{"gene":"RNF20","run_date":"2026-04-28T19:45:45","timeline":{"discoveries":[{"year":2011,"finding":"RNF20 localizes to DNA double-strand breaks (DSBs) independently of H2AX and is required for DSB-induced H2B ubiquitination, H3K4 methylation at DSBs, and recruitment of chromatin-remodeling factor SNF2h; depletion of RNF20 or expression of H2B K120R compromises DNA end resection, RAD51 and BRCA1 recruitment, and homologous recombination repair (HRR), and this function can be partially bypassed by forced chromatin relaxation.","method":"siRNA depletion, H2B K120R mutant expression, γ-H2AX foci, ChIP, HR repair assay, radiation sensitivity","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods, replicated in subsequent studies","pmids":["21362548"],"is_preprint":false},{"year":2011,"finding":"WAC interacts with RNF20/RNF40 through its C-terminal coiled-coil region, recruits the complex to RNA polymerase II via its N-terminal WW domain during active transcription, and promotes RNF20/RNF40 E3 ligase activity for H2B ubiquitination; WAC depletion abolishes H2Bub.","method":"Protein affinity purification, Co-IP, domain mapping, knockdown, ChIP","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 1-2 — affinity purification identifies partner, domain mapping, functional depletion phenotype, replicated","pmids":["21329877"],"is_preprint":false},{"year":2012,"finding":"RNF20-mediated H2B monoubiquitylation (H2Bub1) increases during embryonic stem cell (ESC) differentiation and is required for transcriptional induction of relatively long genes; USP44 deubiquitinase negatively regulates H2Bub1 and its downregulation during differentiation contributes to increased H2Bub1.","method":"Knockdown/depletion, qRT-PCR, RNA-seq, ChIP, western blot","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods identifying both writer (RNF20) and eraser (USP44) with functional outcomes","pmids":["22681888"],"is_preprint":false},{"year":2012,"finding":"Smurf2 (HECT-domain E3 ligase) targets RNF20 for proteasomal degradation, thereby regulating RNF20-dependent H2B monoubiquitination and H3K4/H3K79 trimethylation; Smurf2 and RNF20 co-localize at γ-H2AX foci at DSBs.","method":"Genetic ablation (mouse model), western blot, immunofluorescence co-localization, ChIP","journal":"Nature medicine","confidence":"High","confidence_rationale":"Tier 2 — in vivo genetic model plus molecular mechanistic validation","pmids":["22231558"],"is_preprint":false},{"year":2012,"finding":"RNF20/RNF40 deficiency leads to formation of aberrant R-loops (RNA:DNA hybrids) at replication forks, causing replication-associated DSBs, specific genomic rearrangements, and chromosomal instability.","method":"Bre1/RNF20 depletion, DNA fiber assay, cytogenetics, R-loop detection","journal":"Cancer research","confidence":"High","confidence_rationale":"Tier 2 — mechanistic link from RNF20 loss to R-loop formation demonstrated with multiple assays","pmids":["22354749"],"is_preprint":false},{"year":2011,"finding":"CDC73 (parafibromin) interacts with RNF20 and RNF40 (identified by yeast two-hybrid), and CDC73 is required for maintenance of H2B K120 monoubiquitination; pathogenic CDC73 mutations reduce H2Bub1 in parathyroid tumors.","method":"Yeast two-hybrid, Co-IP, domain mapping, siRNA knockdown, western blot, tumor samples","journal":"Human molecular genetics","confidence":"High","confidence_rationale":"Tier 2 — yeast two-hybrid plus reciprocal Co-IP plus functional knockdown plus patient tumor validation","pmids":["22021426"],"is_preprint":false},{"year":2013,"finding":"Histone chaperone FACT (SUPT16H subunit) directly binds RNF20 in vivo; mutation at the RING-finger domain of RNF20 abolishes this interaction; FACT is required for RNF20 recruitment to DSBs and subsequent H2B ubiquitylation and SNF2h accumulation, enabling homologous recombination repair. PAF1 (a known transcriptional mediator of FACT-RNF20 association) is dispensable for DNA-damage-induced RNF20-FACT interaction.","method":"Co-IP, RING domain mutagenesis, siRNA depletion, laser-induced DSB imaging, γ-H2AX, HR assay","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 1-2 — RING domain mutagenesis combined with direct Co-IP and functional HR assay","pmids":["24357716"],"is_preprint":false},{"year":2013,"finding":"RNF20 is required for MLL-fusion-mediated leukemogenesis; Rnf20 knockdown reduces H2B ubiquitination enriched in MLL-fusion target gene bodies, reduces local H3K79 methylation by Dot1l at Hoxa9 and Meis1, and suppresses MLL-fusion target gene expression, indicating co-transcriptional RNF20 recruitment amplifies Dot1l-mediated H3K79me at oncogenic targets.","method":"shRNA knockdown, ChIP-seq, RNA-seq, in vivo mouse leukemia model","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 — ChIP-seq plus in vivo functional data with epistasis to Dot1l","pmids":["23412334"],"is_preprint":false},{"year":2013,"finding":"Human adenovirus E1A protein binds RNF20 (hBre1) and repurposes it from a ubiquitin ligase to a scaffold that recruits hPaf1 to activate viral early gene transcription, while simultaneously blocking H2B monoubiquitination to suppress the interferon response.","method":"Co-IP, reporter assay, knockdown, ChIP","journal":"PLoS pathogens","confidence":"Medium","confidence_rationale":"Tier 2 — Co-IP plus functional transcription assays in a single study","pmids":["23785282"],"is_preprint":false},{"year":2014,"finding":"Arsenite binds directly to the RING finger domain cysteine residues of RNF20 and RNF40 in vitro and in cells, inhibiting H2B ubiquitination and impairing BRCA1/RAD51 recruitment to DSBs and DNA repair.","method":"In vitro binding assay, cell-based H2Bub1 assay, laser-induced DSB recruitment, comet assay","journal":"Journal of the American Chemical Society","confidence":"High","confidence_rationale":"Tier 1-2 — direct in vitro binding to RING domain plus cellular functional consequences","pmids":["25170678"],"is_preprint":false},{"year":2016,"finding":"RNF20/RNF40 complex monoubiquitinates and stabilizes the mitotic kinesin motor protein Eg5, interacting with it during mitosis; loss of RNF20/RNF40 causes spindle assembly defects, cell cycle arrest, and apoptosis.","method":"Co-IP, ubiquitination assay, siRNA depletion, immunofluorescence, in vivo tumor models","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 — Co-IP plus ubiquitination assay plus functional spindle phenotype replicated in vivo","pmids":["27557628"],"is_preprint":false},{"year":2016,"finding":"Downregulation of RNF20 and H2Bub1 favors recruitment of p65-containing NF-κB dimers over repressive p50 homodimers and decreases H3K9me3 on a subset of NF-κB target genes, augmenting their transcription and promoting inflammation-associated colorectal cancer.","method":"siRNA knockdown, ChIP, NF-κB reporter, mouse genetic model (RNF20+/- mice), human patient samples","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 2 — ChIP demonstrates mechanistic chromatin changes, validated in mouse genetic model and human specimens","pmids":["26854224"],"is_preprint":false},{"year":2016,"finding":"Crystal structure of the RNF20 RING domain reveals it forms a homodimer that specifically interacts with the Ube2B~Ub conjugate; heterodimer of RNF20 and RNF40 RING domains is active; mutagenesis of E3-E2 and E3-ubiquitin interfaces identifies key contacts required for Ube2B~Ub interaction.","method":"X-ray crystallography, mutagenesis, in vitro ubiquitin transfer assay","journal":"Journal of molecular biology","confidence":"High","confidence_rationale":"Tier 1 — crystal structure plus mutagenesis plus in vitro reconstitution","pmids":["27569044"],"is_preprint":false},{"year":2017,"finding":"RNF20 suppresses tumorigenesis in clear cell renal cell carcinoma by inhibiting SREBP1c, which regulates cell cycle progression through PTTG1; RNF20 overexpression represses lipogenesis and cell proliferation.","method":"Overexpression, knockdown, western blot, xenograft tumor models, pharmacological SREBP1 inhibition","journal":"Molecular and cellular biology","confidence":"Medium","confidence_rationale":"Tier 2 — multiple cell and in vivo models, but molecular mechanism of SREBP1c repression by RNF20 not fully resolved","pmids":["28827316"],"is_preprint":false},{"year":2011,"finding":"RNF20 and RNF40 physically and functionally interact with the androgen receptor (AR) and modulate AR-dependent transcription; ChIP shows androgen induction of FKBP51 and PSA is accompanied by dynamic increase in H2Bub1 within transcribed regions.","method":"Co-IP, reporter assay, ChIP, siRNA depletion","journal":"Molecular and cellular endocrinology","confidence":"Medium","confidence_rationale":"Tier 2 — reciprocal Co-IP plus ChIP demonstrating chromatin-level mechanism","pmids":["22155569"],"is_preprint":false},{"year":2019,"finding":"RNF20/RNF40 complex associates with p53 on chromatin; p53 mediates recruitment of RNF20/RNF40 to p21 and PUMA loci; RNF20/RNF40-dependent H2B ubiquitination (ubH2B) is recognized by the ubiquitin-binding motif of PRPF8 (a spliceosome subunit), which is required for maturation of p21 and PUMA mRNAs, linking H2Bub1 to mRNA splicing.","method":"Protein affinity purification, Co-IP, ChIP, knockdown, RT-PCR mRNA splicing assay","journal":"Journal of molecular cell biology","confidence":"High","confidence_rationale":"Tier 1-2 — unbiased purification plus Co-IP plus functional splicing assay revealing novel mechanistic link","pmids":["31152661"],"is_preprint":false},{"year":2019,"finding":"RNF20/RNF40 in conjunction with RAD6 monoubiquitinates lysine 381 of eEF1BδL (a heat shock transcription factor); monoubiquitylated eEF1BδL increases protein accumulation and potentiates p-TEFb recruitment to heat shock-responsive gene promoters, enhancing their transcription; cooperative physical interactions among eEF1BδL, RNF20/RNF40, and HSF1 are required.","method":"In vitro ubiquitylation assay with purified factors, Co-IP, ChIP, siRNA knockdown, qRT-PCR","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 1 — in vitro reconstitution with purified components plus cell-based validation","pmids":["30649429"],"is_preprint":false},{"year":2019,"finding":"RNF20 promotes adipogenesis by acting as a transcriptional coactivator for PPARγ through proteasomal degradation of nuclear corepressor NCoR1; Rnf20 adipocyte-specific knockout mice show reduced fat mass and impaired PPARγ target gene expression.","method":"Conditional knockout mice, quantitative proteomics, western blot, reporter assay, adipogenesis assay","journal":"Diabetes","confidence":"High","confidence_rationale":"Tier 2 — in vivo genetic model plus proteomics plus mechanistic NCoR1 degradation assay","pmids":["31604693"],"is_preprint":false},{"year":2013,"finding":"RNF20 interacts with and promotes polyubiquitination and proteasome-dependent degradation of AP-2α transcription factor; overexpression of RNF20 inhibits AP-2α activity and rescues C/EBPα expression repressed by AP-2α.","method":"Co-IP, mass spectrometry, ubiquitination assay, overexpression, reporter assay","journal":"Acta biochimica et biophysica Sinica","confidence":"Medium","confidence_rationale":"Tier 2 — Co-IP plus ubiquitination assay plus functional rescue in a single study","pmids":["24374663"],"is_preprint":false},{"year":2021,"finding":"SARS-CoV-2 main protease 3Clpro cleaves RNF20 at a conserved Gln521, which prevents SREBP1 from RNF20-mediated degradation and promotes viral replication; RNAi-mediated depletion of RNF20 or RNF40 enhances viral replication, indicating antiviral activity.","method":"Bioinformatics prediction, protease cleavage assay, catalytic mutant (3ClproC145A), RNAi knockdown, viral replication assay","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1-2 — direct cleavage assay with catalytic mutant control plus functional replication assay","pmids":["34452991"],"is_preprint":false},{"year":2023,"finding":"RNF20/RNF40-RAD6A (hRAD6A) ubiquitin transfer structures were captured by chemical trapping and solved by cryo-EM; RNF40 directly binds nucleosomal DNA; the interface shows conserved E3/E2/nucleosome interaction from yeast to humans; an unconventional non-hydrophobic contact in the Bre1 RING-Rad6 interface positions E2 directly above the target H2B lysine.","method":"Chemical trapping, cryo-EM structure determination, mutagenesis","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 1 — cryo-EM structure with chemical trapping and mutagenesis validation","pmids":["37633270"],"is_preprint":false},{"year":2023,"finding":"Replication protein A (RPA) interacts with RNF20 and recruits it to mitotic centromeres in a centromeric R-loop-dependent manner and to chromosomal breaks upon DNA damage; disruption of RPA-RNF20 interaction increases mitotic lagging chromosomes and impairs BRCA1/RAD51 loading and HR repair; mechanistically, the RPA-RNF20 pathway promotes local H2Bub, H3K4 dimethylation, and SNF2H recruitment, ensuring Aurora B kinase activation at centromeres.","method":"Co-IP, domain mapping, RPA-RNF20 interaction disruption mutants, ChIP, immunofluorescence, HR assay","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods, pathway epistasis to SNF2H and Aurora B","pmids":["37155876"],"is_preprint":false},{"year":2023,"finding":"Bre1/RNF20 functions as a recombination mediator independent of its ligase activity: it interacts with Rad51, directs Rad51 to ssDNA, facilitates Rad51-ssDNA filament assembly and strand exchange in vitro, and counteracts the Srs2/FBH1 helicases that disrupt Rad51 filaments; this function is additive to Rad52 (yeast) or BRCA2 (human).","method":"In vitro Rad51 filament assembly and strand exchange reconstitution, Co-IP, genetic epistasis, cell-based HR assay","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1 — in vitro reconstitution of Rad51 filament assembly/strand exchange plus genetic epistasis","pmids":["37230987"],"is_preprint":false},{"year":2018,"finding":"RNF20 interacts with Snail and G9a; RNF20 and H3K9me2 are enriched on the E-cadherin promoter in a Snail-dependent manner; RNF20 knockdown reduces this enrichment, linking RNF20 to Snail-mediated epigenetic repression of E-cadherin during EMT.","method":"Co-IP, ChIP, siRNA knockdown, migration/invasion assay","journal":"Frontiers in oncology","confidence":"Medium","confidence_rationale":"Tier 2-3 — Co-IP plus ChIP but single lab, mechanism of how RNF20 contributes to H3K9me2 not fully resolved","pmids":["33364200"],"is_preprint":false},{"year":2017,"finding":"Fbxl19 (CxxC domain-containing protein) interacts with Rnf20 and recruits it to CpG island-containing gene promoters; Fbxl19 downregulation reduces H2Bub1 genome-wide at CpG island targets; this interaction is required for proper ES cell differentiation.","method":"Co-IP, genome-wide ChIP-seq, siRNA knockdown, differentiation assay","journal":"Nucleic acids research","confidence":"Medium","confidence_rationale":"Tier 2 — Co-IP plus genome-wide ChIP-seq demonstrating recruitment mechanism","pmids":["28453857"],"is_preprint":false},{"year":2024,"finding":"RNF20 stabilizes LSD1 via K29-linked ubiquitination in a CDK9-phosphorylation-dependent manner; this CDK9/RNF20-dependent LSD1 stabilization promotes H3K4 demethylation, suppresses endogenous retrovirus expression and interferon response, leading to epigenetic immunosuppression; loss of RNF20 sensitizes cancer cells to anti-PD-1 immunotherapy.","method":"Co-IP, ubiquitination assay (K29-specific), CDK9 inhibition, ectopic LSD1 rescue, in vivo anti-PD-1 treatment","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 — mechanistic ubiquitination assay with specific linkage plus CDK9 dependence plus in vivo rescue experiment","pmids":["38315842"],"is_preprint":false},{"year":2024,"finding":"HIF-1 recruits both FACT and RNF20/RNF40 to hypoxia response elements (HREs); FACT and RNF20/RNF40 stabilize HIF-1 binding at HREs and each other; hypoxia induces H2B K120 monoubiquitination at HIF-1 target genes in an HIF-1-dependent manner; FACT and RNF20/RNF40 are required for transcription initiation and elongation at HIF-1 target genes.","method":"ChIP, siRNA knockdown, RNA-seq, Co-IP","journal":"Cell reports","confidence":"Medium","confidence_rationale":"Tier 2 — ChIP and Co-IP in multiple cell lines but single lab","pmids":["38517892"],"is_preprint":false},{"year":2024,"finding":"RNF20 mediates RNA Polymerase II promoter-proximal pausing at highly paused endothelial cell genes; RNF20 restricts ERG-dependent Pol II pause release at these genes while binding Notch1 to promote H2Bub1 at Notch target genes; loss of Rnf20 leads to uncontrolled tip cell specification; RNF20 also orchestrates VEGFA mRNA splicing.","method":"CRISPR/Cas9 knockout, ChIP-seq, PRO-seq/GRO-seq (transcriptional pausing), RNA-seq, Co-IP with Notch1, in vivo angiogenesis assay","journal":"Nature cardiovascular research","confidence":"High","confidence_rationale":"Tier 1-2 — genome-wide PRO-seq plus Co-IP plus in vivo functional data with mechanistic detail","pmids":["39322771"],"is_preprint":false},{"year":2025,"finding":"RNF20 localizes to replicating sites and promotes H2Bub there; RNF20 knockdown leads to MRE11/DNA2-dependent stalled fork degradation; RNF20 facilitates RAD51 and RAD51C loading at stalled forks; RING domain (catalytic) and ATR-mediated phosphorylation of RNF20 are essential for these replication stress responses; chromatin relaxation rescues fork protection defects in RNF20-depleted cells.","method":"DNA fiber assay, siRNA depletion, RING domain mutant, phospho-deficient mutant, Co-IP, immunofluorescence at replication forks, chromatin relaxation rescue","journal":"EMBO reports","confidence":"High","confidence_rationale":"Tier 1-2 — RING domain mutagenesis plus phospho-mutant plus epistasis to fork remodelers","pmids":["40495033"],"is_preprint":false},{"year":2024,"finding":"RNF20 regulates oocyte acentrosomal spindle assembly by recruiting tropomyosin 3 (TPM3) to centromeres and spindle poles via its coiled-coil motif; this function is independent of RNF20's E3 ligase activity; RNF20-depleted oocytes show abnormal spindle and chromosome misalignment.","method":"Oocyte-specific depletion, immunofluorescence, Co-IP, coiled-coil domain mapping, E3 ligase dead mutant","journal":"Advanced science","confidence":"Medium","confidence_rationale":"Tier 2 — domain mapping with ligase-dead mutant plus Co-IP, but single lab","pmids":["38240347"],"is_preprint":false},{"year":2018,"finding":"RNF20 interacts with ZSCAN4 protein; RNF20 depletion increases ZSCAN4 protein levels and stabilizes its half-life without affecting ZSCAN4 mRNA, indicating RNF20 negatively regulates ZSCAN4 protein stability via ubiquitin-proteasome degradation.","method":"Co-IP, cycloheximide chase, siRNA knockdown, K48-polyubiquitination assay","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2-3 — Co-IP plus protein stability assay, single lab","pmids":["29477841"],"is_preprint":false},{"year":2023,"finding":"Conditional knockout of Rnf20 in Sertoli cells causes H2BK120ub loss, impairs transcription elongation of Cldn11 (tight junction component), disrupts cell adhesion and seminiferous tubule organization, and leads to apoptosis of spermatogonia and spermatocytes, causing male infertility recapitulating Sertoli cell-only syndrome.","method":"Conditional knockout mouse (Amh-Cre), ChIP, RNA-seq, western blot, histology","journal":"Cell & bioscience","confidence":"High","confidence_rationale":"Tier 2 — conditional KO with ChIP demonstrating specific gene (Cldn11) as downstream effector","pmids":["37024990"],"is_preprint":false},{"year":2021,"finding":"RNF20 and RNF40 are required for cardiomyocyte maturation; in vivo CRISPR screen identifies RNF20/40 as regulators; the H2BK120 monoubiquitination epigenetic mark controls dynamic gene expression changes required for cardiomyocyte maturation.","method":"In vivo somatic CRISPR screen, conditional validation, RNA-seq, ChIP","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 — unbiased in vivo screen followed by mechanistic validation","pmids":["34290256"],"is_preprint":false},{"year":2021,"finding":"Intestine-specific deletion of Rnf20 or Rnf40 causes spontaneous colorectal inflammation; RNF20/RNF40 controls H3K4me3 occupancy and transcription of the Vitamin D Receptor (Vdr) gene and VDR target genes; loss of H2Bub1 promotes IBD-associated gene expression programs.","method":"Conditional knockout mice, ChIP-seq, RNA-seq, patient ChIP-seq","journal":"Cell death and differentiation","confidence":"High","confidence_rationale":"Tier 2 — conditional KO plus ChIP-seq demonstrating VDR as direct downstream target","pmids":["34088983"],"is_preprint":false},{"year":2019,"finding":"RNF20 and RNF40 are required for DSB repair leading to both homologous recombination and class switch recombination (NHEJ) in mouse B cells; DSBs induce a global increase in H2Bub independent of H2AX phosphorylation; ATM and ATR jointly regulate IR-induced H2Bub.","method":"Conditional knockout B cells, class switch recombination assay, HR assay, western blot for H2Bub, γ-H2AX","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 — genetic KO with mechanistic epistasis to ATM/ATR and H2AX","pmids":["30692271"],"is_preprint":false},{"year":2023,"finding":"Ferritinophagy (selective autophagy) induced by iron deficiency causes RNF20 degradation through the autophagy-lysosomal pathway; reduced RNF20 decreases H2Bub1 at promoters of myogenic markers MyoD and MyoG, inhibiting myogenic differentiation and skeletal muscle regeneration.","method":"Iron deficiency model, lysosomal inhibitor, conditional NCOA4 KO, ChIP, myogenic differentiation assay","journal":"Science advances","confidence":"High","confidence_rationale":"Tier 2 — conditional KO plus ChIP showing H2Bub1 at specific gene loci plus rescue experiments","pmids":["37976359"],"is_preprint":false},{"year":2024,"finding":"RNF20 controls Rbx1 expression and thereby the activity of the VHL ubiquitin ligase complex and HIF1α protein levels; RNF20 haploinsufficiency results in inadequate tumor suppression via the Rnf20-H2Bub1-p53 axis and induces metabolic rewiring through HIF1α-mediated RNAPII promoter-proximal pause release (independent of H2Bub1).","method":"Mouse haploinsufficiency model, RNA-seq, ChIP-seq, western blot, metabolomics","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 — in vivo genetic model plus ChIP-seq separating H2Bub1-dependent and independent mechanisms","pmids":["40436847"],"is_preprint":false},{"year":2024,"finding":"Upon cold stimuli in brown adipose tissue (BAT), RNF20 is rapidly downregulated allowing GABPα protein to accumulate (by controlling its stability), stimulating thermogenic gene expression; in inguinal white adipose tissue (iWAT), RNF20 is gradually upregulated upon prolonged cold and promotes beige adipogenesis via NCoR1 protein degradation to activate PPARγ (depot-specific substrates).","method":"Adipose tissue-specific KO mice, cold challenge experiments, Co-IP, protein stability assay, western blot, gene expression","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 — depot-specific conditional KO with identification of distinct substrates (GABPα vs NCoR1) in each depot","pmids":["38296968"],"is_preprint":false},{"year":2023,"finding":"KSHV RTA protein interacts with the RNF20/RNF40 complex; this interaction is necessary for RTA-induced KSHV lytic reactivation.","method":"Proteomics, Co-IP, siRNA knockdown, lytic reactivation assay","journal":"Journal of virology","confidence":"Medium","confidence_rationale":"Tier 2-3 — proteomics identification plus Co-IP plus functional lytic assay, single lab","pmids":["37888983"],"is_preprint":false},{"year":2026,"finding":"H2B S112 GlcNAcylation allosterically stimulates RNF20/RNF40-RAD6A-mediated H2B K120 monoubiquitylation by interacting with the E2 enzyme RAD6A (not the E3 ligase); cryo-EM structure of trapped RNF20/RNF40-RAD6A-Ub-H2BS112GlcNAc nucleosome complex reveals the GlcNAc moiety enhances nucleophilicity of H2B K120; the C2 N-acetyl group and β-configuration of C1 are essential.","method":"Chemical synthesis of modified nucleosomes, cryo-EM, kinetics assays, mutagenesis, structure-activity relationship analysis","journal":"Nature chemical biology","confidence":"High","confidence_rationale":"Tier 1 — cryo-EM structure plus mutagenesis plus quantitative kinetics with chemically synthesized substrates","pmids":["41495224"],"is_preprint":false},{"year":2026,"finding":"Crystal structure of the Bre1-Lge1 complex and AlphaFold model of RNF20/RNF40-WAC reveal extensive interaction interfaces; the RNF20/RNF40-WAC interaction is critical for the H2BUb1 reaction in vitro and in vivo; key electrostatic interactions at these interfaces encode binding specificity.","method":"X-ray crystallography, AlphaFold structural modeling, in vitro ubiquitination assay, in vivo functional assay","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 1 — crystal structure plus in vitro reconstitution plus mutagenesis of interface residues","pmids":["41533567"],"is_preprint":false},{"year":2023,"finding":"RNF20 promotes STAT3 ubiquitination and degradation; exosomal miR-let-7b-5p targets RNF20, reducing it and thereby allowing STAT3/FOXO1 axis activation and insulin resistance in skeletal muscle cells; ectopic RNF20 expression attenuates this insulin resistance.","method":"Luciferase reporter (let-7b-5p target), Co-IP, ubiquitination assay, rescue experiment with RNF20 overexpression","journal":"Diabetes, metabolic syndrome and obesity","confidence":"Medium","confidence_rationale":"Tier 2-3 — Co-IP plus ubiquitination assay plus functional rescue, single lab","pmids":["37842335"],"is_preprint":false},{"year":2010,"finding":"RNF20 (hBre1) is localized to the IRF1 gene locus in both uninduced and IFNγ-induced states; RNAi-mediated depletion of RNF20 lowers both H2Bub and H3K4me3 but unexpectedly upregulates IFNγ-induced IRF1 transcription, indicating that H2Bub can exert a repressive effect on inducible transcription at this locus, possibly by affecting RNA Pol II CTD phosphorylation cycling.","method":"ChIP, RNAi depletion, qRT-PCR","journal":"Epigenetics & chromatin","confidence":"Medium","confidence_rationale":"Tier 2-3 — ChIP plus functional RNAi with unexpected direction of effect, single lab","pmids":["20825659"],"is_preprint":false}],"current_model":"RNF20 is a RING-type E3 ubiquitin ligase that forms a heterodimeric complex with RNF40 and, together with E2 enzyme hRAD6A, monoubiquitinates histone H2B at lysine 120 (H2Bub1) on nucleosomal DNA—a modification whose structural mechanism has been resolved by cryo-EM; this activity requires the WAC adaptor that bridges the complex to RNA Pol II, is allosterically stimulated by H2B S112 GlcNAcylation acting on RAD6A, and is antagonized by USP44 deubiquitinase; H2Bub1 promotes downstream H3K4 and H3K79 methylation, facilitates transcription elongation, and drives SNF2h-dependent chromatin remodeling at DSBs—enabling DNA end resection, RAD51/BRCA1 recruitment, homologous recombination, and stalled fork protection (the last requiring ATR-mediated phosphorylation of RNF20); beyond histone modification, RNF20 ubiquitinates non-histone substrates including Eg5 (mitotic kinesin), LSD1 (K29-linked, CDK9-dependent), STAT3, AP-2α, and ZSCAN4, and its own stability is regulated by Smurf2-mediated degradation and by SARS-CoV-2 Mpro cleavage at Gln521; RNF20 is recruited to chromatin by context-specific adaptors (RPA at centromeres/DSBs, FACT at DSBs, HIF-1 at hypoxia response elements, Fbxl19 at CpG islands, p53 at target genes) and its activity underpins diverse processes including stem cell differentiation, cardiomyocyte maturation, adipogenesis, angiogenesis, NF-κB-driven inflammation, and antiviral signaling, acting as a broadly context-dependent tumor suppressor."},"narrative":{"teleology":[{"year":2010,"claim":"Initial chromatin studies revealed that RNF20 occupies gene loci and maintains H2Bub1 and H3K4me3, but that H2Bub1 can repress as well as activate inducible transcription, establishing RNF20 as a context-dependent transcriptional regulator rather than a simple activator.","evidence":"ChIP and RNAi depletion at the IRF1 locus showing unexpected transcriptional derepression upon RNF20 loss","pmids":["20825659"],"confidence":"Medium","gaps":["Single locus studied; genome-wide scope of repressive versus activating H2Bub1 effects unknown","Mechanism of repression (Pol II CTD cycling hypothesis) not directly tested"]},{"year":2011,"claim":"Multiple studies converged to define RNF20 as the principal H2B K120 ubiquitin ligase operating in both transcription and DNA repair: WAC was identified as the adaptor bridging RNF20/RNF40 to elongating RNA Pol II, CDC73/parafibromin was shown to maintain H2Bub1 levels, and RNF20 was demonstrated to be essential for DSB-induced chromatin remodeling (via SNF2h), DNA end resection, RAD51/BRCA1 recruitment, and homologous recombination.","evidence":"Co-IP/domain mapping for WAC and CDC73, ChIP at DSBs, HR repair assays, H2B K120R mutant, radiation sensitivity","pmids":["21329877","22021426","21362548","22155569"],"confidence":"High","gaps":["Structural basis of WAC-RNF20 interaction unresolved at atomic level","Whether CDC73 mutations cause disease through H2Bub1 loss versus other mechanisms unclear"]},{"year":2012,"claim":"RNF20 was established as a regulated enzyme whose levels are controlled by Smurf2-mediated proteasomal degradation, whose activity is opposed by USP44 deubiquitinase, and whose loss causes R-loop accumulation and genomic instability—linking H2Bub1 to both differentiation (ESC exit) and genome integrity.","evidence":"Smurf2 KO mouse model, USP44 depletion during ESC differentiation, R-loop detection and cytogenetics upon RNF20 depletion","pmids":["22231558","22681888","22354749"],"confidence":"High","gaps":["How Smurf2 itself is regulated at DSBs not determined","Whether R-loops are a direct consequence of H2Bub1 loss or secondary to transcription-replication conflicts unclear"]},{"year":2013,"claim":"The chromatin adaptor FACT was identified as a DSB-specific recruiter of RNF20 (distinct from transcription-associated recruitment via PAF1), and RNF20 was shown to be co-opted by MLL-fusion oncoproteins to amplify Dot1l-dependent H3K79 methylation at leukemogenic target genes, revealing RNF20's role in both repair and oncogenesis.","evidence":"RING domain mutagenesis abolishing FACT interaction, PAF1-independence at DSBs, ChIP-seq/RNA-seq in MLL-fusion leukemia models, in vivo mouse leukemia","pmids":["24357716","23412334"],"confidence":"High","gaps":["How FACT is recruited to DSBs independently of PAF1 not fully resolved","Whether pharmacological targeting of RNF20 in MLL leukemia is feasible unknown"]},{"year":2013,"claim":"RNF20's substrate range was expanded beyond histones: it ubiquitinates AP-2α for proteasomal degradation, and adenovirus E1A was found to hijack RNF20 as a scaffold for viral transcription while blocking H2Bub1 to suppress interferon responses, establishing RNF20 as a target of viral immune evasion.","evidence":"Co-IP, ubiquitination assay for AP-2α; Co-IP and transcription reporter for E1A-RNF20 interaction","pmids":["24374663","23785282"],"confidence":"Medium","gaps":["Ubiquitin chain type on AP-2α not characterized","Whether E1A-RNF20 interaction is conserved across adenovirus serotypes not tested","Single-lab findings for each substrate"]},{"year":2014,"claim":"Arsenite was shown to directly bind RING finger cysteine residues of RNF20/RNF40, establishing the RING domain as a druggable interface and revealing a chemical mechanism for environmental carcinogenesis through H2Bub1 loss and DNA repair impairment.","evidence":"In vitro arsenite binding assay to RING domain, cellular H2Bub1 loss, impaired BRCA1/RAD51 recruitment","pmids":["25170678"],"confidence":"High","gaps":["Whether arsenic concentrations used reflect physiological exposure levels not fully addressed","Selectivity of arsenite for RNF20 versus other RING E3 ligases not compared"]},{"year":2016,"claim":"The structural basis of E3 activity was revealed by crystal structures of the RNF20 RING domain with Ube2B~Ub conjugate, and the non-histone substrate Eg5 was identified as an RNF20/RNF40 target whose ubiquitination is required for mitotic spindle assembly, while loss of H2Bub1 was linked to NF-κB-driven inflammation and colorectal cancer.","evidence":"X-ray crystallography and mutagenesis of RING-E2 interface; Co-IP and ubiquitination assay for Eg5 with spindle phenotypes; ChIP and mouse haploinsufficiency model for NF-κB target regulation","pmids":["27569044","27557628","26854224"],"confidence":"High","gaps":["Whether Eg5 ubiquitination is mono- or polyubiquitination and the specific lysine(s) not mapped","How H2Bub1 loss shifts NF-κB dimer composition mechanistically not resolved"]},{"year":2017,"claim":"Fbxl19 was identified as a CpG-island-directed recruiter of RNF20 to gene promoters in ES cells, and RNF20 was shown to suppress renal cell carcinoma through SREBP1c inhibition, broadening the repertoire of chromatin recruitment mechanisms and tumor-suppressive outputs.","evidence":"Co-IP and genome-wide ChIP-seq for Fbxl19-RNF20; xenograft models and pharmacological SREBP1 inhibition for tumor suppression","pmids":["28453857","28827316"],"confidence":"Medium","gaps":["Direct ubiquitination of SREBP1c by RNF20 not demonstrated","Single-lab ChIP-seq for Fbxl19; independent validation needed"]},{"year":2019,"claim":"Multiple non-histone substrates and adaptor mechanisms were defined: p53 recruits RNF20/RNF40 to p21/PUMA loci where H2Bub1 is read by spliceosome subunit PRPF8 for mRNA maturation; eEF1BδL is monoubiquitinated by RNF20/RAD6 to recruit p-TEFb for heat shock gene transcription; and RNF20 promotes adipogenesis by degrading NCoR1 to activate PPARγ. Additionally, RNF20/RNF40 was shown essential for both HR and NHEJ (class switch recombination) in B cells under ATM/ATR control.","evidence":"In vitro reconstituted ubiquitination for eEF1BδL; affinity purification revealing PRPF8; adipocyte-conditional KO mice; conditional KO B cells with CSR assays","pmids":["31152661","30649429","31604693","30692271"],"confidence":"High","gaps":["Whether PRPF8-H2Bub1 interaction is general or gene-specific not resolved genome-wide","ATR versus ATM individual contributions to H2Bub induction not separated cleanly"]},{"year":2021,"claim":"SARS-CoV-2 Mpro was shown to cleave RNF20 at Gln521, stabilizing SREBP1 and enhancing viral replication, establishing RNF20 as an antiviral factor; independently, unbiased in vivo CRISPR screens identified RNF20/RNF40 as required for cardiomyocyte maturation, and intestine-specific deletion revealed RNF20 controls VDR expression to prevent spontaneous colitis.","evidence":"Protease cleavage assay with catalytic mutant control; somatic CRISPR screen in mouse heart; intestinal conditional KO with ChIP-seq for VDR","pmids":["34452991","34290256","34088983"],"confidence":"High","gaps":["Whether Mpro cleavage occurs at endogenous viral loads not demonstrated","Whether cardiomyocyte maturation defect is H2Bub1-dependent or involves non-histone substrates unclear"]},{"year":2023,"claim":"A ligase-independent function was discovered: RNF20/Bre1 directly binds Rad51, facilitates Rad51-ssDNA filament assembly and strand exchange, and counteracts anti-recombinases Srs2/FBH1, acting as a bona fide recombination mediator additive to BRCA2; simultaneously, RPA was identified as a recruiter of RNF20 to centromeric R-loops and DSBs, enabling Aurora B activation and chromosome segregation fidelity.","evidence":"In vitro reconstitution of Rad51 filament assembly/strand exchange; Co-IP and domain mapping for RPA-RNF20; genetic epistasis","pmids":["37230987","37155876"],"confidence":"High","gaps":["Which domain of RNF20 mediates Rad51 binding not mapped","Whether RPA-dependent RNF20 centromeric function is conserved in human meiosis unknown"]},{"year":2023,"claim":"Tissue-specific conditional knockouts demonstrated RNF20's essential roles in spermatogenesis (via Cldn11 transcription elongation), myogenesis (where ferritinophagy-mediated RNF20 degradation impairs MyoD/MyoG expression), and viral biology (KSHV RTA interaction for lytic reactivation).","evidence":"Sertoli cell-specific KO with ChIP for Cldn11; iron deficiency model with NCOA4 KO for ferritinophagy; proteomics and Co-IP for KSHV RTA","pmids":["37024990","37976359","37888983"],"confidence":"High","gaps":["Whether ferritinophagy-RNF20 axis is relevant to iron deficiency anemia-associated myopathies in humans unknown","KSHV RTA-RNF20 interaction: single lab, mechanism of exploitation unclear"]},{"year":2024,"claim":"CDK9-dependent K29-linked ubiquitination of LSD1 by RNF20 was identified as a mechanism suppressing endogenous retrovirus expression and interferon responses, creating epigenetic immunosuppression reversible by anti-PD-1 therapy; RNF20 was also shown to regulate Pol II promoter-proximal pausing at endothelial genes and to control angiogenesis through Notch1 interaction; depot-specific adipose substrates (GABPα in BAT, NCoR1 in iWAT) were defined; and RNF20 haploinsufficiency was linked to HIF1α-mediated metabolic rewiring independent of H2Bub1.","evidence":"K29-specific ubiquitination assay with CDK9 inhibition; PRO-seq/GRO-seq for Pol II pausing; depot-specific adipose KO mice; mouse haploinsufficiency model with ChIP-seq","pmids":["38315842","39322771","38296968","40436847"],"confidence":"High","gaps":["Whether K29-linked LSD1 ubiquitination is unique to RNF20 or shared with other E3 ligases not tested","Structural basis of Notch1-RNF20 interaction unknown","H2Bub1-independent mechanisms at HIF1α remain poorly characterized molecularly"]},{"year":2025,"claim":"RNF20 was established as a replication fork protector: it localizes to replicating sites, promotes H2Bub there, facilitates RAD51/RAD51C loading at stalled forks, and prevents MRE11/DNA2-dependent fork degradation—functions requiring both RING domain catalytic activity and ATR-mediated phosphorylation.","evidence":"DNA fiber assay with RING and phospho-deficient mutants, epistasis to fork remodelers, chromatin relaxation rescue","pmids":["40495033"],"confidence":"High","gaps":["ATR phosphorylation site(s) on RNF20 not mapped","Whether fork protection involves non-histone substrates not addressed"]},{"year":2026,"claim":"Cryo-EM structures of the full RNF20/RNF40-RAD6A-nucleosome complex revealed that H2B S112 GlcNAcylation allosterically stimulates ubiquitin transfer by contacting RAD6A (not the E3), enhancing K120 nucleophilicity; crystal structures of Bre1-Lge1 and AlphaFold models of the RNF20/RNF40-WAC complex defined the extensive adaptor interfaces essential for H2Bub1 in vivo.","evidence":"Cryo-EM with chemically synthesized GlcNAc-nucleosomes and kinetics; X-ray crystallography of Bre1-Lge1 with in vitro/in vivo mutagenesis","pmids":["41495224","41533567"],"confidence":"High","gaps":["Whether GlcNAcylation-mediated stimulation occurs at all genomic loci or specific chromatin contexts unknown","Full-length RNF20/RNF40 structure on nucleosome not yet determined"]},{"year":null,"claim":"Key open questions include: how RNF20 selects between histone and diverse non-histone substrates in different cellular contexts; the structural basis for ligase-independent Rad51 mediator activity; whether RNF20 mutations cause Mendelian disease in humans; and how ATR phosphorylation sites regulate RNF20 activity at replication forks versus DSBs.","evidence":"","pmids":[],"confidence":"Low","gaps":["No substrate-selection mechanism resolved","No full-length structure of human RNF20/RNF40 heterodimer","No Mendelian disease association established by direct genetic evidence"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,2,10,12,16,17,18,20,25,30,39,41]},{"term_id":"GO:0042393","term_label":"histone binding","supporting_discovery_ids":[0,2,12,20,39]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[22,29]}],"localization":[{"term_id":"GO:0005694","term_label":"chromosome","supporting_discovery_ids":[0,6,7,21,26,28]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[0,1,7,15,42]}],"pathway":[{"term_id":"R-HSA-4839726","term_label":"Chromatin organization","supporting_discovery_ids":[0,2,7,12,20,39,40]},{"term_id":"R-HSA-73894","term_label":"DNA Repair","supporting_discovery_ids":[0,6,9,21,22,34]},{"term_id":"R-HSA-74160","term_label":"Gene expression (Transcription)","supporting_discovery_ids":[1,7,15,26,27,42]},{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[10,28]},{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[2,24,31,32]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[12,17,20,25,39,40]},{"term_id":"R-HSA-8953854","term_label":"Metabolism of RNA","supporting_discovery_ids":[15,27]}],"complexes":["RNF20/RNF40 heterodimer","RNF20/RNF40-RAD6A (hBre1-hRAD6) E3-E2 complex"],"partners":["RNF40","RAD6A","WAC","FACT","RPA","RAD51","SMURF2","NOTCH1"],"other_free_text":[]},"mechanistic_narrative":"RNF20 is a RING-type E3 ubiquitin ligase that, as a heterodimer with RNF40, catalyzes histone H2B lysine 120 monoubiquitination (H2Bub1)—a chromatin mark central to transcription elongation, mRNA splicing, DNA double-strand break repair, replication fork protection, and cell differentiation. The RNF20/RNF40 complex is recruited to chromatin by context-specific adaptors including WAC (linking it to RNA Pol II), RPA (at centromeres and DNA damage sites), FACT (at DSBs and hypoxia response elements), p53, HIF-1, and Fbxl19 (at CpG islands); H2Bub1 stimulates downstream H3K4 and H3K79 methylation, recruits SNF2h chromatin remodelers at DSBs and centromeres, and is read by the spliceosome component PRPF8 to couple transcription with mRNA processing [PMID:21329877, PMID:37155876, PMID:24357716, PMID:31152661, PMID:38517892, PMID:28453857]. Beyond histone modification, RNF20 ubiquitinates non-histone substrates—Eg5 (mitotic kinesin stabilization), LSD1 (K29-linked, CDK9-dependent stabilization suppressing endogenous retrovirus expression), NCoR1 (proteasomal degradation enabling PPARγ-driven adipogenesis), STAT3, AP-2α, and ZSCAN4—and possesses a ligase-independent recombination mediator function that directly facilitates Rad51 filament assembly on ssDNA [PMID:27557628, PMID:38315842, PMID:31604693, PMID:37230987, PMID:24374663]. RNF20 stability and activity are regulated by Smurf2-mediated proteasomal degradation, ferritinophagy-dependent lysosomal degradation, SARS-CoV-2 Mpro cleavage at Gln521, ATR-mediated phosphorylation during replication stress, and allosteric stimulation by H2B S112 GlcNAcylation acting on the E2 enzyme RAD6A [PMID:22231558, PMID:37976359, PMID:34452991, PMID:40495033, PMID:41495224]."},"prefetch_data":{"uniprot":{"accession":"Q5VTR2","full_name":"E3 ubiquitin-protein ligase BRE1A","aliases":["RING finger protein 20","RING-type E3 ubiquitin transferase BRE1A"],"length_aa":975,"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 inb 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. Recruited to the MDM2 promoter, probably by being recruited by p53/TP53, and thereby acts as a transcriptional coactivator. Mediates the polyubiquitination of isoform 2 of PA2G4 in cancer cells leading to its proteasome-mediated degradation (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/Q5VTR2/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":true,"resolved_as":"","url":"https://depmap.org/portal/gene/RNF20","classification":"Common Essential","n_dependent_lines":1069,"n_total_lines":1208,"dependency_fraction":0.8849337748344371},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"RNF40","stoichiometry":10.0},{"gene":"PRPF19","stoichiometry":0.2},{"gene":"RBM12","stoichiometry":0.2},{"gene":"FAM117B","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/RNF20","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":"612860","title":"QUIESCIN Q6 SULFHYDRYL OXIDASE 2; QSOX2","url":"https://www.omim.org/entry/612860"},{"mim_id":"612116","title":"UBIQUITIN-SPECIFIC PROTEASE 22; USP22","url":"https://www.omim.org/entry/612116"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Enhanced","locations":[{"location":"Nucleoplasm","reliability":"Enhanced"},{"location":"Cytosol","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/RNF20"},"hgnc":{"alias_symbol":["FLJ20382","FLJ11189","KAIA2779","BRE1A","hBRE1","BRE1"],"prev_symbol":[]},"alphafold":{"accession":"Q5VTR2","domains":[{"cath_id":"-","chopping":"63-112_153-295","consensus_level":"medium","plddt":84.8974,"start":63,"end":295},{"cath_id":"3.30.40.10","chopping":"907-970","consensus_level":"high","plddt":87.1144,"start":907,"end":970}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q5VTR2","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q5VTR2-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q5VTR2-F1-predicted_aligned_error_v6.png","plddt_mean":75.38},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=RNF20","jax_strain_url":"https://www.jax.org/strain/search?query=RNF20"},"sequence":{"accession":"Q5VTR2","fasta_url":"https://rest.uniprot.org/uniprotkb/Q5VTR2.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q5VTR2/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q5VTR2"}},"corpus_meta":[{"pmid":"21362548","id":"PMC_21362548","title":"Regulation of homologous recombination by RNF20-dependent H2B ubiquitination.","date":"2011","source":"Molecular cell","url":"https://pubmed.ncbi.nlm.nih.gov/21362548","citation_count":299,"is_preprint":false},{"pmid":"22681888","id":"PMC_22681888","title":"RNF20 and USP44 regulate stem cell differentiation by modulating H2B monoubiquitylation.","date":"2012","source":"Molecular cell","url":"https://pubmed.ncbi.nlm.nih.gov/22681888","citation_count":175,"is_preprint":false},{"pmid":"22231558","id":"PMC_22231558","title":"A tumor suppressor function of Smurf2 associated with controlling chromatin landscape and genome stability through RNF20.","date":"2012","source":"Nature medicine","url":"https://pubmed.ncbi.nlm.nih.gov/22231558","citation_count":136,"is_preprint":false},{"pmid":"21329877","id":"PMC_21329877","title":"WAC, a functional partner of RNF20/40, regulates histone H2B ubiquitination and gene transcription.","date":"2011","source":"Molecular 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Repair.","date":"2019","source":"Molecular and cellular biology","url":"https://pubmed.ncbi.nlm.nih.gov/30692271","citation_count":58,"is_preprint":false},{"pmid":"24357716","id":"PMC_24357716","title":"Histone chaperone FACT regulates homologous recombination by chromatin remodeling through interaction with RNF20.","date":"2013","source":"Journal of cell science","url":"https://pubmed.ncbi.nlm.nih.gov/24357716","citation_count":51,"is_preprint":false},{"pmid":"34290256","id":"PMC_34290256","title":"Massively parallel in vivo CRISPR screening identifies RNF20/40 as epigenetic regulators of cardiomyocyte maturation.","date":"2021","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/34290256","citation_count":51,"is_preprint":false},{"pmid":"28690313","id":"PMC_28690313","title":"The HDAC inhibitor panobinostat (LBH589) exerts in vivo anti-leukaemic activity against MLL-rearranged acute lymphoblastic leukaemia and involves the RNF20/RNF40/WAC-H2B ubiquitination axis.","date":"2017","source":"Leukemia","url":"https://pubmed.ncbi.nlm.nih.gov/28690313","citation_count":48,"is_preprint":false},{"pmid":"25170678","id":"PMC_25170678","title":"Arsenite binds to the RING finger domains of RNF20-RNF40 histone E3 ubiquitin ligase and inhibits DNA double-strand break repair.","date":"2014","source":"Journal of the American Chemical Society","url":"https://pubmed.ncbi.nlm.nih.gov/25170678","citation_count":47,"is_preprint":false},{"pmid":"28157208","id":"PMC_28157208","title":"RNF20 and histone H2B ubiquitylation exert opposing effects in Basal-Like versus luminal breast cancer.","date":"2017","source":"Cell death and differentiation","url":"https://pubmed.ncbi.nlm.nih.gov/28157208","citation_count":46,"is_preprint":false},{"pmid":"28827316","id":"PMC_28827316","title":"RNF20 Suppresses Tumorigenesis by Inhibiting the SREBP1c-PTTG1 Axis in Kidney Cancer.","date":"2017","source":"Molecular and cellular 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bypassed by forced chromatin relaxation.\",\n      \"method\": \"siRNA depletion, H2B K120R mutant expression, γ-H2AX foci, ChIP, HR repair assay, radiation sensitivity\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods, replicated in subsequent studies\",\n      \"pmids\": [\"21362548\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"WAC interacts with RNF20/RNF40 through its C-terminal coiled-coil region, recruits the complex to RNA polymerase II via its N-terminal WW domain during active transcription, and promotes RNF20/RNF40 E3 ligase activity for H2B ubiquitination; WAC depletion abolishes H2Bub.\",\n      \"method\": \"Protein affinity purification, Co-IP, domain mapping, knockdown, ChIP\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — affinity purification identifies partner, domain mapping, functional depletion phenotype, replicated\",\n      \"pmids\": [\"21329877\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"RNF20-mediated H2B monoubiquitylation (H2Bub1) increases during embryonic stem cell (ESC) differentiation and is required for transcriptional induction of relatively long genes; USP44 deubiquitinase negatively regulates H2Bub1 and its downregulation during differentiation contributes to increased H2Bub1.\",\n      \"method\": \"Knockdown/depletion, qRT-PCR, RNA-seq, ChIP, western blot\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods identifying both writer (RNF20) and eraser (USP44) with functional outcomes\",\n      \"pmids\": [\"22681888\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Smurf2 (HECT-domain E3 ligase) targets RNF20 for proteasomal degradation, thereby regulating RNF20-dependent H2B monoubiquitination and H3K4/H3K79 trimethylation; Smurf2 and RNF20 co-localize at γ-H2AX foci at DSBs.\",\n      \"method\": \"Genetic ablation (mouse model), western blot, immunofluorescence co-localization, ChIP\",\n      \"journal\": \"Nature medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — in vivo genetic model plus molecular mechanistic validation\",\n      \"pmids\": [\"22231558\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"RNF20/RNF40 deficiency leads to formation of aberrant R-loops (RNA:DNA hybrids) at replication forks, causing replication-associated DSBs, specific genomic rearrangements, and chromosomal instability.\",\n      \"method\": \"Bre1/RNF20 depletion, DNA fiber assay, cytogenetics, R-loop detection\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — mechanistic link from RNF20 loss to R-loop formation demonstrated with multiple assays\",\n      \"pmids\": [\"22354749\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"CDC73 (parafibromin) interacts with RNF20 and RNF40 (identified by yeast two-hybrid), and CDC73 is required for maintenance of H2B K120 monoubiquitination; pathogenic CDC73 mutations reduce H2Bub1 in parathyroid tumors.\",\n      \"method\": \"Yeast two-hybrid, Co-IP, domain mapping, siRNA knockdown, western blot, tumor samples\",\n      \"journal\": \"Human molecular genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — yeast two-hybrid plus reciprocal Co-IP plus functional knockdown plus patient tumor validation\",\n      \"pmids\": [\"22021426\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Histone chaperone FACT (SUPT16H subunit) directly binds RNF20 in vivo; mutation at the RING-finger domain of RNF20 abolishes this interaction; FACT is required for RNF20 recruitment to DSBs and subsequent H2B ubiquitylation and SNF2h accumulation, enabling homologous recombination repair. PAF1 (a known transcriptional mediator of FACT-RNF20 association) is dispensable for DNA-damage-induced RNF20-FACT interaction.\",\n      \"method\": \"Co-IP, RING domain mutagenesis, siRNA depletion, laser-induced DSB imaging, γ-H2AX, HR assay\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — RING domain mutagenesis combined with direct Co-IP and functional HR assay\",\n      \"pmids\": [\"24357716\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"RNF20 is required for MLL-fusion-mediated leukemogenesis; Rnf20 knockdown reduces H2B ubiquitination enriched in MLL-fusion target gene bodies, reduces local H3K79 methylation by Dot1l at Hoxa9 and Meis1, and suppresses MLL-fusion target gene expression, indicating co-transcriptional RNF20 recruitment amplifies Dot1l-mediated H3K79me at oncogenic targets.\",\n      \"method\": \"shRNA knockdown, ChIP-seq, RNA-seq, in vivo mouse leukemia model\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — ChIP-seq plus in vivo functional data with epistasis to Dot1l\",\n      \"pmids\": [\"23412334\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Human adenovirus E1A protein binds RNF20 (hBre1) and repurposes it from a ubiquitin ligase to a scaffold that recruits hPaf1 to activate viral early gene transcription, while simultaneously blocking H2B monoubiquitination to suppress the interferon response.\",\n      \"method\": \"Co-IP, reporter assay, knockdown, ChIP\",\n      \"journal\": \"PLoS pathogens\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — Co-IP plus functional transcription assays in a single study\",\n      \"pmids\": [\"23785282\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Arsenite binds directly to the RING finger domain cysteine residues of RNF20 and RNF40 in vitro and in cells, inhibiting H2B ubiquitination and impairing BRCA1/RAD51 recruitment to DSBs and DNA repair.\",\n      \"method\": \"In vitro binding assay, cell-based H2Bub1 assay, laser-induced DSB recruitment, comet assay\",\n      \"journal\": \"Journal of the American Chemical Society\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — direct in vitro binding to RING domain plus cellular functional consequences\",\n      \"pmids\": [\"25170678\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"RNF20/RNF40 complex monoubiquitinates and stabilizes the mitotic kinesin motor protein Eg5, interacting with it during mitosis; loss of RNF20/RNF40 causes spindle assembly defects, cell cycle arrest, and apoptosis.\",\n      \"method\": \"Co-IP, ubiquitination assay, siRNA depletion, immunofluorescence, in vivo tumor models\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — Co-IP plus ubiquitination assay plus functional spindle phenotype replicated in vivo\",\n      \"pmids\": [\"27557628\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Downregulation of RNF20 and H2Bub1 favors recruitment of p65-containing NF-κB dimers over repressive p50 homodimers and decreases H3K9me3 on a subset of NF-κB target genes, augmenting their transcription and promoting inflammation-associated colorectal cancer.\",\n      \"method\": \"siRNA knockdown, ChIP, NF-κB reporter, mouse genetic model (RNF20+/- mice), human patient samples\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — ChIP demonstrates mechanistic chromatin changes, validated in mouse genetic model and human specimens\",\n      \"pmids\": [\"26854224\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Crystal structure of the RNF20 RING domain reveals it forms a homodimer that specifically interacts with the Ube2B~Ub conjugate; heterodimer of RNF20 and RNF40 RING domains is active; mutagenesis of E3-E2 and E3-ubiquitin interfaces identifies key contacts required for Ube2B~Ub interaction.\",\n      \"method\": \"X-ray crystallography, mutagenesis, in vitro ubiquitin transfer assay\",\n      \"journal\": \"Journal of molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — crystal structure plus mutagenesis plus in vitro reconstitution\",\n      \"pmids\": [\"27569044\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"RNF20 suppresses tumorigenesis in clear cell renal cell carcinoma by inhibiting SREBP1c, which regulates cell cycle progression through PTTG1; RNF20 overexpression represses lipogenesis and cell proliferation.\",\n      \"method\": \"Overexpression, knockdown, western blot, xenograft tumor models, pharmacological SREBP1 inhibition\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple cell and in vivo models, but molecular mechanism of SREBP1c repression by RNF20 not fully resolved\",\n      \"pmids\": [\"28827316\"],\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 AR-dependent transcription; ChIP shows androgen induction of FKBP51 and PSA is accompanied by dynamic increase in H2Bub1 within transcribed regions.\",\n      \"method\": \"Co-IP, reporter assay, ChIP, siRNA depletion\",\n      \"journal\": \"Molecular and cellular endocrinology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal Co-IP plus ChIP demonstrating chromatin-level mechanism\",\n      \"pmids\": [\"22155569\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"RNF20/RNF40 complex associates with p53 on chromatin; p53 mediates recruitment of RNF20/RNF40 to p21 and PUMA loci; RNF20/RNF40-dependent H2B ubiquitination (ubH2B) is recognized by the ubiquitin-binding motif of PRPF8 (a spliceosome subunit), which is required for maturation of p21 and PUMA mRNAs, linking H2Bub1 to mRNA splicing.\",\n      \"method\": \"Protein affinity purification, Co-IP, ChIP, knockdown, RT-PCR mRNA splicing assay\",\n      \"journal\": \"Journal of molecular cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — unbiased purification plus Co-IP plus functional splicing assay revealing novel mechanistic link\",\n      \"pmids\": [\"31152661\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"RNF20/RNF40 in conjunction with RAD6 monoubiquitinates lysine 381 of eEF1BδL (a heat shock transcription factor); monoubiquitylated eEF1BδL increases protein accumulation and potentiates p-TEFb recruitment to heat shock-responsive gene promoters, enhancing their transcription; cooperative physical interactions among eEF1BδL, RNF20/RNF40, and HSF1 are required.\",\n      \"method\": \"In vitro ubiquitylation assay with purified factors, Co-IP, ChIP, siRNA knockdown, qRT-PCR\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro reconstitution with purified components plus cell-based validation\",\n      \"pmids\": [\"30649429\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"RNF20 promotes adipogenesis by acting as a transcriptional coactivator for PPARγ through proteasomal degradation of nuclear corepressor NCoR1; Rnf20 adipocyte-specific knockout mice show reduced fat mass and impaired PPARγ target gene expression.\",\n      \"method\": \"Conditional knockout mice, quantitative proteomics, western blot, reporter assay, adipogenesis assay\",\n      \"journal\": \"Diabetes\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — in vivo genetic model plus proteomics plus mechanistic NCoR1 degradation assay\",\n      \"pmids\": [\"31604693\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"RNF20 interacts with and promotes polyubiquitination and proteasome-dependent degradation of AP-2α transcription factor; overexpression of RNF20 inhibits AP-2α activity and rescues C/EBPα expression repressed by AP-2α.\",\n      \"method\": \"Co-IP, mass spectrometry, ubiquitination assay, overexpression, reporter assay\",\n      \"journal\": \"Acta biochimica et biophysica Sinica\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — Co-IP plus ubiquitination assay plus functional rescue in a single study\",\n      \"pmids\": [\"24374663\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"SARS-CoV-2 main protease 3Clpro cleaves RNF20 at a conserved Gln521, which prevents SREBP1 from RNF20-mediated degradation and promotes viral replication; RNAi-mediated depletion of RNF20 or RNF40 enhances viral replication, indicating antiviral activity.\",\n      \"method\": \"Bioinformatics prediction, protease cleavage assay, catalytic mutant (3ClproC145A), RNAi knockdown, viral replication assay\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — direct cleavage assay with catalytic mutant control plus functional replication assay\",\n      \"pmids\": [\"34452991\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"RNF20/RNF40-RAD6A (hRAD6A) ubiquitin transfer structures were captured by chemical trapping and solved by cryo-EM; RNF40 directly binds nucleosomal DNA; the interface shows conserved E3/E2/nucleosome interaction from yeast to humans; an unconventional non-hydrophobic contact in the Bre1 RING-Rad6 interface positions E2 directly above the target H2B lysine.\",\n      \"method\": \"Chemical trapping, 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\",\n      \"pmids\": [\"37633270\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Replication protein A (RPA) interacts with RNF20 and recruits it to mitotic centromeres in a centromeric R-loop-dependent manner and to chromosomal breaks upon DNA damage; disruption of RPA-RNF20 interaction increases mitotic lagging chromosomes and impairs BRCA1/RAD51 loading and HR repair; mechanistically, the RPA-RNF20 pathway promotes local H2Bub, H3K4 dimethylation, and SNF2H recruitment, ensuring Aurora B kinase activation at centromeres.\",\n      \"method\": \"Co-IP, domain mapping, RPA-RNF20 interaction disruption mutants, ChIP, immunofluorescence, HR assay\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods, pathway epistasis to SNF2H and Aurora B\",\n      \"pmids\": [\"37155876\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Bre1/RNF20 functions as a recombination mediator independent of its ligase activity: it interacts with Rad51, directs Rad51 to ssDNA, facilitates Rad51-ssDNA filament assembly and strand exchange in vitro, and counteracts the Srs2/FBH1 helicases that disrupt Rad51 filaments; this function is additive to Rad52 (yeast) or BRCA2 (human).\",\n      \"method\": \"In vitro Rad51 filament assembly and strand exchange reconstitution, Co-IP, genetic epistasis, cell-based HR assay\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro reconstitution of Rad51 filament assembly/strand exchange plus genetic epistasis\",\n      \"pmids\": [\"37230987\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"RNF20 interacts with Snail and G9a; RNF20 and H3K9me2 are enriched on the E-cadherin promoter in a Snail-dependent manner; RNF20 knockdown reduces this enrichment, linking RNF20 to Snail-mediated epigenetic repression of E-cadherin during EMT.\",\n      \"method\": \"Co-IP, ChIP, siRNA knockdown, migration/invasion assay\",\n      \"journal\": \"Frontiers in oncology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — Co-IP plus ChIP but single lab, mechanism of how RNF20 contributes to H3K9me2 not fully resolved\",\n      \"pmids\": [\"33364200\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Fbxl19 (CxxC domain-containing protein) interacts with Rnf20 and recruits it to CpG island-containing gene promoters; Fbxl19 downregulation reduces H2Bub1 genome-wide at CpG island targets; this interaction is required for proper ES cell differentiation.\",\n      \"method\": \"Co-IP, genome-wide ChIP-seq, siRNA knockdown, differentiation assay\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — Co-IP plus genome-wide ChIP-seq demonstrating recruitment mechanism\",\n      \"pmids\": [\"28453857\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"RNF20 stabilizes LSD1 via K29-linked ubiquitination in a CDK9-phosphorylation-dependent manner; this CDK9/RNF20-dependent LSD1 stabilization promotes H3K4 demethylation, suppresses endogenous retrovirus expression and interferon response, leading to epigenetic immunosuppression; loss of RNF20 sensitizes cancer cells to anti-PD-1 immunotherapy.\",\n      \"method\": \"Co-IP, ubiquitination assay (K29-specific), CDK9 inhibition, ectopic LSD1 rescue, in vivo anti-PD-1 treatment\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — mechanistic ubiquitination assay with specific linkage plus CDK9 dependence plus in vivo rescue experiment\",\n      \"pmids\": [\"38315842\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"HIF-1 recruits both FACT and RNF20/RNF40 to hypoxia response elements (HREs); FACT and RNF20/RNF40 stabilize HIF-1 binding at HREs and each other; hypoxia induces H2B K120 monoubiquitination at HIF-1 target genes in an HIF-1-dependent manner; FACT and RNF20/RNF40 are required for transcription initiation and elongation at HIF-1 target genes.\",\n      \"method\": \"ChIP, siRNA knockdown, RNA-seq, Co-IP\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — ChIP and Co-IP in multiple cell lines but single lab\",\n      \"pmids\": [\"38517892\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"RNF20 mediates RNA Polymerase II promoter-proximal pausing at highly paused endothelial cell genes; RNF20 restricts ERG-dependent Pol II pause release at these genes while binding Notch1 to promote H2Bub1 at Notch target genes; loss of Rnf20 leads to uncontrolled tip cell specification; RNF20 also orchestrates VEGFA mRNA splicing.\",\n      \"method\": \"CRISPR/Cas9 knockout, ChIP-seq, PRO-seq/GRO-seq (transcriptional pausing), RNA-seq, Co-IP with Notch1, in vivo angiogenesis assay\",\n      \"journal\": \"Nature cardiovascular research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — genome-wide PRO-seq plus Co-IP plus in vivo functional data with mechanistic detail\",\n      \"pmids\": [\"39322771\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"RNF20 localizes to replicating sites and promotes H2Bub there; RNF20 knockdown leads to MRE11/DNA2-dependent stalled fork degradation; RNF20 facilitates RAD51 and RAD51C loading at stalled forks; RING domain (catalytic) and ATR-mediated phosphorylation of RNF20 are essential for these replication stress responses; chromatin relaxation rescues fork protection defects in RNF20-depleted cells.\",\n      \"method\": \"DNA fiber assay, siRNA depletion, RING domain mutant, phospho-deficient mutant, Co-IP, immunofluorescence at replication forks, chromatin relaxation rescue\",\n      \"journal\": \"EMBO reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — RING domain mutagenesis plus phospho-mutant plus epistasis to fork remodelers\",\n      \"pmids\": [\"40495033\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"RNF20 regulates oocyte acentrosomal spindle assembly by recruiting tropomyosin 3 (TPM3) to centromeres and spindle poles via its coiled-coil motif; this function is independent of RNF20's E3 ligase activity; RNF20-depleted oocytes show abnormal spindle and chromosome misalignment.\",\n      \"method\": \"Oocyte-specific depletion, immunofluorescence, Co-IP, coiled-coil domain mapping, E3 ligase dead mutant\",\n      \"journal\": \"Advanced science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — domain mapping with ligase-dead mutant plus Co-IP, but single lab\",\n      \"pmids\": [\"38240347\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"RNF20 interacts with ZSCAN4 protein; RNF20 depletion increases ZSCAN4 protein levels and stabilizes its half-life without affecting ZSCAN4 mRNA, indicating RNF20 negatively regulates ZSCAN4 protein stability via ubiquitin-proteasome degradation.\",\n      \"method\": \"Co-IP, cycloheximide chase, siRNA knockdown, K48-polyubiquitination assay\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — Co-IP plus protein stability assay, single lab\",\n      \"pmids\": [\"29477841\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Conditional knockout of Rnf20 in Sertoli cells causes H2BK120ub loss, impairs transcription elongation of Cldn11 (tight junction component), disrupts cell adhesion and seminiferous tubule organization, and leads to apoptosis of spermatogonia and spermatocytes, causing male infertility recapitulating Sertoli cell-only syndrome.\",\n      \"method\": \"Conditional knockout mouse (Amh-Cre), ChIP, RNA-seq, western blot, histology\",\n      \"journal\": \"Cell & bioscience\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — conditional KO with ChIP demonstrating specific gene (Cldn11) as downstream effector\",\n      \"pmids\": [\"37024990\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"RNF20 and RNF40 are required for cardiomyocyte maturation; in vivo CRISPR screen identifies RNF20/40 as regulators; the H2BK120 monoubiquitination epigenetic mark controls dynamic gene expression changes required for cardiomyocyte maturation.\",\n      \"method\": \"In vivo somatic CRISPR screen, conditional validation, RNA-seq, ChIP\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — unbiased in vivo screen followed by mechanistic validation\",\n      \"pmids\": [\"34290256\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Intestine-specific deletion of Rnf20 or Rnf40 causes spontaneous colorectal inflammation; RNF20/RNF40 controls H3K4me3 occupancy and transcription of the Vitamin D Receptor (Vdr) gene and VDR target genes; loss of H2Bub1 promotes IBD-associated gene expression programs.\",\n      \"method\": \"Conditional knockout mice, ChIP-seq, RNA-seq, patient ChIP-seq\",\n      \"journal\": \"Cell death and differentiation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — conditional KO plus ChIP-seq demonstrating VDR as direct downstream target\",\n      \"pmids\": [\"34088983\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"RNF20 and RNF40 are required for DSB repair leading to both homologous recombination and class switch recombination (NHEJ) in mouse B cells; DSBs induce a global increase in H2Bub independent of H2AX phosphorylation; ATM and ATR jointly regulate IR-induced H2Bub.\",\n      \"method\": \"Conditional knockout B cells, class switch recombination assay, HR assay, western blot for H2Bub, γ-H2AX\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic KO with mechanistic epistasis to ATM/ATR and H2AX\",\n      \"pmids\": [\"30692271\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Ferritinophagy (selective autophagy) induced by iron deficiency causes RNF20 degradation through the autophagy-lysosomal pathway; reduced RNF20 decreases H2Bub1 at promoters of myogenic markers MyoD and MyoG, inhibiting myogenic differentiation and skeletal muscle regeneration.\",\n      \"method\": \"Iron deficiency model, lysosomal inhibitor, conditional NCOA4 KO, ChIP, myogenic differentiation assay\",\n      \"journal\": \"Science advances\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — conditional KO plus ChIP showing H2Bub1 at specific gene loci plus rescue experiments\",\n      \"pmids\": [\"37976359\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"RNF20 controls Rbx1 expression and thereby the activity of the VHL ubiquitin ligase complex and HIF1α protein levels; RNF20 haploinsufficiency results in inadequate tumor suppression via the Rnf20-H2Bub1-p53 axis and induces metabolic rewiring through HIF1α-mediated RNAPII promoter-proximal pause release (independent of H2Bub1).\",\n      \"method\": \"Mouse haploinsufficiency model, RNA-seq, ChIP-seq, western blot, metabolomics\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — in vivo genetic model plus ChIP-seq separating H2Bub1-dependent and independent mechanisms\",\n      \"pmids\": [\"40436847\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Upon cold stimuli in brown adipose tissue (BAT), RNF20 is rapidly downregulated allowing GABPα protein to accumulate (by controlling its stability), stimulating thermogenic gene expression; in inguinal white adipose tissue (iWAT), RNF20 is gradually upregulated upon prolonged cold and promotes beige adipogenesis via NCoR1 protein degradation to activate PPARγ (depot-specific substrates).\",\n      \"method\": \"Adipose tissue-specific KO mice, cold challenge experiments, Co-IP, protein stability assay, western blot, gene expression\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — depot-specific conditional KO with identification of distinct substrates (GABPα vs NCoR1) in each depot\",\n      \"pmids\": [\"38296968\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"KSHV RTA protein interacts with the RNF20/RNF40 complex; this interaction is necessary for RTA-induced KSHV lytic reactivation.\",\n      \"method\": \"Proteomics, Co-IP, siRNA knockdown, lytic reactivation assay\",\n      \"journal\": \"Journal of virology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — proteomics identification plus Co-IP plus functional lytic assay, single lab\",\n      \"pmids\": [\"37888983\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"H2B S112 GlcNAcylation allosterically stimulates RNF20/RNF40-RAD6A-mediated H2B K120 monoubiquitylation by interacting with the E2 enzyme RAD6A (not the E3 ligase); cryo-EM structure of trapped RNF20/RNF40-RAD6A-Ub-H2BS112GlcNAc nucleosome complex reveals the GlcNAc moiety enhances nucleophilicity of H2B K120; the C2 N-acetyl group and β-configuration of C1 are essential.\",\n      \"method\": \"Chemical synthesis of modified nucleosomes, cryo-EM, kinetics assays, mutagenesis, structure-activity relationship analysis\",\n      \"journal\": \"Nature chemical biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — cryo-EM structure plus mutagenesis plus quantitative kinetics with chemically synthesized substrates\",\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 reveal extensive interaction interfaces; the RNF20/RNF40-WAC interaction is critical for the H2BUb1 reaction in vitro and in vivo; key electrostatic interactions at these interfaces encode binding specificity.\",\n      \"method\": \"X-ray crystallography, AlphaFold structural modeling, in vitro ubiquitination assay, in vivo functional assay\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — crystal structure plus in vitro reconstitution plus mutagenesis of interface residues\",\n      \"pmids\": [\"41533567\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"RNF20 promotes STAT3 ubiquitination and degradation; exosomal miR-let-7b-5p targets RNF20, reducing it and thereby allowing STAT3/FOXO1 axis activation and insulin resistance in skeletal muscle cells; ectopic RNF20 expression attenuates this insulin resistance.\",\n      \"method\": \"Luciferase reporter (let-7b-5p target), Co-IP, ubiquitination assay, rescue experiment with RNF20 overexpression\",\n      \"journal\": \"Diabetes, metabolic syndrome and obesity\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — Co-IP plus ubiquitination assay plus functional rescue, single lab\",\n      \"pmids\": [\"37842335\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"RNF20 (hBre1) is localized to the IRF1 gene locus in both uninduced and IFNγ-induced states; RNAi-mediated depletion of RNF20 lowers both H2Bub and H3K4me3 but unexpectedly upregulates IFNγ-induced IRF1 transcription, indicating that H2Bub can exert a repressive effect on inducible transcription at this locus, possibly by affecting RNA Pol II CTD phosphorylation cycling.\",\n      \"method\": \"ChIP, RNAi depletion, qRT-PCR\",\n      \"journal\": \"Epigenetics & chromatin\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — ChIP plus functional RNAi with unexpected direction of effect, single lab\",\n      \"pmids\": [\"20825659\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"RNF20 is a RING-type E3 ubiquitin ligase that forms a heterodimeric complex with RNF40 and, together with E2 enzyme hRAD6A, monoubiquitinates histone H2B at lysine 120 (H2Bub1) on nucleosomal DNA—a modification whose structural mechanism has been resolved by cryo-EM; this activity requires the WAC adaptor that bridges the complex to RNA Pol II, is allosterically stimulated by H2B S112 GlcNAcylation acting on RAD6A, and is antagonized by USP44 deubiquitinase; H2Bub1 promotes downstream H3K4 and H3K79 methylation, facilitates transcription elongation, and drives SNF2h-dependent chromatin remodeling at DSBs—enabling DNA end resection, RAD51/BRCA1 recruitment, homologous recombination, and stalled fork protection (the last requiring ATR-mediated phosphorylation of RNF20); beyond histone modification, RNF20 ubiquitinates non-histone substrates including Eg5 (mitotic kinesin), LSD1 (K29-linked, CDK9-dependent), STAT3, AP-2α, and ZSCAN4, and its own stability is regulated by Smurf2-mediated degradation and by SARS-CoV-2 Mpro cleavage at Gln521; RNF20 is recruited to chromatin by context-specific adaptors (RPA at centromeres/DSBs, FACT at DSBs, HIF-1 at hypoxia response elements, Fbxl19 at CpG islands, p53 at target genes) and its activity underpins diverse processes including stem cell differentiation, cardiomyocyte maturation, adipogenesis, angiogenesis, NF-κB-driven inflammation, and antiviral signaling, acting as a broadly context-dependent tumor suppressor.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"RNF20 is a RING-type E3 ubiquitin ligase that, as a heterodimer with RNF40, catalyzes histone H2B lysine 120 monoubiquitination (H2Bub1)—a chromatin mark central to transcription elongation, mRNA splicing, DNA double-strand break repair, replication fork protection, and cell differentiation. The RNF20/RNF40 complex is recruited to chromatin by context-specific adaptors including WAC (linking it to RNA Pol II), RPA (at centromeres and DNA damage sites), FACT (at DSBs and hypoxia response elements), p53, HIF-1, and Fbxl19 (at CpG islands); H2Bub1 stimulates downstream H3K4 and H3K79 methylation, recruits SNF2h chromatin remodelers at DSBs and centromeres, and is read by the spliceosome component PRPF8 to couple transcription with mRNA processing [PMID:21329877, PMID:37155876, PMID:24357716, PMID:31152661, PMID:38517892, PMID:28453857]. Beyond histone modification, RNF20 ubiquitinates non-histone substrates—Eg5 (mitotic kinesin stabilization), LSD1 (K29-linked, CDK9-dependent stabilization suppressing endogenous retrovirus expression), NCoR1 (proteasomal degradation enabling PPARγ-driven adipogenesis), STAT3, AP-2α, and ZSCAN4—and possesses a ligase-independent recombination mediator function that directly facilitates Rad51 filament assembly on ssDNA [PMID:27557628, PMID:38315842, PMID:31604693, PMID:37230987, PMID:24374663]. RNF20 stability and activity are regulated by Smurf2-mediated proteasomal degradation, ferritinophagy-dependent lysosomal degradation, SARS-CoV-2 Mpro cleavage at Gln521, ATR-mediated phosphorylation during replication stress, and allosteric stimulation by H2B S112 GlcNAcylation acting on the E2 enzyme RAD6A [PMID:22231558, PMID:37976359, PMID:34452991, PMID:40495033, PMID:41495224].\",\n  \"teleology\": [\n    {\n      \"year\": 2010,\n      \"claim\": \"Initial chromatin studies revealed that RNF20 occupies gene loci and maintains H2Bub1 and H3K4me3, but that H2Bub1 can repress as well as activate inducible transcription, establishing RNF20 as a context-dependent transcriptional regulator rather than a simple activator.\",\n      \"evidence\": \"ChIP and RNAi depletion at the IRF1 locus showing unexpected transcriptional derepression upon RNF20 loss\",\n      \"pmids\": [\"20825659\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single locus studied; genome-wide scope of repressive versus activating H2Bub1 effects unknown\", \"Mechanism of repression (Pol II CTD cycling hypothesis) not directly tested\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Multiple studies converged to define RNF20 as the principal H2B K120 ubiquitin ligase operating in both transcription and DNA repair: WAC was identified as the adaptor bridging RNF20/RNF40 to elongating RNA Pol II, CDC73/parafibromin was shown to maintain H2Bub1 levels, and RNF20 was demonstrated to be essential for DSB-induced chromatin remodeling (via SNF2h), DNA end resection, RAD51/BRCA1 recruitment, and homologous recombination.\",\n      \"evidence\": \"Co-IP/domain mapping for WAC and CDC73, ChIP at DSBs, HR repair assays, H2B K120R mutant, radiation sensitivity\",\n      \"pmids\": [\"21329877\", \"22021426\", \"21362548\", \"22155569\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of WAC-RNF20 interaction unresolved at atomic level\", \"Whether CDC73 mutations cause disease through H2Bub1 loss versus other mechanisms unclear\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"RNF20 was established as a regulated enzyme whose levels are controlled by Smurf2-mediated proteasomal degradation, whose activity is opposed by USP44 deubiquitinase, and whose loss causes R-loop accumulation and genomic instability—linking H2Bub1 to both differentiation (ESC exit) and genome integrity.\",\n      \"evidence\": \"Smurf2 KO mouse model, USP44 depletion during ESC differentiation, R-loop detection and cytogenetics upon RNF20 depletion\",\n      \"pmids\": [\"22231558\", \"22681888\", \"22354749\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How Smurf2 itself is regulated at DSBs not determined\", \"Whether R-loops are a direct consequence of H2Bub1 loss or secondary to transcription-replication conflicts unclear\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"The chromatin adaptor FACT was identified as a DSB-specific recruiter of RNF20 (distinct from transcription-associated recruitment via PAF1), and RNF20 was shown to be co-opted by MLL-fusion oncoproteins to amplify Dot1l-dependent H3K79 methylation at leukemogenic target genes, revealing RNF20's role in both repair and oncogenesis.\",\n      \"evidence\": \"RING domain mutagenesis abolishing FACT interaction, PAF1-independence at DSBs, ChIP-seq/RNA-seq in MLL-fusion leukemia models, in vivo mouse leukemia\",\n      \"pmids\": [\"24357716\", \"23412334\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How FACT is recruited to DSBs independently of PAF1 not fully resolved\", \"Whether pharmacological targeting of RNF20 in MLL leukemia is feasible unknown\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"RNF20's substrate range was expanded beyond histones: it ubiquitinates AP-2α for proteasomal degradation, and adenovirus E1A was found to hijack RNF20 as a scaffold for viral transcription while blocking H2Bub1 to suppress interferon responses, establishing RNF20 as a target of viral immune evasion.\",\n      \"evidence\": \"Co-IP, ubiquitination assay for AP-2α; Co-IP and transcription reporter for E1A-RNF20 interaction\",\n      \"pmids\": [\"24374663\", \"23785282\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Ubiquitin chain type on AP-2α not characterized\", \"Whether E1A-RNF20 interaction is conserved across adenovirus serotypes not tested\", \"Single-lab findings for each substrate\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Arsenite was shown to directly bind RING finger cysteine residues of RNF20/RNF40, establishing the RING domain as a druggable interface and revealing a chemical mechanism for environmental carcinogenesis through H2Bub1 loss and DNA repair impairment.\",\n      \"evidence\": \"In vitro arsenite binding assay to RING domain, cellular H2Bub1 loss, impaired BRCA1/RAD51 recruitment\",\n      \"pmids\": [\"25170678\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether arsenic concentrations used reflect physiological exposure levels not fully addressed\", \"Selectivity of arsenite for RNF20 versus other RING E3 ligases not compared\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"The structural basis of E3 activity was revealed by crystal structures of the RNF20 RING domain with Ube2B~Ub conjugate, and the non-histone substrate Eg5 was identified as an RNF20/RNF40 target whose ubiquitination is required for mitotic spindle assembly, while loss of H2Bub1 was linked to NF-κB-driven inflammation and colorectal cancer.\",\n      \"evidence\": \"X-ray crystallography and mutagenesis of RING-E2 interface; Co-IP and ubiquitination assay for Eg5 with spindle phenotypes; ChIP and mouse haploinsufficiency model for NF-κB target regulation\",\n      \"pmids\": [\"27569044\", \"27557628\", \"26854224\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether Eg5 ubiquitination is mono- or polyubiquitination and the specific lysine(s) not mapped\", \"How H2Bub1 loss shifts NF-κB dimer composition mechanistically not resolved\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Fbxl19 was identified as a CpG-island-directed recruiter of RNF20 to gene promoters in ES cells, and RNF20 was shown to suppress renal cell carcinoma through SREBP1c inhibition, broadening the repertoire of chromatin recruitment mechanisms and tumor-suppressive outputs.\",\n      \"evidence\": \"Co-IP and genome-wide ChIP-seq for Fbxl19-RNF20; xenograft models and pharmacological SREBP1 inhibition for tumor suppression\",\n      \"pmids\": [\"28453857\", \"28827316\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct ubiquitination of SREBP1c by RNF20 not demonstrated\", \"Single-lab ChIP-seq for Fbxl19; independent validation needed\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Multiple non-histone substrates and adaptor mechanisms were defined: p53 recruits RNF20/RNF40 to p21/PUMA loci where H2Bub1 is read by spliceosome subunit PRPF8 for mRNA maturation; eEF1BδL is monoubiquitinated by RNF20/RAD6 to recruit p-TEFb for heat shock gene transcription; and RNF20 promotes adipogenesis by degrading NCoR1 to activate PPARγ. Additionally, RNF20/RNF40 was shown essential for both HR and NHEJ (class switch recombination) in B cells under ATM/ATR control.\",\n      \"evidence\": \"In vitro reconstituted ubiquitination for eEF1BδL; affinity purification revealing PRPF8; adipocyte-conditional KO mice; conditional KO B cells with CSR assays\",\n      \"pmids\": [\"31152661\", \"30649429\", \"31604693\", \"30692271\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether PRPF8-H2Bub1 interaction is general or gene-specific not resolved genome-wide\", \"ATR versus ATM individual contributions to H2Bub induction not separated cleanly\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"SARS-CoV-2 Mpro was shown to cleave RNF20 at Gln521, stabilizing SREBP1 and enhancing viral replication, establishing RNF20 as an antiviral factor; independently, unbiased in vivo CRISPR screens identified RNF20/RNF40 as required for cardiomyocyte maturation, and intestine-specific deletion revealed RNF20 controls VDR expression to prevent spontaneous colitis.\",\n      \"evidence\": \"Protease cleavage assay with catalytic mutant control; somatic CRISPR screen in mouse heart; intestinal conditional KO with ChIP-seq for VDR\",\n      \"pmids\": [\"34452991\", \"34290256\", \"34088983\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether Mpro cleavage occurs at endogenous viral loads not demonstrated\", \"Whether cardiomyocyte maturation defect is H2Bub1-dependent or involves non-histone substrates unclear\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"A ligase-independent function was discovered: RNF20/Bre1 directly binds Rad51, facilitates Rad51-ssDNA filament assembly and strand exchange, and counteracts anti-recombinases Srs2/FBH1, acting as a bona fide recombination mediator additive to BRCA2; simultaneously, RPA was identified as a recruiter of RNF20 to centromeric R-loops and DSBs, enabling Aurora B activation and chromosome segregation fidelity.\",\n      \"evidence\": \"In vitro reconstitution of Rad51 filament assembly/strand exchange; Co-IP and domain mapping for RPA-RNF20; genetic epistasis\",\n      \"pmids\": [\"37230987\", \"37155876\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Which domain of RNF20 mediates Rad51 binding not mapped\", \"Whether RPA-dependent RNF20 centromeric function is conserved in human meiosis unknown\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Tissue-specific conditional knockouts demonstrated RNF20's essential roles in spermatogenesis (via Cldn11 transcription elongation), myogenesis (where ferritinophagy-mediated RNF20 degradation impairs MyoD/MyoG expression), and viral biology (KSHV RTA interaction for lytic reactivation).\",\n      \"evidence\": \"Sertoli cell-specific KO with ChIP for Cldn11; iron deficiency model with NCOA4 KO for ferritinophagy; proteomics and Co-IP for KSHV RTA\",\n      \"pmids\": [\"37024990\", \"37976359\", \"37888983\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether ferritinophagy-RNF20 axis is relevant to iron deficiency anemia-associated myopathies in humans unknown\", \"KSHV RTA-RNF20 interaction: single lab, mechanism of exploitation unclear\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"CDK9-dependent K29-linked ubiquitination of LSD1 by RNF20 was identified as a mechanism suppressing endogenous retrovirus expression and interferon responses, creating epigenetic immunosuppression reversible by anti-PD-1 therapy; RNF20 was also shown to regulate Pol II promoter-proximal pausing at endothelial genes and to control angiogenesis through Notch1 interaction; depot-specific adipose substrates (GABPα in BAT, NCoR1 in iWAT) were defined; and RNF20 haploinsufficiency was linked to HIF1α-mediated metabolic rewiring independent of H2Bub1.\",\n      \"evidence\": \"K29-specific ubiquitination assay with CDK9 inhibition; PRO-seq/GRO-seq for Pol II pausing; depot-specific adipose KO mice; mouse haploinsufficiency model with ChIP-seq\",\n      \"pmids\": [\"38315842\", \"39322771\", \"38296968\", \"40436847\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether K29-linked LSD1 ubiquitination is unique to RNF20 or shared with other E3 ligases not tested\", \"Structural basis of Notch1-RNF20 interaction unknown\", \"H2Bub1-independent mechanisms at HIF1α remain poorly characterized molecularly\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"RNF20 was established as a replication fork protector: it localizes to replicating sites, promotes H2Bub there, facilitates RAD51/RAD51C loading at stalled forks, and prevents MRE11/DNA2-dependent fork degradation—functions requiring both RING domain catalytic activity and ATR-mediated phosphorylation.\",\n      \"evidence\": \"DNA fiber assay with RING and phospho-deficient mutants, epistasis to fork remodelers, chromatin relaxation rescue\",\n      \"pmids\": [\"40495033\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"ATR phosphorylation site(s) on RNF20 not mapped\", \"Whether fork protection involves non-histone substrates not addressed\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Cryo-EM structures of the full RNF20/RNF40-RAD6A-nucleosome complex revealed that H2B S112 GlcNAcylation allosterically stimulates ubiquitin transfer by contacting RAD6A (not the E3), enhancing K120 nucleophilicity; crystal structures of Bre1-Lge1 and AlphaFold models of the RNF20/RNF40-WAC complex defined the extensive adaptor interfaces essential for H2Bub1 in vivo.\",\n      \"evidence\": \"Cryo-EM with chemically synthesized GlcNAc-nucleosomes and kinetics; X-ray crystallography of Bre1-Lge1 with in vitro/in vivo mutagenesis\",\n      \"pmids\": [\"41495224\", \"41533567\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether GlcNAcylation-mediated stimulation occurs at all genomic loci or specific chromatin contexts unknown\", \"Full-length RNF20/RNF40 structure on nucleosome not yet determined\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Key open questions include: how RNF20 selects between histone and diverse non-histone substrates in different cellular contexts; the structural basis for ligase-independent Rad51 mediator activity; whether RNF20 mutations cause Mendelian disease in humans; and how ATR phosphorylation sites regulate RNF20 activity at replication forks versus DSBs.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No substrate-selection mechanism resolved\", \"No full-length structure of human RNF20/RNF40 heterodimer\", \"No Mendelian disease association established by direct genetic evidence\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 2, 10, 12, 16, 17, 18, 20, 25, 30, 39, 41]},\n      {\"term_id\": \"GO:0042393\", \"supporting_discovery_ids\": [0, 2, 12, 20, 39]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [22, 29]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005694\", \"supporting_discovery_ids\": [0, 6, 7, 21, 26, 28]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [0, 1, 7, 15, 42]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-4839726\", \"supporting_discovery_ids\": [0, 2, 7, 12, 20, 39, 40]},\n      {\"term_id\": \"R-HSA-73894\", \"supporting_discovery_ids\": [0, 6, 9, 21, 22, 34]},\n      {\"term_id\": \"R-HSA-74160\", \"supporting_discovery_ids\": [1, 7, 15, 26, 27, 42]},\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [10, 28]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [2, 24, 31, 32]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [12, 17, 20, 25, 39, 40]},\n      {\"term_id\": \"R-HSA-8953854\", \"supporting_discovery_ids\": [15, 27]}\n    ],\n    \"complexes\": [\n      \"RNF20/RNF40 heterodimer\",\n      \"RNF20/RNF40-RAD6A (hBre1-hRAD6) E3-E2 complex\"\n    ],\n    \"partners\": [\n      \"RNF40\",\n      \"RAD6A\",\n      \"WAC\",\n      \"FACT\",\n      \"RPA\",\n      \"RAD51\",\n      \"Smurf2\",\n      \"Notch1\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}