rather involved in signaling cellular stress","subcellular_location":"Nucleus, PML body","url":"https://www.uniprot.org/uniprotkb/Q96EP1/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/CHFR","classification":"Not Classified","n_dependent_lines":4,"n_total_lines":1208,"dependency_fraction":0.0033112582781456954},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"PARP1","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/CHFR","total_profiled":1310},"omim":[{"mim_id":"611035","title":"APRATAXIN- AND PNKP-LIKE FACTOR; APLF","url":"https://www.omim.org/entry/611035"},{"mim_id":"605209","title":"CHECKPOINT PROTEIN WITH FHA AND RING FINGER DOMAINS; CHFR","url":"https://www.omim.org/entry/605209"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Uncertain","locations":[{"location":"Nuclear bodies","reliability":"Uncertain"},{"location":"Microtubules","reliability":"Uncertain"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/CHFR"},"hgnc":{"alias_symbol":["FLJ10796","RNF196"],"prev_symbol":[]},"alphafold":{"accession":"Q96EP1","domains":[{"cath_id":"2.60.200.20","chopping":"18-123","consensus_level":"high","plddt":87.8324,"start":18,"end":123},{"cath_id":"3.30.40.10","chopping":"300-367","consensus_level":"high","plddt":92.7059,"start":300,"end":367},{"cath_id":"3.30.40.140","chopping":"482-534","consensus_level":"high","plddt":94.9221,"start":482,"end":534},{"cath_id":"-","chopping":"555-653","consensus_level":"high","plddt":96.9751,"start":555,"end":653}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q96EP1","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q96EP1-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q96EP1-F1-predicted_aligned_error_v6.png","plddt_mean":72.0},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=CHFR","jax_strain_url":"https://www.jax.org/strain/search?query=CHFR"},"sequence":{"accession":"Q96EP1","fasta_url":"https://rest.uniprot.org/uniprotkb/Q96EP1.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q96EP1/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q96EP1"}},"corpus_meta":[{"pmid":"10935642","id":"PMC_10935642","title":"Chfr 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the miR-214-3p/PAPPA axis.","date":"2022","source":"Clinical hemorheology and microcirculation","url":"https://pubmed.ncbi.nlm.nih.gov/34842180","citation_count":10,"is_preprint":false},{"pmid":"21768102","id":"PMC_21768102","title":"The auto-ubiquitylation of E3 ubiquitin-protein ligase Chfr at G2 phase is required for accumulation of polo-like kinase 1 and mitotic entry in mammalian cells.","date":"2011","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/21768102","citation_count":10,"is_preprint":false},{"pmid":"30854101","id":"PMC_30854101","title":"CHFR Promoter Hypermethylation Is Associated with Gastric Cancer and Plays a Protective Role in Gastric Cancer Process.","date":"2019","source":"Journal of Cancer","url":"https://pubmed.ncbi.nlm.nih.gov/30854101","citation_count":9,"is_preprint":false},{"pmid":"35093868","id":"PMC_35093868","title":"CHFR-Promoter-Methylation Status Is Predictive of Response to Irinotecan-based Systemic Chemotherapy in Advanced Colorectal Cancer.","date":"2022","source":"Anticancer research","url":"https://pubmed.ncbi.nlm.nih.gov/35093868","citation_count":9,"is_preprint":false},{"pmid":"19326084","id":"PMC_19326084","title":"Nuclear localization of Chfr is crucial for its checkpoint function.","date":"2009","source":"Molecules and cells","url":"https://pubmed.ncbi.nlm.nih.gov/19326084","citation_count":9,"is_preprint":false},{"pmid":"25477232","id":"PMC_25477232","title":"Molecular characteristics of non-small cell lung cancer with reduced CHFR expression in The Cancer Genome Atlas (TCGA) project.","date":"2014","source":"Respiratory medicine","url":"https://pubmed.ncbi.nlm.nih.gov/25477232","citation_count":9,"is_preprint":false},{"pmid":"20855974","id":"PMC_20855974","title":"Alternative efficacy-predicting markers for paclitaxel instead of CHFR in non-small-cell lung cancer.","date":"2010","source":"Cancer biology & 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cancer","url":"https://pubmed.ncbi.nlm.nih.gov/20109344","citation_count":5,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":49429,"output_tokens":9692,"usd":0.146834,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":19872,"output_tokens":6251,"usd":0.127818,"stage2_stop_reason":"end_turn"},"total_usd":0.274652,"stage1_batch_id":"msgbatch_01ACAxJQabDsht39JXeWG22d","stage2_batch_id":"msgbatch_01QEU22A9U5sbPTgsMzTmvSf","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2000,\n      \"finding\": \"CHFR defines a mitotic stress checkpoint that delays entry into metaphase (prophase checkpoint). Cells expressing wild-type CHFR delay chromosome condensation in response to microtubule poisons (nocodazole, taxol); cells lacking CHFR expression enter metaphase without delay. Ectopic expression of wild-type CHFR restored the cell cycle delay and increased cell survival under mitotic stress.\",\n      \"method\": \"Ectopic expression of wild-type vs. mutant CHFR in human cancer cell lines; cell cycle analysis after microtubule poison treatment\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — founding study with clear loss-of-function and rescue experiments; replicated extensively by subsequent labs\",\n      \"pmids\": [\"10935642\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"CHFR is an E3 ubiquitin ligase; its RING finger domain is required for ubiquitin ligase activity. Chfr auto-ubiquitinates in vitro and in vivo, and mutations of conserved RING-finger residues abolish ligase activity. Chfr delays mitotic entry by negatively regulating Cdc2 kinase activation at the G2-M transition by prolonging phosphorylation of Cdc2 tyrosine 15. The direct target of the Chfr pathway is Polo-like kinase 1 (Plk1): Chfr ubiquitinates Plk1, delaying activation of Cdc25C and inactivation of Wee1, thereby delaying Cdc2 activation. The Chfr pathway requires ubiquitin-dependent protein degradation (inactivating RING-domain mutations, poly-ubiquitination interference, or proteasome inhibition all abolish the delay).\",\n      \"method\": \"In vitro ubiquitination assays; in vivo overexpression in HEK293T; Xenopus cell-free extracts; RING-domain mutagenesis; proteasome inhibitor experiments\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution, mutagenesis, cell-free system, multiple orthogonal methods in one study; Plk1 as substrate replicated by subsequent labs\",\n      \"pmids\": [\"11807090\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"CHFR RING finger domain is required for ubiquitin ligase activity (auto-ubiquitination in vitro with E1, Ubc4/Ubc5, and ubiquitin). RING domain deletion mutant is defective in inhibiting chromosome condensation after taxol treatment. CHFR expression is cell cycle regulated and dependent on its ubiquitin ligase activity.\",\n      \"method\": \"In vitro ubiquitination assay with purified components; stable cell lines expressing wild-type or RING-deleted CHFR; taxol treatment cell cycle assay\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution and cell-based mutagenesis; independent confirmation of RING-domain requirement\",\n      \"pmids\": [\"11912157\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"Crystal structure of the CHFR FHA domain (native and in complex with tungstate, a phosphate analog) revealed a beta-sandwich fold similar to Rad53 FHA domains but crystallizing as a segment-swapped dimer. The ability to recognize tungstate suggests the CHFR FHA domain shares the ability of other FHA domains to bind phosphoproteins, though sequence/structure differences indicate distinct binding properties across FHA domain families.\",\n      \"method\": \"X-ray crystallography (structure determination of FHA domain native and tungstate-bound forms)\",\n      \"journal\": \"Structure (London, England : 1993)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — crystal structure with ligand complex; single lab but structural method is definitive\",\n      \"pmids\": [\"12121644\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"CHFR catalyzes the formation of non-canonical Lys63-linked polyubiquitin chains using Ubc13-Mms2 as the ubiquitin-conjugating enzyme. Ubc13-Mms2-dependent Lys63 chains are not associated with proteasomal targeting but with stress signaling. In mitosis, Chfr undergoes a phosphorylation-dependent electrophoretic mobility shift with no change in overall protein levels (contrary to a prior report suggesting Chfr undergoes proteasome-dependent degradation in mitosis).\",\n      \"method\": \"In vitro ubiquitination assay with Ubc13-Mms2; polyubiquitin chain linkage analysis; cell cycle protein level analysis\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution identifying Lys63-linkage; mechanistically distinct from Lys48 chains; single lab but biochemically rigorous\",\n      \"pmids\": [\"14562038\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"In mammalian cells, the antephase checkpoint (blocking mitotic entry in response to microtubule poisons) is primarily mediated by the p38 stress kinases and requires the Chfr protein. The checkpoint requires ubiquitylation but not proteasome activity, consistent with Chfr forming Lys63-linked (non-degradative) ubiquitin chains.\",\n      \"method\": \"Genetic/pharmacological inhibition of p38 kinases; checkpoint assays in Chfr-deficient vs. Chfr-expressing cell lines; proteasome inhibitor experiments\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — epistasis experiments placing Chfr downstream of p38; proteasome independence confirmed with inhibitors; published in high-quality journal\",\n      \"pmids\": [\"15302856\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"PML bodies control the nuclear dynamics and function of CHFR. CHFR is a dynamic component of PML bodies; FRET analysis identified a fraction of CHFR that interacts with PML in living cells. A trans-dominant CHFR mutant that inhibits checkpoint function also prevents colocalization/interaction with PML. In PML−/− cells, CHFR distribution and mobility are perturbed, accompanied by aberrations in mitotic entry and response to spindle depolymerization.\",\n      \"method\": \"Live-cell FRET; fluorescence recovery analysis; PML-knockout cells; checkpoint assays\",\n      \"journal\": \"Nature structural & molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — FRET in living cells, PML-KO rescue experiments, direct localization tied to checkpoint function\",\n      \"pmids\": [\"15467728\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"CHFR physically interacts with Aurora A and ubiquitinates Aurora A both in vitro and in vivo, controlling Aurora A expression levels. Chfr-deficient mice are cancer-prone with chromosomal instability in embryonic fibroblasts, establishing Chfr as a tumor suppressor that maintains chromosomal stability partly through control of Aurora A.\",\n      \"method\": \"Co-immunoprecipitation; in vitro ubiquitination assay; Chfr knockout mouse model; MEF chromosome analysis\",\n      \"journal\": \"Nature genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — in vitro ubiquitination + co-IP + KO mouse model; replicated by other labs\",\n      \"pmids\": [\"15793587\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"CHFR delays chromosome condensation in prophase by excluding Cyclin B1 from the nucleus. In CHFR-expressing cells arrested by microtubule poisons, Cyclin A/Cdc2 is activated but Aurora-A, Aurora-B, and Cyclin B1/Cdc2 are inactive; Cyclin B1 is excluded from the nucleus. Ectopic expression of Cyclin B1 with a mutant nuclear export sequence induces chromosome condensation and overrides the CHFR checkpoint.\",\n      \"method\": \"Stable cell lines expressing wild-type CHFR; immunofluorescence and cell cycle marker analysis after microtubule poison treatment; ectopic Cyclin B1 nuclear export mutant expression\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — mechanistic rescue experiment with Cyclin B1 NES mutant; clear functional phenotype readout; single lab but multiple orthogonal approaches\",\n      \"pmids\": [\"15674323\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"USP7/HAUSP is a deubiquitinase that interacts with Chfr and removes ubiquitin moieties from auto-ubiquitinated Chfr both in vivo and in vitro, resulting in Chfr accumulation. USP7-mediated deubiquitination of Chfr leads to its stabilization and may be a key regulatory step for Chfr activation.\",\n      \"method\": \"Immunoaffinity purification and mass spectrometry; co-immunoprecipitation; in vitro deubiquitination assay\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP and in vitro deubiquitination assay; single lab with two orthogonal methods\",\n      \"pmids\": [\"17442268\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Endogenous CHFR localizes to the cytoplasm and to the mitotic spindle during mitosis. CHFR interacts with TCTP and beta-tubulin (confirmed by biochemical analysis of endogenous proteins). The Chfr-TCTP interaction is stable throughout the cell cycle but is diminished by complete depolymerization of microtubules, suggesting Chfr may sense microtubule disruption through this interaction to activate the prophase checkpoint. High-level ectopic Chfr overexpression shifts localization to the nucleus/PML bodies and causes protein degradation in an autoubiquitination/proteasome-dependent manner.\",\n      \"method\": \"Two-hybrid screen; co-immunoprecipitation of endogenous proteins; immunofluorescence with three different anti-Chfr antibodies; nocodazole microtubule depolymerization assay\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — endogenous Co-IP and direct localization experiments; single lab; localization tied to functional interpretation\",\n      \"pmids\": [\"18504434\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"The FHA domain of CHFR is critical for its anti-proliferative and G2/M checkpoint functions; the RING finger and cysteine-rich domains are required for E3 ligase activity but not for growth suppression. Domain deletion analysis established that checkpoint function (FHA domain) and ubiquitin ligase activity (RING + Cys domains) are separable activities.\",\n      \"method\": \"Domain deletion mutagenesis; cell proliferation assays; cell cycle checkpoint assays; in vitro/in vivo ubiquitination assays\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — mutagenesis combined with functional cell cycle assays; single lab\",\n      \"pmids\": [\"18335050\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"CHFR interacts with alpha-tubulin and can regulate its ubiquitination in response to nocodazole and control the amount of acetylated alpha-tubulin (a spindle component). CHFR also interacts with the spindle checkpoint protein MAD2; decreased CHFR expression results in mislocalization of MAD2 and BUBR1 during mitosis and impaired MAD2/CDC20 complex formation, leading to multipolar spindles and genomic instability.\",\n      \"method\": \"siRNA knockdown of CHFR in MCF10A; co-immunoprecipitation (CHFR with Aurora A, alpha-tubulin, MAD2); ubiquitination assays; fluorescence microscopy of mitotic spindle; chromosome counting\",\n      \"journal\": \"Neoplasia (New York, N.Y.)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — Co-IP plus functional KD phenotype; single lab; multiple interacting partners identified\",\n      \"pmids\": [\"18592005\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"CHFR binds and ubiquitinates HDAC1 (histone deacetylase 1) both in vitro and in vivo, targeting it for degradation. Ectopic expression of CHFR in cancer cells leads to downregulation of HDAC1, upregulation of p21(CIP1/WAF1), KAI1, and E-cadherin, G1 arrest, and reduced invasiveness. Identified via affinity purification/mass spectrometry.\",\n      \"method\": \"Affinity purification combined with mass spectrometry; co-immunoprecipitation; in vitro ubiquitination assay; invasion assays; xenograft model\",\n      \"journal\": \"Nature cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — AP-MS identification + in vitro ubiquitination + co-IP + functional cell and in vivo assays; published in high-quality journal\",\n      \"pmids\": [\"19182791\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"CHFR interacts with Kif22 (a chromokinesin that binds DNA and microtubules) and ubiquitinates Kif22 as a novel substrate, leading to Kif22 downregulation. Chfr-mediated Kif22 down-regulation is critical for the maintenance of chromosome stability.\",\n      \"method\": \"Co-immunoprecipitation; in vitro ubiquitination assay; siRNA knockdown; chromosome stability assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus in vitro ubiquitination of substrate; single lab with two orthogonal methods\",\n      \"pmids\": [\"19321445\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Activated PKB/Akt phosphorylates CHFR in vitro and in vivo. Expression of a CHFR mutant that cannot be phosphorylated by PKB results in reduced Plk1 levels and inhibition of mitotic entry. Thus, PKB facilitates mitotic entry after DNA damage by phosphorylating and inhibiting CHFR checkpoint function.\",\n      \"method\": \"In vitro kinase assay (PKB phosphorylating CHFR); phospho-mutant CHFR expression; Plk1 level analysis; mitotic entry assay\",\n      \"journal\": \"Molecular cancer research : MCR\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro kinase assay plus phospho-mutant functional rescue; single lab\",\n      \"pmids\": [\"14638868\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Nuclear localization of CHFR is mediated by a nuclear localization signal (NLS); a lysine-rich stretch (KKK) at amino acids 257-259 is essential for nuclear localization. Nuclear localization is essential for CHFR checkpoint function but not for its stability.\",\n      \"method\": \"NLS mutagenesis (KKK→AAA); immunofluorescence localization; checkpoint functional assays\",\n      \"journal\": \"Molecules and cells\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — site-directed mutagenesis linked to localization and functional checkpoint readout; single lab\",\n      \"pmids\": [\"19326084\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Crystal structure of the cysteine-rich region of human CHFR (residues 425–664) at 1.9 Å resolution revealed a multizinc binding domain with elaborate topology containing the PAR-binding zinc finger (PBZ). Co-crystal structures with PAR-like ligands (ADP-ribose, AMP, diadenosine pyrophosphate) defined the PBZ-PAR interaction: the PBZ recognizes two adenine-containing subunits of PAR and the connecting phosphate backbone. Site-specific mutagenesis and surface plasmon resonance validated this interaction. The PBZ-PAR interaction is required for antephase checkpoint function.\",\n      \"method\": \"X-ray crystallography (1.9 Å); co-crystal structures with PAR analogs; site-specific mutagenesis; surface plasmon resonance\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure + co-crystal with ligands + mutagenesis + SPR binding validation in one study\",\n      \"pmids\": [\"20880844\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"CHFR functions as a ubiquitin ligase for HLTF (helicase-like transcription factor), binding to and ubiquitinating HLTF to target it for proteasomal degradation. CHFR-mediated HLTF downregulation reduces PAI-1 expression, inhibiting cell migration.\",\n      \"method\": \"Co-immunoprecipitation; in vitro ubiquitination assay; cell migration assay\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — Co-IP and in vitro ubiquitination; functional migration readout; single lab\",\n      \"pmids\": [\"20388495\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"STIL promotes Chfr auto-ubiquitination and reduces its protein stability. In cells lacking STIL (MEFs, E9.5 embryos), Chfr protein levels are elevated and Plk1 (a Chfr substrate) is reduced. Knockdown of Chfr or overexpression of Plk1 reverses the abnormal mitotic phenotypes of STIL-deficient fibroblasts. STIL is thus identified as the first negative regulator of the Chfr mitotic checkpoint.\",\n      \"method\": \"Genetic KO (Stil-/- mice); siRNA knockdown; protein stability assays; rescue by Chfr KD or Plk1 OE; flow cytometry\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic KO + functional rescue experiments; single lab\",\n      \"pmids\": [\"21245198\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"RNF8 and CHFR synergistically regulate histone ubiquitination to control histone H4 Lys16 acetylation through MRG15-dependent acetyltransferase complexes. This chromatin remodeling function is required for ATM activation after DNA damage. Double-knockout (RNF8/Chfr DKO) cells show suppressed DNA damage-induced ATM kinase activation, hypersensitivity to ionizing radiation, and DKO mice develop thymic lymphomas with clonal chromosome translocations.\",\n      \"method\": \"Double-knockout mouse model; chromatin fractionation; histone ubiquitination and acetylation assays; ATM activation assays; radiation sensitivity assays\",\n      \"journal\": \"Nature structural & molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — KO mouse model + biochemical histone modification assays + ATM activation readout; published in high-quality journal\",\n      \"pmids\": [\"21706008\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Auto-ubiquitylation of Chfr at G2 phase is required for Plk1 accumulation and mitotic entry. Chfr auto-ubiquitylation mutants (Chfr-K2A) do not undergo cell cycle-dependent degradation; elevated Chfr-K2A levels at G2/M reduce Plk1 levels and delay mitotic entry by suppressing cyclin B1/Cdk1 kinase activity. Overexpression of Plk1 in Chfr-K2A cells restores kinase activity and promotes mitotic entry. Aurora A levels are not affected, identifying Plk1 as the major Chfr target in controlling mitotic entry timing.\",\n      \"method\": \"Site-directed mutagenesis of Chfr auto-ubiquitylation sites; cell cycle analysis; Plk1 and Aurora A level measurement; cyclin B1/Cdk1 kinase assay; Plk1 overexpression rescue\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — mutagenesis + functional rescue experiment + kinase assay; single lab with multiple orthogonal methods\",\n      \"pmids\": [\"21768102\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"CHFR is recruited to DNA double-strand breaks (DSBs) via poly(ADP-ribose) (PAR). At DSBs, CHFR regulates the first wave of protein ubiquitination. CHFR ubiquitinates PARP1 and regulates chromatin-associated PARP1 in vivo, establishing CHFR as an important E3 ligase in the early DNA damage response that mediates crosstalk between ubiquitination and poly-ADP-ribosylation.\",\n      \"method\": \"Recruitment assay to laser-induced DSBs; PAR-binding experiments; in vivo ubiquitination assay; chromatin fractionation\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct DSB recruitment assay and in vivo ubiquitination; single lab\",\n      \"pmids\": [\"23268447\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"CHFR binds MAD2 via its cysteine-rich domain; deletion of this domain abolishes the CHFR/MAD2 interaction. The cysteine-rich domain is required for proper MAD2 localization and for promoting the MAD2/CDC20 interaction, thus enabling spindle assembly checkpoint function.\",\n      \"method\": \"Domain deletion mutagenesis; co-immunoprecipitation; MAD2 localization by immunofluorescence; MAD2/CDC20 interaction assay\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — domain deletion + Co-IP + localization; single lab with multiple readouts\",\n      \"pmids\": [\"21575600\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"CHFR interacts with PARP-1; mitotic stress induces PARP-1 auto-PARylation, which enhances the CHFR-PARP-1 interaction and increases polyubiquitination/degradation of PARP-1. Decreased PARP-1 protein levels promote cell cycle arrest at prophase. In CHFR-silenced cells, PARP-1 is not degraded after mitotic stress and cells progress into mitosis. Chfr knockout mice and CHFR-silenced primary gastric cancer tissues have higher PARP-1 protein levels.\",\n      \"method\": \"Affinity purification/mass spectrometry; co-immunoprecipitation; in vitro ubiquitination; Chfr KO mouse tissue analysis; cell cycle assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — AP-MS identification + in vitro ubiquitination + Co-IP + KO mouse validation; multiple orthogonal methods\",\n      \"pmids\": [\"22337872\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"SRG3/mBAF155 blocks CHFR-mediated ubiquitination and degradation of core SWI/SNF-BAF complex components (BRG1, SNF5, BAF60a). CHFR interacts with BRG1, SNF5, and BAF60a and ubiquitinates them for proteasomal degradation; SRG3/mBAF155 stabilizes these components by blocking their interaction with CHFR.\",\n      \"method\": \"Co-immunoprecipitation; in vitro ubiquitination assay; protein stability assays\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — Co-IP and in vitro ubiquitination of multiple substrates; single lab\",\n      \"pmids\": [\"22285184\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"CHFR ubiquitinates and regulates TOPK/PBK (a serine/threonine kinase) levels as part of its checkpoint function. TOPK in turn phosphorylates and inactivates PTEN, which activates Akt, leading to proper G2/M progression. TOPK and PTEN are identified as new players in CHFR-mediated mitotic checkpoint signaling.\",\n      \"method\": \"Co-immunoprecipitation; in vitro ubiquitination assay; kinase assay (TOPK phosphorylation of PTEN); CHFR knockdown/overexpression\",\n      \"journal\": \"Cellular signalling\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — Co-IP + in vitro ubiquitination + kinase assay; single lab; pathway placement by epistasis\",\n      \"pmids\": [\"24012691\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"The CHFR PBZ domain mediates a protein interaction with poly-ADP-ribosylated PARP1, leading to stabilization of CHFR. Disruption of the CHFR-PARP1 interaction (via PARP1 knockdown or PBZ peptide overexpression) causes loss of CHFR protein expression. A small-molecule inhibitor ('A3') targeting the CHFR PBZ domain disrupts the CHFR-PARP1 interaction and inhibits mitotic checkpoint function, showing therapeutic synergy with docetaxel in vitro and in xenograft models.\",\n      \"method\": \"shRNA knockdown; PBZ domain peptide overexpression; computational docking screen; in vitro protein-protein interaction assay; cell viability and colony formation assays; xenograft mouse model\",\n      \"journal\": \"Oncotarget\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — functional domain disruption + in vivo xenograft; single lab; confirms PAR-binding mechanistic role\",\n      \"pmids\": [\"26356822\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"CHFR functions as an E3 ubiquitin ligase for SIRT1, responsible for its proteasomal degradation under oxidative stress. CHFR interacts with SIRT1 and ubiquitinates it. CHFR-mediated SIRT1 degradation is enhanced when SIRT1 is phosphorylated by JNK1 upon oxidative stress, leading to increased p53 acetylation and apoptosis. JNK inhibition prevents SIRT1 phosphorylation and CHFR-mediated degradation.\",\n      \"method\": \"Co-immunoprecipitation; in vivo ubiquitination assay; protein stability assay; JNK inhibitor experiments; p53 acetylation assay\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — Co-IP plus ubiquitination and functional apoptosis readout; single lab\",\n      \"pmids\": [\"27883020\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"CHFR associates with and ubiquitinates ZEB1 in a ubiquitination-dependent manner, leading to decreased ZEB1 expression in TNBC. CHFR represses FASN expression through ZEB1, causing cell death under chemotherapy. HDAC inhibitor TSA increases CHFR expression independent of histone acetylation, destabilizing ZEB1 and sensitizing TNBC cells to chemotherapy.\",\n      \"method\": \"Affinity purification/mass spectrometry; co-immunoprecipitation; in vivo ubiquitination assay; CHFR overexpression/knockdown; cell viability assays\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — AP-MS identification + Co-IP + ubiquitination assay; single lab\",\n      \"pmids\": [\"34462429\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"PARP1 interacts with and poly(ADP-ribosyl)ates RNF126, which then recruits CHFR (via its PAR-binding PBZ domain) to ubiquitinate and degrade RNF126. RNF126 is required for ATR-Chk1 signaling activation by IR or PARP inhibitors; CHFR-mediated RNF126 degradation thus modulates DNA damage checkpoint signaling.\",\n      \"method\": \"Co-immunoprecipitation; in vitro and in vivo ubiquitination assay; PAR-binding assays; siRNA knockdown; cell viability assays\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — Co-IP and ubiquitination assay identifying new substrate; single lab\",\n      \"pmids\": [\"34388456\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"CHFR is required for ubiquitylation-dependent degradation of VE-cadherin in endothelial cells. Endothelial-restricted Chfr knockout (ChfrΔEC) mice show augmented VE-cadherin expression at adherens junctions and abrogated LPS-induced VE-cadherin degradation. LPS-induced lung endothelial barrier breakdown, inflammatory neutrophil extravasation, and mortality are suppressed in ChfrΔEC mice. FoxO1 transcription factor is identified as an upstream regulator of CHFR expression.\",\n      \"method\": \"Endothelial-specific conditional Chfr knockout mice; immunofluorescence; VE-cadherin ubiquitination assay; LPS challenge model; endothelial permeability assay\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — conditional KO mouse with specific vascular phenotype + mechanistic ubiquitination assay; single lab but in vivo validation\",\n      \"pmids\": [\"37852964\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"In S. cerevisiae Chfr homologs (Chf1/Chf2), Ubc4 is required for G1 cell cycle delay and Chf protein turnover (Lys48-type modification), while Ubc13/Mms2 is required for G2 delay and does not contribute to protein turnover (Lys63-type modification). Site-specific mutagenesis identified the Lys residues modified by each E2. This dual E2 specificity accounts for different cell cycle functions.\",\n      \"method\": \"Yeast genetic assays; in vitro reconstitution with purified components; tandem mass spectrometry linkage analysis; site-directed mutagenesis\",\n      \"journal\": \"Cell cycle (Georgetown, Tex.)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution + MS linkage analysis + mutagenesis; functional dissection of two E2 specificities in ortholog\",\n      \"pmids\": [\"18202552\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"S. cerevisiae Dma1 and Dma2 (budding yeast Chfr homologs) are involved in proper spindle positioning, likely by regulating septin ring deposition at the bud neck. DMA2 overexpression causes defects in septin ring disassembly and cytokinesis that can be rescued by eliminating the spindle position checkpoint protein Bub2 or overproducing Tem1. dma1Δdma2Δ cells fail to activate the spindle position checkpoint in response to dynein loss.\",\n      \"method\": \"Genetic deletion analysis; DMA2 overexpression; epistasis with Bub2/Tem1; spindle position checkpoint assays\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis in yeast ortholog; functional checkpoint assays; single lab\",\n      \"pmids\": [\"15146058\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"CHFR interacts with LRP1 in human vascular smooth muscle cells. Aggregated LDL (agLDL) downregulates CHFR expression and reduces the CHFR-LRP1 physical interaction, leading to reduced LRP1 ubiquitination and prolonged LRP1 half-life. CHFR silencing increases VSMC LRP1 expression specifically (not classical LDLR), indicating CHFR targets LRP1 for ubiquitin-mediated degradation.\",\n      \"method\": \"Co-immunoprecipitation; pulse-chase analysis; cycloheximide chase; CHFR siRNA silencing; 2D proteomics\",\n      \"journal\": \"Arteriosclerosis, thrombosis, and vascular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — Co-IP + protein stability assay + KD phenotype; single lab; substrate identification without full in vitro reconstitution\",\n      \"pmids\": [\"23241409\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"CHFR is a multifunctional E3 ubiquitin ligase that (1) defines an antephase/prophase checkpoint by ubiquitinating Plk1 (via Lys48-linked chains with Ubc4/Ubc5 for proteasomal degradation) to suppress Cdc25C/Wee1 and thereby exclude Cyclin B1 from the nucleus, delaying chromosome condensation in response to mitotic stress through a pathway requiring p38 kinases; (2) forms non-degradative Lys63-linked polyubiquitin chains with Ubc13-Mms2 for stress signaling; (3) ubiquitinates additional mitotic substrates including Aurora A, HDAC1, PARP1, Kif22, TOPK, SIRT1, ZEB1, HLTF, BAF complex components (BRG1, SNF5, BAF60a), RNF126, and VE-cadherin to regulate diverse processes including chromosomal stability, transcription, DNA damage response, and endothelial barrier integrity; (4) is recruited to DNA double-strand breaks via PAR through its PBZ zinc-finger domain, where it mediates the first wave of damage-site ubiquitination and cooperates with RNF8 to regulate histone H4K16 acetylation and ATM activation; (5) localizes to the cytoplasm/mitotic spindle (interacting with TCTP and beta-tubulin) and to the nucleus via a KKK NLS (residues 257–259), with nuclear localization required for checkpoint function; (6) is stabilized by USP7/HAUSP-mediated deubiquitination and regulated by PKB/Akt phosphorylation (which inhibits checkpoint function); and (7) functions as a tumor suppressor whose loss is associated with chromosomal instability, cancer predisposition, and sensitivity to taxanes.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"CHFR is a RING-domain E3 ubiquitin ligase and tumor suppressor that enforces a mitotic stress (antephase/prophase) checkpoint, delaying chromosome condensation in response to microtubule poisons [#0]. Its checkpoint activity is genetically separable from its catalytic activity: the FHA domain mediates growth suppression and checkpoint function while the RING and cysteine-rich regions are required for ubiquitin ligase activity [#11]. The principal effector of the checkpoint is Polo-like kinase 1 (Plk1), which CHFR ubiquitinates to prolong inhibitory Cdc2 tyrosine-15 phosphorylation by delaying Cdc25C activation and Wee1 inactivation, thereby excluding Cyclin B1 from the nucleus and blocking mitotic entry [#1, #8, #21], a checkpoint that operates downstream of the p38 stress kinases [#5]. CHFR builds distinct chain types with distinct E2s — Lys48-linked chains via Ubc4/Ubc5 that target substrates for proteasomal degradation, and non-degradative Lys63-linked chains via Ubc13-Mms2 for stress signaling [#2, #4, #32]. Beyond Plk1, CHFR ubiquitinates a broad substrate set to maintain chromosomal stability and regulate transcription, migration, and apoptosis, including Aurora A [#7], HDAC1 [#13], Kif22 [#14], PARP1 [#22, #24], HLTF [#18], SIRT1 [#28], ZEB1 [#29], SWI/SNF-BAF components [#25], RNF126 [#30], and VE-cadherin in endothelial barrier control [#31]. CHFR is recruited to DNA double-strand breaks through poly(ADP-ribose) binding by its cysteine-rich PAR-binding zinc finger (PBZ), whose structure and PAR-recognition mode are defined, where it directs the first wave of damage-site ubiquitination and cooperates with RNF8 to control histone H4K16 acetylation and ATM activation [#17, #20, #22]. CHFR activity is set by counter-regulators: USP7/HAUSP deubiquitination stabilizes it [#9], STIL promotes its auto-ubiquitination and turnover [#19], and PKB/Akt phosphorylation inhibits its checkpoint function [#15]. Loss of CHFR causes chromosomal instability and cancer predisposition in knockout mice [#7, #20].\"\n,\n  \"teleology\": [\n    {\n      \"year\": 2000,\n      \"claim\": \"Established that CHFR exists and defines a previously unrecognized mitotic stress checkpoint controlling the timing of chromosome condensation.\",\n      \"evidence\": \"Ectopic wild-type vs. mutant CHFR expression and cell-cycle analysis after microtubule poisons in cancer cell lines\",\n      \"pmids\": [\"10935642\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular activity of CHFR unknown at this stage\", \"Direct substrates unidentified\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Defined CHFR as a RING-domain E3 ubiquitin ligase and identified Plk1 as the checkpoint substrate, explaining how the checkpoint delays Cdc2 activation.\",\n      \"evidence\": \"In vitro ubiquitination, RING mutagenesis, Xenopus extracts, and proteasome inhibition; independent confirmation of RING requirement and cell-cycle-regulated expression\",\n      \"pmids\": [\"11807090\", \"11912157\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Chain linkage type not yet resolved\", \"Whether Plk1 degradation is the sole mechanism unclear\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Determined the FHA domain structure, indicating a phosphopeptide-recognition module consistent with FHA family proteins.\",\n      \"evidence\": \"X-ray crystallography of FHA domain native and tungstate-bound forms\",\n      \"pmids\": [\"12121644\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Physiological phosphoprotein ligand of the FHA domain not identified\", \"Crystallized as segment-swapped dimer of uncertain in vivo relevance\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Showed CHFR can build non-degradative Lys63-linked chains via Ubc13-Mms2, distinguishing a signaling function from proteasomal targeting.\",\n      \"evidence\": \"In vitro ubiquitination with Ubc13-Mms2 and chain linkage analysis; mitotic mobility-shift analysis\",\n      \"pmids\": [\"14562038\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo substrates of K63 chains not defined\", \"Reconciliation with degradative K48 activity left open\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Placed CHFR downstream of p38 stress kinases and confirmed the antephase checkpoint requires ubiquitylation but not the proteasome.\",\n      \"evidence\": \"p38 inhibition, checkpoint assays in Chfr-deficient cells, proteasome inhibitor experiments\",\n      \"pmids\": [\"15302856\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct link between p38 and CHFR activation not biochemically defined\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Linked CHFR nuclear dynamics and checkpoint function to PML bodies.\",\n      \"evidence\": \"Live-cell FRET, fluorescence recovery, PML-knockout cells, checkpoint assays\",\n      \"pmids\": [\"15467728\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism by which PML bodies regulate CHFR activity unresolved\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Identified Aurora A as a substrate and established CHFR as a chromosomal-stability tumor suppressor in vivo.\",\n      \"evidence\": \"Co-IP, in vitro ubiquitination, Chfr-knockout mice, MEF chromosome analysis\",\n      \"pmids\": [\"15793587\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Relative contribution of Aurora A vs. Plk1 to tumor suppression unclear\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Defined the downstream output of the checkpoint as nuclear exclusion of Cyclin B1, preventing chromosome condensation.\",\n      \"evidence\": \"Wild-type CHFR cell lines, cell-cycle marker immunofluorescence, Cyclin B1 NES-mutant override experiment\",\n      \"pmids\": [\"15674323\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism connecting Plk1 ubiquitination to Cyclin B1 localization not fully traced\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Separated CHFR's checkpoint function (FHA domain) from its catalytic activity (RING + cysteine-rich domains).\",\n      \"evidence\": \"Domain deletion mutagenesis with proliferation, checkpoint, and ubiquitination assays\",\n      \"pmids\": [\"18335050\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"How FHA-dependent checkpoint function operates without requiring ligase activity not explained\", \"Single lab\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Localized endogenous CHFR to the cytoplasm and mitotic spindle and proposed microtubule sensing through TCTP/tubulin interactions.\",\n      \"evidence\": \"Two-hybrid screen, endogenous Co-IP, immunofluorescence with three antibodies, nocodazole depolymerization\",\n      \"pmids\": [\"18504434\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Causal role of TCTP/tubulin binding in checkpoint activation not demonstrated\", \"Ectopic overexpression shifts localization, complicating interpretation\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Extended CHFR's spindle role through alpha-tubulin and MAD2 regulation affecting spindle assembly checkpoint integrity.\",\n      \"evidence\": \"siRNA knockdown, Co-IP, ubiquitination assays, mitotic spindle microscopy, chromosome counting\",\n      \"pmids\": [\"18592005\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct vs. indirect effects on MAD2/BUBR1 localization not distinguished\", \"Single lab\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Expanded the substrate repertoire to transcriptional and motility regulators (HDAC1, Kif22, HLTF), linking CHFR to gene expression, invasion, and chromosome stability.\",\n      \"evidence\": \"AP-MS, Co-IP, in vitro ubiquitination, invasion/migration assays, xenograft model\",\n      \"pmids\": [\"19182791\", \"19321445\", \"20388495\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Physiological conditions selecting each substrate undefined\", \"Some readouts rely on overexpression\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Identified upstream regulation by PKB/Akt phosphorylation that inhibits checkpoint function and a KKK NLS required for nuclear checkpoint activity.\",\n      \"evidence\": \"In vitro kinase assay, phospho-mutant rescue, NLS mutagenesis, localization and checkpoint assays\",\n      \"pmids\": [\"14638868\", \"19326084\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Phosphosite mapping limited\", \"Single-lab findings\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Defined the PBZ zinc finger structure and its PAR-recognition mode, establishing the molecular basis for PAR-dependent recruitment and antephase checkpoint function.\",\n      \"evidence\": \"1.9 Å crystal and co-crystal structures with PAR analogs, mutagenesis, SPR binding validation\",\n      \"pmids\": [\"20880844\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo PAR sources driving CHFR recruitment in different contexts not fully mapped\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Established CHFR's role in the early DNA damage response: PAR-dependent recruitment to DSBs, PARP1 ubiquitination, and RNF8-cooperative control of H4K16 acetylation and ATM activation.\",\n      \"evidence\": \"Laser-induced DSB recruitment, PAR binding, in vivo ubiquitination, RNF8/Chfr double-knockout mice, histone modification and ATM assays\",\n      \"pmids\": [\"21706008\", \"23268447\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Order of events between CHFR and RNF8 at chromatin not fully resolved\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Clarified the role of CHFR auto-ubiquitylation in normal cell-cycle timing, showing auto-ubiquitylation-driven turnover permits Plk1 accumulation for mitotic entry, with Plk1 the dominant target over Aurora A.\",\n      \"evidence\": \"Auto-ubiquitylation site mutants, cell-cycle analysis, Plk1/Aurora A measurement, Cyclin B1/Cdk1 kinase assay, Plk1 rescue\",\n      \"pmids\": [\"21768102\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Trigger controlling timing of auto-ubiquitylation not defined\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Identified STIL as the first negative regulator promoting CHFR auto-ubiquitination and turnover.\",\n      \"evidence\": \"Stil-knockout mice/MEFs, siRNA, stability assays, rescue by Chfr KD or Plk1 OE\",\n      \"pmids\": [\"21245198\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether STIL acts directly on CHFR not established\", \"Single lab\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Showed PARP1 auto-PARylation under mitotic stress enhances CHFR-PARP1 interaction and PARP1 degradation, coupling PAR signaling to checkpoint arrest.\",\n      \"evidence\": \"AP-MS, Co-IP, in vitro ubiquitination, Chfr-KO mouse tissue, cell-cycle assays\",\n      \"pmids\": [\"22337872\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Quantitative contribution of PARP1 degradation to arrest vs. other substrates unclear\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Connected CHFR to additional checkpoint and metabolic signaling via TOPK/PTEN/Akt and BAF complex stabilization by SRG3/mBAF155.\",\n      \"evidence\": \"Co-IP, in vitro ubiquitination, kinase assays, protein stability assays\",\n      \"pmids\": [\"24012691\", \"22285184\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Pathway placements rest on epistasis in single labs\", \"Physiological substrate selection conditions undefined\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Demonstrated that PBZ-mediated CHFR-PARP1 interaction stabilizes CHFR and is a druggable node, with a PBZ-targeting compound synergizing with docetaxel.\",\n      \"evidence\": \"shRNA, PBZ peptide overexpression, docking screen, protein-interaction and viability assays, xenograft model\",\n      \"pmids\": [\"26356822\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Compound specificity and off-target effects not fully characterized\", \"Single lab\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Identified SIRT1 as an oxidative-stress substrate, linking CHFR to JNK1-dependent p53 acetylation and apoptosis.\",\n      \"evidence\": \"Co-IP, in vivo ubiquitination, stability assays, JNK inhibition, p53 acetylation assay\",\n      \"pmids\": [\"27883020\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct ubiquitination not reconstituted in vitro\", \"Single lab\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Extended CHFR substrate range to ZEB1 (chemo-sensitization in TNBC) and RNF126 (DNA damage checkpoint modulation), the latter via PAR-dependent recruitment.\",\n      \"evidence\": \"AP-MS, Co-IP, in vivo/in vitro ubiquitination, PAR-binding assays, viability assays\",\n      \"pmids\": [\"34462429\", \"34388456\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Context-dependence of these substrate relationships not defined\", \"Single-lab studies\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Established a non-mitotic physiological role for CHFR in endothelial barrier integrity through VE-cadherin degradation, downstream of FoxO1.\",\n      \"evidence\": \"Endothelial-specific conditional Chfr knockout mice, immunofluorescence, VE-cadherin ubiquitination assay, LPS challenge, permeability assays\",\n      \"pmids\": [\"37852964\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether VE-cadherin is a direct ubiquitination substrate in vitro not shown\", \"Link to mitotic functions unclear\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How CHFR integrates upstream stress signals (p38, JNK, Akt, PAR) to select among its many substrates and switch between K48 and K63 chain outputs in specific contexts remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No unified model linking signal input to substrate/chain-type choice\", \"FHA domain physiological phospholigand unidentified\", \"Mechanism coupling cytoplasmic microtubule sensing to nuclear checkpoint output incomplete\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0016874\", \"supporting_discovery_ids\": [1, 2, 7, 13, 22, 24, 32]},\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [1, 7, 13, 14, 22, 24, 28, 31]},\n      {\"term_id\": \"GO:0003723\", \"supporting_discovery_ids\": [17, 22, 27]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [6, 16]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [10]},\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [10, 12]},\n      {\"term_id\": \"GO:0000228\", \"supporting_discovery_ids\": [20, 22]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [0, 1, 8, 21]},\n      {\"term_id\": \"R-HSA-73894\", \"supporting_discovery_ids\": [20, 22, 30]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [1, 13, 24]},\n      {\"term_id\": \"R-HSA-4839726\", \"supporting_discovery_ids\": [20]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"PLK1\", \"AURKA\", \"HDAC1\", \"PARP1\", \"USP7\", \"MAD2L1\", \"TPT1\", \"RNF8\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":8,"faith_total":8,"faith_pct":100.0}}