{"gene":"RIOX2","run_date":"2026-06-10T06:43:36","timeline":{"discoveries":[{"year":2002,"finding":"mina53 (RIOX2) is a direct transcriptional target of c-Myc: c-Myc protein binds the mina53 promoter E-box sites in vivo, and ectopic c-Myc (but not a transactivation-domain mutant) induces mina53 mRNA even in the presence of protein synthesis inhibitors. RNAi knockdown of mina53 severely suppresses cell proliferation.","method":"Chromatin immunoprecipitation (ChIP) of c-Myc at mina53 promoter; promoter-reporter assays; c-MycER activation with cycloheximide; RNA interference with proliferation readout","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (ChIP, reporter assay, cycloheximide block, RNAi) in a single focused study, replicated in multiple cell contexts","pmids":["12091391"],"is_preprint":false},{"year":2005,"finding":"NO52 (RIOX2) localizes constitutively to the granular component of nucleoli, is present in free preribosomal particles but absent from cytoplasmic ribosomes, and co-immunoprecipitates with ribosomal proteins and non-ribosomal nucleolar proteins. Its nucleolar accumulation depends on ongoing rRNA transcription and the metabolic state of the cell.","method":"Immunolocalization; subcellular fractionation; co-immunoprecipitation followed by MALDI-MS; treatment with RNase A, actinomycin D, and serum starvation","journal":"European journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal co-IP + MS identification of interactors + functional perturbations (RNase A, actinomycin D, serum starvation) in one study","pmids":["15819408"],"is_preprint":false},{"year":2009,"finding":"mdig (RIOX2) demethylates tri-methyl lysine 9 of histone H3 (H3K9me3): overexpression reduces H3K9me3 at the rRNA gene promoter and increases RNA Pol I occupancy and rRNA transcription, while gene silencing has the opposite effect.","method":"Gene overexpression and siRNA knockdown; chromatin immunoprecipitation (ChIP) for H3K9me3 and RNA Pol I; rRNA expression assays","journal":"Cell cycle (Georgetown, Tex.)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ChIP with gain- and loss-of-function in the same study, single lab, no in vitro reconstitution","pmids":["19502796"],"is_preprint":false},{"year":2013,"finding":"mdig (RIOX2) demethylates H3K9me3 at the promoter of the imprinted H19 lncRNA gene: overexpression reduces H3K9me3 at the H19 promoter and activates H19 transcription; shRNA/siRNA knockdown increases H3K9me3 and reduces H19 expression. In vitro demethylation assay with immunoprecipitated mdig and an H3K9me3 peptide confirmed catalytic activity.","method":"Overexpression and shRNA/siRNA knockdown with ChIP for H3K9me3; in vitro demethylation assay using immunoprecipitated mdig protein and histone H3 peptide substrate","journal":"Oncotarget","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro demethylation assay plus cellular ChIP, single lab, two orthogonal methods","pmids":["23965803"],"is_preprint":false},{"year":2013,"finding":"Mina53-deficient mice show reduced Th17 cell infiltration into airways and increased Treg cell infiltration following allergen (house dust mite) challenge, with lower IL-4 and IL-5 levels, demonstrating that Mina53 (RIOX2) regulates the Th17/Treg balance and allergic airway responses.","method":"Mina53-knockout mouse model; intranasal allergen challenge; bronchoalveolar lavage cell differential counts; ELISA for cytokines; airway hyperresponsiveness measurement","journal":"Cell structure and function","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean KO mouse with defined cellular and cytokine phenotype, single lab","pmids":["23748603"],"is_preprint":false},{"year":2015,"finding":"mdig (RIOX2) protein co-immunoprecipitates with DNA double-strand break repair and chromatin-binding proteins XRCC5, XRCC6, RBBP4, CBX8, PRMT5, and TDRD in lung cancer (A549) and bronchial epithelial (BEAS-2B) cells, validated by reciprocal co-immunoprecipitation.","method":"Co-immunoprecipitation with anti-mdig antibody followed by nanoESI-MS/MS proteomics (Orbitrap); reciprocal co-IP validation; four independent experiments","journal":"Oncotarget","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal co-IP plus mass spectrometry in two cell lines, single lab","pmids":["26293673"],"is_preprint":false},{"year":2015,"finding":"mdig (RIOX2) regulates cell cycle progression through p27(KIP1): knockdown of mdig increases p27(KIP1) mRNA and protein and inhibits phosphorylation of p27 at Thr187, causing cell cycle arrest; in human lung cancer tissues, mdig upregulation inversely correlates with p27(KIP1) levels.","method":"siRNA knockdown in A549 cells; MTT proliferation assay; cell cycle analysis; RT-qPCR for cell cycle regulators; Western blot for p27(KIP1) and phospho-p27(KIP1) isoforms; Western blot on human lung cancer tissue samples","journal":"Tumour biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — loss-of-function with multiple molecular readouts (mRNA, total protein, phospho-protein) plus in vivo tissue correlation, single lab","pmids":["25851349"],"is_preprint":false},{"year":2016,"finding":"mdig (RIOX2) directly interacts with c-Myc and JAK1 in multiple myeloma cell lines, contributing to hyperactivation of the IL-6-JAK-STAT3 signaling pathway; genetic silencing of mdig reduces activity of downstream effectors in this pathway.","method":"Co-immunoprecipitation; integrative genomics and proteomics; siRNA knockdown with pathway activity readouts (Western blot for STAT3 phosphorylation)","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP plus proteomics plus functional siRNA knockdown, single lab","pmids":["27833099"],"is_preprint":false},{"year":2017,"finding":"MDIG (RIOX2) regulates H3K9me3 at the p21(CIP1/WAF1) promoter in hepatocellular carcinoma: MDIG overexpression reduces H3K9me3 and activates p21 expression; knockdown has the opposite effect, influencing HCC cell proliferation and migration.","method":"Gain- and loss-of-function experiments (overexpression and siRNA knockdown) in HCC cells; ChIP for H3K9me3; Western blot for p21; xenograft tumor model","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ChIP plus gain/loss-of-function plus in vivo xenograft, single lab","pmids":["28471446"],"is_preprint":false},{"year":2018,"finding":"MINA53 (RIOX2) regulates expression of the CDC45-MCM-GINS (CMG) complex genes required for DNA replication initiation; knockdown reduces CMG gene expression, induces DNA replication stress, diminishes ATM/ATR-H2AX DNA damage response, and leads to glioblastoma cell apoptosis.","method":"siRNA knockdown in glioblastoma cells; DNA replication initiation assays; RT-qPCR and Western blot for CMG genes; flow cytometry for apoptosis; Western blot for ATM/ATR-H2AX pathway components","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — loss-of-function with multiple molecular readouts (replication initiation, DDR pathway, apoptosis), single lab","pmids":["30333481"],"is_preprint":false},{"year":2019,"finding":"MINA53 (RIOX2) preferentially demethylates H3K36me3 in vitro, and its depletion by RNAi increases local H3K36me3 levels at the HIV-1 LTR, promoting HIV-1 latency reversal. The pan-JmjC inhibitor JIB-04 inhibits MINA53-mediated H3K36me3 demethylation and synergizes with latency-reversing agents to reactivate latent HIV-1.","method":"CRISPR/Cas9 screen; RNAi depletion; in vitro histone demethylation assay; ChIP for H3K36me3 at LTR; HIV-1 latency reactivation assays; JIB-04 inhibitor treatment","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — in vitro demethylation assay plus cellular ChIP plus CRISPR functional screen, multiple orthogonal methods","pmids":["31165872"],"is_preprint":false},{"year":2018,"finding":"Loss of mdig (RIOX2) expression enhances global DNA methylation and H3K9me3 heterochromatin, and increases migration and invasion of triple-negative breast cancer cells, indicating mdig acts as an inhibitor of DNA and histone methylation and suppresses metastatic behavior in advanced breast cancer.","method":"siRNA/shRNA knockdown; global DNA methylation assay; chromatin accessibility assay; ChIP for H3K9me3; Transwell migration/invasion assays","journal":"Signal transduction and targeted therapy","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal epigenetic assays plus functional migration readout, single lab","pmids":["30254753"],"is_preprint":false},{"year":2020,"finding":"ZNF143 promotes expression of MDIG (RIOX2) by direct transcriptional activation; MDIG in turn reduces H3K9me3 at the CDC6 promoter to activate CDC6 transcription and accelerate HCC cell-cycle progression, establishing a ZNF143-MDIG-CDC6 oncoprotein axis.","method":"ChIP for ZNF143 at MDIG promoter; gain- and loss-of-function experiments; ChIP for H3K9me3 at CDC6 promoter; Western blot; in vitro and in vivo tumor growth assays","journal":"Cancer research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ChIP-based pathway placement plus gain/loss-of-function plus in vivo xenograft, single lab","pmids":["32312832"],"is_preprint":false},{"year":2018,"finding":"Phylogenetic and structural analyses show that RIOX2 (MINA53) and RIOX1 (NO66) share conserved active-site residues within their JmjC domains, a dimerization domain, and a winged-helix domain; the proteins catalyze C-3 histidine hydroxylation in ribosomal proteins (Rpl27a for MINA53). RIOX2 has a distinct subnuclear localization in Hydra compared to human, indicating evolutionary adaptation.","method":"Phylogenomic analysis of 49 metazoan species; domain architecture comparison; immunofluorescence for subnuclear localization in HeLa cells and Hydra","journal":"BMC evolutionary biology","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — localization by immunofluorescence plus evolutionary structural analysis; histidine hydroxylase activity inferred from conservation but directly assayed in companion biochemical literature","pmids":["29914368"],"is_preprint":false},{"year":2023,"finding":"MINA53 (RIOX2) is a histidine hydroxylase with narrow substrate selectivity: it catalyzes C-3 hydroxylation of a histidine residue in ribosomal protein Rpl27a. Inhibition assays with histidine analogues in Rpl peptides showed that MINA53 activity can be inhibited by competition with non-oxidized peptides containing acyclic side-chain analogues (Asn, Gln, homoGln).","method":"In vitro biochemical assays with natural and unnatural histidine analogues incorporated into Rpl peptides; substrate selectivity assays; inhibition assays","journal":"RSC chemical biology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution with defined peptide substrates and mutagenic analogues, rigorous biochemical characterization, single lab","pmids":["36908702"],"is_preprint":false},{"year":2023,"finding":"MDIG (RIOX2) demethylates H3K9me3 at the OTX2 promoter to increase chromatin accessibility, allowing OTX2 transcription factor expression; OTX2 then binds the Myc promoter to activate Myc expression, forming a positive feedback loop that sustains hepatocyte proliferation during liver regeneration.","method":"Liver-specific MDIG knockout mice; partial hepatectomy and CCl4 injury models; ChIP for H3K9me3 at OTX2 promoter; ATAC-seq for chromatin accessibility; ChIP for OTX2 at Myc promoter; RNA-seq","journal":"Signal transduction and targeted therapy","confidence":"High","confidence_rationale":"Tier 2 / Strong — tissue-specific KO mouse model with multiple orthogonal genomic methods (ChIP, ATAC-seq, RNA-seq) establishing mechanistic pathway, single lab","pmids":["37709738"],"is_preprint":false},{"year":2024,"finding":"Mina53 (RIOX2) is a bona fide H4R3me2a (asymmetric di-methylation at arginine 3 of histone H4) eraser: identified as an H4R3me2a interactor by photoaffinity capture, it demethylates H4R3me2a in vitro and in cells. In a transgenic mouse with neural stem/progenitor cell-specific Mina53 deletion, failure to demethylate H4R3me2a dysregulates genes for NSC proliferation and differentiation, impairing cognitive function.","method":"Photoaffinity capture for interactor identification; in vitro demethylation biochemical assay; cellular H4R3me2a quantification; molecular dynamics simulation; transgenic conditional KO mouse with cognitive testing","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution of demethylase activity + photoaffinity capture + molecular dynamics + conditional KO mouse with functional phenotype, multiple orthogonal methods in one study","pmids":["39587091"],"is_preprint":false},{"year":2025,"finding":"Mina53 (RIOX2) is an arginine demethylase that removes asymmetric dimethylation at arginine 337 of p53 (p53 R337me2a): demethylation reduces p53 protein stability and oligomerization, alters chromatin modifications at p53 target gene promoters, and suppresses p53-mediated transcriptional activation and cell-cycle arrest, thereby promoting tumor growth in mouse xenograft and spontaneous tumor models.","method":"In vitro demethylation assay; mass spectrometry for p53 arginine methylation status; co-immunoprecipitation for oligomerization; ChIP for chromatin modifications at p53 target promoters; xenograft and spontaneous tumor mouse models; loss-of-function experiments","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution of demethylase activity on p53 R337me2a + MS validation + structural stability assays + in vivo tumor models, multiple orthogonal methods","pmids":["39864061"],"is_preprint":false},{"year":2017,"finding":"mdig (RIOX2) suppresses epithelial-mesenchymal transition (EMT) in NSCLC by inhibiting GSK-3β phosphorylation, which promotes β-catenin phosphorylation and destabilization, downregulating slug, snail, and ZEB1 transcription factors, thereby increasing epithelial markers (E-cadherin, claudin-1) and decreasing mesenchymal markers (vimentin, N-cadherin).","method":"Lentiviral overexpression and knockdown in A549 and HUVEC cells; Transwell invasion/migration assays; Western blot for GSK-3β, β-catenin, EMT markers and transcription factors","journal":"International journal of oncology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — gain- and loss-of-function with molecular pathway readouts in two cell lines, single lab","pmids":["29039479"],"is_preprint":false},{"year":2019,"finding":"MDIG (RIOX2) promotes cisplatin resistance in lung adenocarcinoma by activating WNT/β-catenin signaling, which upregulates ABC transporter expression (ABCB1, ABCC1, ABCG2); overexpression increases IC50 for cisplatin while knockdown reduces it.","method":"siRNA knockdown and overexpression in A549 and A549/DDP cisplatin-resistant cells; RT-qPCR and Western blot for MDIG, ABC transporters, WNT pathway components; IC50 determination","journal":"Oncology letters","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — gain- and loss-of-function with molecular pathway and functional drug resistance readouts, single lab","pmids":["31579066"],"is_preprint":false},{"year":2021,"finding":"mdig (RIOX2) depletion by CRISPR-Cas9 in bronchial epithelial cells reduces expression of SARS-CoV-2 receptors NRP1 and NRP2, cathepsins, and glycan metabolism genes, associated with enrichment of H3K9me3 and/or H3K27me3 at these loci as determined by ChIP-seq.","method":"CRISPR-Cas9 gene editing; ChIP-seq for H3K9me3/H3K27me3; RT-qPCR and Western blot for NRP1, NRP2, cathepsins; gene ontology analysis","journal":"Theranostics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — CRISPR KO with genome-wide ChIP-seq and gene expression validation, single lab","pmids":["34335974"],"is_preprint":false},{"year":2022,"finding":"Mdig (RIOX2) deletion in triple-negative breast cancer MDA-MB-231 cells enhances global H3K36me3 and upregulates X-chromosome-linked genes for cell motility and invasion (including MAGED2); silencing MAGED2 partially reverses the invasive migration of mdig-KO cells, establishing H3K36me3/MAGED2 as a downstream effector axis.","method":"CRISPR-Cas9 mdig knockout; ChIP-seq for H3K36me3; RNA-seq; siRNA knockdown of MAGED2; in vivo orthotopic xenograft; Transwell migration assay","journal":"iScience","confidence":"High","confidence_rationale":"Tier 2 / Strong — CRISPR KO with genome-wide ChIP-seq + RNA-seq + in vivo xenograft + rescue experiment (MAGED2 knockdown), multiple orthogonal methods in one study","pmids":["36124233"],"is_preprint":false}],"current_model":"RIOX2 (MINA53/mdig) is a multifunctional JmjC-domain oxygenase that acts as a histone eraser — catalyzing demethylation of H3K9me3, H3K36me3, and H4R3me2a — and as an arginine demethylase for p53 (removing R337me2a to destabilize p53 and suppress cell-cycle arrest); it also hydroxylates the C-3 position of a histidine in ribosomal protein Rpl27a, and is a direct transcriptional target of c-Myc that localizes to nucleolar preribosomal particles, interacts with DNA repair factors (XRCC5, XRCC6, RBBP4) and c-Myc/JAK1, and controls cell proliferation, EMT, immune Th17/Treg balance, and liver regeneration through epigenetic regulation of key target gene promoters."},"narrative":{"mechanistic_narrative":"RIOX2 (MINA53/mdig) is a JmjC-domain 2-oxoglutarate oxygenase that couples chromatin modification to cell proliferation, acting both as a histone-modification \"eraser\" and as a protein hydroxylase [PMID:19502796, PMID:36908702, PMID:39587091]. As a demethylase it removes repressive H3K9me3 at target promoters and preferentially demethylates H3K36me3 in vitro, and it also functions as an arginine demethylase for histone H4R3me2a [PMID:19502796, PMID:31165872, PMID:39587091]. Through H3K9me3 erasure it derepresses growth-promoting loci including rRNA genes, the H19 lncRNA, p21(CIP1/WAF1), CDC6, and OTX2, the last sustaining a self-reinforcing OTX2–Myc loop that drives hepatocyte proliferation during liver regeneration [PMID:19502796, PMID:23965803, PMID:28471446, PMID:32312832, PMID:37709738]. Beyond histones, RIOX2 is an arginine demethylase for p53, removing R337me2a to reduce p53 stability and oligomerization and thereby suppress p53-dependent cell-cycle arrest and promote tumor growth [PMID:39864061]. It is a histidine hydroxylase that catalyzes C-3 hydroxylation of a specific histidine in ribosomal protein Rpl27a [PMID:36908702]. Consistent with these activities, RIOX2 is a direct c-Myc transcriptional target whose expression is required for proliferation, localizes constitutively to the nucleolar granular component and preribosomal particles, and modulates cell-cycle progression, EMT, replication, drug resistance, and Th17/Treg immune balance [PMID:12091391, PMID:15819408, PMID:25851349, PMID:30333481, PMID:29039479]. Its catalytic loss broadly reshapes the epigenome — deletion raises global H3K9me3, H3K36me3, and DNA methylation, altering motility and invasion programs in cancer cells [PMID:30254753, PMID:36124233].","teleology":[{"year":2002,"claim":"Establishing what drives RIOX2 expression and why it matters: defining it as a direct c-Myc effector linked the gene to proliferative control from the outset.","evidence":"ChIP, promoter-reporter, c-MycER/cycloheximide block, and RNAi with proliferation readout in cultured cells","pmids":["12091391"],"confidence":"High","gaps":["Did not define the biochemical activity of the protein","Mechanism linking expression to proliferation unresolved"]},{"year":2005,"claim":"Localizing the protein answered where it acts: constitutive nucleolar/preribosomal residence tied RIOX2 to ribosome biogenesis and rRNA metabolism.","evidence":"Immunolocalization, fractionation, co-IP/MALDI-MS, and RNase A/actinomycin D/serum-starvation perturbations","pmids":["15819408"],"confidence":"High","gaps":["Interactor identities not resolved to specific functional partners","No catalytic activity demonstrated"]},{"year":2009,"claim":"First assignment of a catalytic function: RIOX2 demethylates H3K9me3 at rRNA gene promoters to license Pol I transcription.","evidence":"Gain/loss-of-function with ChIP for H3K9me3 and Pol I plus rRNA assays","pmids":["19502796"],"confidence":"Medium","gaps":["No in vitro reconstitution in this study","Direct vs indirect demethylation not separated"]},{"year":2013,"claim":"Confirmed direct H3K9me3 demethylase activity and extended it to an imprinted locus, showing catalysis is intrinsic to RIOX2.","evidence":"In vitro demethylation with immunoprecipitated mdig plus cellular ChIP at the H19 promoter","pmids":["23965803"],"confidence":"Medium","gaps":["Immunoprecipitated (not purified recombinant) enzyme used","Substrate specificity versus other marks not addressed"]},{"year":2013,"claim":"Defined a physiological role in immunity: RIOX2 controls Th17/Treg balance and allergic airway responses in vivo.","evidence":"Mina53-knockout mice with allergen challenge, BAL differentials, cytokine ELISA, and airway hyperresponsiveness","pmids":["23748603"],"confidence":"Medium","gaps":["Molecular target in T cells not identified","Catalytic dependence of the phenotype untested"]},{"year":2015,"claim":"Mapped the protein interactome, placing RIOX2 with DNA repair and chromatin factors.","evidence":"Reciprocal co-IP plus nanoESI-MS/MS in A549 and BEAS-2B cells (XRCC5, XRCC6, RBBP4, CBX8, PRMT5, TDRD)","pmids":["26293673"],"confidence":"Medium","gaps":["Functional consequence of each interaction undefined","Direct vs complex-mediated binding not resolved"]},{"year":2015,"claim":"Connected RIOX2 to cell-cycle regulation through p27(KIP1), explaining its proliferative requirement.","evidence":"siRNA knockdown with proliferation/cell-cycle assays, p27 and phospho-p27 Western blots, and tumor-tissue correlation","pmids":["25851349"],"confidence":"Medium","gaps":["Whether p27 regulation is epigenetic or indirect unclear","No catalytic-dead control"]},{"year":2016,"claim":"Identified signaling partners, showing RIOX2 binds c-Myc and JAK1 to amplify IL-6–JAK–STAT3 signaling.","evidence":"Co-IP, integrative genomics/proteomics, and siRNA knockdown with STAT3 phosphorylation readout in myeloma cells","pmids":["27833099"],"confidence":"Medium","gaps":["Direct binding interface not mapped","Catalytic involvement in signaling untested"]},{"year":2017,"claim":"Extended the H3K9me3-demethylase paradigm to tumor-suppressor control via p21 in hepatocellular carcinoma.","evidence":"Gain/loss-of-function, ChIP for H3K9me3 at the p21 promoter, and xenograft model","pmids":["28471446"],"confidence":"Medium","gaps":["Single-lab observation","Other p21-controlling inputs not excluded"]},{"year":2017,"claim":"Defined a context-dependent anti-EMT role through GSK-3β/β-catenin signaling in NSCLC.","evidence":"Overexpression/knockdown with invasion assays and Western blots for EMT markers and transcription factors","pmids":["29039479"],"confidence":"Medium","gaps":["Link between catalytic activity and β-catenin signaling unestablished","Cell-line restricted"]},{"year":2018,"claim":"Provided structural/evolutionary framing and assigned histidine hydroxylase activity on Rpl27a, distinguishing RIOX2 catalysis from pure demethylation.","evidence":"Phylogenomics across 49 species, domain comparison, and immunofluorescence localization (human vs Hydra)","pmids":["29914368"],"confidence":"Medium","gaps":["Hydroxylase activity inferred here, assayed directly later","Functional consequence of Rpl27a hydroxylation unknown"]},{"year":2018,"claim":"Showed catalytic loss reshapes the epigenome and metastatic behavior, casting RIOX2 as a suppressor of DNA/histone methylation in TNBC.","evidence":"Knockdown with global DNA-methylation, chromatin accessibility, H3K9me3 ChIP, and migration/invasion assays","pmids":["30254753"],"confidence":"Medium","gaps":["Direct vs indirect effects on global methylation not separated"]},{"year":2018,"claim":"Linked RIOX2 to DNA replication licensing, explaining proliferation arrest upon loss.","evidence":"siRNA knockdown in glioblastoma with replication-initiation assays, CMG gene expression, DDR Western blots, and apoptosis flow cytometry","pmids":["30333481"],"confidence":"Medium","gaps":["Whether CMG genes are direct chromatin targets untested"]},{"year":2019,"claim":"Established H3K36me3 as a preferred substrate in vitro and a druggable role in HIV-1 latency.","evidence":"CRISPR screen, RNAi, in vitro demethylation assay, LTR ChIP, latency reactivation, and JIB-04 inhibition","pmids":["31165872"],"confidence":"High","gaps":["Relative cellular preference for H3K9me3 vs H3K36me3 not quantified"]},{"year":2019,"claim":"Connected RIOX2 to chemoresistance via WNT/β-catenin-driven ABC transporter expression.","evidence":"Knockdown/overexpression in cisplatin-resistant lung cells with IC50, ABC transporter, and WNT pathway readouts","pmids":["31579066"],"confidence":"Medium","gaps":["Catalytic dependence and direct target genes unresolved"]},{"year":2020,"claim":"Placed RIOX2 in an upstream-downstream transcriptional axis: ZNF143 induces MDIG, which derepresses CDC6 via H3K9me3 removal.","evidence":"ChIP at MDIG and CDC6 promoters, gain/loss-of-function, and in vitro/in vivo tumor assays in HCC","pmids":["32312832"],"confidence":"Medium","gaps":["Single-lab pathway placement","Direct CDC6 promoter binding not shown"]},{"year":2021,"claim":"Used genome-wide profiling to show RIOX2 controls host genes for viral entry through H3K9me3/H3K27me3 chromatin states.","evidence":"CRISPR-Cas9 KO with ChIP-seq and expression validation of NRP1/NRP2, cathepsins, glycan genes","pmids":["34335974"],"confidence":"Medium","gaps":["Direct demethylase action vs indirect chromatin effects not distinguished"]},{"year":2022,"claim":"Defined an H3K36me3-dependent invasion program with a rescuable downstream effector (MAGED2).","evidence":"CRISPR KO with H3K36me3 ChIP-seq, RNA-seq, MAGED2 knockdown rescue, orthotopic xenograft, and migration assays in TNBC","pmids":["36124233"],"confidence":"High","gaps":["Why X-linked loci are preferentially affected unexplained"]},{"year":2023,"claim":"Rigorously characterized RIOX2 as a substrate-selective histidine hydroxylase on Rpl27a, with defined inhibitory analogues.","evidence":"In vitro biochemistry with natural/unnatural histidine analogue peptides and inhibition assays","pmids":["36908702"],"confidence":"High","gaps":["Cellular consequence of Rpl27a hydroxylation on translation unresolved"]},{"year":2023,"claim":"Established a physiological demethylase circuit in vivo: MDIG erases H3K9me3 at OTX2 to drive an OTX2–Myc loop sustaining liver regeneration.","evidence":"Liver-specific KO mice, hepatectomy/CCl4 injury, ChIP, ATAC-seq, OTX2 ChIP at Myc, and RNA-seq","pmids":["37709738"],"confidence":"High","gaps":["Whether the same loop operates in non-hepatic tissues untested"]},{"year":2024,"claim":"Expanded the substrate repertoire to arginine methylation: RIOX2 is a bona fide H4R3me2a eraser important for neural stem cell programs and cognition.","evidence":"Photoaffinity capture, in vitro and cellular demethylation, molecular dynamics, and NSC-specific conditional KO with cognitive testing","pmids":["39587091"],"confidence":"High","gaps":["Genome-wide H4R3me2a targets not fully mapped"]},{"year":2025,"claim":"Identified a non-histone arginine demethylase substrate, p53 R337me2a, linking RIOX2 catalysis directly to tumor-suppressor inactivation.","evidence":"In vitro demethylation, MS validation, oligomerization co-IP, target-promoter ChIP, and xenograft/spontaneous tumor models","pmids":["39864061"],"confidence":"High","gaps":["Crosstalk between p53 demethylation and histone activities unresolved"]},{"year":null,"claim":"How RIOX2 selects among its multiple chemistries (H3K9me3, H3K36me3, H4R3me2a, p53 R337me2a demethylation, and Rpl27a hydroxylation) and is targeted to specific substrates and loci in a given context remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structural model integrating substrate selectivity","Recruitment/targeting mechanism to specific promoters unknown","Relative contribution of catalytic vs scaffolding functions unquantified"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140098","term_label":"catalytic activity, acting on RNA","supporting_discovery_ids":[2,3,10,16,17]},{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[14]},{"term_id":"GO:0016491","term_label":"oxidoreductase activity","supporting_discovery_ids":[2,10,14,16]},{"term_id":"GO:0140110","term_label":"transcription regulator activity","supporting_discovery_ids":[0,12,15]}],"localization":[{"term_id":"GO:0005730","term_label":"nucleolus","supporting_discovery_ids":[1,13]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[2,3,8]}],"pathway":[{"term_id":"R-HSA-4839726","term_label":"Chromatin organization","supporting_discovery_ids":[2,3,8,15,16]},{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[6,9,12]},{"term_id":"R-HSA-74160","term_label":"Gene expression (Transcription)","supporting_discovery_ids":[0,12,15]}],"complexes":[],"partners":["MYC","JAK1","XRCC5","XRCC6","RBBP4","CBX8","PRMT5"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q8IUF8","full_name":"Ribosomal oxygenase 2","aliases":["60S ribosomal protein L27a histidine hydroxylase","Bifunctional lysine-specific demethylase and histidyl-hydroxylase MINA","Histone lysine demethylase MINA","MYC-induced nuclear antigen","Mineral dust-induced gene protein","Nucleolar protein 52","Ribosomal oxygenase MINA","ROX"],"length_aa":465,"mass_kda":52.8,"function":"Oxygenase that can act as both a histone lysine demethylase and a ribosomal histidine hydroxylase. Is involved in the demethylation of trimethylated 'Lys-9' on histone H3 (H3K9me3), leading to an increase in ribosomal RNA expression. Also catalyzes the hydroxylation of 60S ribosomal protein L27a on 'His-39'. May play an important role in cell growth and survival. May be involved in ribosome biogenesis, most likely during the assembly process of pre-ribosomal particles","subcellular_location":"Nucleus; Nucleus, nucleolus","url":"https://www.uniprot.org/uniprotkb/Q8IUF8/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/RIOX2","classification":"Not Classified","n_dependent_lines":5,"n_total_lines":1208,"dependency_fraction":0.0041390728476821195},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"NPM1","stoichiometry":0.2},{"gene":"RPS16","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/RIOX2","total_profiled":1310},"omim":[{"mim_id":"612049","title":"RIBOSOMAL OXYGENASE 2; RIOX2","url":"https://www.omim.org/entry/612049"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Nucleoplasm","reliability":"Supported"},{"location":"Nucleoli","reliability":"Supported"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in 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mineral dust-induced gene, mdig, regulates angiogenesis and lymphangiogenesis in lung adenocarcinoma by modulating the expression of VEGF-A/C/D via EGFR and HIF-1α signaling.","date":"2021","source":"Oncology reports","url":"https://pubmed.ncbi.nlm.nih.gov/33760153","citation_count":12,"is_preprint":false},{"pmid":"29039479","id":"PMC_29039479","title":"Mdig suppresses epithelial-mesenchymal transition and inhibits the invasion and metastasis of non‑small cell lung cancer via regulating GSK-3β/β-catenin signaling.","date":"2017","source":"International journal of oncology","url":"https://pubmed.ncbi.nlm.nih.gov/29039479","citation_count":11,"is_preprint":false},{"pmid":"34335974","id":"PMC_34335974","title":"Environmentally-induced mdig contributes to the severity of COVID-19 through fostering expression of SARS-CoV-2 receptor NRPs and glycan 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c-Myc protein binds the mina53 promoter E-box sites in vivo, and ectopic c-Myc (but not a transactivation-domain mutant) induces mina53 mRNA even in the presence of protein synthesis inhibitors. RNAi knockdown of mina53 severely suppresses cell proliferation.\",\n \"method\": \"Chromatin immunoprecipitation (ChIP) of c-Myc at mina53 promoter; promoter-reporter assays; c-MycER activation with cycloheximide; RNA interference with proliferation readout\",\n \"journal\": \"The Journal of biological chemistry\",\n \"confidence\": \"High\",\n \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (ChIP, reporter assay, cycloheximide block, RNAi) in a single focused study, replicated in multiple cell contexts\",\n \"pmids\": [\"12091391\"],\n \"is_preprint\": false\n },\n {\n \"year\": 2005,\n \"finding\": \"NO52 (RIOX2) localizes constitutively to the granular component of nucleoli, is present in free preribosomal particles but absent from cytoplasmic ribosomes, and co-immunoprecipitates with ribosomal proteins and non-ribosomal nucleolar proteins. Its nucleolar accumulation depends on ongoing rRNA transcription and the metabolic state of the cell.\",\n \"method\": \"Immunolocalization; subcellular fractionation; co-immunoprecipitation followed by MALDI-MS; treatment with RNase A, actinomycin D, and serum starvation\",\n \"journal\": \"European journal of cell biology\",\n \"confidence\": \"High\",\n \"confidence_rationale\": \"Tier 2 / Strong — reciprocal co-IP + MS identification of interactors + functional perturbations (RNase A, actinomycin D, serum starvation) in one study\",\n \"pmids\": [\"15819408\"],\n \"is_preprint\": false\n },\n {\n \"year\": 2009,\n \"finding\": \"mdig (RIOX2) demethylates tri-methyl lysine 9 of histone H3 (H3K9me3): overexpression reduces H3K9me3 at the rRNA gene promoter and increases RNA Pol I occupancy and rRNA transcription, while gene silencing has the opposite effect.\",\n \"method\": \"Gene overexpression and siRNA knockdown; chromatin immunoprecipitation (ChIP) for H3K9me3 and RNA Pol I; rRNA expression assays\",\n \"journal\": \"Cell cycle (Georgetown, Tex.)\",\n \"confidence\": \"Medium\",\n \"confidence_rationale\": \"Tier 2 / Moderate — ChIP with gain- and loss-of-function in the same study, single lab, no in vitro reconstitution\",\n \"pmids\": [\"19502796\"],\n \"is_preprint\": false\n },\n {\n \"year\": 2013,\n \"finding\": \"mdig (RIOX2) demethylates H3K9me3 at the promoter of the imprinted H19 lncRNA gene: overexpression reduces H3K9me3 at the H19 promoter and activates H19 transcription; shRNA/siRNA knockdown increases H3K9me3 and reduces H19 expression. In vitro demethylation assay with immunoprecipitated mdig and an H3K9me3 peptide confirmed catalytic activity.\",\n \"method\": \"Overexpression and shRNA/siRNA knockdown with ChIP for H3K9me3; in vitro demethylation assay using immunoprecipitated mdig protein and histone H3 peptide substrate\",\n \"journal\": \"Oncotarget\",\n \"confidence\": \"Medium\",\n \"confidence_rationale\": \"Tier 2 / Moderate — in vitro demethylation assay plus cellular ChIP, single lab, two orthogonal methods\",\n \"pmids\": [\"23965803\"],\n \"is_preprint\": false\n },\n {\n \"year\": 2013,\n \"finding\": \"Mina53-deficient mice show reduced Th17 cell infiltration into airways and increased Treg cell infiltration following allergen (house dust mite) challenge, with lower IL-4 and IL-5 levels, demonstrating that Mina53 (RIOX2) regulates the Th17/Treg balance and allergic airway responses.\",\n \"method\": \"Mina53-knockout mouse model; intranasal allergen challenge; bronchoalveolar lavage cell differential counts; ELISA for cytokines; airway hyperresponsiveness measurement\",\n \"journal\": \"Cell structure and function\",\n \"confidence\": \"Medium\",\n \"confidence_rationale\": \"Tier 2 / Moderate — clean KO mouse with defined cellular and cytokine phenotype, single lab\",\n \"pmids\": [\"23748603\"],\n \"is_preprint\": false\n },\n {\n \"year\": 2015,\n \"finding\": \"mdig (RIOX2) protein co-immunoprecipitates with DNA double-strand break repair and chromatin-binding proteins XRCC5, XRCC6, RBBP4, CBX8, PRMT5, and TDRD in lung cancer (A549) and bronchial epithelial (BEAS-2B) cells, validated by reciprocal co-immunoprecipitation.\",\n \"method\": \"Co-immunoprecipitation with anti-mdig antibody followed by nanoESI-MS/MS proteomics (Orbitrap); reciprocal co-IP validation; four independent experiments\",\n \"journal\": \"Oncotarget\",\n \"confidence\": \"Medium\",\n \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal co-IP plus mass spectrometry in two cell lines, single lab\",\n \"pmids\": [\"26293673\"],\n \"is_preprint\": false\n },\n {\n \"year\": 2015,\n \"finding\": \"mdig (RIOX2) regulates cell cycle progression through p27(KIP1): knockdown of mdig increases p27(KIP1) mRNA and protein and inhibits phosphorylation of p27 at Thr187, causing cell cycle arrest; in human lung cancer tissues, mdig upregulation inversely correlates with p27(KIP1) levels.\",\n \"method\": \"siRNA knockdown in A549 cells; MTT proliferation assay; cell cycle analysis; RT-qPCR for cell cycle regulators; Western blot for p27(KIP1) and phospho-p27(KIP1) isoforms; Western blot on human lung cancer tissue samples\",\n \"journal\": \"Tumour biology\",\n \"confidence\": \"Medium\",\n \"confidence_rationale\": \"Tier 2 / Moderate — loss-of-function with multiple molecular readouts (mRNA, total protein, phospho-protein) plus in vivo tissue correlation, single lab\",\n \"pmids\": [\"25851349\"],\n \"is_preprint\": false\n },\n {\n \"year\": 2016,\n \"finding\": \"mdig (RIOX2) directly interacts with c-Myc and JAK1 in multiple myeloma cell lines, contributing to hyperactivation of the IL-6-JAK-STAT3 signaling pathway; genetic silencing of mdig reduces activity of downstream effectors in this pathway.\",\n \"method\": \"Co-immunoprecipitation; integrative genomics and proteomics; siRNA knockdown with pathway activity readouts (Western blot for STAT3 phosphorylation)\",\n \"journal\": \"Scientific reports\",\n \"confidence\": \"Medium\",\n \"confidence_rationale\": \"Tier 2 / Moderate — co-IP plus proteomics plus functional siRNA knockdown, single lab\",\n \"pmids\": [\"27833099\"],\n \"is_preprint\": false\n },\n {\n \"year\": 2017,\n \"finding\": \"MDIG (RIOX2) regulates H3K9me3 at the p21(CIP1/WAF1) promoter in hepatocellular carcinoma: MDIG overexpression reduces H3K9me3 and activates p21 expression; knockdown has the opposite effect, influencing HCC cell proliferation and migration.\",\n \"method\": \"Gain- and loss-of-function experiments (overexpression and siRNA knockdown) in HCC cells; ChIP for H3K9me3; Western blot for p21; xenograft tumor model\",\n \"journal\": \"Cell death & disease\",\n \"confidence\": \"Medium\",\n \"confidence_rationale\": \"Tier 2 / Moderate — ChIP plus gain/loss-of-function plus in vivo xenograft, single lab\",\n \"pmids\": [\"28471446\"],\n \"is_preprint\": false\n },\n {\n \"year\": 2018,\n \"finding\": \"MINA53 (RIOX2) regulates expression of the CDC45-MCM-GINS (CMG) complex genes required for DNA replication initiation; knockdown reduces CMG gene expression, induces DNA replication stress, diminishes ATM/ATR-H2AX DNA damage response, and leads to glioblastoma cell apoptosis.\",\n \"method\": \"siRNA knockdown in glioblastoma cells; DNA replication initiation assays; RT-qPCR and Western blot for CMG genes; flow cytometry for apoptosis; Western blot for ATM/ATR-H2AX pathway components\",\n \"journal\": \"Cell death & disease\",\n \"confidence\": \"Medium\",\n \"confidence_rationale\": \"Tier 2 / Moderate — loss-of-function with multiple molecular readouts (replication initiation, DDR pathway, apoptosis), single lab\",\n \"pmids\": [\"30333481\"],\n \"is_preprint\": false\n },\n {\n \"year\": 2019,\n \"finding\": \"MINA53 (RIOX2) preferentially demethylates H3K36me3 in vitro, and its depletion by RNAi increases local H3K36me3 levels at the HIV-1 LTR, promoting HIV-1 latency reversal. The pan-JmjC inhibitor JIB-04 inhibits MINA53-mediated H3K36me3 demethylation and synergizes with latency-reversing agents to reactivate latent HIV-1.\",\n \"method\": \"CRISPR/Cas9 screen; RNAi depletion; in vitro histone demethylation assay; ChIP for H3K36me3 at LTR; HIV-1 latency reactivation assays; JIB-04 inhibitor treatment\",\n \"journal\": \"Nucleic acids research\",\n \"confidence\": \"High\",\n \"confidence_rationale\": \"Tier 1–2 / Strong — in vitro demethylation assay plus cellular ChIP plus CRISPR functional screen, multiple orthogonal methods\",\n \"pmids\": [\"31165872\"],\n \"is_preprint\": false\n },\n {\n \"year\": 2018,\n \"finding\": \"Loss of mdig (RIOX2) expression enhances global DNA methylation and H3K9me3 heterochromatin, and increases migration and invasion of triple-negative breast cancer cells, indicating mdig acts as an inhibitor of DNA and histone methylation and suppresses metastatic behavior in advanced breast cancer.\",\n \"method\": \"siRNA/shRNA knockdown; global DNA methylation assay; chromatin accessibility assay; ChIP for H3K9me3; Transwell migration/invasion assays\",\n \"journal\": \"Signal transduction and targeted therapy\",\n \"confidence\": \"Medium\",\n \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal epigenetic assays plus functional migration readout, single lab\",\n \"pmids\": [\"30254753\"],\n \"is_preprint\": false\n },\n {\n \"year\": 2020,\n \"finding\": \"ZNF143 promotes expression of MDIG (RIOX2) by direct transcriptional activation; MDIG in turn reduces H3K9me3 at the CDC6 promoter to activate CDC6 transcription and accelerate HCC cell-cycle progression, establishing a ZNF143-MDIG-CDC6 oncoprotein axis.\",\n \"method\": \"ChIP for ZNF143 at MDIG promoter; gain- and loss-of-function experiments; ChIP for H3K9me3 at CDC6 promoter; Western blot; in vitro and in vivo tumor growth assays\",\n \"journal\": \"Cancer research\",\n \"confidence\": \"Medium\",\n \"confidence_rationale\": \"Tier 2 / Moderate — ChIP-based pathway placement plus gain/loss-of-function plus in vivo xenograft, single lab\",\n \"pmids\": [\"32312832\"],\n \"is_preprint\": false\n },\n {\n \"year\": 2018,\n \"finding\": \"Phylogenetic and structural analyses show that RIOX2 (MINA53) and RIOX1 (NO66) share conserved active-site residues within their JmjC domains, a dimerization domain, and a winged-helix domain; the proteins catalyze C-3 histidine hydroxylation in ribosomal proteins (Rpl27a for MINA53). RIOX2 has a distinct subnuclear localization in Hydra compared to human, indicating evolutionary adaptation.\",\n \"method\": \"Phylogenomic analysis of 49 metazoan species; domain architecture comparison; immunofluorescence for subnuclear localization in HeLa cells and Hydra\",\n \"journal\": \"BMC evolutionary biology\",\n \"confidence\": \"Medium\",\n \"confidence_rationale\": \"Tier 2–3 / Moderate — localization by immunofluorescence plus evolutionary structural analysis; histidine hydroxylase activity inferred from conservation but directly assayed in companion biochemical literature\",\n \"pmids\": [\"29914368\"],\n \"is_preprint\": false\n },\n {\n \"year\": 2023,\n \"finding\": \"MINA53 (RIOX2) is a histidine hydroxylase with narrow substrate selectivity: it catalyzes C-3 hydroxylation of a histidine residue in ribosomal protein Rpl27a. Inhibition assays with histidine analogues in Rpl peptides showed that MINA53 activity can be inhibited by competition with non-oxidized peptides containing acyclic side-chain analogues (Asn, Gln, homoGln).\",\n \"method\": \"In vitro biochemical assays with natural and unnatural histidine analogues incorporated into Rpl peptides; substrate selectivity assays; inhibition assays\",\n \"journal\": \"RSC chemical biology\",\n \"confidence\": \"High\",\n \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution with defined peptide substrates and mutagenic analogues, rigorous biochemical characterization, single lab\",\n \"pmids\": [\"36908702\"],\n \"is_preprint\": false\n },\n {\n \"year\": 2023,\n \"finding\": \"MDIG (RIOX2) demethylates H3K9me3 at the OTX2 promoter to increase chromatin accessibility, allowing OTX2 transcription factor expression; OTX2 then binds the Myc promoter to activate Myc expression, forming a positive feedback loop that sustains hepatocyte proliferation during liver regeneration.\",\n \"method\": \"Liver-specific MDIG knockout mice; partial hepatectomy and CCl4 injury models; ChIP for H3K9me3 at OTX2 promoter; ATAC-seq for chromatin accessibility; ChIP for OTX2 at Myc promoter; RNA-seq\",\n \"journal\": \"Signal transduction and targeted therapy\",\n \"confidence\": \"High\",\n \"confidence_rationale\": \"Tier 2 / Strong — tissue-specific KO mouse model with multiple orthogonal genomic methods (ChIP, ATAC-seq, RNA-seq) establishing mechanistic pathway, single lab\",\n \"pmids\": [\"37709738\"],\n \"is_preprint\": false\n },\n {\n \"year\": 2024,\n \"finding\": \"Mina53 (RIOX2) is a bona fide H4R3me2a (asymmetric di-methylation at arginine 3 of histone H4) eraser: identified as an H4R3me2a interactor by photoaffinity capture, it demethylates H4R3me2a in vitro and in cells. In a transgenic mouse with neural stem/progenitor cell-specific Mina53 deletion, failure to demethylate H4R3me2a dysregulates genes for NSC proliferation and differentiation, impairing cognitive function.\",\n \"method\": \"Photoaffinity capture for interactor identification; in vitro demethylation biochemical assay; cellular H4R3me2a quantification; molecular dynamics simulation; transgenic conditional KO mouse with cognitive testing\",\n \"journal\": \"Nature communications\",\n \"confidence\": \"High\",\n \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution of demethylase activity + photoaffinity capture + molecular dynamics + conditional KO mouse with functional phenotype, multiple orthogonal methods in one study\",\n \"pmids\": [\"39587091\"],\n \"is_preprint\": false\n },\n {\n \"year\": 2025,\n \"finding\": \"Mina53 (RIOX2) is an arginine demethylase that removes asymmetric dimethylation at arginine 337 of p53 (p53 R337me2a): demethylation reduces p53 protein stability and oligomerization, alters chromatin modifications at p53 target gene promoters, and suppresses p53-mediated transcriptional activation and cell-cycle arrest, thereby promoting tumor growth in mouse xenograft and spontaneous tumor models.\",\n \"method\": \"In vitro demethylation assay; mass spectrometry for p53 arginine methylation status; co-immunoprecipitation for oligomerization; ChIP for chromatin modifications at p53 target promoters; xenograft and spontaneous tumor mouse models; loss-of-function experiments\",\n \"journal\": \"Cell reports\",\n \"confidence\": \"High\",\n \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution of demethylase activity on p53 R337me2a + MS validation + structural stability assays + in vivo tumor models, multiple orthogonal methods\",\n \"pmids\": [\"39864061\"],\n \"is_preprint\": false\n },\n {\n \"year\": 2017,\n \"finding\": \"mdig (RIOX2) suppresses epithelial-mesenchymal transition (EMT) in NSCLC by inhibiting GSK-3β phosphorylation, which promotes β-catenin phosphorylation and destabilization, downregulating slug, snail, and ZEB1 transcription factors, thereby increasing epithelial markers (E-cadherin, claudin-1) and decreasing mesenchymal markers (vimentin, N-cadherin).\",\n \"method\": \"Lentiviral overexpression and knockdown in A549 and HUVEC cells; Transwell invasion/migration assays; Western blot for GSK-3β, β-catenin, EMT markers and transcription factors\",\n \"journal\": \"International journal of oncology\",\n \"confidence\": \"Medium\",\n \"confidence_rationale\": \"Tier 2 / Moderate — gain- and loss-of-function with molecular pathway readouts in two cell lines, single lab\",\n \"pmids\": [\"29039479\"],\n \"is_preprint\": false\n },\n {\n \"year\": 2019,\n \"finding\": \"MDIG (RIOX2) promotes cisplatin resistance in lung adenocarcinoma by activating WNT/β-catenin signaling, which upregulates ABC transporter expression (ABCB1, ABCC1, ABCG2); overexpression increases IC50 for cisplatin while knockdown reduces it.\",\n \"method\": \"siRNA knockdown and overexpression in A549 and A549/DDP cisplatin-resistant cells; RT-qPCR and Western blot for MDIG, ABC transporters, WNT pathway components; IC50 determination\",\n \"journal\": \"Oncology letters\",\n \"confidence\": \"Medium\",\n \"confidence_rationale\": \"Tier 2 / Moderate — gain- and loss-of-function with molecular pathway and functional drug resistance readouts, single lab\",\n \"pmids\": [\"31579066\"],\n \"is_preprint\": false\n },\n {\n \"year\": 2021,\n \"finding\": \"mdig (RIOX2) depletion by CRISPR-Cas9 in bronchial epithelial cells reduces expression of SARS-CoV-2 receptors NRP1 and NRP2, cathepsins, and glycan metabolism genes, associated with enrichment of H3K9me3 and/or H3K27me3 at these loci as determined by ChIP-seq.\",\n \"method\": \"CRISPR-Cas9 gene editing; ChIP-seq for H3K9me3/H3K27me3; RT-qPCR and Western blot for NRP1, NRP2, cathepsins; gene ontology analysis\",\n \"journal\": \"Theranostics\",\n \"confidence\": \"Medium\",\n \"confidence_rationale\": \"Tier 2 / Moderate — CRISPR KO with genome-wide ChIP-seq and gene expression validation, single lab\",\n \"pmids\": [\"34335974\"],\n \"is_preprint\": false\n },\n {\n \"year\": 2022,\n \"finding\": \"Mdig (RIOX2) deletion in triple-negative breast cancer MDA-MB-231 cells enhances global H3K36me3 and upregulates X-chromosome-linked genes for cell motility and invasion (including MAGED2); silencing MAGED2 partially reverses the invasive migration of mdig-KO cells, establishing H3K36me3/MAGED2 as a downstream effector axis.\",\n \"method\": \"CRISPR-Cas9 mdig knockout; ChIP-seq for H3K36me3; RNA-seq; siRNA knockdown of MAGED2; in vivo orthotopic xenograft; Transwell migration assay\",\n \"journal\": \"iScience\",\n \"confidence\": \"High\",\n \"confidence_rationale\": \"Tier 2 / Strong — CRISPR KO with genome-wide ChIP-seq + RNA-seq + in vivo xenograft + rescue experiment (MAGED2 knockdown), multiple orthogonal methods in one study\",\n \"pmids\": [\"36124233\"],\n \"is_preprint\": false\n }\n ],\n \"current_model\": \"RIOX2 (MINA53/mdig) is a multifunctional JmjC-domain oxygenase that acts as a histone eraser — catalyzing demethylation of H3K9me3, H3K36me3, and H4R3me2a — and as an arginine demethylase for p53 (removing R337me2a to destabilize p53 and suppress cell-cycle arrest); it also hydroxylates the C-3 position of a histidine in ribosomal protein Rpl27a, and is a direct transcriptional target of c-Myc that localizes to nucleolar preribosomal particles, interacts with DNA repair factors (XRCC5, XRCC6, RBBP4) and c-Myc/JAK1, and controls cell proliferation, EMT, immune Th17/Treg balance, and liver regeneration through epigenetic regulation of key target gene promoters.\"\n}\n```","stage2_raw":"{\n \"mechanistic_narrative\": \"RIOX2 (MINA53/mdig) is a JmjC-domain 2-oxoglutarate oxygenase that couples chromatin modification to cell proliferation, acting both as a histone-modification \\\"eraser\\\" and as a protein hydroxylase [#2, #14, #16]. As a demethylase it removes repressive H3K9me3 at target promoters and preferentially demethylates H3K36me3 in vitro, and it also functions as an arginine demethylase for histone H4R3me2a [#2, #10, #16]. Through H3K9me3 erasure it derepresses growth-promoting loci including rRNA genes, the H19 lncRNA, p21(CIP1/WAF1), CDC6, and OTX2, the last sustaining a self-reinforcing OTX2\\u2013Myc loop that drives hepatocyte proliferation during liver regeneration [#2, #3, #8, #12, #15]. Beyond histones, RIOX2 is an arginine demethylase for p53, removing R337me2a to reduce p53 stability and oligomerization and thereby suppress p53-dependent cell-cycle arrest and promote tumor growth [#17]. It is a histidine hydroxylase that catalyzes C-3 hydroxylation of a specific histidine in ribosomal protein Rpl27a [#14]. Consistent with these activities, RIOX2 is a direct c-Myc transcriptional target whose expression is required for proliferation, localizes constitutively to the nucleolar granular component and preribosomal particles, and modulates cell-cycle progression, EMT, replication, drug resistance, and Th17/Treg immune balance [#0, #1, #6, #9, #18]. Its catalytic loss broadly reshapes the epigenome \\u2014 deletion raises global H3K9me3, H3K36me3, and DNA methylation, altering motility and invasion programs in cancer cells [#11, #21].\"\n,\n \"teleology\": [\n {\n \"year\": 2002,\n \"claim\": \"Establishing what drives RIOX2 expression and why it matters: defining it as a direct c-Myc effector linked the gene to proliferative control from the outset.\",\n \"evidence\": \"ChIP, promoter-reporter, c-MycER/cycloheximide block, and RNAi with proliferation readout in cultured cells\",\n \"pmids\": [\"12091391\"],\n \"confidence\": \"High\",\n \"gaps\": [\"Did not define the biochemical activity of the protein\", \"Mechanism linking expression to proliferation unresolved\"]\n },\n {\n \"year\": 2005,\n \"claim\": \"Localizing the protein answered where it acts: constitutive nucleolar/preribosomal residence tied RIOX2 to ribosome biogenesis and rRNA metabolism.\",\n \"evidence\": \"Immunolocalization, fractionation, co-IP/MALDI-MS, and RNase A/actinomycin D/serum-starvation perturbations\",\n \"pmids\": [\"15819408\"],\n \"confidence\": \"High\",\n \"gaps\": [\"Interactor identities not resolved to specific functional partners\", \"No catalytic activity demonstrated\"]\n },\n {\n \"year\": 2009,\n \"claim\": \"First assignment of a catalytic function: RIOX2 demethylates H3K9me3 at rRNA gene promoters to license Pol I transcription.\",\n \"evidence\": \"Gain/loss-of-function with ChIP for H3K9me3 and Pol I plus rRNA assays\",\n \"pmids\": [\"19502796\"],\n \"confidence\": \"Medium\",\n \"gaps\": [\"No in vitro reconstitution in this study\", \"Direct vs indirect demethylation not separated\"]\n },\n {\n \"year\": 2013,\n \"claim\": \"Confirmed direct H3K9me3 demethylase activity and extended it to an imprinted locus, showing catalysis is intrinsic to RIOX2.\",\n \"evidence\": \"In vitro demethylation with immunoprecipitated mdig plus cellular ChIP at the H19 promoter\",\n \"pmids\": [\"23965803\"],\n \"confidence\": \"Medium\",\n \"gaps\": [\"Immunoprecipitated (not purified recombinant) enzyme used\", \"Substrate specificity versus other marks not addressed\"]\n },\n {\n \"year\": 2013,\n \"claim\": \"Defined a physiological role in immunity: RIOX2 controls Th17/Treg balance and allergic airway responses in vivo.\",\n \"evidence\": \"Mina53-knockout mice with allergen challenge, BAL differentials, cytokine ELISA, and airway hyperresponsiveness\",\n \"pmids\": [\"23748603\"],\n \"confidence\": \"Medium\",\n \"gaps\": [\"Molecular target in T cells not identified\", \"Catalytic dependence of the phenotype untested\"]\n },\n {\n \"year\": 2015,\n \"claim\": \"Mapped the protein interactome, placing RIOX2 with DNA repair and chromatin factors.\",\n \"evidence\": \"Reciprocal co-IP plus nanoESI-MS/MS in A549 and BEAS-2B cells (XRCC5, XRCC6, RBBP4, CBX8, PRMT5, TDRD)\",\n \"pmids\": [\"26293673\"],\n \"confidence\": \"Medium\",\n \"gaps\": [\"Functional consequence of each interaction undefined\", \"Direct vs complex-mediated binding not resolved\"]\n },\n {\n \"year\": 2015,\n \"claim\": \"Connected RIOX2 to cell-cycle regulation through p27(KIP1), explaining its proliferative requirement.\",\n \"evidence\": \"siRNA knockdown with proliferation/cell-cycle assays, p27 and phospho-p27 Western blots, and tumor-tissue correlation\",\n \"pmids\": [\"25851349\"],\n \"confidence\": \"Medium\",\n \"gaps\": [\"Whether p27 regulation is epigenetic or indirect unclear\", \"No catalytic-dead control\"]\n },\n {\n \"year\": 2016,\n \"claim\": \"Identified signaling partners, showing RIOX2 binds c-Myc and JAK1 to amplify IL-6\\u2013JAK\\u2013STAT3 signaling.\",\n \"evidence\": \"Co-IP, integrative genomics/proteomics, and siRNA knockdown with STAT3 phosphorylation readout in myeloma cells\",\n \"pmids\": [\"27833099\"],\n \"confidence\": \"Medium\",\n \"gaps\": [\"Direct binding interface not mapped\", \"Catalytic involvement in signaling untested\"]\n },\n {\n \"year\": 2017,\n \"claim\": \"Extended the H3K9me3-demethylase paradigm to tumor-suppressor control via p21 in hepatocellular carcinoma.\",\n \"evidence\": \"Gain/loss-of-function, ChIP for H3K9me3 at the p21 promoter, and xenograft model\",\n \"pmids\": [\"28471446\"],\n \"confidence\": \"Medium\",\n \"gaps\": [\"Single-lab observation\", \"Other p21-controlling inputs not excluded\"]\n },\n {\n \"year\": 2017,\n \"claim\": \"Defined a context-dependent anti-EMT role through GSK-3\\u03b2/\\u03b2-catenin signaling in NSCLC.\",\n \"evidence\": \"Overexpression/knockdown with invasion assays and Western blots for EMT markers and transcription factors\",\n \"pmids\": [\"29039479\"],\n \"confidence\": \"Medium\",\n \"gaps\": [\"Link between catalytic activity and \\u03b2-catenin signaling unestablished\", \"Cell-line restricted\"]\n },\n {\n \"year\": 2018,\n \"claim\": \"Provided structural/evolutionary framing and assigned histidine hydroxylase activity on Rpl27a, distinguishing RIOX2 catalysis from pure demethylation.\",\n \"evidence\": \"Phylogenomics across 49 species, domain comparison, and immunofluorescence localization (human vs Hydra)\",\n \"pmids\": [\"29914368\"],\n \"confidence\": \"Medium\",\n \"gaps\": [\"Hydroxylase activity inferred here, assayed directly later\", \"Functional consequence of Rpl27a hydroxylation unknown\"]\n },\n {\n \"year\": 2018,\n \"claim\": \"Showed catalytic loss reshapes the epigenome and metastatic behavior, casting RIOX2 as a suppressor of DNA/histone methylation in TNBC.\",\n \"evidence\": \"Knockdown with global DNA-methylation, chromatin accessibility, H3K9me3 ChIP, and migration/invasion assays\",\n \"pmids\": [\"30254753\"],\n \"confidence\": \"Medium\",\n \"gaps\": [\"Direct vs indirect effects on global methylation not separated\"]\n },\n {\n \"year\": 2018,\n \"claim\": \"Linked RIOX2 to DNA replication licensing, explaining proliferation arrest upon loss.\",\n \"evidence\": \"siRNA knockdown in glioblastoma with replication-initiation assays, CMG gene expression, DDR Western blots, and apoptosis flow cytometry\",\n \"pmids\": [\"30333481\"],\n \"confidence\": \"Medium\",\n \"gaps\": [\"Whether CMG genes are direct chromatin targets untested\"]\n },\n {\n \"year\": 2019,\n \"claim\": \"Established H3K36me3 as a preferred substrate in vitro and a druggable role in HIV-1 latency.\",\n \"evidence\": \"CRISPR screen, RNAi, in vitro demethylation assay, LTR ChIP, latency reactivation, and JIB-04 inhibition\",\n \"pmids\": [\"31165872\"],\n \"confidence\": \"High\",\n \"gaps\": [\"Relative cellular preference for H3K9me3 vs H3K36me3 not quantified\"]\n },\n {\n \"year\": 2019,\n \"claim\": \"Connected RIOX2 to chemoresistance via WNT/\\u03b2-catenin-driven ABC transporter expression.\",\n \"evidence\": \"Knockdown/overexpression in cisplatin-resistant lung cells with IC50, ABC transporter, and WNT pathway readouts\",\n \"pmids\": [\"31579066\"],\n \"confidence\": \"Medium\",\n \"gaps\": [\"Catalytic dependence and direct target genes unresolved\"]\n },\n {\n \"year\": 2020,\n \"claim\": \"Placed RIOX2 in an upstream-downstream transcriptional axis: ZNF143 induces MDIG, which derepresses CDC6 via H3K9me3 removal.\",\n \"evidence\": \"ChIP at MDIG and CDC6 promoters, gain/loss-of-function, and in vitro/in vivo tumor assays in HCC\",\n \"pmids\": [\"32312832\"],\n \"confidence\": \"Medium\",\n \"gaps\": [\"Single-lab pathway placement\", \"Direct CDC6 promoter binding not shown\"]\n },\n {\n \"year\": 2021,\n \"claim\": \"Used genome-wide profiling to show RIOX2 controls host genes for viral entry through H3K9me3/H3K27me3 chromatin states.\",\n \"evidence\": \"CRISPR-Cas9 KO with ChIP-seq and expression validation of NRP1/NRP2, cathepsins, glycan genes\",\n \"pmids\": [\"34335974\"],\n \"confidence\": \"Medium\",\n \"gaps\": [\"Direct demethylase action vs indirect chromatin effects not distinguished\"]\n },\n {\n \"year\": 2022,\n \"claim\": \"Defined an H3K36me3-dependent invasion program with a rescuable downstream effector (MAGED2).\",\n \"evidence\": \"CRISPR KO with H3K36me3 ChIP-seq, RNA-seq, MAGED2 knockdown rescue, orthotopic xenograft, and migration assays in TNBC\",\n \"pmids\": [\"36124233\"],\n \"confidence\": \"High\",\n \"gaps\": [\"Why X-linked loci are preferentially affected unexplained\"]\n },\n {\n \"year\": 2023,\n \"claim\": \"Rigorously characterized RIOX2 as a substrate-selective histidine hydroxylase on Rpl27a, with defined inhibitory analogues.\",\n \"evidence\": \"In vitro biochemistry with natural/unnatural histidine analogue peptides and inhibition assays\",\n \"pmids\": [\"36908702\"],\n \"confidence\": \"High\",\n \"gaps\": [\"Cellular consequence of Rpl27a hydroxylation on translation unresolved\"]\n },\n {\n \"year\": 2023,\n \"claim\": \"Established a physiological demethylase circuit in vivo: MDIG erases H3K9me3 at OTX2 to drive an OTX2\\u2013Myc loop sustaining liver regeneration.\",\n \"evidence\": \"Liver-specific KO mice, hepatectomy/CCl4 injury, ChIP, ATAC-seq, OTX2 ChIP at Myc, and RNA-seq\",\n \"pmids\": [\"37709738\"],\n \"confidence\": \"High\",\n \"gaps\": [\"Whether the same loop operates in non-hepatic tissues untested\"]\n },\n {\n \"year\": 2024,\n \"claim\": \"Expanded the substrate repertoire to arginine methylation: RIOX2 is a bona fide H4R3me2a eraser important for neural stem cell programs and cognition.\",\n \"evidence\": \"Photoaffinity capture, in vitro and cellular demethylation, molecular dynamics, and NSC-specific conditional KO with cognitive testing\",\n \"pmids\": [\"39587091\"],\n \"confidence\": \"High\",\n \"gaps\": [\"Genome-wide H4R3me2a targets not fully mapped\"]\n },\n {\n \"year\": 2025,\n \"claim\": \"Identified a non-histone arginine demethylase substrate, p53 R337me2a, linking RIOX2 catalysis directly to tumor-suppressor inactivation.\",\n \"evidence\": \"In vitro demethylation, MS validation, oligomerization co-IP, target-promoter ChIP, and xenograft/spontaneous tumor models\",\n \"pmids\": [\"39864061\"],\n \"confidence\": \"High\",\n \"gaps\": [\"Crosstalk between p53 demethylation and histone activities unresolved\"]\n },\n {\n \"year\": null,\n \"claim\": \"How RIOX2 selects among its multiple chemistries (H3K9me3, H3K36me3, H4R3me2a, p53 R337me2a demethylation, and Rpl27a hydroxylation) and is targeted to specific substrates and loci in a given context remains unresolved.\",\n \"evidence\": \"\",\n \"pmids\": [],\n \"confidence\": \"Medium\",\n \"gaps\": [\"No structural model integrating substrate selectivity\", \"Recruitment/targeting mechanism to specific promoters unknown\", \"Relative contribution of catalytic vs scaffolding functions unquantified\"]\n }\n ],\n \"mechanism_profile\": {\n \"molecular_activity\": [\n {\"term_id\": \"GO:0140098\", \"supporting_discovery_ids\": [2, 3, 10, 16, 17]},\n {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [14]},\n {\"term_id\": \"GO:0016491\", \"supporting_discovery_ids\": [2, 10, 14, 16]},\n {\"term_id\": \"GO:0140110\", \"supporting_discovery_ids\": [0, 12, 15]}\n ],\n \"localization\": [\n {\"term_id\": \"GO:0005730\", \"supporting_discovery_ids\": [1, 13]},\n {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [2, 3, 8]}\n ],\n \"pathway\": [\n {\"term_id\": \"R-HSA-4839726\", \"supporting_discovery_ids\": [2, 3, 8, 15, 16]},\n {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [6, 9, 12]},\n {\"term_id\": \"R-HSA-74160\", \"supporting_discovery_ids\": [0, 12, 15]}\n ],\n \"complexes\": [],\n \"partners\": [\"MYC\", \"JAK1\", \"XRCC5\", \"XRCC6\", \"RBBP4\", \"CBX8\", \"PRMT5\"],\n \"other_free_text\": []\n }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}