{"gene":"RNF40","run_date":"2026-04-28T19:45:45","timeline":{"discoveries":[{"year":2023,"finding":"Cryo-EM structure of the RNF20/RNF40-hRAD6A-Ub-nucleosome complex (captured via chemical trapping) revealed that RNF40 directly binds nucleosomal DNA and exhibits a conserved E3/E2/nucleosome interaction pattern for H2B monoubiquitylation at K120; an uncanonical non-hydrophobic contact in the RING-E2 interface positions RAD6A directly above the target H2B lysine residue.","method":"Chemical trapping strategy + cryo-EM structure determination + mutagenesis","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 1 — cryo-EM structure with chemical trapping and mutagenesis validation in a single rigorous study","pmids":["37633270"],"is_preprint":false},{"year":2016,"finding":"Crystal structure of the RNF20 RING domain shows it forms a homodimer that specifically interacts with the Ube2B~Ub conjugate; the RING domains of RNF20 and RNF40 can form a stable heterodimer that is catalytically active; key contacts at the E3-E2 and E3-ubiquitin interfaces were identified by mutagenesis.","method":"X-ray crystallography + mutagenesis of E3-E2 and E3-ubiquitin interfaces + in vitro ubiquitination assay","journal":"Journal of molecular biology","confidence":"High","confidence_rationale":"Tier 1 — crystal structure combined with mutagenesis and functional assay in one study","pmids":["27569044"],"is_preprint":false},{"year":2026,"finding":"Cryo-EM structure of RNF20/RNF40-RAD6A-Ub-H2BS112GlcNAc nucleosome complex showed that H2B S112 GlcNAcylation interacts with the E2 enzyme RAD6A (not the E3 RNF20/RNF40), allosterically enhancing the nucleophilicity of H2B K120 to stimulate ubiquitin transfer; structure-activity relationship identified the C2 N-acetyl group and β-configuration of C1 as essential.","method":"Chemical synthesis of modified nucleosomes + cryo-EM + mutagenesis + kinetics assays","journal":"Nature chemical biology","confidence":"High","confidence_rationale":"Tier 1 — cryo-EM structure with mutagenesis and kinetics, multiple orthogonal methods","pmids":["41495224"],"is_preprint":false},{"year":2026,"finding":"Crystal structure of the Bre1-Lge1 complex and AlphaFold model of RNF20/RNF40-WAC revealed extensive interaction interfaces; in vitro and in vivo experiments demonstrated that RNF20/RNF40-WAC interactions are critical for H2B monoubiquitination, with different electrostatic contacts encoding binding specificity compared to yeast Bre1-Lge1.","method":"X-ray crystallography (Bre1-Lge1) + AlphaFold modeling (RNF20/RNF40-WAC) + in vitro ubiquitination assay + in vivo functional experiments","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 1/2 — crystal structure plus orthogonal in vitro and in vivo validation","pmids":["41533567"],"is_preprint":false},{"year":2012,"finding":"RNF20/RNF40 (mammalian Bre1 complex) functions as the E3 ubiquitin ligase for histone H2B monoubiquitination; deficiency leads to aberrant R-loop formation causing replication-associated double-strand breaks, combined with a defect in homologous recombination, resulting in chromosomal instability.","method":"Loss-of-function (Bre1-deficient cells) + detection of R-loops, DSBs, and genomic rearrangements","journal":"Cancer research","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal readouts in defined KO cells, replicated finding","pmids":["22354749"],"is_preprint":false},{"year":2011,"finding":"RNF20 and RNF40 form a heterodimeric E3 ubiquitin ligase complex that monoubiquitinates histone H2B at lysine 120; the tumor suppressor CDC73 interacts with both RNF20 and RNF40 at discrete but closely located residues and is required for maintenance of H2Bub1 levels.","method":"Yeast two-hybrid + co-immunoprecipitation + knockdown of CDC73 + western blot for H2Bub1","journal":"Human molecular genetics","confidence":"High","confidence_rationale":"Tier 2 — reciprocal interaction confirmed by multiple methods, functional consequence demonstrated","pmids":["22021426"],"is_preprint":false},{"year":2011,"finding":"RNF40 depletion results in sustained γH2AX phosphorylation, decreased rapid cell cycle checkpoint activation, decreased H3K56ac, and decreased recruitment of the FACT complex (specifically SUPT16H) to chromatin following DNA DSBs; RNF40 and SUPT16H cooperate to promote DNA end resection and timely DNA repair.","method":"siRNA knockdown + immunofluorescence for γH2AX + western blot for H3K56ac + chromatin fractionation + DNA end resection assay","journal":"Cell cycle","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods, phenocopy by SUPT16H knockdown establishes pathway","pmids":["22031019"],"is_preprint":false},{"year":2014,"finding":"Arsenite directly binds to the RING finger domains of RNF20 and RNF40 via cysteine residues in vitro and in cells, inhibiting H2B ubiquitination and impairing recruitment of BRCA1 and RAD51 to DNA DSB sites, thereby compromising DNA DSB repair.","method":"In vitro binding assay + cellular arsenite treatment + ChIP for BRCA1/RAD51 recruitment + comet assay/western blot for DSB repair","journal":"Journal of the American Chemical Society","confidence":"High","confidence_rationale":"Tier 1/2 — direct in vitro binding demonstrated plus multiple cellular functional readouts","pmids":["25170678"],"is_preprint":false},{"year":2019,"finding":"RNF20 and RNF40 are required for DSB repair via both homologous recombination and class switch recombination (NHEJ) in mouse B cells; DSBs induce a global increase in H2Bub1 regulated jointly by ATM and ATR kinases, independently of H2AX phosphorylation.","method":"RNF20/RNF40 knockout in mouse B cells + class switch recombination assay + HR assay + western blot + ATM/ATR inhibitor treatment","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 — genetic KO with multiple functional DSB repair assays and defined signaling pathway","pmids":["30692271"],"is_preprint":false},{"year":2011,"finding":"RNF20 and RNF40 physically and functionally interact with the androgen receptor (AR) and modulate its transcriptional activity; androgen induction of FKBP51 and PSA is accompanied by dynamic increases in H2Bub1 within transcribed regions of these loci.","method":"Co-immunoprecipitation + reporter assay + ChIP for H2Bub1 at AR target loci","journal":"Molecular and cellular endocrinology","confidence":"Medium","confidence_rationale":"Tier 2/3 — co-IP plus ChIP, single lab","pmids":["22155569"],"is_preprint":false},{"year":2017,"finding":"RNF20, RNF40, and WAC form an E3 ligase complex that mediates H2B ubiquitination; knockdown of WAC phenocopies loss of H2B ubiquitination and causes cell death in MLL-rearranged ALL cells.","method":"siRNA knockdown + western blot for H2Bub1 + cell viability assay","journal":"Leukemia","confidence":"Medium","confidence_rationale":"Tier 2/3 — knockdown phenocopy establishes WAC as complex component, single lab","pmids":["28690313"],"is_preprint":false},{"year":2019,"finding":"RNF40 loss decreases nuclear localization of NF-κB following TNF-α treatment in colorectal cancer cells, reducing induction of NF-κB-associated cytokines; colon-specific Rnf40 deletion exerts a protective effect in a murine model of acute colitis.","method":"siRNA knockdown + immunofluorescence for NF-κB nuclear localization + mRNA-seq + in vivo colitis model with colon-specific Rnf40 knockout","journal":"Journal of Crohn's & colitis","confidence":"Medium","confidence_rationale":"Tier 2 — direct localization experiment plus in vivo genetic model, moderate evidence","pmids":["30321325"],"is_preprint":false},{"year":2019,"finding":"RNF40 depletion in colorectal cancer cells increases apoptosis via elevated caspase 3/7 activity, associated with reduced anti-apoptotic and elevated pro-apoptotic BCL2 family member expression; H2Bub1 directly occupies transcribed regions of anti-apoptotic genes.","method":"siRNA knockdown + flow cytometry (cell cycle) + caspase 3/7 assay + Annexin V + ChIP-seq for H2Bub1 + mRNA-seq + caspase inhibitor rescue","journal":"Clinical epigenetics","confidence":"Medium","confidence_rationale":"Tier 2 — multiple orthogonal methods with rescue experiment, single lab","pmids":["31266541"],"is_preprint":false},{"year":2020,"finding":"RNF40-driven H2B monoubiquitination is essential for transcriptional activation of RHO/ROCK/LIMK pathway components and proper actin-cytoskeleton dynamics through trans-histone crosstalk with H3K4me3 in HER2+ breast cancer.","method":"Tissue-specific Rnf40 deletion (mouse mammary carcinoma model) + ChIP for H2Bub1 and H3K4me3 + transcriptome analysis + actin dynamics assays","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 — in vivo genetic model with ChIP and transcriptome, single lab","pmids":["33070155"],"is_preprint":false},{"year":2020,"finding":"RNF40 loss impairs early gene activation during somatic cell reprogramming to iPSCs; mechanistically, RNF40 controls expression of the PRC2 component EZH2 and promotes resolution of H3K4me3/H3K27me3 bivalency on H2Bub1-occupied pluripotency genes.","method":"RNF40 knockdown + iPSC reprogramming assay + ChIP-seq for H3K4me3, H3K27me3, H2Bub1 + mRNA-seq","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 — ChIP-seq with functional reprogramming readout, single lab","pmids":["32341358"],"is_preprint":false},{"year":2020,"finding":"Osteoblast-specific RNF40 deletion in mice impairs early lineage specification, decreases osteoclast number and function via reduced RANKL (Tnfsf11) expression in osteoblasts; H2B monoubiquitination directly occupies the Tnfsf11 locus.","method":"Conditional osteoblast-specific Rnf40 knockout mouse + bone phenotype analysis + ChIP for H2Bub1 at Tnfsf11 + RANKL expression analysis","journal":"Cell death and differentiation","confidence":"Medium","confidence_rationale":"Tier 2 — in vivo genetic model with ChIP showing direct target gene regulation","pmids":["32901120"],"is_preprint":false},{"year":2021,"finding":"Intestine-specific deletion of Rnf20 or Rnf40 in mice causes spontaneous colorectal inflammation; mechanistically, RNF20/RNF40-mediated H2Bub1 controls H3K4me3 occupancy and transcription of the Vitamin D Receptor (Vdr) gene and its target genes in intestinal epithelial cells.","method":"Conditional intestinal Rnf20/Rnf40 knockout + mRNA-seq + ChIP-seq for H2Bub1 and H3K4me3 + in vivo colitis monitoring","journal":"Cell death and differentiation","confidence":"Medium","confidence_rationale":"Tier 2 — in vivo genetic model with ChIP-seq, single lab","pmids":["34088983"],"is_preprint":false},{"year":2024,"finding":"RNF40 mediates ubiquitination and proteasome-dependent degradation of LIMA1 (an actin-binding protein involved in cholesterol absorption) as a non-histone substrate; the 1-166aa fragment of LIMA1 is required for interaction with RNF40, and RNF40 overexpression reduces cellular lipid content in a LIMA1-dependent manner.","method":"Co-immunoprecipitation + domain mapping + ubiquitination assay + proteasome inhibitor treatment + lipid content measurement + rescue by LIMA1 overexpression","journal":"Cell death discovery","confidence":"Medium","confidence_rationale":"Tier 2/3 — co-IP with functional rescue, single lab","pmids":["38909032"],"is_preprint":false},{"year":2025,"finding":"RNF40 catalyzes K6- and K11-linked polyubiquitination of KDM6A, targeting it for autophagic degradation via TAX1BP1 recognition; this contrasts with USP7-mediated deubiquitination (K48-linked) that prevents KDM6A proteasomal degradation, forming an antagonistic ubiquitin-switching circuit regulating KDM6A stability and coronavirus receptor expression.","method":"Genetic and pharmacological inhibition + ubiquitination linkage analysis + co-immunoprecipitation + autophagic degradation assay + viral infection functional assay","journal":"Advanced science","confidence":"Medium","confidence_rationale":"Tier 2 — ubiquitin linkage analysis plus functional rescue, single lab","pmids":["41691482"],"is_preprint":false},{"year":2025,"finding":"RNF40 promotes K48-linked polyubiquitination and proteasomal degradation of Parkin in cerebrovascular endothelial cells, thereby inhibiting mitophagy; Rnf40 knockdown in spontaneously hypertensive rats restored mitophagy, improved tight junction proteins, and protected the blood-brain barrier.","method":"In vivo Rnf40 knockdown (pAAV-shRnf40 in SHRs) + ubiquitination assay (K48 linkage) + mitophagy assay + tight junction protein western blot + cerebral blood flow measurement","journal":"CNS neuroscience & therapeutics","confidence":"Medium","confidence_rationale":"Tier 2 — in vivo genetic model with ubiquitination linkage analysis, single lab","pmids":["39777866"],"is_preprint":false},{"year":2023,"finding":"The RNF40/H2Bub1 axis positively regulates peroxisome-related gene expression (PRDX5, PEX6, PMVK); loss of RNF20/RNF40 impairs peroxisomal biogenesis and ROS metabolism, leading to increased lipid peroxidation and ferroptotic cell death in cervical cancer cells.","method":"RNF20/RNF40 knockdown + ChIP-qPCR for H2Bub1 + transcriptome analysis + lipid peroxidation assay + flow cytometry for ferroptosis markers + in vivo CAM assay","journal":"Cell communication and signaling","confidence":"Medium","confidence_rationale":"Tier 2/3 — ChIP with transcriptome and ferroptosis functional assays, single lab","pmids":["40597205"],"is_preprint":false},{"year":2023,"finding":"The RNF40/H2Bub1 axis promotes cancer stem cell properties and glycolytic program in triple-negative breast cancer by supporting YAP1 signaling.","method":"RNF40 knockdown/overexpression + sphere formation assay + ChIP-seq for H2Bub1 + transcriptome analysis + YAP1 pathway analysis","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2/3 — ChIP-seq with functional stem cell assays, single lab","pmids":["37770435"],"is_preprint":false},{"year":2025,"finding":"PAF1C-driven transcription restart after DNA damage is independent of RNF20/RNF40-mediated H2B-K120 ubiquitination; H2Bub1 levels do not correlate with transcription restoration following DNA lesions, and RTF1-stimulated H2Bub1 does not contribute to restart.","method":"RNF20/RNF40 depletion + transcription restart assay after DNA damage + H2Bub1 ChIP","journal":"bioRxiv","confidence":"Low","confidence_rationale":"Tier 2 but preprint, single lab, awaiting peer review","pmids":["bio_10.1101_2025.07.23.666359"],"is_preprint":true},{"year":2024,"finding":"RNF40 physically interacts with RTF1 and is required for histone H2B monoubiquitination that supports Th17 cell differentiation; RNF40-deficient cells phenocopy RTF1-deficient cells in impaired Th17 differentiation.","method":"RNF40 knockout in T cells + Th17 differentiation assay + H2Bub1 ChIP-seq + genetic epistasis with RTF1","journal":"bioRxiv","confidence":"Low","confidence_rationale":"Tier 2/3 but preprint, single lab","pmids":["bio_10.1101_2024.11.08.622668"],"is_preprint":true}],"current_model":"RNF40 forms a stable heterodimeric E3 ubiquitin ligase complex with RNF20, and together with the E2 enzyme hRAD6A (and cofactor WAC), monoubiquitinates histone H2B at lysine 120 (H2Bub1) by directly binding nucleosomal DNA — a mechanism structurally characterized by cryo-EM and crystallography — which in turn promotes transcriptional elongation, facilitates DNA double-strand break repair (via FACT recruitment, HR, and NHEJ), maintains genomic stability by suppressing R-loops, and regulates diverse cellular processes including NF-κB signaling, apoptosis, bone remodeling, and pluripotency; additionally, RNF40 ubiquitinates non-histone substrates including LIMA1, Parkin, and KDM6A to regulate lipid metabolism, mitophagy, and viral receptor stability."},"narrative":{"teleology":[{"year":2011,"claim":"Establishing that RNF20 and RNF40 form a stable heterodimeric E3 ligase for H2B K120 monoubiquitination, with the tumor suppressor CDC73 required for maintaining H2Bub1 levels, defined the core enzymatic identity of the complex.","evidence":"Yeast two-hybrid, co-IP, knockdown, and western blot in human cells","pmids":["22021426"],"confidence":"High","gaps":["Structural basis of heterodimer assembly not yet resolved","Catalytic contribution of each RING domain unknown","Mechanism by which CDC73 promotes H2Bub1 unclear"]},{"year":2011,"claim":"Demonstrating that RNF40 depletion impairs FACT recruitment, H3K56 acetylation, and DNA end resection after DSBs established a direct role for H2Bub1 in the DNA damage response beyond transcription.","evidence":"siRNA knockdown with γH2AX immunofluorescence, chromatin fractionation, and end resection assays","pmids":["22031019"],"confidence":"High","gaps":["Whether RNF40 is recruited to damage sites or acts globally was unresolved","Relative contributions of HR vs NHEJ pathways not separated"]},{"year":2012,"claim":"Linking RNF20/RNF40 deficiency to aberrant R-loop formation and replication-associated DSBs revealed that H2Bub1 maintains genomic stability by suppressing co-transcriptional R-loops.","evidence":"Loss-of-function cells with R-loop detection, DSB assays, and genomic rearrangement analysis","pmids":["22354749"],"confidence":"High","gaps":["Mechanism by which H2Bub1 suppresses R-loops not determined","Whether R-loop suppression requires FACT or other downstream effectors unknown"]},{"year":2016,"claim":"Solving the crystal structure of the RNF20 RING domain and demonstrating that the RNF20/RNF40 RING heterodimer is catalytically active with UBE2B provided the first atomic-level view of the E3 architecture.","evidence":"X-ray crystallography with mutagenesis and in vitro ubiquitination assays","pmids":["27569044"],"confidence":"High","gaps":["Structure of the full heterodimer on a nucleosome substrate not yet available","Role of WAC in E3 activity not structurally characterized"]},{"year":2019,"claim":"Genetic knockout of RNF20/RNF40 in mouse B cells confirmed their requirement for both HR and class-switch recombination (NHEJ), and showed that DSB-induced global H2Bub1 increase is regulated by ATM/ATR independently of γH2AX.","evidence":"Conditional KO in mouse B cells with CSR, HR assays, and kinase inhibitor treatment","pmids":["30692271"],"confidence":"High","gaps":["Identity of RNF20/RNF40 phosphorylation sites regulated by ATM/ATR unknown","Whether this pathway operates identically in non-lymphoid cells untested"]},{"year":2019,"claim":"Demonstrating that RNF40 loss reduces NF-κB nuclear translocation and that intestinal Rnf40 deletion protects against colitis connected H2Bub1 to inflammatory signaling in vivo.","evidence":"siRNA knockdown with NF-κB immunofluorescence, mRNA-seq, and colon-specific Rnf40 KO colitis model","pmids":["30321325"],"confidence":"Medium","gaps":["Direct vs indirect mechanism linking H2Bub1 to NF-κB nuclear import unresolved","Contribution of non-histone RNF40 substrates to the colitis phenotype not examined"]},{"year":2020,"claim":"Multiple tissue-specific knockout studies established that RNF40-mediated H2Bub1 drives trans-histone crosstalk with H3K4me3 to control lineage-specific gene programs in osteoblasts, mammary epithelium, and during iPSC reprogramming.","evidence":"Conditional KO mice (osteoblast, mammary) and knockdown in reprogramming with ChIP-seq for H2Bub1/H3K4me3/H3K27me3 and transcriptomics","pmids":["32901120","33070155","32341358"],"confidence":"Medium","gaps":["How H2Bub1 mechanistically promotes H3K4 methylation (direct recruitment of COMPASS vs chromatin opening) remains unresolved","Whether all H2Bub1-dependent genes require the same downstream methyltransferase complex unknown"]},{"year":2023,"claim":"The cryo-EM structure of the RNF20/RNF40–RAD6A–Ub–nucleosome complex revealed that RNF40 directly contacts nucleosomal DNA and identified an uncanonical RING–E2 interface that positions RAD6A above H2B K120, providing the definitive structural mechanism for substrate targeting.","evidence":"Chemical trapping strategy with cryo-EM at near-atomic resolution and mutagenesis validation","pmids":["37633270"],"confidence":"High","gaps":["How WAC binding modulates the nucleosome-engaged conformation not structurally resolved","Dynamics of the catalytic cycle (product release, processivity) not captured"]},{"year":2024,"claim":"Identification of LIMA1 as a non-histone RNF40 substrate targeted for proteasomal degradation expanded RNF40 function beyond chromatin to regulation of cholesterol absorption and lipid metabolism.","evidence":"Co-IP, domain mapping, ubiquitination assay, proteasome inhibitor rescue, and lipid content measurement","pmids":["38909032"],"confidence":"Medium","gaps":["Ubiquitin chain type on LIMA1 not determined","In vivo relevance for lipid metabolism not tested in animal models","Whether RNF20 is required as co-E3 for LIMA1 ubiquitination unknown"]},{"year":2025,"claim":"Discovery that RNF40 catalyzes K6/K11-linked polyubiquitination of KDM6A for autophagic degradation and K48-linked polyubiquitination of Parkin for proteasomal degradation revealed substrate-specific ubiquitin chain-type selectivity in its non-histone functions.","evidence":"Ubiquitin linkage analysis, co-IP, autophagy/mitophagy assays, and in vivo Rnf40 knockdown in hypertensive rats","pmids":["41691482","39777866"],"confidence":"Medium","gaps":["How RNF40 switches between monoubiquitination (H2B) and polyubiquitination (non-histone substrates) is mechanistically unexplained","Whether different E2 enzymes direct chain-type specificity for non-histone substrates unknown","Each non-histone substrate identified by a single lab; independent replication pending"]},{"year":2026,"claim":"Structural characterization of H2B S112 GlcNAcylation's allosteric enhancement of H2Bub1 and the WAC–RNF20/RNF40 interaction interface refined the catalytic mechanism by revealing crosstalk between histone modifications and cofactor-mediated complex assembly.","evidence":"Cryo-EM of GlcNAc-modified nucleosome complex with kinetics; crystal structure of Bre1-Lge1 with AlphaFold modeling of RNF20/RNF40-WAC and in vitro/in vivo validation","pmids":["41495224","41533567"],"confidence":"High","gaps":["Physiological regulation of H2B S112 GlcNAcylation in vivo not established","Full-length RNF20/RNF40-WAC complex structure on nucleosome not yet solved experimentally"]},{"year":null,"claim":"The mechanism by which RNF40 switches between monoubiquitination of histones and polyubiquitination of non-histone substrates with different chain types, and the identity of the full repertoire of non-histone substrates, remain open questions.","evidence":"","pmids":[],"confidence":"Low","gaps":["E2 enzyme(s) used for non-histone substrate polyubiquitination not identified","No systematic unbiased substrate screen for RNF40 published","Structural basis for non-histone substrate recognition unknown"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,1,2,3,4,5,17,18,19]},{"term_id":"GO:0016874","term_label":"ligase activity","supporting_discovery_ids":[0,1,5]},{"term_id":"GO:0003677","term_label":"DNA binding","supporting_discovery_ids":[0]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[0,4,5,6]},{"term_id":"GO:0005694","term_label":"chromosome","supporting_discovery_ids":[0,2,6]}],"pathway":[{"term_id":"R-HSA-4839726","term_label":"Chromatin organization","supporting_discovery_ids":[0,1,2,3,4,5]},{"term_id":"R-HSA-73894","term_label":"DNA Repair","supporting_discovery_ids":[4,6,7,8]},{"term_id":"R-HSA-74160","term_label":"Gene expression (Transcription)","supporting_discovery_ids":[9,13,14,15,16]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[8,11,16]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[12,20]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[17,18,19]}],"complexes":["RNF20/RNF40 (BRE1) E3 ligase complex","RNF20/RNF40/WAC complex"],"partners":["RNF20","WAC","UBE2B","CDC73","RTF1","LIMA1","PRKN","KDM6A"],"other_free_text":[]},"mechanistic_narrative":"RNF40 is an E3 ubiquitin ligase that heterodimerizes with RNF20 to catalyze monoubiquitination of histone H2B at lysine 120 (H2Bub1), a modification central to transcriptional elongation, chromatin remodeling, and DNA double-strand break repair. Cryo-EM and crystal structures show that the RNF20/RNF40 RING heterodimer engages the E2 enzyme RAD6A (UBE2B) and directly contacts nucleosomal DNA, positioning the E2 above the target lysine through an uncanonical RING–E2 interface; the cofactor WAC is integral to complex activity [PMID:37633270, PMID:27569044, PMID:41533567]. Loss of RNF40 causes aberrant R-loop accumulation and replication-associated DNA breaks, impairs homologous recombination and class-switch recombination via reduced FACT recruitment, and dysregulates trans-histone crosstalk with H3K4me3 at genes controlling differentiation, apoptosis, NF-κB signaling, and peroxisomal biogenesis [PMID:22354749, PMID:22031019, PMID:30692271, PMID:33070155, PMID:30321325]. Beyond histones, RNF40 polyubiquitinates non-histone substrates including LIMA1 (proteasomal degradation, lipid metabolism), Parkin (K48-linked, inhibiting mitophagy), and KDM6A (K6/K11-linked, autophagic degradation regulating viral receptor expression) [PMID:38909032, PMID:39777866, PMID:41691482]."},"prefetch_data":{"uniprot":{"accession":"O75150","full_name":"E3 ubiquitin-protein ligase BRE1B","aliases":["95 kDa retinoblastoma-associated protein","RBP95","RING finger protein 40","RING-type E3 ubiquitin transferase BRE1B"],"length_aa":1001,"mass_kda":113.7,"function":"Component of the RNF20/40 E3 ubiquitin-protein ligase complex that mediates monoubiquitination of 'Lys-120' of histone H2B (H2BK120ub1). H2BK120ub1 gives a specific tag for epigenetic transcriptional activation and is also prerequisite for histone H3 'Lys-4' and 'Lys-79' methylation (H3K4me and H3K79me, respectively). It thereby plays a central role in histone code and gene regulation. The RNF20/40 complex forms a H2B ubiquitin ligase complex in cooperation with the E2 enzyme UBE2A or UBE2B; reports about the cooperation with UBE2E1/UBCH are contradictory. Required for transcriptional activation of Hox genes (Microbial infection) Promotes the human herpesvirus 8 (KSHV) lytic cycle by inducing the expression of lytic viral genes including the latency switch gene RTA/ORF50","subcellular_location":"Nucleus","url":"https://www.uniprot.org/uniprotkb/O75150/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":true,"resolved_as":"","url":"https://depmap.org/portal/gene/RNF40","classification":"Common Essential","n_dependent_lines":668,"n_total_lines":1208,"dependency_fraction":0.5529801324503312},"opencell":{"profiled":true,"resolved_as":"","ensg_id":"ENSG00000103549","cell_line_id":"CID001763","localizations":[{"compartment":"nucleoplasm","grade":3},{"compartment":"nuclear_punctae","grade":2}],"interactors":[{"gene":"WAC","stoichiometry":10.0},{"gene":"RNF20","stoichiometry":10.0},{"gene":"METTL3","stoichiometry":4.0},{"gene":"C9ORF16","stoichiometry":0.2},{"gene":"NOMO1","stoichiometry":0.2},{"gene":"PRPF19","stoichiometry":0.2},{"gene":"RBM12","stoichiometry":0.2},{"gene":"SNRNP200","stoichiometry":0.2},{"gene":"PLRG1","stoichiometry":0.2},{"gene":"HNRNPR","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/target/CID001763","total_profiled":1310},"omim":[{"mim_id":"617233","title":"WD REPEAT-CONTAINING PROTEIN 70; WDR70","url":"https://www.omim.org/entry/617233"},{"mim_id":"614802","title":"MSL COMPLEX SUBUNIT 2; MSL2","url":"https://www.omim.org/entry/614802"},{"mim_id":"614801","title":"MSL COMPLEX SUBUNIT 1; MSL1","url":"https://www.omim.org/entry/614801"},{"mim_id":"610506","title":"PAF1 HOMOLOG, PAF1/RNA POLYMERASE II COMPLEX COMPONENT; PAF1","url":"https://www.omim.org/entry/610506"},{"mim_id":"607700","title":"RING FINGER PROTEIN 40; RNF40","url":"https://www.omim.org/entry/607700"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Nucleoplasm","reliability":"Supported"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/RNF40"},"hgnc":{"alias_symbol":["KIAA0661","RBP95","BRE1B","STARING"],"prev_symbol":[]},"alphafold":{"accession":"O75150","domains":[{"cath_id":"3.30.40.10","chopping":"945-998","consensus_level":"high","plddt":88.0089,"start":945,"end":998}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/O75150","model_url":"https://alphafold.ebi.ac.uk/files/AF-O75150-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-O75150-F1-predicted_aligned_error_v6.png","plddt_mean":72.5},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=RNF40","jax_strain_url":"https://www.jax.org/strain/search?query=RNF40"},"sequence":{"accession":"O75150","fasta_url":"https://rest.uniprot.org/uniprotkb/O75150.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/O75150/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/O75150"}},"corpus_meta":[{"pmid":"22354749","id":"PMC_22354749","title":"Deficiency 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RNF20/RNF40-hRAD6A-Ub-nucleosome complex (captured via chemical trapping) revealed that RNF40 directly binds nucleosomal DNA and exhibits a conserved E3/E2/nucleosome interaction pattern for H2B monoubiquitylation at K120; an uncanonical non-hydrophobic contact in the RING-E2 interface positions RAD6A directly above the target H2B lysine residue.\",\n      \"method\": \"Chemical trapping strategy + cryo-EM structure determination + mutagenesis\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — cryo-EM structure with chemical trapping and mutagenesis validation in a single rigorous study\",\n      \"pmids\": [\"37633270\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Crystal structure of the RNF20 RING domain shows it forms a homodimer that specifically interacts with the Ube2B~Ub conjugate; the RING domains of RNF20 and RNF40 can form a stable heterodimer that is catalytically active; key contacts at the E3-E2 and E3-ubiquitin interfaces were identified by mutagenesis.\",\n      \"method\": \"X-ray crystallography + mutagenesis of E3-E2 and E3-ubiquitin interfaces + in vitro ubiquitination assay\",\n      \"journal\": \"Journal of molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — crystal structure combined with mutagenesis and functional assay in one study\",\n      \"pmids\": [\"27569044\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"Cryo-EM structure of RNF20/RNF40-RAD6A-Ub-H2BS112GlcNAc nucleosome complex showed that H2B S112 GlcNAcylation interacts with the E2 enzyme RAD6A (not the E3 RNF20/RNF40), allosterically enhancing the nucleophilicity of H2B K120 to stimulate ubiquitin transfer; structure-activity relationship identified the C2 N-acetyl group and β-configuration of C1 as essential.\",\n      \"method\": \"Chemical synthesis of modified nucleosomes + cryo-EM + mutagenesis + kinetics assays\",\n      \"journal\": \"Nature chemical biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — cryo-EM structure with mutagenesis and kinetics, multiple orthogonal methods\",\n      \"pmids\": [\"41495224\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"Crystal structure of the Bre1-Lge1 complex and AlphaFold model of RNF20/RNF40-WAC revealed extensive interaction interfaces; in vitro and in vivo experiments demonstrated that RNF20/RNF40-WAC interactions are critical for H2B monoubiquitination, with different electrostatic contacts encoding binding specificity compared to yeast Bre1-Lge1.\",\n      \"method\": \"X-ray crystallography (Bre1-Lge1) + AlphaFold modeling (RNF20/RNF40-WAC) + in vitro ubiquitination assay + in vivo functional experiments\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1/2 — crystal structure plus orthogonal in vitro and in vivo validation\",\n      \"pmids\": [\"41533567\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"RNF20/RNF40 (mammalian Bre1 complex) functions as the E3 ubiquitin ligase for histone H2B monoubiquitination; deficiency leads to aberrant R-loop formation causing replication-associated double-strand breaks, combined with a defect in homologous recombination, resulting in chromosomal instability.\",\n      \"method\": \"Loss-of-function (Bre1-deficient cells) + detection of R-loops, DSBs, and genomic rearrangements\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal readouts in defined KO cells, replicated finding\",\n      \"pmids\": [\"22354749\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"RNF20 and RNF40 form a heterodimeric E3 ubiquitin ligase complex that monoubiquitinates histone H2B at lysine 120; the tumor suppressor CDC73 interacts with both RNF20 and RNF40 at discrete but closely located residues and is required for maintenance of H2Bub1 levels.\",\n      \"method\": \"Yeast two-hybrid + co-immunoprecipitation + knockdown of CDC73 + western blot for H2Bub1\",\n      \"journal\": \"Human molecular genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal interaction confirmed by multiple methods, functional consequence demonstrated\",\n      \"pmids\": [\"22021426\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"RNF40 depletion results in sustained γH2AX phosphorylation, decreased rapid cell cycle checkpoint activation, decreased H3K56ac, and decreased recruitment of the FACT complex (specifically SUPT16H) to chromatin following DNA DSBs; RNF40 and SUPT16H cooperate to promote DNA end resection and timely DNA repair.\",\n      \"method\": \"siRNA knockdown + immunofluorescence for γH2AX + western blot for H3K56ac + chromatin fractionation + DNA end resection assay\",\n      \"journal\": \"Cell cycle\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods, phenocopy by SUPT16H knockdown establishes pathway\",\n      \"pmids\": [\"22031019\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Arsenite directly binds to the RING finger domains of RNF20 and RNF40 via cysteine residues in vitro and in cells, inhibiting H2B ubiquitination and impairing recruitment of BRCA1 and RAD51 to DNA DSB sites, thereby compromising DNA DSB repair.\",\n      \"method\": \"In vitro binding assay + cellular arsenite treatment + ChIP for BRCA1/RAD51 recruitment + comet assay/western blot for DSB repair\",\n      \"journal\": \"Journal of the American Chemical Society\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1/2 — direct in vitro binding demonstrated plus multiple cellular functional readouts\",\n      \"pmids\": [\"25170678\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"RNF20 and RNF40 are required for DSB repair via both homologous recombination and class switch recombination (NHEJ) in mouse B cells; DSBs induce a global increase in H2Bub1 regulated jointly by ATM and ATR kinases, independently of H2AX phosphorylation.\",\n      \"method\": \"RNF20/RNF40 knockout in mouse B cells + class switch recombination assay + HR assay + western blot + ATM/ATR inhibitor treatment\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic KO with multiple functional DSB repair assays and defined signaling pathway\",\n      \"pmids\": [\"30692271\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"RNF20 and RNF40 physically and functionally interact with the androgen receptor (AR) and modulate its transcriptional activity; androgen induction of FKBP51 and PSA is accompanied by dynamic increases in H2Bub1 within transcribed regions of these loci.\",\n      \"method\": \"Co-immunoprecipitation + reporter assay + ChIP for H2Bub1 at AR target loci\",\n      \"journal\": \"Molecular and cellular endocrinology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2/3 — co-IP plus ChIP, single lab\",\n      \"pmids\": [\"22155569\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"RNF20, RNF40, and WAC form an E3 ligase complex that mediates H2B ubiquitination; knockdown of WAC phenocopies loss of H2B ubiquitination and causes cell death in MLL-rearranged ALL cells.\",\n      \"method\": \"siRNA knockdown + western blot for H2Bub1 + cell viability assay\",\n      \"journal\": \"Leukemia\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2/3 — knockdown phenocopy establishes WAC as complex component, single lab\",\n      \"pmids\": [\"28690313\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"RNF40 loss decreases nuclear localization of NF-κB following TNF-α treatment in colorectal cancer cells, reducing induction of NF-κB-associated cytokines; colon-specific Rnf40 deletion exerts a protective effect in a murine model of acute colitis.\",\n      \"method\": \"siRNA knockdown + immunofluorescence for NF-κB nuclear localization + mRNA-seq + in vivo colitis model with colon-specific Rnf40 knockout\",\n      \"journal\": \"Journal of Crohn's & colitis\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — direct localization experiment plus in vivo genetic model, moderate evidence\",\n      \"pmids\": [\"30321325\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"RNF40 depletion in colorectal cancer cells increases apoptosis via elevated caspase 3/7 activity, associated with reduced anti-apoptotic and elevated pro-apoptotic BCL2 family member expression; H2Bub1 directly occupies transcribed regions of anti-apoptotic genes.\",\n      \"method\": \"siRNA knockdown + flow cytometry (cell cycle) + caspase 3/7 assay + Annexin V + ChIP-seq for H2Bub1 + mRNA-seq + caspase inhibitor rescue\",\n      \"journal\": \"Clinical epigenetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods with rescue experiment, single lab\",\n      \"pmids\": [\"31266541\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"RNF40-driven H2B monoubiquitination is essential for transcriptional activation of RHO/ROCK/LIMK pathway components and proper actin-cytoskeleton dynamics through trans-histone crosstalk with H3K4me3 in HER2+ breast cancer.\",\n      \"method\": \"Tissue-specific Rnf40 deletion (mouse mammary carcinoma model) + ChIP for H2Bub1 and H3K4me3 + transcriptome analysis + actin dynamics assays\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — in vivo genetic model with ChIP and transcriptome, single lab\",\n      \"pmids\": [\"33070155\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"RNF40 loss impairs early gene activation during somatic cell reprogramming to iPSCs; mechanistically, RNF40 controls expression of the PRC2 component EZH2 and promotes resolution of H3K4me3/H3K27me3 bivalency on H2Bub1-occupied pluripotency genes.\",\n      \"method\": \"RNF40 knockdown + iPSC reprogramming assay + ChIP-seq for H3K4me3, H3K27me3, H2Bub1 + mRNA-seq\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — ChIP-seq with functional reprogramming readout, single lab\",\n      \"pmids\": [\"32341358\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Osteoblast-specific RNF40 deletion in mice impairs early lineage specification, decreases osteoclast number and function via reduced RANKL (Tnfsf11) expression in osteoblasts; H2B monoubiquitination directly occupies the Tnfsf11 locus.\",\n      \"method\": \"Conditional osteoblast-specific Rnf40 knockout mouse + bone phenotype analysis + ChIP for H2Bub1 at Tnfsf11 + RANKL expression analysis\",\n      \"journal\": \"Cell death and differentiation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — in vivo genetic model with ChIP showing direct target gene regulation\",\n      \"pmids\": [\"32901120\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Intestine-specific deletion of Rnf20 or Rnf40 in mice causes spontaneous colorectal inflammation; mechanistically, RNF20/RNF40-mediated H2Bub1 controls H3K4me3 occupancy and transcription of the Vitamin D Receptor (Vdr) gene and its target genes in intestinal epithelial cells.\",\n      \"method\": \"Conditional intestinal Rnf20/Rnf40 knockout + mRNA-seq + ChIP-seq for H2Bub1 and H3K4me3 + in vivo colitis monitoring\",\n      \"journal\": \"Cell death and differentiation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — in vivo genetic model with ChIP-seq, single lab\",\n      \"pmids\": [\"34088983\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"RNF40 mediates ubiquitination and proteasome-dependent degradation of LIMA1 (an actin-binding protein involved in cholesterol absorption) as a non-histone substrate; the 1-166aa fragment of LIMA1 is required for interaction with RNF40, and RNF40 overexpression reduces cellular lipid content in a LIMA1-dependent manner.\",\n      \"method\": \"Co-immunoprecipitation + domain mapping + ubiquitination assay + proteasome inhibitor treatment + lipid content measurement + rescue by LIMA1 overexpression\",\n      \"journal\": \"Cell death discovery\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2/3 — co-IP with functional rescue, single lab\",\n      \"pmids\": [\"38909032\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"RNF40 catalyzes K6- and K11-linked polyubiquitination of KDM6A, targeting it for autophagic degradation via TAX1BP1 recognition; this contrasts with USP7-mediated deubiquitination (K48-linked) that prevents KDM6A proteasomal degradation, forming an antagonistic ubiquitin-switching circuit regulating KDM6A stability and coronavirus receptor expression.\",\n      \"method\": \"Genetic and pharmacological inhibition + ubiquitination linkage analysis + co-immunoprecipitation + autophagic degradation assay + viral infection functional assay\",\n      \"journal\": \"Advanced science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — ubiquitin linkage analysis plus functional rescue, single lab\",\n      \"pmids\": [\"41691482\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"RNF40 promotes K48-linked polyubiquitination and proteasomal degradation of Parkin in cerebrovascular endothelial cells, thereby inhibiting mitophagy; Rnf40 knockdown in spontaneously hypertensive rats restored mitophagy, improved tight junction proteins, and protected the blood-brain barrier.\",\n      \"method\": \"In vivo Rnf40 knockdown (pAAV-shRnf40 in SHRs) + ubiquitination assay (K48 linkage) + mitophagy assay + tight junction protein western blot + cerebral blood flow measurement\",\n      \"journal\": \"CNS neuroscience & therapeutics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — in vivo genetic model with ubiquitination linkage analysis, single lab\",\n      \"pmids\": [\"39777866\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"The RNF40/H2Bub1 axis positively regulates peroxisome-related gene expression (PRDX5, PEX6, PMVK); loss of RNF20/RNF40 impairs peroxisomal biogenesis and ROS metabolism, leading to increased lipid peroxidation and ferroptotic cell death in cervical cancer cells.\",\n      \"method\": \"RNF20/RNF40 knockdown + ChIP-qPCR for H2Bub1 + transcriptome analysis + lipid peroxidation assay + flow cytometry for ferroptosis markers + in vivo CAM assay\",\n      \"journal\": \"Cell communication and signaling\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2/3 — ChIP with transcriptome and ferroptosis functional assays, single lab\",\n      \"pmids\": [\"40597205\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"The RNF40/H2Bub1 axis promotes cancer stem cell properties and glycolytic program in triple-negative breast cancer by supporting YAP1 signaling.\",\n      \"method\": \"RNF40 knockdown/overexpression + sphere formation assay + ChIP-seq for H2Bub1 + transcriptome analysis + YAP1 pathway analysis\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2/3 — ChIP-seq with functional stem cell assays, single lab\",\n      \"pmids\": [\"37770435\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"PAF1C-driven transcription restart after DNA damage is independent of RNF20/RNF40-mediated H2B-K120 ubiquitination; H2Bub1 levels do not correlate with transcription restoration following DNA lesions, and RTF1-stimulated H2Bub1 does not contribute to restart.\",\n      \"method\": \"RNF20/RNF40 depletion + transcription restart assay after DNA damage + H2Bub1 ChIP\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 2 but preprint, single lab, awaiting peer review\",\n      \"pmids\": [\"bio_10.1101_2025.07.23.666359\"],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"RNF40 physically interacts with RTF1 and is required for histone H2B monoubiquitination that supports Th17 cell differentiation; RNF40-deficient cells phenocopy RTF1-deficient cells in impaired Th17 differentiation.\",\n      \"method\": \"RNF40 knockout in T cells + Th17 differentiation assay + H2Bub1 ChIP-seq + genetic epistasis with RTF1\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 2/3 but preprint, single lab\",\n      \"pmids\": [\"bio_10.1101_2024.11.08.622668\"],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"RNF40 forms a stable heterodimeric E3 ubiquitin ligase complex with RNF20, and together with the E2 enzyme hRAD6A (and cofactor WAC), monoubiquitinates histone H2B at lysine 120 (H2Bub1) by directly binding nucleosomal DNA — a mechanism structurally characterized by cryo-EM and crystallography — which in turn promotes transcriptional elongation, facilitates DNA double-strand break repair (via FACT recruitment, HR, and NHEJ), maintains genomic stability by suppressing R-loops, and regulates diverse cellular processes including NF-κB signaling, apoptosis, bone remodeling, and pluripotency; additionally, RNF40 ubiquitinates non-histone substrates including LIMA1, Parkin, and KDM6A to regulate lipid metabolism, mitophagy, and viral receptor stability.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"RNF40 is an E3 ubiquitin ligase that heterodimerizes with RNF20 to catalyze monoubiquitination of histone H2B at lysine 120 (H2Bub1), a modification central to transcriptional elongation, chromatin remodeling, and DNA double-strand break repair. Cryo-EM and crystal structures show that the RNF20/RNF40 RING heterodimer engages the E2 enzyme RAD6A (UBE2B) and directly contacts nucleosomal DNA, positioning the E2 above the target lysine through an uncanonical RING–E2 interface; the cofactor WAC is integral to complex activity [PMID:37633270, PMID:27569044, PMID:41533567]. Loss of RNF40 causes aberrant R-loop accumulation and replication-associated DNA breaks, impairs homologous recombination and class-switch recombination via reduced FACT recruitment, and dysregulates trans-histone crosstalk with H3K4me3 at genes controlling differentiation, apoptosis, NF-κB signaling, and peroxisomal biogenesis [PMID:22354749, PMID:22031019, PMID:30692271, PMID:33070155, PMID:30321325]. Beyond histones, RNF40 polyubiquitinates non-histone substrates including LIMA1 (proteasomal degradation, lipid metabolism), Parkin (K48-linked, inhibiting mitophagy), and KDM6A (K6/K11-linked, autophagic degradation regulating viral receptor expression) [PMID:38909032, PMID:39777866, PMID:41691482].\",\n  \"teleology\": [\n    {\n      \"year\": 2011,\n      \"claim\": \"Establishing that RNF20 and RNF40 form a stable heterodimeric E3 ligase for H2B K120 monoubiquitination, with the tumor suppressor CDC73 required for maintaining H2Bub1 levels, defined the core enzymatic identity of the complex.\",\n      \"evidence\": \"Yeast two-hybrid, co-IP, knockdown, and western blot in human cells\",\n      \"pmids\": [\"22021426\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Structural basis of heterodimer assembly not yet resolved\",\n        \"Catalytic contribution of each RING domain unknown\",\n        \"Mechanism by which CDC73 promotes H2Bub1 unclear\"\n      ]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Demonstrating that RNF40 depletion impairs FACT recruitment, H3K56 acetylation, and DNA end resection after DSBs established a direct role for H2Bub1 in the DNA damage response beyond transcription.\",\n      \"evidence\": \"siRNA knockdown with γH2AX immunofluorescence, chromatin fractionation, and end resection assays\",\n      \"pmids\": [\"22031019\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Whether RNF40 is recruited to damage sites or acts globally was unresolved\",\n        \"Relative contributions of HR vs NHEJ pathways not separated\"\n      ]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Linking RNF20/RNF40 deficiency to aberrant R-loop formation and replication-associated DSBs revealed that H2Bub1 maintains genomic stability by suppressing co-transcriptional R-loops.\",\n      \"evidence\": \"Loss-of-function cells with R-loop detection, DSB assays, and genomic rearrangement analysis\",\n      \"pmids\": [\"22354749\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Mechanism by which H2Bub1 suppresses R-loops not determined\",\n        \"Whether R-loop suppression requires FACT or other downstream effectors unknown\"\n      ]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Solving the crystal structure of the RNF20 RING domain and demonstrating that the RNF20/RNF40 RING heterodimer is catalytically active with UBE2B provided the first atomic-level view of the E3 architecture.\",\n      \"evidence\": \"X-ray crystallography with mutagenesis and in vitro ubiquitination assays\",\n      \"pmids\": [\"27569044\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Structure of the full heterodimer on a nucleosome substrate not yet available\",\n        \"Role of WAC in E3 activity not structurally characterized\"\n      ]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Genetic knockout of RNF20/RNF40 in mouse B cells confirmed their requirement for both HR and class-switch recombination (NHEJ), and showed that DSB-induced global H2Bub1 increase is regulated by ATM/ATR independently of γH2AX.\",\n      \"evidence\": \"Conditional KO in mouse B cells with CSR, HR assays, and kinase inhibitor treatment\",\n      \"pmids\": [\"30692271\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Identity of RNF20/RNF40 phosphorylation sites regulated by ATM/ATR unknown\",\n        \"Whether this pathway operates identically in non-lymphoid cells untested\"\n      ]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Demonstrating that RNF40 loss reduces NF-κB nuclear translocation and that intestinal Rnf40 deletion protects against colitis connected H2Bub1 to inflammatory signaling in vivo.\",\n      \"evidence\": \"siRNA knockdown with NF-κB immunofluorescence, mRNA-seq, and colon-specific Rnf40 KO colitis model\",\n      \"pmids\": [\"30321325\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Direct vs indirect mechanism linking H2Bub1 to NF-κB nuclear import unresolved\",\n        \"Contribution of non-histone RNF40 substrates to the colitis phenotype not examined\"\n      ]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Multiple tissue-specific knockout studies established that RNF40-mediated H2Bub1 drives trans-histone crosstalk with H3K4me3 to control lineage-specific gene programs in osteoblasts, mammary epithelium, and during iPSC reprogramming.\",\n      \"evidence\": \"Conditional KO mice (osteoblast, mammary) and knockdown in reprogramming with ChIP-seq for H2Bub1/H3K4me3/H3K27me3 and transcriptomics\",\n      \"pmids\": [\"32901120\", \"33070155\", \"32341358\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"How H2Bub1 mechanistically promotes H3K4 methylation (direct recruitment of COMPASS vs chromatin opening) remains unresolved\",\n        \"Whether all H2Bub1-dependent genes require the same downstream methyltransferase complex unknown\"\n      ]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"The cryo-EM structure of the RNF20/RNF40–RAD6A–Ub–nucleosome complex revealed that RNF40 directly contacts nucleosomal DNA and identified an uncanonical RING–E2 interface that positions RAD6A above H2B K120, providing the definitive structural mechanism for substrate targeting.\",\n      \"evidence\": \"Chemical trapping strategy with cryo-EM at near-atomic resolution and mutagenesis validation\",\n      \"pmids\": [\"37633270\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"How WAC binding modulates the nucleosome-engaged conformation not structurally resolved\",\n        \"Dynamics of the catalytic cycle (product release, processivity) not captured\"\n      ]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Identification of LIMA1 as a non-histone RNF40 substrate targeted for proteasomal degradation expanded RNF40 function beyond chromatin to regulation of cholesterol absorption and lipid metabolism.\",\n      \"evidence\": \"Co-IP, domain mapping, ubiquitination assay, proteasome inhibitor rescue, and lipid content measurement\",\n      \"pmids\": [\"38909032\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Ubiquitin chain type on LIMA1 not determined\",\n        \"In vivo relevance for lipid metabolism not tested in animal models\",\n        \"Whether RNF20 is required as co-E3 for LIMA1 ubiquitination unknown\"\n      ]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Discovery that RNF40 catalyzes K6/K11-linked polyubiquitination of KDM6A for autophagic degradation and K48-linked polyubiquitination of Parkin for proteasomal degradation revealed substrate-specific ubiquitin chain-type selectivity in its non-histone functions.\",\n      \"evidence\": \"Ubiquitin linkage analysis, co-IP, autophagy/mitophagy assays, and in vivo Rnf40 knockdown in hypertensive rats\",\n      \"pmids\": [\"41691482\", \"39777866\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"How RNF40 switches between monoubiquitination (H2B) and polyubiquitination (non-histone substrates) is mechanistically unexplained\",\n        \"Whether different E2 enzymes direct chain-type specificity for non-histone substrates unknown\",\n        \"Each non-histone substrate identified by a single lab; independent replication pending\"\n      ]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Structural characterization of H2B S112 GlcNAcylation's allosteric enhancement of H2Bub1 and the WAC–RNF20/RNF40 interaction interface refined the catalytic mechanism by revealing crosstalk between histone modifications and cofactor-mediated complex assembly.\",\n      \"evidence\": \"Cryo-EM of GlcNAc-modified nucleosome complex with kinetics; crystal structure of Bre1-Lge1 with AlphaFold modeling of RNF20/RNF40-WAC and in vitro/in vivo validation\",\n      \"pmids\": [\"41495224\", \"41533567\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Physiological regulation of H2B S112 GlcNAcylation in vivo not established\",\n        \"Full-length RNF20/RNF40-WAC complex structure on nucleosome not yet solved experimentally\"\n      ]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The mechanism by which RNF40 switches between monoubiquitination of histones and polyubiquitination of non-histone substrates with different chain types, and the identity of the full repertoire of non-histone substrates, remain open questions.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\n        \"E2 enzyme(s) used for non-histone substrate polyubiquitination not identified\",\n        \"No systematic unbiased substrate screen for RNF40 published\",\n        \"Structural basis for non-histone substrate recognition unknown\"\n      ]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 1, 2, 3, 4, 5, 17, 18, 19]},\n      {\"term_id\": \"GO:0016874\", \"supporting_discovery_ids\": [0, 1, 5]},\n      {\"term_id\": \"GO:0003677\", \"supporting_discovery_ids\": [0]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [0, 4, 5, 6]},\n      {\"term_id\": \"GO:0005694\", \"supporting_discovery_ids\": [0, 2, 6]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-4839726\", \"supporting_discovery_ids\": [0, 1, 2, 3, 4, 5]},\n      {\"term_id\": \"R-HSA-73894\", \"supporting_discovery_ids\": [4, 6, 7, 8]},\n      {\"term_id\": \"R-HSA-74160\", \"supporting_discovery_ids\": [9, 13, 14, 15, 16]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [8, 11, 16]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [12, 20]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [17, 18, 19]}\n    ],\n    \"complexes\": [\n      \"RNF20/RNF40 (BRE1) E3 ligase complex\",\n      \"RNF20/RNF40/WAC complex\"\n    ],\n    \"partners\": [\n      \"RNF20\",\n      \"WAC\",\n      \"UBE2B\",\n      \"CDC73\",\n      \"RTF1\",\n      \"LIMA1\",\n      \"PRKN\",\n      \"KDM6A\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}