{"gene":"SETD6","run_date":"2026-06-10T07:46:31","timeline":{"discoveries":[{"year":2010,"finding":"SETD6 monomethylates the NF-κB subunit RelA at Lys310 (RelAK310me1), rendering RelA transcriptionally inert and attenuating NF-κB-driven inflammatory gene programs. RelAK310me1 is recognized by the ankyrin repeat of histone methyltransferase GLP, which promotes a repressed chromatin state at RelA target genes via GLP-mediated H3K9 methylation. Phosphorylation of RelA at Ser311 by PKC-ζ blocks GLP binding to RelAK310me1 and relieves transcriptional repression.","method":"Biochemical screen of >40 PKMTs, in vitro methylation assays, Co-IP, ChIP, primary immune cell gene expression analysis, site-directed mutagenesis","journal":"Nature immunology","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — in vitro enzymatic assay plus reciprocal Co-IP plus ChIP plus mutagenesis, replicated across multiple labs","pmids":["21131967"],"is_preprint":false},{"year":2011,"finding":"Crystal structure of SETD6 in complex with RelA peptide and SAM revealed a V-like protein structure and the molecular basis for substrate recognition; Ser311 phosphorylation sterically inhibits Lys310 methylation by SETD6 and binding of Lys310me1 by GLP ankyrin repeats, establishing a methylation-phosphorylation switch at adjacent residues.","method":"X-ray crystallography, structural modeling, biochemical binding assays","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure with functional validation of phosphorylation-methylation switch, consistent with independent findings in PMID:21131967","pmids":["21515635"],"is_preprint":false},{"year":2013,"finding":"SETD6 monomethylates histone variant H2AZ at Lys7 (H2AZK7me1). H2AZK7me1 and H3K27me3 co-occupy transcriptional start sites of differentiation genes in mESCs; upon retinoic acid-induced differentiation both marks are removed. Setd6 depletion in mESCs leads to spontaneous differentiation, compromised self-renewal, and poor clonogenicity.","method":"In vitro methylation assay, ChIP, siRNA knockdown, clonogenicity assay, differentiation assays in mESCs","journal":"Epigenetics","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — in vitro enzymatic assay plus ChIP plus genetic loss-of-function with defined cellular phenotype, single lab with multiple orthogonal methods","pmids":["23324626"],"is_preprint":false},{"year":2016,"finding":"SETD6 methylates PAK4 in vitro and at chromatin in cells. SETD6-mediated PAK4 methylation enhances physical interaction between PAK4 and β-catenin, promoting transcription of Wnt/β-catenin target genes. Depletion of SETD6 significantly hinders Wnt/β-catenin target gene activation.","method":"In vitro methylation assay, Co-IP, ChIP, siRNA knockdown, luciferase reporter assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — in vitro assay plus reciprocal Co-IP plus ChIP plus reporter assays, single lab with multiple orthogonal methods","pmids":["26841865"],"is_preprint":false},{"year":2014,"finding":"SETD6 associates with estrogen receptor α (ERα), HDAC1, MTA2, and TRRAP. SETD6 acts as a transcriptional repressor in reporter assays but functions as a co-activator of estrogen-responsive genes (PGR, TFF1). SETD6 silencing in breast carcinoma cells induces proliferation defects, enhanced CDKN1A expression, and apoptosis.","method":"Co-immunoprecipitation/mass spectrometry, luciferase reporter assay, siRNA knockdown, cell proliferation and apoptosis assays","journal":"Epigenetics","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — reciprocal Co-IP plus reporter assay plus KD phenotype, single lab","pmids":["24751716"],"is_preprint":false},{"year":2016,"finding":"SETD6 interacts with oxidative stress sensor DJ1 both in vitro and in cells at chromatin. Through this catalytically independent interaction, SETD6 inhibits DJ1 activity and represses Nrf2-dependent antioxidant gene transcription. Under oxidative stress, SETD6 protein levels decrease, weakening the SETD6-DJ1 interaction and de-repressing Nrf2 target genes. SETD6 does not methylate DJ1.","method":"In vitro pulldown, Co-IP, ChIP, siRNA knockdown, qRT-PCR gene expression analysis","journal":"Biochimica et biophysica acta","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — in vitro pulldown plus Co-IP plus ChIP plus KD, single lab with multiple methods","pmids":["26780326"],"is_preprint":false},{"year":2019,"finding":"SETD6 binds and methylates PLK1 at K209 and K413 during mitosis. Loss of these methylation sites increases PLK1 kinase activity, leading to accelerated mitotic progression and faster cellular proliferation. SETD6-deficient cells similarly progress faster through mitotic steps toward cytokinesis.","method":"In vitro methylation assay, kinase activity assay, SETD6 KO/KD cell lines, live-cell imaging of mitosis, site-directed mutagenesis of PLK1 methylation sites","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — in vitro methylation plus kinase assay plus mutagenesis plus KO cell phenotype, single lab with multiple orthogonal methods","pmids":["30622182"],"is_preprint":false},{"year":2017,"finding":"A truncating SETD6 mutation (p.Met264IlefsTer3) found in familial colorectal cancer type X lacks methyltransferase activity while retaining expression, localization, and substrate-binding. The truncated protein competes with wild-type SETD6 for substrates, acting as a dominant negative inhibitor of SETD6 function.","method":"Whole-exome sequencing, in vitro methylation assay, localization analysis, competition binding assays, dominant-negative characterization","journal":"Human molecular genetics","confidence":"Medium","confidence_rationale":"Tier 1–2 / Moderate — enzymatic assay plus localization plus dominant-negative competition experiments, single lab","pmids":["28973356"],"is_preprint":false},{"year":2018,"finding":"SETD6 forms high-molecular-weight oligomeric structures (monomer, dimer, trimer) stabilized by SAM. SETD6 auto-methylates at K39, K179, and K372. A K179 point mutation in the SET domain impairs trimer formation. Auto-methylation at K39 and K179 increases SETD6 catalytic rate in vitro, linking auto-methylation to oligomerization and enzymatic activation.","method":"SEC-MALS, in vitro radioactive methylation assay, mass spectrometry, site-directed mutagenesis, kinetic analysis","journal":"Journal of molecular biology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro biochemical reconstitution plus mutagenesis plus mass spectrometry plus kinetics, single lab with multiple orthogonal methods","pmids":["30189201"],"is_preprint":false},{"year":2021,"finding":"SETD6 methylates BRD4 at Lys99 on chromatin. BRD4 methylation negatively regulates expression of genes involved in mRNA translation and inhibits total mRNA translation in cells. Mechanistically, BRD4 K99 methylation does not affect BRD4 association with acetylated histone H4 but specifically determines recruitment of transcription factor E2F1 to translation-related target genes.","method":"In vitro methylation assay, ChIP, RNA-seq, polysome profiling, Co-IP, site-directed mutagenesis (BRD4 K99R)","journal":"Science advances","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — in vitro assay plus ChIP plus RNA-seq plus mutagenesis plus polysome profiling, single lab with multiple orthogonal methods","pmids":["34039605"],"is_preprint":false},{"year":2020,"finding":"SETD6 methylates PAK4 at Lys473 as the primary methylation site. PAK4 K473 methylation activates β-catenin transcriptional activity and inhibits cell adhesion by attenuating paxillin localization to focal adhesions, reducing filopodia and actin structures, and decreasing cell migration and invasion.","method":"In vitro methylation assay, site-directed mutagenesis (PAK4 K473R), β-catenin reporter assay, immunofluorescence of focal adhesions, migration/invasion assays","journal":"Scientific reports","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — in vitro methylation plus mutagenesis plus cellular localization plus functional phenotype, single lab with multiple orthogonal methods","pmids":["33051544"],"is_preprint":false},{"year":2022,"finding":"SETD6 methylates TWIST1 at Lys33 on chromatin. TWIST1 methylation represses transcription of the long non-coding RNA LINC-PINT by increasing EZH2 occupancy and H3K27me3 at the LINC-PINT locus. Unmethylated TWIST1 dissociates from the LINC-PINT locus, allowing LINC-PINT expression, which promotes cell adhesion and reduces cell migration, thereby antagonizing EMT in glioma.","method":"In vitro methylation assay, ChIP, RNA-seq, SETD6/TWIST1 KD, site-directed mutagenesis, migration and adhesion assays","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — in vitro assay plus ChIP plus RNA-seq plus mutagenesis plus functional phenotype, single lab with multiple orthogonal methods","pmids":["35694846"],"is_preprint":false},{"year":2018,"finding":"SETD6 monomethylates WDR5 at Lys207 and Lys325. Disruption of these methylation sites by K207R/K325R double mutation attenuates WDR5-promoted breast cancer cell proliferation and migration. WDR5 K207/K325 methylation partially maintains global H3K4me3 levels but does not affect MLL/SET1 complex assembly.","method":"In vitro methylation assay, site-directed mutagenesis (K207R/K325R), western blot for H3K4me3, cell proliferation and migration assays, Co-IP for complex assembly","journal":"Oncology reports","confidence":"Medium","confidence_rationale":"Tier 1–2 / Weak — in vitro assay plus mutagenesis plus cellular phenotype, single lab, limited independent validation","pmids":["30226578"],"is_preprint":false},{"year":2019,"finding":"SETD6 is necessary for RelA K310 methylation and associated H3K9me2 increases in the dorsal hippocampus during memory consolidation. Setd6 knockdown in rat dorsal hippocampus interferes with memory consolidation, alters gene expression, and disrupts dendritic spine morphology.","method":"siRNA knockdown in vivo (rat dorsal hippocampus), biochemical assays, ChIP, electrophysiology, behavioral memory tests, spine morphology imaging","journal":"Biological psychiatry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vivo KD plus biochemical plus ChIP plus behavioral phenotype, single lab with multiple methods","pmids":["31378303"],"is_preprint":false},{"year":2017,"finding":"A cell-penetrating peptide (vp22-RelA302-316) based on the SETD6 methylation site of RelA directly and specifically binds SETD6 in vitro, inhibits its methyltransferase activity, and upon cellular treatment leads to induced cellular migration and proliferation consistent with SETD6 catalytic blockade.","method":"In vitro binding assay, in vitro methylation inhibition assay, cell-penetrating peptide treatment, migration and proliferation assays","journal":"Oncotarget","confidence":"Medium","confidence_rationale":"Tier 1–3 / Moderate — in vitro enzymatic inhibition plus cellular functional readout, single lab","pmids":["29435148"],"is_preprint":false},{"year":2023,"finding":"SETD6 monomethylates transcription factor E2F1 at Lys117 in vitro and in cells. E2F1 binds to the SETD6 promoter and drives SETD6 mRNA expression; E2F1 K117 methylation by SETD6 in turn positively enhances SETD6 mRNA levels, establishing a positive feedback loop. A K117R E2F1 mutant that cannot be methylated reverses this effect.","method":"In vitro methylation assay, ChIP, promoter-binding assay, qRT-PCR, site-directed mutagenesis (E2F1 K117R), siRNA KD","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — in vitro methylation plus ChIP plus mutagenesis plus KD rescue, single lab with multiple orthogonal methods","pmids":["37690684"],"is_preprint":false},{"year":2025,"finding":"SETD6 binds and methylates RAD18 at K73 and K406. SETD6 KO cells show increased nuclear RAD18 localization, elevated γH2AX, and increased DNA breaks (comet assay). Restoration of SETD6 enzymatic activity reduces DNA damage, while a catalytically inactive mutant does not, demonstrating that SETD6-mediated methylation of RAD18 regulates its nuclear/cytoplasmic localization and attenuates DNA breaks.","method":"Protein microarray, ELISA, Co-IP, mass spectrometry, site-directed mutagenesis, immunofluorescence (nuclear localization), γH2AX western blot, comet assay, SETD6 KO cell lines","journal":"Scientific reports","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — in vitro biochemical identification plus MS plus mutagenesis plus KO phenotype plus localization, single lab with multiple orthogonal methods","pmids":["40866490"],"is_preprint":false},{"year":2025,"finding":"Peptide SPOT array and AlphaFold3 docking experiments reveal that SETD6 substrate recognition relies on sequence preferences at positions -1 (glycine or large aliphatic), +2 (Ile/Val), and +3 (Lys) relative to the target lysine. These preferences are context-dependent and variable across substrates; SETD6 residue L260 contacts the +2 position in the SETD6-RELA complex structure, and L260 mutation shows substrate-specific effects, revealing a versatile, multispecific mode of substrate recognition.","method":"AlphaFold3 structural docking, peptide SPOT array methylation assay, site-directed mutagenesis (L260), in vitro methylation","journal":"Life (Basel, Switzerland)","confidence":"Medium","confidence_rationale":"Tier 1–3 / Moderate — SPOT array plus mutagenesis plus structural modeling, single lab","pmids":["41157251"],"is_preprint":false},{"year":2025,"finding":"BRD4 K99 methylation by SETD6 in melanoma cells determines BRD4 genomic occupancy. SETD6 KO or BRD4 K99R mutation disrupts BRD4 chromatin distribution. SETD6 also interacts with MITF and influences MITF genomic distribution. BRD4 and MITF form a chromatin-localized complex dependent on both SETD6-mediated BRD4 methylation and MITF acetylation.","method":"ChIP-seq, Co-IP, SETD6 KO, BRD4 K99R mutagenesis, immunoprecipitation, chromatin fractionation","journal":"NAR cancer","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ChIP-seq plus Co-IP plus KO plus mutagenesis, single lab","pmids":["40809945"],"is_preprint":false},{"year":2026,"finding":"SETD6-mediated K117 monomethylation of E2F1 disrupts BRD4-E2F1 interaction by preventing K117 acetylation, establishing a methylation/acetylation switch at K117 that controls bromodomain binding to E2F1. This switch modulates E2F1 chromatin occupancy and target gene expression, affecting oncogenic phenotypes in prostate cancer cells.","method":"Biochemical binding assays, ChIP-seq, RNA-seq, Co-IP, site-directed mutagenesis (E2F1 K117R, acetylation-deficient mutants), cellular oncogenic phenotype assays","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — biochemical assays plus ChIP-seq plus mutagenesis plus cellular phenotype, single lab with multiple orthogonal methods","pmids":["41540805"],"is_preprint":false},{"year":2024,"finding":"SETD6 binds and methylates Aurora-B kinase on two adjacent lysine residues. SETD6-depleted HeLa cells display chromatin bridges, actin patches, and increased multinucleation under replication stress — hallmarks of chromosomal instability. Aurora-B methylation by SETD6 increases under replication stress but is abolished when the two target lysines are mutated. SETD6 KO cells show reduced Aurora-B kinase activity during cytokinesis, suggesting that SETD6-mediated methylation sustains Aurora-B activity to ensure proper cytokinesis.","method":"Proteomic screen, in vitro methylation assay, site-directed mutagenesis, SETD6 KO live-cell imaging, kinase activity assay, immunofluorescence","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 1–2 / Weak — in vitro assay plus mutagenesis plus KO phenotype, single lab, preprint not yet peer-reviewed","pmids":["bio_10.1101_2024.12.22.629973"],"is_preprint":true}],"current_model":"SETD6 is a monomethyltransferase that modifies multiple histone and non-histone substrates (RelA/K310, H2AZ/K7, PAK4/K473, PLK1/K209+K413, BRD4/K99, TWIST1/K33, WDR5/K207+K325, E2F1/K117, RAD18/K73+K406, Aurora-B) at chromatin; these methylation events regulate downstream signaling cascades (NF-κB, Wnt/β-catenin, oxidative stress/Nrf2, Wnt-dependent cell adhesion, mRNA translation, EMT, DNA damage repair, and cell cycle progression) through mechanisms that include creating methyl-lysine docking sites for effector proteins (e.g., GLP ankyrin repeats reading RelAK310me1), imposing methyl/phospho or methyl/acetyl switches at adjacent residues that gate effector binding, modulating kinase activities (PLK1, Aurora-B), controlling nuclear-cytoplasmic shuttling of substrates (RAD18), and auto-methylation-driven oligomerization that positively feeds back on its own catalytic rate."},"narrative":{"mechanistic_narrative":"SETD6 is a protein lysine monomethyltransferase that controls diverse signaling and transcriptional programs by installing single methyl marks on chromatin-associated histone and non-histone substrates [PMID:21131967, PMID:23324626, PMID:26841865]. Its founding activity is monomethylation of the NF-κB subunit RelA at Lys310, which renders RelA transcriptionally inert and recruits the methyltransferase GLP, whose ankyrin repeat reads RelAK310me1 to impose a repressive H3K9-methylated chromatin state; adjacent Ser311 phosphorylation by PKC-ζ blocks both the methyl mark and GLP binding, defining a methyl/phospho switch resolved structurally in a SETD6–RelA–SAM complex [PMID:21131967, PMID:21515635]. SETD6 substrate recognition is multispecific, governed by sequence preferences flanking the target lysine and by SETD6 residue L260, which contacts the +2 position [PMID:41157251]. Beyond RelA, SETD6 methylates histone variant H2AZ at Lys7 to maintain mES cell self-renewal [PMID:23324626], PAK4 to potentiate β-catenin–driven Wnt target gene transcription and restrain cell adhesion and migration [PMID:26841865, PMID:33051544], PLK1 (K209/K413) to restrain its kinase activity and slow mitotic progression [PMID:30622182], BRD4 at Lys99 to repress translation-related genes and direct BRD4 genomic occupancy and an E2F1/MITF chromatin complex [PMID:34039605, PMID:40809945], TWIST1 at Lys33 to repress LINC-PINT via EZH2/H3K27me3 and antagonize EMT [PMID:35694846], E2F1 at Lys117 in a positive autoregulatory loop that disrupts E2F1 acetylation and bromodomain binding [PMID:37690684, PMID:41540805], and RAD18 to control its nuclear localization and limit DNA breaks [PMID:40866490]. SETD6 also engages substrates non-catalytically, inhibiting the oxidative-stress sensor DJ1 to repress Nrf2-dependent antioxidant transcription without methylating it [PMID:26780326]. SETD6 forms SAM-stabilized oligomers and auto-methylates at K39, K179, and K372, with auto-methylation enhancing its own catalytic rate [PMID:30189201]. A truncating SETD6 mutation lacking methyltransferase activity acts as a dominant negative and is associated with familial colorectal cancer type X [PMID:28973356].","teleology":[{"year":2010,"claim":"Establishing that SETD6 has a defined non-histone substrate answered whether this enzyme had a specific biological function: it monomethylates RelA at K310 to silence NF-κB transcription via GLP-dependent chromatin repression.","evidence":"Biochemical PKMT screen, in vitro methylation, Co-IP, ChIP and primary immune cell expression analysis","pmids":["21131967"],"confidence":"High","gaps":["Did not establish the full repertoire of SETD6 substrates","Physiological context of NF-κB regulation in vivo not addressed"]},{"year":2011,"claim":"The crystal structure resolved how SETD6 recognizes its substrate and gave a structural basis for the methyl/phospho switch, explaining how a neighboring phosphorylation event can toggle methylation-dependent repression.","evidence":"X-ray crystallography of SETD6 with RelA peptide and SAM plus biochemical binding assays","pmids":["21515635"],"confidence":"High","gaps":["Single-substrate structure; did not explain multispecificity","No structure of the catalytically active oligomer"]},{"year":2013,"claim":"Identification of H2AZ K7 as a histone substrate showed SETD6 acts directly on chromatin to maintain pluripotency, extending its role from signaling to epigenetic gene regulation.","evidence":"In vitro methylation, ChIP, siRNA knockdown and differentiation/clonogenicity assays in mESCs","pmids":["23324626"],"confidence":"High","gaps":["Reader of H2AZK7me1 not identified","Mechanistic link between mark removal and differentiation incompletely defined"]},{"year":2014,"claim":"Mapping SETD6 into an ERα/HDAC1/MTA2/TRRAP transcriptional context showed it can act as both repressor and estrogen-responsive co-activator, broadening its transcriptional roles in cancer cells.","evidence":"Co-IP/mass spectrometry, luciferase reporters, siRNA knockdown and proliferation/apoptosis assays in breast carcinoma cells","pmids":["24751716"],"confidence":"Medium","gaps":["Whether ERα or complex members are SETD6 substrates not resolved","Single lab, context-specific dual function not mechanistically explained"]},{"year":2016,"claim":"PAK4 methylation linked SETD6 to Wnt/β-catenin signaling, showing methylation enhances PAK4–β-catenin interaction to drive Wnt target genes.","evidence":"In vitro methylation, Co-IP, ChIP, siRNA and luciferase reporter assays","pmids":["26841865"],"confidence":"High","gaps":["Exact methylation site not defined in this study","Effect on PAK4 kinase activity not addressed"]},{"year":2016,"claim":"The DJ1 interaction revealed a catalysis-independent mode: SETD6 can repress Nrf2 antioxidant transcription by binding and inhibiting DJ1 without methylating it, expanding how SETD6 controls stress responses.","evidence":"In vitro pulldown, Co-IP, ChIP, siRNA and qRT-PCR","pmids":["26780326"],"confidence":"Medium","gaps":["Structural basis of the non-catalytic interaction unknown","Mechanism of oxidative-stress-induced SETD6 downregulation not defined"]},{"year":2017,"claim":"A truncating SETD6 mutation in familial colorectal cancer type X established disease relevance and an unexpected dominant-negative mechanism: a catalytically dead but substrate-binding protein competes with wild-type SETD6.","evidence":"Whole-exome sequencing, in vitro methylation, localization and competition/dominant-negative binding assays","pmids":["28973356"],"confidence":"Medium","gaps":["Causality vs. association in CRC-X families not fully established","In vivo consequences of the dominant-negative not tested"]},{"year":2018,"claim":"Discovery of SAM-stabilized oligomerization and activating auto-methylation answered how SETD6 activity is intrinsically regulated, revealing a positive feedback on its own catalytic rate.","evidence":"SEC-MALS, in vitro radioactive methylation, mass spectrometry, mutagenesis and kinetic analysis","pmids":["30189201"],"confidence":"High","gaps":["Whether oligomerization/auto-methylation is regulated in cells unknown","No structure of the oligomeric active form"]},{"year":2018,"claim":"WDR5 methylation at K207/K325 connected SETD6 to H3K4me3 maintenance and breast cancer proliferation/migration, while showing the mark does not alter MLL/SET1 complex assembly.","evidence":"In vitro methylation, K207R/K325R mutagenesis, H3K4me3 western blot, Co-IP and proliferation/migration assays","pmids":["30226578"],"confidence":"Medium","gaps":["Mechanism linking WDR5 methylation to H3K4me3 unclear","Limited independent validation"]},{"year":2019,"claim":"PLK1 methylation showed SETD6 directly restrains a mitotic kinase, with loss of methylation accelerating mitosis and proliferation, placing SETD6 in cell-cycle control.","evidence":"In vitro methylation, kinase assay, KO/KD cell lines, live-cell imaging and PLK1 site mutagenesis","pmids":["30622182"],"confidence":"High","gaps":["How methylation reduces PLK1 catalytic activity structurally unknown","Upstream signals controlling mitotic SETD6 activity unclear"]},{"year":2019,"claim":"In vivo hippocampal work showed SETD6-dependent RelA K310 methylation is required for memory consolidation, demonstrating physiological relevance of the RelA axis in the brain.","evidence":"In vivo siRNA knockdown in rat dorsal hippocampus, ChIP, electrophysiology, behavioral and spine morphology assays","pmids":["31378303"],"confidence":"Medium","gaps":["Direct causal chain from RelA methylation to spine morphology not isolated","Single lab, single model system"]},{"year":2021,"claim":"BRD4 K99 methylation revealed SETD6 controls mRNA translation programs by selectively gating E2F1 recruitment without disrupting BRD4–acetyl-histone binding.","evidence":"In vitro methylation, ChIP, RNA-seq, polysome profiling, Co-IP and BRD4 K99R mutagenesis","pmids":["34039605"],"confidence":"High","gaps":["Reader interpreting BRD4 K99me1 not defined","Generality across cell types not established at the time"]},{"year":2020,"claim":"Defining K473 as the primary PAK4 methylation site connected SETD6 to focal adhesion and migration control, showing methylation both activates β-catenin and suppresses adhesion.","evidence":"In vitro methylation, PAK4 K473R mutagenesis, β-catenin reporter, focal adhesion immunofluorescence and migration/invasion assays","pmids":["33051544"],"confidence":"High","gaps":["Molecular link between PAK4 methylation and paxillin mislocalization unresolved","Relationship to PAK4 kinase function unclear"]},{"year":2022,"claim":"TWIST1 K33 methylation showed SETD6 represses LINC-PINT via EZH2/H3K27me3 to antagonize EMT in glioma, linking SETD6 to lncRNA-mediated migration control.","evidence":"In vitro methylation, ChIP, RNA-seq, knockdown, mutagenesis and migration/adhesion assays","pmids":["35694846"],"confidence":"High","gaps":["How TWIST1 methylation recruits EZH2 mechanistically unclear","In vivo glioma relevance not tested"]},{"year":2023,"claim":"E2F1 K117 methylation defined a transcriptional feedback loop, where E2F1 drives SETD6 expression and its methylation reinforces SETD6 levels, explaining a self-sustaining regulatory circuit.","evidence":"In vitro methylation, ChIP, promoter-binding assay, qRT-PCR, E2F1 K117R mutagenesis and siRNA","pmids":["37690684"],"confidence":"High","gaps":["Downstream effector reading E2F1 K117me1 not identified here","Physiological setting of the feedback loop undefined"]},{"year":2025,"claim":"RAD18 methylation showed SETD6 controls genome stability by regulating RAD18 nuclear/cytoplasmic localization to limit DNA breaks, extending SETD6 into the DNA damage response.","evidence":"Protein microarray, MS, Co-IP, mutagenesis, immunofluorescence, γH2AX western blot and comet assay in SETD6 KO cells","pmids":["40866490"],"confidence":"High","gaps":["Mechanism by which methylation alters RAD18 shuttling unknown","Impact on RAD18-dependent DNA repair pathways not detailed"]},{"year":2025,"claim":"Systematic peptide-array and docking analysis answered how one enzyme methylates many unrelated substrates, defining a context-dependent, multispecific recognition code anchored by L260 contacting the +2 position.","evidence":"AlphaFold3 docking, peptide SPOT array methylation and L260 mutagenesis","pmids":["41157251"],"confidence":"Medium","gaps":["Predictive code not validated genome-wide","Computational docking lacks experimental complex structures for most substrates"]},{"year":2025,"claim":"BRD4 K99 methylation and SETD6–MITF interaction were shown to organize a chromatin BRD4–MITF complex in melanoma, connecting SETD6 methylation to genomic occupancy of both factors.","evidence":"ChIP-seq, Co-IP, SETD6 KO, BRD4 K99R mutagenesis and chromatin fractionation","pmids":["40809945"],"confidence":"Medium","gaps":["Whether MITF is a SETD6 substrate not established","Single lab, melanoma-specific context"]},{"year":2026,"claim":"Resolving the E2F1 K117 methyl/acetyl switch explained how SETD6 methylation prevents acetylation and blocks bromodomain binding to control E2F1 chromatin occupancy and oncogenic output.","evidence":"Biochemical binding assays, ChIP-seq, RNA-seq, Co-IP and E2F1 K117R/acetylation-deficient mutagenesis in prostate cancer cells","pmids":["41540805"],"confidence":"High","gaps":["Generalizability of methyl/acetyl switch to other substrates unclear","Acetyltransferase competing at K117 not identified"]},{"year":null,"claim":"A unifying question remains: how SETD6 substrate selection, oligomerization, and auto-methylation are coordinated in vivo to determine which of its many targets are methylated in a given cell state.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No in-cell regulation of substrate choice defined","No structures of SETD6 with most substrates or in its active oligomeric form","Reader proteins for most methyl marks unidentified"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0016740","term_label":"transferase activity","supporting_discovery_ids":[0,2,3,6,8,9,10,11,15,16]},{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,6,8,9,10,11,15,16]},{"term_id":"GO:0042393","term_label":"histone binding","supporting_discovery_ids":[2]},{"term_id":"GO:0140110","term_label":"transcription regulator activity","supporting_discovery_ids":[4]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[0,2,9,11,16]},{"term_id":"GO:0000228","term_label":"nuclear chromosome","supporting_discovery_ids":[0,3,9,11,18]}],"pathway":[{"term_id":"R-HSA-74160","term_label":"Gene expression (Transcription)","supporting_discovery_ids":[0,9,11,15,19]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[3,5,10]},{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[6,20]},{"term_id":"R-HSA-73894","term_label":"DNA Repair","supporting_discovery_ids":[16]},{"term_id":"R-HSA-4839726","term_label":"Chromatin organization","supporting_discovery_ids":[0,2,11]}],"complexes":[],"partners":["RELA","GLP","PAK4","PLK1","BRD4","E2F1","RAD18","TWIST1"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q8TBK2","full_name":"N-lysine methyltransferase SETD6","aliases":["SET domain-containing protein 6"],"length_aa":473,"mass_kda":53.2,"function":"Protein-lysine N-methyltransferase. Monomethylates 'Lys-310' of the RELA subunit of NF-kappa-B complex, leading to down-regulation of NF-kappa-B transcription factor activity (PubMed:21131967, PubMed:21515635, PubMed:30189201). Monomethylates 'Lys-8' of H2AZ (H2AZK8me1) (PubMed:23324626). Required for the maintenance of embryonic stem cell self-renewal (By similarity). Methylates PAK4","subcellular_location":"Nucleus","url":"https://www.uniprot.org/uniprotkb/Q8TBK2/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/SETD6","classification":"Not Classified","n_dependent_lines":0,"n_total_lines":1208,"dependency_fraction":0.0},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/SETD6","total_profiled":1310},"omim":[{"mim_id":"618505","title":"STOLERMAN NEURODEVELOPMENTAL SYNDROME; NEDSST","url":"https://www.omim.org/entry/618505"},{"mim_id":"616424","title":"SET DOMAIN-CONTAINING PROTEIN 6; SETD6","url":"https://www.omim.org/entry/616424"},{"mim_id":"611577","title":"LYSINE DEMETHYLASE 6B; KDM6B","url":"https://www.omim.org/entry/611577"},{"mim_id":"606594","title":"SET DOMAIN-CONTAINING PROTEIN 7; SETD7","url":"https://www.omim.org/entry/606594"},{"mim_id":"142763","title":"H2A.Z VARIANT HISTONE 1; H2AZ1","url":"https://www.omim.org/entry/142763"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Nucleoplasm","reliability":"Supported"},{"location":"Cytosol","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in 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methylation of the NF-κB subunit RelA by SETD6 couples activity of the histone methyltransferase GLP at chromatin to tonic repression of NF-κB signaling.","date":"2010","source":"Nature immunology","url":"https://pubmed.ncbi.nlm.nih.gov/21131967","citation_count":221,"is_preprint":false},{"pmid":"21515635","id":"PMC_21515635","title":"Structural basis of SETD6-mediated regulation of the NF-kB network via methyl-lysine signaling.","date":"2011","source":"Nucleic acids research","url":"https://pubmed.ncbi.nlm.nih.gov/21515635","citation_count":59,"is_preprint":false},{"pmid":"23324626","id":"PMC_23324626","title":"SETD6 monomethylates H2AZ on lysine 7 and is required for the maintenance of embryonic stem cell self-renewal.","date":"2013","source":"Epigenetics","url":"https://pubmed.ncbi.nlm.nih.gov/23324626","citation_count":57,"is_preprint":false},{"pmid":"26841865","id":"PMC_26841865","title":"PAK4 Methylation by SETD6 Promotes the Activation of the Wnt/β-Catenin Pathway.","date":"2016","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/26841865","citation_count":56,"is_preprint":false},{"pmid":"30622182","id":"PMC_30622182","title":"The methyltransferase SETD6 regulates Mitotic progression through PLK1 methylation.","date":"2019","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/30622182","citation_count":34,"is_preprint":false},{"pmid":"24751716","id":"PMC_24751716","title":"SETD6 controls the expression of estrogen-responsive genes and proliferation of breast carcinoma cells.","date":"2014","source":"Epigenetics","url":"https://pubmed.ncbi.nlm.nih.gov/24751716","citation_count":31,"is_preprint":false},{"pmid":"34039605","id":"PMC_34039605","title":"BRD4 methylation by the methyltransferase SETD6 regulates selective transcription to control mRNA translation.","date":"2021","source":"Science 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palmitic acid-induced apoptosis, and mitochondrial dysfunction via activating Nrf2-Keap1 signaling pathway in diabetic nephropathy.","date":"2020","source":"Journal of molecular histology","url":"https://pubmed.ncbi.nlm.nih.gov/32803470","citation_count":19,"is_preprint":false},{"pmid":"31081088","id":"PMC_31081088","title":"MiR-411 inhibits gastric cancer proliferation and migration through targeting SETD6.","date":"2019","source":"European review for medical and pharmacological sciences","url":"https://pubmed.ncbi.nlm.nih.gov/31081088","citation_count":18,"is_preprint":false},{"pmid":"33051544","id":"PMC_33051544","title":"PAK4 methylation by the methyltransferase SETD6 attenuates cell adhesion.","date":"2020","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/33051544","citation_count":17,"is_preprint":false},{"pmid":"35694846","id":"PMC_35694846","title":"TWIST1 methylation by SETD6 selectively antagonizes LINC-PINT expression in glioma.","date":"2022","source":"Nucleic acids research","url":"https://pubmed.ncbi.nlm.nih.gov/35694846","citation_count":16,"is_preprint":false},{"pmid":"30226578","id":"PMC_30226578","title":"Lysines 207 and 325 methylation of WDR5 catalyzed by SETD6 promotes breast cancer cell proliferation and migration.","date":"2018","source":"Oncology reports","url":"https://pubmed.ncbi.nlm.nih.gov/30226578","citation_count":14,"is_preprint":false},{"pmid":"29435148","id":"PMC_29435148","title":"Peptide inhibition of the SETD6 methyltransferase catalytic activity.","date":"2017","source":"Oncotarget","url":"https://pubmed.ncbi.nlm.nih.gov/29435148","citation_count":13,"is_preprint":false},{"pmid":"36300937","id":"PMC_36300937","title":"SETD6 Regulates E2-Dependent Human Papillomavirus Transcription.","date":"2022","source":"Journal of virology","url":"https://pubmed.ncbi.nlm.nih.gov/36300937","citation_count":9,"is_preprint":false},{"pmid":"30189201","id":"PMC_30189201","title":"Oligomerization and Auto-methylation of the Human Lysine Methyltransferase SETD6.","date":"2018","source":"Journal of molecular biology","url":"https://pubmed.ncbi.nlm.nih.gov/30189201","citation_count":9,"is_preprint":false},{"pmid":"37690684","id":"PMC_37690684","title":"Methylation of the transcription factor E2F1 by SETD6 regulates SETD6 expression via a positive feedback mechanism.","date":"2023","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/37690684","citation_count":8,"is_preprint":false},{"pmid":"35550916","id":"PMC_35550916","title":"Structure-function conservation between the methyltransferases SETD3 and SETD6.","date":"2022","source":"Biochimie","url":"https://pubmed.ncbi.nlm.nih.gov/35550916","citation_count":6,"is_preprint":false},{"pmid":"33710605","id":"PMC_33710605","title":"Silencing of SETD6 inhibits the tumorigenesis of oral squamous cell carcinoma by inhibiting methylation of PAK4 and RelA.","date":"2021","source":"Histology and 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adenocarcinoma.","date":"2023","source":"BMC cancer","url":"https://pubmed.ncbi.nlm.nih.gov/36604642","citation_count":3,"is_preprint":false},{"pmid":"40866490","id":"PMC_40866490","title":"RAD18 methylation by the methyltransferase SETD6 attenuates DNA breaks.","date":"2025","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/40866490","citation_count":1,"is_preprint":false},{"pmid":"41157251","id":"PMC_41157251","title":"Mechanisms of Substrate Recognition by the Multispecific Protein Lysine Methyltransferase SETD6.","date":"2025","source":"Life (Basel, Switzerland)","url":"https://pubmed.ncbi.nlm.nih.gov/41157251","citation_count":1,"is_preprint":false},{"pmid":"40809945","id":"PMC_40809945","title":"SETD6 mediates selective interaction and genomic occupancy of BRD4 and MITF in melanoma cells.","date":"2025","source":"NAR cancer","url":"https://pubmed.ncbi.nlm.nih.gov/40809945","citation_count":0,"is_preprint":false},{"pmid":"41540805","id":"PMC_41540805","title":"E2F1 K117 methylation by SETD6 disrupts BRD4-E2F1 binding and modulates E2F1 chromatin binding and gene regulation in prostate cancer cells.","date":"2026","source":"Nucleic acids research","url":"https://pubmed.ncbi.nlm.nih.gov/41540805","citation_count":0,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2024.12.22.629973","title":"AuroraB-kinase methylation by SETD6 regulates cytokinesis and protects cells from chromosomal instability","date":"2024-12-22","source":"bioRxiv","url":"https://doi.org/10.1101/2024.12.22.629973","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":15222,"output_tokens":5733,"usd":0.065831,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":14240,"output_tokens":5919,"usd":0.109588,"stage2_stop_reason":"end_turn"},"total_usd":0.175419,"stage1_batch_id":"msgbatch_013LWZZT6MWaqB9qyTSeQBmU","stage2_batch_id":"msgbatch_0194ZzQaTGNpruoNJBbEMbt3","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2010,\n      \"finding\": \"SETD6 monomethylates the NF-κB subunit RelA at Lys310 (RelAK310me1), rendering RelA transcriptionally inert and attenuating NF-κB-driven inflammatory gene programs. RelAK310me1 is recognized by the ankyrin repeat of histone methyltransferase GLP, which promotes a repressed chromatin state at RelA target genes via GLP-mediated H3K9 methylation. Phosphorylation of RelA at Ser311 by PKC-ζ blocks GLP binding to RelAK310me1 and relieves transcriptional repression.\",\n      \"method\": \"Biochemical screen of >40 PKMTs, in vitro methylation assays, Co-IP, ChIP, primary immune cell gene expression analysis, site-directed mutagenesis\",\n      \"journal\": \"Nature immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — in vitro enzymatic assay plus reciprocal Co-IP plus ChIP plus mutagenesis, replicated across multiple labs\",\n      \"pmids\": [\"21131967\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Crystal structure of SETD6 in complex with RelA peptide and SAM revealed a V-like protein structure and the molecular basis for substrate recognition; Ser311 phosphorylation sterically inhibits Lys310 methylation by SETD6 and binding of Lys310me1 by GLP ankyrin repeats, establishing a methylation-phosphorylation switch at adjacent residues.\",\n      \"method\": \"X-ray crystallography, structural modeling, biochemical binding assays\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure with functional validation of phosphorylation-methylation switch, consistent with independent findings in PMID:21131967\",\n      \"pmids\": [\"21515635\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"SETD6 monomethylates histone variant H2AZ at Lys7 (H2AZK7me1). H2AZK7me1 and H3K27me3 co-occupy transcriptional start sites of differentiation genes in mESCs; upon retinoic acid-induced differentiation both marks are removed. Setd6 depletion in mESCs leads to spontaneous differentiation, compromised self-renewal, and poor clonogenicity.\",\n      \"method\": \"In vitro methylation assay, ChIP, siRNA knockdown, clonogenicity assay, differentiation assays in mESCs\",\n      \"journal\": \"Epigenetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — in vitro enzymatic assay plus ChIP plus genetic loss-of-function with defined cellular phenotype, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"23324626\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"SETD6 methylates PAK4 in vitro and at chromatin in cells. SETD6-mediated PAK4 methylation enhances physical interaction between PAK4 and β-catenin, promoting transcription of Wnt/β-catenin target genes. Depletion of SETD6 significantly hinders Wnt/β-catenin target gene activation.\",\n      \"method\": \"In vitro methylation assay, Co-IP, ChIP, siRNA knockdown, luciferase reporter assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — in vitro assay plus reciprocal Co-IP plus ChIP plus reporter assays, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"26841865\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"SETD6 associates with estrogen receptor α (ERα), HDAC1, MTA2, and TRRAP. SETD6 acts as a transcriptional repressor in reporter assays but functions as a co-activator of estrogen-responsive genes (PGR, TFF1). SETD6 silencing in breast carcinoma cells induces proliferation defects, enhanced CDKN1A expression, and apoptosis.\",\n      \"method\": \"Co-immunoprecipitation/mass spectrometry, luciferase reporter assay, siRNA knockdown, cell proliferation and apoptosis assays\",\n      \"journal\": \"Epigenetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — reciprocal Co-IP plus reporter assay plus KD phenotype, single lab\",\n      \"pmids\": [\"24751716\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"SETD6 interacts with oxidative stress sensor DJ1 both in vitro and in cells at chromatin. Through this catalytically independent interaction, SETD6 inhibits DJ1 activity and represses Nrf2-dependent antioxidant gene transcription. Under oxidative stress, SETD6 protein levels decrease, weakening the SETD6-DJ1 interaction and de-repressing Nrf2 target genes. SETD6 does not methylate DJ1.\",\n      \"method\": \"In vitro pulldown, Co-IP, ChIP, siRNA knockdown, qRT-PCR gene expression analysis\",\n      \"journal\": \"Biochimica et biophysica acta\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — in vitro pulldown plus Co-IP plus ChIP plus KD, single lab with multiple methods\",\n      \"pmids\": [\"26780326\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"SETD6 binds and methylates PLK1 at K209 and K413 during mitosis. Loss of these methylation sites increases PLK1 kinase activity, leading to accelerated mitotic progression and faster cellular proliferation. SETD6-deficient cells similarly progress faster through mitotic steps toward cytokinesis.\",\n      \"method\": \"In vitro methylation assay, kinase activity assay, SETD6 KO/KD cell lines, live-cell imaging of mitosis, site-directed mutagenesis of PLK1 methylation sites\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — in vitro methylation plus kinase assay plus mutagenesis plus KO cell phenotype, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"30622182\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"A truncating SETD6 mutation (p.Met264IlefsTer3) found in familial colorectal cancer type X lacks methyltransferase activity while retaining expression, localization, and substrate-binding. The truncated protein competes with wild-type SETD6 for substrates, acting as a dominant negative inhibitor of SETD6 function.\",\n      \"method\": \"Whole-exome sequencing, in vitro methylation assay, localization analysis, competition binding assays, dominant-negative characterization\",\n      \"journal\": \"Human molecular genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — enzymatic assay plus localization plus dominant-negative competition experiments, single lab\",\n      \"pmids\": [\"28973356\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"SETD6 forms high-molecular-weight oligomeric structures (monomer, dimer, trimer) stabilized by SAM. SETD6 auto-methylates at K39, K179, and K372. A K179 point mutation in the SET domain impairs trimer formation. Auto-methylation at K39 and K179 increases SETD6 catalytic rate in vitro, linking auto-methylation to oligomerization and enzymatic activation.\",\n      \"method\": \"SEC-MALS, in vitro radioactive methylation assay, mass spectrometry, site-directed mutagenesis, kinetic analysis\",\n      \"journal\": \"Journal of molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro biochemical reconstitution plus mutagenesis plus mass spectrometry plus kinetics, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"30189201\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"SETD6 methylates BRD4 at Lys99 on chromatin. BRD4 methylation negatively regulates expression of genes involved in mRNA translation and inhibits total mRNA translation in cells. Mechanistically, BRD4 K99 methylation does not affect BRD4 association with acetylated histone H4 but specifically determines recruitment of transcription factor E2F1 to translation-related target genes.\",\n      \"method\": \"In vitro methylation assay, ChIP, RNA-seq, polysome profiling, Co-IP, site-directed mutagenesis (BRD4 K99R)\",\n      \"journal\": \"Science advances\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — in vitro assay plus ChIP plus RNA-seq plus mutagenesis plus polysome profiling, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"34039605\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"SETD6 methylates PAK4 at Lys473 as the primary methylation site. PAK4 K473 methylation activates β-catenin transcriptional activity and inhibits cell adhesion by attenuating paxillin localization to focal adhesions, reducing filopodia and actin structures, and decreasing cell migration and invasion.\",\n      \"method\": \"In vitro methylation assay, site-directed mutagenesis (PAK4 K473R), β-catenin reporter assay, immunofluorescence of focal adhesions, migration/invasion assays\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — in vitro methylation plus mutagenesis plus cellular localization plus functional phenotype, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"33051544\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"SETD6 methylates TWIST1 at Lys33 on chromatin. TWIST1 methylation represses transcription of the long non-coding RNA LINC-PINT by increasing EZH2 occupancy and H3K27me3 at the LINC-PINT locus. Unmethylated TWIST1 dissociates from the LINC-PINT locus, allowing LINC-PINT expression, which promotes cell adhesion and reduces cell migration, thereby antagonizing EMT in glioma.\",\n      \"method\": \"In vitro methylation assay, ChIP, RNA-seq, SETD6/TWIST1 KD, site-directed mutagenesis, migration and adhesion assays\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — in vitro assay plus ChIP plus RNA-seq plus mutagenesis plus functional phenotype, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"35694846\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"SETD6 monomethylates WDR5 at Lys207 and Lys325. Disruption of these methylation sites by K207R/K325R double mutation attenuates WDR5-promoted breast cancer cell proliferation and migration. WDR5 K207/K325 methylation partially maintains global H3K4me3 levels but does not affect MLL/SET1 complex assembly.\",\n      \"method\": \"In vitro methylation assay, site-directed mutagenesis (K207R/K325R), western blot for H3K4me3, cell proliferation and migration assays, Co-IP for complex assembly\",\n      \"journal\": \"Oncology reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1–2 / Weak — in vitro assay plus mutagenesis plus cellular phenotype, single lab, limited independent validation\",\n      \"pmids\": [\"30226578\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"SETD6 is necessary for RelA K310 methylation and associated H3K9me2 increases in the dorsal hippocampus during memory consolidation. Setd6 knockdown in rat dorsal hippocampus interferes with memory consolidation, alters gene expression, and disrupts dendritic spine morphology.\",\n      \"method\": \"siRNA knockdown in vivo (rat dorsal hippocampus), biochemical assays, ChIP, electrophysiology, behavioral memory tests, spine morphology imaging\",\n      \"journal\": \"Biological psychiatry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo KD plus biochemical plus ChIP plus behavioral phenotype, single lab with multiple methods\",\n      \"pmids\": [\"31378303\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"A cell-penetrating peptide (vp22-RelA302-316) based on the SETD6 methylation site of RelA directly and specifically binds SETD6 in vitro, inhibits its methyltransferase activity, and upon cellular treatment leads to induced cellular migration and proliferation consistent with SETD6 catalytic blockade.\",\n      \"method\": \"In vitro binding assay, in vitro methylation inhibition assay, cell-penetrating peptide treatment, migration and proliferation assays\",\n      \"journal\": \"Oncotarget\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1–3 / Moderate — in vitro enzymatic inhibition plus cellular functional readout, single lab\",\n      \"pmids\": [\"29435148\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"SETD6 monomethylates transcription factor E2F1 at Lys117 in vitro and in cells. E2F1 binds to the SETD6 promoter and drives SETD6 mRNA expression; E2F1 K117 methylation by SETD6 in turn positively enhances SETD6 mRNA levels, establishing a positive feedback loop. A K117R E2F1 mutant that cannot be methylated reverses this effect.\",\n      \"method\": \"In vitro methylation assay, ChIP, promoter-binding assay, qRT-PCR, site-directed mutagenesis (E2F1 K117R), siRNA KD\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — in vitro methylation plus ChIP plus mutagenesis plus KD rescue, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"37690684\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"SETD6 binds and methylates RAD18 at K73 and K406. SETD6 KO cells show increased nuclear RAD18 localization, elevated γH2AX, and increased DNA breaks (comet assay). Restoration of SETD6 enzymatic activity reduces DNA damage, while a catalytically inactive mutant does not, demonstrating that SETD6-mediated methylation of RAD18 regulates its nuclear/cytoplasmic localization and attenuates DNA breaks.\",\n      \"method\": \"Protein microarray, ELISA, Co-IP, mass spectrometry, site-directed mutagenesis, immunofluorescence (nuclear localization), γH2AX western blot, comet assay, SETD6 KO cell lines\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — in vitro biochemical identification plus MS plus mutagenesis plus KO phenotype plus localization, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"40866490\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Peptide SPOT array and AlphaFold3 docking experiments reveal that SETD6 substrate recognition relies on sequence preferences at positions -1 (glycine or large aliphatic), +2 (Ile/Val), and +3 (Lys) relative to the target lysine. These preferences are context-dependent and variable across substrates; SETD6 residue L260 contacts the +2 position in the SETD6-RELA complex structure, and L260 mutation shows substrate-specific effects, revealing a versatile, multispecific mode of substrate recognition.\",\n      \"method\": \"AlphaFold3 structural docking, peptide SPOT array methylation assay, site-directed mutagenesis (L260), in vitro methylation\",\n      \"journal\": \"Life (Basel, Switzerland)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1–3 / Moderate — SPOT array plus mutagenesis plus structural modeling, single lab\",\n      \"pmids\": [\"41157251\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"BRD4 K99 methylation by SETD6 in melanoma cells determines BRD4 genomic occupancy. SETD6 KO or BRD4 K99R mutation disrupts BRD4 chromatin distribution. SETD6 also interacts with MITF and influences MITF genomic distribution. BRD4 and MITF form a chromatin-localized complex dependent on both SETD6-mediated BRD4 methylation and MITF acetylation.\",\n      \"method\": \"ChIP-seq, Co-IP, SETD6 KO, BRD4 K99R mutagenesis, immunoprecipitation, chromatin fractionation\",\n      \"journal\": \"NAR cancer\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP-seq plus Co-IP plus KO plus mutagenesis, single lab\",\n      \"pmids\": [\"40809945\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"SETD6-mediated K117 monomethylation of E2F1 disrupts BRD4-E2F1 interaction by preventing K117 acetylation, establishing a methylation/acetylation switch at K117 that controls bromodomain binding to E2F1. This switch modulates E2F1 chromatin occupancy and target gene expression, affecting oncogenic phenotypes in prostate cancer cells.\",\n      \"method\": \"Biochemical binding assays, ChIP-seq, RNA-seq, Co-IP, site-directed mutagenesis (E2F1 K117R, acetylation-deficient mutants), cellular oncogenic phenotype assays\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — biochemical assays plus ChIP-seq plus mutagenesis plus cellular phenotype, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"41540805\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"SETD6 binds and methylates Aurora-B kinase on two adjacent lysine residues. SETD6-depleted HeLa cells display chromatin bridges, actin patches, and increased multinucleation under replication stress — hallmarks of chromosomal instability. Aurora-B methylation by SETD6 increases under replication stress but is abolished when the two target lysines are mutated. SETD6 KO cells show reduced Aurora-B kinase activity during cytokinesis, suggesting that SETD6-mediated methylation sustains Aurora-B activity to ensure proper cytokinesis.\",\n      \"method\": \"Proteomic screen, in vitro methylation assay, site-directed mutagenesis, SETD6 KO live-cell imaging, kinase activity assay, immunofluorescence\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1–2 / Weak — in vitro assay plus mutagenesis plus KO phenotype, single lab, preprint not yet peer-reviewed\",\n      \"pmids\": [\"bio_10.1101_2024.12.22.629973\"],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"SETD6 is a monomethyltransferase that modifies multiple histone and non-histone substrates (RelA/K310, H2AZ/K7, PAK4/K473, PLK1/K209+K413, BRD4/K99, TWIST1/K33, WDR5/K207+K325, E2F1/K117, RAD18/K73+K406, Aurora-B) at chromatin; these methylation events regulate downstream signaling cascades (NF-κB, Wnt/β-catenin, oxidative stress/Nrf2, Wnt-dependent cell adhesion, mRNA translation, EMT, DNA damage repair, and cell cycle progression) through mechanisms that include creating methyl-lysine docking sites for effector proteins (e.g., GLP ankyrin repeats reading RelAK310me1), imposing methyl/phospho or methyl/acetyl switches at adjacent residues that gate effector binding, modulating kinase activities (PLK1, Aurora-B), controlling nuclear-cytoplasmic shuttling of substrates (RAD18), and auto-methylation-driven oligomerization that positively feeds back on its own catalytic rate.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"SETD6 is a protein lysine monomethyltransferase that controls diverse signaling and transcriptional programs by installing single methyl marks on chromatin-associated histone and non-histone substrates [#0, #2, #3]. Its founding activity is monomethylation of the NF-\\u03baB subunit RelA at Lys310, which renders RelA transcriptionally inert and recruits the methyltransferase GLP, whose ankyrin repeat reads RelAK310me1 to impose a repressive H3K9-methylated chromatin state; adjacent Ser311 phosphorylation by PKC-\\u03b6 blocks both the methyl mark and GLP binding, defining a methyl/phospho switch resolved structurally in a SETD6\\u2013RelA\\u2013SAM complex [#0, #1]. SETD6 substrate recognition is multispecific, governed by sequence preferences flanking the target lysine and by SETD6 residue L260, which contacts the +2 position [#17]. Beyond RelA, SETD6 methylates histone variant H2AZ at Lys7 to maintain mES cell self-renewal [#2], PAK4 to potentiate \\u03b2-catenin\\u2013driven Wnt target gene transcription and restrain cell adhesion and migration [#3, #10], PLK1 (K209/K413) to restrain its kinase activity and slow mitotic progression [#6], BRD4 at Lys99 to repress translation-related genes and direct BRD4 genomic occupancy and an E2F1/MITF chromatin complex [#9, #18], TWIST1 at Lys33 to repress LINC-PINT via EZH2/H3K27me3 and antagonize EMT [#11], E2F1 at Lys117 in a positive autoregulatory loop that disrupts E2F1 acetylation and bromodomain binding [#15, #19], and RAD18 to control its nuclear localization and limit DNA breaks [#16]. SETD6 also engages substrates non-catalytically, inhibiting the oxidative-stress sensor DJ1 to repress Nrf2-dependent antioxidant transcription without methylating it [#5]. SETD6 forms SAM-stabilized oligomers and auto-methylates at K39, K179, and K372, with auto-methylation enhancing its own catalytic rate [#8]. A truncating SETD6 mutation lacking methyltransferase activity acts as a dominant negative and is associated with familial colorectal cancer type X [#7].\",\n  \"teleology\": [\n    {\n      \"year\": 2010,\n      \"claim\": \"Establishing that SETD6 has a defined non-histone substrate answered whether this enzyme had a specific biological function: it monomethylates RelA at K310 to silence NF-\\u03baB transcription via GLP-dependent chromatin repression.\",\n      \"evidence\": \"Biochemical PKMT screen, in vitro methylation, Co-IP, ChIP and primary immune cell expression analysis\",\n      \"pmids\": [\"21131967\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not establish the full repertoire of SETD6 substrates\", \"Physiological context of NF-\\u03baB regulation in vivo not addressed\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"The crystal structure resolved how SETD6 recognizes its substrate and gave a structural basis for the methyl/phospho switch, explaining how a neighboring phosphorylation event can toggle methylation-dependent repression.\",\n      \"evidence\": \"X-ray crystallography of SETD6 with RelA peptide and SAM plus biochemical binding assays\",\n      \"pmids\": [\"21515635\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Single-substrate structure; did not explain multispecificity\", \"No structure of the catalytically active oligomer\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Identification of H2AZ K7 as a histone substrate showed SETD6 acts directly on chromatin to maintain pluripotency, extending its role from signaling to epigenetic gene regulation.\",\n      \"evidence\": \"In vitro methylation, ChIP, siRNA knockdown and differentiation/clonogenicity assays in mESCs\",\n      \"pmids\": [\"23324626\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Reader of H2AZK7me1 not identified\", \"Mechanistic link between mark removal and differentiation incompletely defined\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Mapping SETD6 into an ER\\u03b1/HDAC1/MTA2/TRRAP transcriptional context showed it can act as both repressor and estrogen-responsive co-activator, broadening its transcriptional roles in cancer cells.\",\n      \"evidence\": \"Co-IP/mass spectrometry, luciferase reporters, siRNA knockdown and proliferation/apoptosis assays in breast carcinoma cells\",\n      \"pmids\": [\"24751716\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether ER\\u03b1 or complex members are SETD6 substrates not resolved\", \"Single lab, context-specific dual function not mechanistically explained\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"PAK4 methylation linked SETD6 to Wnt/\\u03b2-catenin signaling, showing methylation enhances PAK4\\u2013\\u03b2-catenin interaction to drive Wnt target genes.\",\n      \"evidence\": \"In vitro methylation, Co-IP, ChIP, siRNA and luciferase reporter assays\",\n      \"pmids\": [\"26841865\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Exact methylation site not defined in this study\", \"Effect on PAK4 kinase activity not addressed\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"The DJ1 interaction revealed a catalysis-independent mode: SETD6 can repress Nrf2 antioxidant transcription by binding and inhibiting DJ1 without methylating it, expanding how SETD6 controls stress responses.\",\n      \"evidence\": \"In vitro pulldown, Co-IP, ChIP, siRNA and qRT-PCR\",\n      \"pmids\": [\"26780326\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Structural basis of the non-catalytic interaction unknown\", \"Mechanism of oxidative-stress-induced SETD6 downregulation not defined\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"A truncating SETD6 mutation in familial colorectal cancer type X established disease relevance and an unexpected dominant-negative mechanism: a catalytically dead but substrate-binding protein competes with wild-type SETD6.\",\n      \"evidence\": \"Whole-exome sequencing, in vitro methylation, localization and competition/dominant-negative binding assays\",\n      \"pmids\": [\"28973356\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Causality vs. association in CRC-X families not fully established\", \"In vivo consequences of the dominant-negative not tested\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Discovery of SAM-stabilized oligomerization and activating auto-methylation answered how SETD6 activity is intrinsically regulated, revealing a positive feedback on its own catalytic rate.\",\n      \"evidence\": \"SEC-MALS, in vitro radioactive methylation, mass spectrometry, mutagenesis and kinetic analysis\",\n      \"pmids\": [\"30189201\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether oligomerization/auto-methylation is regulated in cells unknown\", \"No structure of the oligomeric active form\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"WDR5 methylation at K207/K325 connected SETD6 to H3K4me3 maintenance and breast cancer proliferation/migration, while showing the mark does not alter MLL/SET1 complex assembly.\",\n      \"evidence\": \"In vitro methylation, K207R/K325R mutagenesis, H3K4me3 western blot, Co-IP and proliferation/migration assays\",\n      \"pmids\": [\"30226578\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism linking WDR5 methylation to H3K4me3 unclear\", \"Limited independent validation\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"PLK1 methylation showed SETD6 directly restrains a mitotic kinase, with loss of methylation accelerating mitosis and proliferation, placing SETD6 in cell-cycle control.\",\n      \"evidence\": \"In vitro methylation, kinase assay, KO/KD cell lines, live-cell imaging and PLK1 site mutagenesis\",\n      \"pmids\": [\"30622182\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How methylation reduces PLK1 catalytic activity structurally unknown\", \"Upstream signals controlling mitotic SETD6 activity unclear\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"In vivo hippocampal work showed SETD6-dependent RelA K310 methylation is required for memory consolidation, demonstrating physiological relevance of the RelA axis in the brain.\",\n      \"evidence\": \"In vivo siRNA knockdown in rat dorsal hippocampus, ChIP, electrophysiology, behavioral and spine morphology assays\",\n      \"pmids\": [\"31378303\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct causal chain from RelA methylation to spine morphology not isolated\", \"Single lab, single model system\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"BRD4 K99 methylation revealed SETD6 controls mRNA translation programs by selectively gating E2F1 recruitment without disrupting BRD4\\u2013acetyl-histone binding.\",\n      \"evidence\": \"In vitro methylation, ChIP, RNA-seq, polysome profiling, Co-IP and BRD4 K99R mutagenesis\",\n      \"pmids\": [\"34039605\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Reader interpreting BRD4 K99me1 not defined\", \"Generality across cell types not established at the time\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Defining K473 as the primary PAK4 methylation site connected SETD6 to focal adhesion and migration control, showing methylation both activates \\u03b2-catenin and suppresses adhesion.\",\n      \"evidence\": \"In vitro methylation, PAK4 K473R mutagenesis, \\u03b2-catenin reporter, focal adhesion immunofluorescence and migration/invasion assays\",\n      \"pmids\": [\"33051544\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular link between PAK4 methylation and paxillin mislocalization unresolved\", \"Relationship to PAK4 kinase function unclear\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"TWIST1 K33 methylation showed SETD6 represses LINC-PINT via EZH2/H3K27me3 to antagonize EMT in glioma, linking SETD6 to lncRNA-mediated migration control.\",\n      \"evidence\": \"In vitro methylation, ChIP, RNA-seq, knockdown, mutagenesis and migration/adhesion assays\",\n      \"pmids\": [\"35694846\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How TWIST1 methylation recruits EZH2 mechanistically unclear\", \"In vivo glioma relevance not tested\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"E2F1 K117 methylation defined a transcriptional feedback loop, where E2F1 drives SETD6 expression and its methylation reinforces SETD6 levels, explaining a self-sustaining regulatory circuit.\",\n      \"evidence\": \"In vitro methylation, ChIP, promoter-binding assay, qRT-PCR, E2F1 K117R mutagenesis and siRNA\",\n      \"pmids\": [\"37690684\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Downstream effector reading E2F1 K117me1 not identified here\", \"Physiological setting of the feedback loop undefined\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"RAD18 methylation showed SETD6 controls genome stability by regulating RAD18 nuclear/cytoplasmic localization to limit DNA breaks, extending SETD6 into the DNA damage response.\",\n      \"evidence\": \"Protein microarray, MS, Co-IP, mutagenesis, immunofluorescence, \\u03b3H2AX western blot and comet assay in SETD6 KO cells\",\n      \"pmids\": [\"40866490\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism by which methylation alters RAD18 shuttling unknown\", \"Impact on RAD18-dependent DNA repair pathways not detailed\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Systematic peptide-array and docking analysis answered how one enzyme methylates many unrelated substrates, defining a context-dependent, multispecific recognition code anchored by L260 contacting the +2 position.\",\n      \"evidence\": \"AlphaFold3 docking, peptide SPOT array methylation and L260 mutagenesis\",\n      \"pmids\": [\"41157251\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Predictive code not validated genome-wide\", \"Computational docking lacks experimental complex structures for most substrates\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"BRD4 K99 methylation and SETD6\\u2013MITF interaction were shown to organize a chromatin BRD4\\u2013MITF complex in melanoma, connecting SETD6 methylation to genomic occupancy of both factors.\",\n      \"evidence\": \"ChIP-seq, Co-IP, SETD6 KO, BRD4 K99R mutagenesis and chromatin fractionation\",\n      \"pmids\": [\"40809945\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether MITF is a SETD6 substrate not established\", \"Single lab, melanoma-specific context\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Resolving the E2F1 K117 methyl/acetyl switch explained how SETD6 methylation prevents acetylation and blocks bromodomain binding to control E2F1 chromatin occupancy and oncogenic output.\",\n      \"evidence\": \"Biochemical binding assays, ChIP-seq, RNA-seq, Co-IP and E2F1 K117R/acetylation-deficient mutagenesis in prostate cancer cells\",\n      \"pmids\": [\"41540805\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Generalizability of methyl/acetyl switch to other substrates unclear\", \"Acetyltransferase competing at K117 not identified\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"A unifying question remains: how SETD6 substrate selection, oligomerization, and auto-methylation are coordinated in vivo to determine which of its many targets are methylated in a given cell state.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No in-cell regulation of substrate choice defined\", \"No structures of SETD6 with most substrates or in its active oligomeric form\", \"Reader proteins for most methyl marks unidentified\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0016740\", \"supporting_discovery_ids\": [0, 2, 3, 6, 8, 9, 10, 11, 15, 16]},\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 6, 8, 9, 10, 11, 15, 16]},\n      {\"term_id\": \"GO:0042393\", \"supporting_discovery_ids\": [2]},\n      {\"term_id\": \"GO:0140110\", \"supporting_discovery_ids\": [4]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [0, 2, 9, 11, 16]},\n      {\"term_id\": \"GO:0000228\", \"supporting_discovery_ids\": [0, 3, 9, 11, 18]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-74160\", \"supporting_discovery_ids\": [0, 9, 11, 15, 19]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [3, 5, 10]},\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [6, 20]},\n      {\"term_id\": \"R-HSA-73894\", \"supporting_discovery_ids\": [16]},\n      {\"term_id\": \"R-HSA-4839726\", \"supporting_discovery_ids\": [0, 2, 11]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"RELA\", \"GLP\", \"PAK4\", \"PLK1\", \"BRD4\", \"E2F1\", \"RAD18\", \"TWIST1\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":7,"faith_pct":85.71428571428571}}