{"gene":"NSD1","run_date":"2026-06-10T05:19:52","timeline":{"discoveries":[{"year":1998,"finding":"NSD1 contains two distinct nuclear receptor interaction domains (NID-L and NID+L): NID-L interacts with unliganded ligand-binding domains (LBDs) of RAR and TR via helix-1-dependent contacts (corepressor-like), while NID+L interacts with liganded LBDs of RAR, TR, RXR, and ER via a novel FxxLL variant of the NR box motif (coactivator-like). NSD1 also contains separate repression and activation domains, establishing it as a bifunctional transcriptional intermediary factor.","method":"Yeast two-hybrid, GST pulldown, LBD mutagenesis, co-transfection reporter assays","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 1 / Strong — multiple orthogonal binding assays combined with domain mutagenesis in a single rigorous study; foundational characterization paper","pmids":["9628876"],"is_preprint":false},{"year":2003,"finding":"NSD1's SET domain possesses intrinsic histone methyltransferase activity with specificity for H3K36 and H4K20. NSD1-null embryos die during gastrulation, demonstrating it is essential for early post-implantation development.","method":"In vitro histone methyltransferase assay with recombinant SET domain; gene-targeted knockout mice","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro enzymatic reconstitution combined with genetic loss-of-function in mice; replicated by subsequent studies","pmids":["12805229"],"is_preprint":false},{"year":2001,"finding":"NSD1 is fused in-frame to NUP98 by the t(5;11)(q35;p15.5) translocation in childhood AML, producing a chimeric NUP98-NSD1 mRNA and the reciprocal NSD1-NUP98 transcript. This was the first implication of NSD1 in human malignancy.","method":"3'-RACE PCR, RT-PCR, FISH","journal":"Blood","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — molecular cloning of fusion transcript confirmed by multiple PCR-based methods in a single study; no functional assay in this paper","pmids":["11493482"],"is_preprint":false},{"year":2001,"finding":"NSD1 (also known as ARA267-alpha) functions as an androgen receptor (AR) coregulator: both its N-terminal and C-terminal regions interact with the AR DNA- and ligand-binding domains, and it enhances AR transactivation in a dihydrotestosterone-dependent manner in prostate cancer cells.","method":"Yeast two-hybrid, GST pulldown, luciferase and CAT reporter assays in PC-3 and H1299 cells","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal binding assays plus functional reporter assays in two cell lines; single lab, two orthogonal methods","pmids":["11509567"],"is_preprint":false},{"year":2007,"finding":"The NUP98-NSD1 fusion induces AML in vivo, sustains myeloid stem cell self-renewal in vitro, and enforces expression of HoxA7, HoxA9, HoxA10, and Meis1. Mechanistically, NUP98-NSD1 binds genomic elements at the Hox-A locus, maintains H3K36 methylation and histone acetylation there, and prevents EZH2-mediated transcriptional repression. Deletion of the NUP98 FG-repeat domain or inactivating mutations of the NSD1 SET domain (H3K36 methyltransferase activity) abolished Hox-A activation and progenitor immortalization.","method":"Retroviral transduction + murine bone marrow transplantation (in vivo AML), methylcellulose colony assay, ChIP, SET-domain point mutagenesis, gene-expression analysis","journal":"Nature cell biology","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — in vivo leukemia model plus SET-domain mutagenesis plus ChIP; multiple orthogonal methods establishing mechanism","pmids":["17589499"],"is_preprint":false},{"year":2009,"finding":"NSD1 methylates p65 (NF-κB subunit) at K218 and K221, activating NF-κB target gene expression. The lysine demethylase FBXL11 reverses this modification. Overexpression of NSD1 activates NF-κB and reverses FBXL11-mediated inhibition; knockdown of NSD1 decreases NF-κB activation. NF-κB-dependent gene expression in mouse embryo fibroblasts relies on K218/K221 methylation.","method":"Overexpression/knockdown in cells, MS-based identification of methylation sites, luciferase reporter assays, MEF gene-expression analysis","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — MS identification of modification sites plus functional reporter assays; single lab, two orthogonal methods","pmids":["20080798"],"is_preprint":false},{"year":2009,"finding":"Epigenetic inactivation of NSD1 by CpG island promoter hypermethylation in neuroblastoma and glioma cells causes specifically diminished H4K20 and H3K36 methylation. Restored NSD1 expression reduces colony formation and inhibits cell growth (tumor-suppressor-like activity). ChIP analysis identified MEIS1 as a direct NSD1 target in neuroblastoma.","method":"Bisulfite sequencing, ChIP, expression microarray, colony formation assay, re-expression experiments in cell lines","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ChIP plus functional rescue; single lab, two orthogonal methods","pmids":["20018718"],"is_preprint":false},{"year":2010,"finding":"The 1.7 Å crystal structure of the NSD1 catalytic domain reveals that a regulatory (autoinhibitory) loop occludes the H3K36 substrate access channel to the bound SAM cofactor. Molecular dynamics and docking show this loop can adopt an active conformation, and that the nucleosome likely stabilizes the active state, explaining NSD1's preference for nucleosomal substrate and its dimethyl-H3K36 product specificity.","method":"X-ray crystallography (1.7 Å), molecular dynamics simulation, computational docking","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — high-resolution crystal structure plus MD simulation; single lab but Tier-1 structural method","pmids":["21196496"],"is_preprint":false},{"year":2010,"finding":"NSD1 binds near promoter elements of target genes (e.g., BMP4, ZFP36L1), regulates H3K36 methylation primarily in the promoter-proximal region, reduces RNA Pol II recruitment to the BMP4 promoter, and causes inappropriate persistence of Ser5-phosphorylated RNAP II with reduced Ser2 phosphorylation within the CTD, linking NSD1-dependent H3K36 methylation to RNAP II elongation control.","method":"ChIP-seq, ChIP-qPCR, siRNA knockdown, Western blot for RNAP II CTD phospho-forms","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ChIP-seq plus RNAP II phospho-state analysis; single lab, two orthogonal methods","pmids":["20837538"],"is_preprint":false},{"year":2011,"finding":"NSD1 PHD domains 1, 4, 5, and 6 bind histone H3 methylated at K4 or K9. Eleven of twelve Sotos-syndrome-associated missense mutations in PHD4, PHD5, and PHD6 disrupt binding to these methylated lysines, and 8 of 9 mutations in PHD4 and PHD6 severely impair binding to the transcription cofactor Nizp1.","method":"Histone peptide binding assays (peptide pulldown), mutagenesis of PHD domains, co-immunoprecipitation with Nizp1","journal":"Human mutation","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — peptide binding assays plus mutagenesis; single lab, two orthogonal methods","pmids":["21972110"],"is_preprint":false},{"year":2004,"finding":"Nizp1 interacts with NSD1 through a novel C2HR zinc-finger motif that docks to the cysteine-rich (C5HCH) domain of NSD1 in a Zn(II)-dependent manner. This interaction is required for Nizp1-mediated transcriptional repression at RNA Pol II promoters. Mutations of the C2HR cysteine/histidine residues or conversion to a canonical C2H2 zinc finger abolish NSD1 binding and transcriptional repression.","method":"Co-immunoprecipitation, GST pulldown, zinc-dependence assay, transcriptional repression assay, domain mutagenesis","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — reciprocal Co-IP plus GST pulldown plus mutagenesis plus functional reporter assay in a single study","pmids":["15169884"],"is_preprint":false},{"year":2014,"finding":"NSD1 preferentially methylates substrates with aromatic/hydrophobic residues at the −2/−1 positions and basic residues at +1/+2 of the target peptide. NSD1 methylates 25 non-histone peptide substrates in vitro; the best protein substrate identified is H1.5 K168. H4K44 is also methylated. Methylation of H4K20 and p65 was NOT observed in this assay. Sotos-syndrome missense mutations in the SET domain inactivate enzymatic activity.","method":"SPOT peptide array assay, in vitro methyltransferase assay with recombinant NSD1, SET-domain mutagenesis","journal":"Chemistry & biology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro enzymatic reconstitution with systematic substrate-specificity profiling plus mutagenesis; single lab, multiple orthogonal methods","pmids":["24412544"],"is_preprint":false},{"year":2019,"finding":"CRISPR/Cas9-mediated NSD1 knockout in hepatocellular carcinoma cells increases H3K27me3 and reduces H3K36me2 at the Wnt10b promoter, suppressing Wnt10b expression and inactivating Wnt/β-catenin signaling, thereby inhibiting cell proliferation, migration, and invasion in vitro and tumor growth in vivo.","method":"CRISPR/Cas9 knockout, ChIP, Western blot, proliferation/migration/invasion assays, xenograft model","journal":"Journal of experimental & clinical cancer research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ChIP plus functional KO phenotype in vitro and in vivo; single lab, two orthogonal methods","pmids":["31727171"],"is_preprint":false},{"year":2020,"finding":"In mouse prospermatogonia (male germline), NSD1 deposits broad H3K36me2 in euchromatic regions and is required for de novo DNA methylation by DNMT3A/DNMT3L, including at imprinted genes. Males with germline NSD1 deficiency show more severe spermatogenesis defects than Dnmt3l-/- males. NSD1 also safeguards a subset of genes against H3K27me3-associated silencing. In oocytes, H3K36me2 is predominantly dependent on SETD2, and NSD1-deficient oocytes support normal female fertility.","method":"Conditional knockout mice, whole-genome bisulfite sequencing, ChIP-seq, RNA-seq","journal":"Nature genetics","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — genetic KO with genome-wide bisulfite sequencing and ChIP-seq; multiple orthogonal methods","pmids":["32929285"],"is_preprint":false},{"year":2020,"finding":"Crystal structure of NSD1 SET domain in complex with a covalently bound inhibitor (BT5) reveals a conformational change in the autoinhibitory loop, exposing a channel-like pocket. The covalent inhibitor inhibits H3K36 dimethylation and downregulates NUP98-NSD1 target genes and impairs colony formation in NUP98-NSD1 patient-derived AML cells.","method":"Fragment-based screening, chemical synthesis, X-ray crystallography, cell-based H3K36me2 assay, colony formation assay","journal":"Nature chemical biology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — crystal structure plus functional cell-based validation; single lab, multiple orthogonal methods","pmids":["32868895"],"is_preprint":false},{"year":2021,"finding":"NSD1 loss in skeletal mesenchymal progenitors (Prx1+) impairs chondrogenic differentiation, skeletal growth, and fracture healing. NSD1 regulates Sox9 expression by modulating H3K36me1 and H3K36me2 at the Sox9 promoter; NSD1 also directly activates HIF1α, which regulates Sox9. Conditional KO in Col2+ chondrocytes does not recapitulate the phenotype, placing NSD1 function upstream in the progenitor compartment.","method":"Conditional knockout mice (Prx1-Cre; Col2-Cre), RNA-seq, ChIP-seq, in vitro differentiation assays","journal":"Bone research","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — conditional KO with ChIP-seq and RNA-seq; two tissue-specific Cre lines providing epistasis; multiple orthogonal methods","pmids":["34099628"],"is_preprint":false},{"year":2022,"finding":"NSD1 antagonizes Polycomb repressor complex (PRC2/EZH2) activity via cooperation with SWI/SNF chromatin remodelers. Loss of NSD1 causes resistance to EZH2 inhibition in SMARCB1-mutant rhabdoid tumor cells; H3K36me2 itself is essential for activation of polycomb target genes. Inhibition of the H3K36me2 demethylase KDM2A restores EZH2 inhibitor efficacy in SWI/SNF-deficient NSD1-null cells.","method":"CRISPR screen, CRISPR KO, KDM2A inhibitor treatment, ChIP-seq, gene expression analysis","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — genome-wide CRISPR screen plus targeted KO plus pharmacological rescue plus ChIP-seq; multiple orthogonal methods","pmids":["35537449"],"is_preprint":false},{"year":2022,"finding":"NUP98-NSD1 fusion protein forms liquid-like phase-separated nuclear condensates dependent on the NUP98 FG-repeat domains. These condensates co-interact with the ISWI-family chromatin remodeler SMARCA5 and BPTF (NURF complex members), which are identified as core interactome partners by AP-MS. SMARCA5 is functionally required for NUP98-NSD1/FLT3-ITD-mediated hematopoietic cell transformation; inhibition of SMARCA5 activity (not condensate formation per se) abrogates transformation.","method":"Affinity purification–mass spectrometry (AP-MS), inducible shRNA knockdown, pharmacological SMARCA5 inhibition, FRAP, proximity ligation assay, methylcellulose colony assay","journal":"Journal of experimental & clinical cancer research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — AP-MS interactome plus functional genetic/pharmacological validation; single lab, multiple orthogonal methods","pmids":["35073946"],"is_preprint":false},{"year":2023,"finding":"NSD1 and H3K36me2 are enriched at cis-regulatory elements, particularly enhancers. NSD1's enhancer association is mediated by a tandem quadruple PHD-PWWP (qPHD-PWWP) module that recognizes p300-catalyzed H3K18ac. Acute NSD1 depletion reduces enhancer activity and impairs RNA Pol II pause release at target genes. Importantly, NSD1 can act as a transcriptional coactivator independently of its catalytic (H3K36 methyltransferase) activity. NSD1 also controls embryonic stem cell multilineage differentiation.","method":"Auxin-inducible degron (acute depletion), ChIP-seq, ATAC-seq, GRO-seq, domain binding assays (PHD-PWWP module), catalytic-dead mutant rescue experiments, ESC differentiation assays","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — acute depletion system with time-resolved multi-omics plus domain binding assays plus catalytic-dead mutagenesis; multiple orthogonal methods in a single rigorous study","pmids":["37402365"],"is_preprint":false},{"year":2023,"finding":"In mouse neurons, NSD1 deposits megabase-scale H3K36me2 that recruits DNMT3A to pattern non-CG DNA methylation. Brain-specific NSD1 deletion causes altered DNA methylation overlapping with DNMT3A-disorder models, driving convergent dysregulation of key neuronal genes. Loss of H3K36me2 (via NSD1 deletion) disrupts the H3K36me2–DNMT3A–non-CG-methylation pathway required for neuronal gene regulation.","method":"Brain-specific conditional KO, ChIP-seq, whole-genome bisulfite sequencing (non-CG methylation), RNA-seq","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — conditional KO with ChIP-seq and bisulfite sequencing; multiple orthogonal genome-wide methods","pmids":["37098340"],"is_preprint":false},{"year":2023,"finding":"NSD1-mediated H3K36me2 in postmitotic neocortical neurons establishes and maintains area- and layer-specific pyramidal neuron identities. Nsd1 conditional KO causes area-shift of all four primary cortical regions, aberrant cortico-thalamic wiring, and progressive mis-expression of deep-layer markers in superficial neurons; neurons remain morphologically and electrophysiologically immature. Loss of Nsd1 causes genome-wide H3K36me2 loss and redistribution of DNA methylation.","method":"Conditional knockout mice, RNA-seq, ChIP-seq, DNA methylation profiling, electrophysiology, axonal tracing","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — conditional KO with ChIP-seq, transcriptomics, methylomics, and electrophysiology; multiple orthogonal methods","pmids":["37995181"],"is_preprint":false},{"year":2023,"finding":"NSD1 inactivation in HNSCC reduces H3K36me2 and elevates H3K27me3 at promoters of T-cell chemokines CXCL9 and CXCL10, suppressing their expression and excluding T cells from the tumor microenvironment. Inhibition of KDM2A (the primary H3K36 demethylase) reverses these altered histone marks, restores CXCL9/10 expression, and rescues T-cell infiltration. KDM2A suppression reduces NSD1-deficient tumor growth in immunocompetent but not immunodeficient mice.","method":"NSD1-mutant HNSCC cell lines/patient samples, ChIP-seq, KDM2A inhibitor treatment, in vivo syngeneic tumor models, flow cytometry for T-cell infiltration","journal":"Cancer research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ChIP-seq plus pharmacological rescue plus in vivo model; single lab, multiple methods","pmids":["37311054"],"is_preprint":false},{"year":2024,"finding":"Among H3K36 methyltransferases, NSD1 is the predominant depositor of intergenic H3K36me2 (with NSD2 contributing partially), while SETD2 deposits most H3K36me3 within gene bodies. Systematic perturbation in mouse mesenchymal stem cells places NSD1 at the top of a K36MT hierarchy: NSD1 > NSD2 > NSD3 > ASH1L for broad H3K36me2 deposition.","method":"CRISPR KO of individual K36MTs, ChIP-seq for H3K36me1/2/3, RNA-seq in mouse mesenchymal stem cells","journal":"Genome biology","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — systematic genetic perturbation of five enzymes with genome-wide ChIP-seq; rigorous comparative approach establishing hierarchy","pmids":["39390582"],"is_preprint":false},{"year":2013,"finding":"Murine NSD1 diminishes caspase-1 activity and downstream IL-1β/IL-18 maturation during macrophage stimulation with listeriolysin O (LLO). Silencing Nsd1 enhances caspase-1 activation, cytokine maturation, and pyroptosis. NSD1 does not affect NF-κB signaling or NLRP3 gene expression at the chromatin or transcriptional level during LLO stimulation.","method":"siRNA knockdown, caspase-1 activity assay, ELISA for cytokines, cell death assay in macrophages","journal":"PloS one","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single knockdown approach, single lab, no direct biochemical mechanism established for how NSD1 modulates the inflammasome","pmids":["24058709"],"is_preprint":false}],"current_model":"NSD1 is a bifunctional nuclear protein that primarily functions as a histone H3K36 mono- and di-methyltransferase (via its SET domain, whose autoinhibitory loop opens upon nucleosome engagement), depositing broad intergenic H3K36me2 that recruits DNMT3A to pattern DNA methylation and antagonizes PRC2-dependent H3K27me3; it also acts as a transcriptional coactivator at enhancers through a PHD-PWWP module that reads p300-catalyzed H3K18ac and facilitates RNA Pol II pause release—in part independent of catalytic activity—while interacting with nuclear receptors (via distinct ligand-dependent and -independent NR-box motifs), the transcriptional co-repressor Nizp1 (via its C5HCH domain), and the androgen receptor, with disease-associated SET-domain mutations abrogating enzymatic activity and PHD-domain mutations disrupting methyl-histone reading and Nizp1 recruitment."},"narrative":{"mechanistic_narrative":"NSD1 is a chromatin-modifying enzyme that functions as the predominant depositor of broad intergenic and enhancer-associated histone H3K36 mono- and di-methylation, organizing the epigenomic landscape during development and differentiation [PMID:32929285, PMID:39390582]. Its SET domain carries intrinsic H3K36 methyltransferase activity gated by a regulatory autoinhibitory loop that occludes the substrate channel in the resting state and opens upon nucleosome engagement, explaining its preference for nucleosomal substrate and its dimethyl product specificity [PMID:21196496, PMID:32868895]; this enzymatic activity is essential for early post-implantation development [PMID:12805229]. The H3K36me2 NSD1 deposits acts as a node that recruits DNMT3A to pattern (including non-CG) DNA methylation and antagonizes PRC2/EZH2-dependent H3K27me3 deposition, in cooperation with SWI/SNF remodelers [PMID:32929285, PMID:37098340, PMID:35537449]. Beyond catalysis, NSD1 reads chromatin and protein partners through dedicated modules: a tandem quadruple PHD-PWWP module recognizes p300-catalyzed H3K18ac to localize NSD1 to enhancers and promote RNA Pol II pause release—a coactivator function that operates partly independent of catalytic activity [PMID:37402365], while its PHD fingers bind methylated H3K4/H3K9 and its cysteine-rich C5HCH domain docks the corepressor Nizp1 via a C2HR zinc-finger motif [PMID:21972110, PMID:15169884]. NSD1 also serves as a bifunctional nuclear-receptor intermediary, contacting both unliganded and liganded receptor LBDs through distinct NR-box motifs and acting as an androgen receptor coregulator [PMID:9628876, PMID:11509567]. These activities translate into tissue-specific roles in spermatogenesis and imprinting, skeletal progenitor differentiation, and the establishment of cortical neuron identity [PMID:32929285, PMID:34099628, PMID:37995181]. NSD1 is recurrently disrupted in cancer: it is fused to NUP98 in childhood AML, where the chimera maintains Hox-A locus activation in a SET-domain-dependent manner [PMID:11493482, PMID:17589499], and Sotos-syndrome-associated SET- and PHD-domain mutations abrogate catalytic activity and methyl-histone/Nizp1 reading, respectively [PMID:24412544, PMID:21972110].","teleology":[{"year":1998,"claim":"Established NSD1 as a bifunctional nuclear-receptor intermediary, answering whether it acts as activator or repressor by showing it does both depending on ligand state.","evidence":"Yeast two-hybrid, GST pulldown, and reporter assays with NID-L and NID+L domains against RAR/TR/RXR/ER LBDs","pmids":["9628876"],"confidence":"High","gaps":["Did not establish enzymatic activity","No endogenous target genes identified","In vivo relevance of NR interactions untested"]},{"year":2001,"claim":"Implicated NSD1 in human malignancy by identifying its in-frame fusion to NUP98 in childhood AML.","evidence":"3'-RACE, RT-PCR, and FISH on patient AML material","pmids":["11493482"],"confidence":"Medium","gaps":["No functional assay of the fusion in this study","Mechanism of transformation unknown"]},{"year":2001,"claim":"Defined NSD1 (ARA267-alpha) as an androgen receptor coregulator, extending its nuclear-receptor role to prostate cancer signaling.","evidence":"Yeast two-hybrid, GST pulldown, and reporter assays in PC-3 and H1299 cells","pmids":["11509567"],"confidence":"Medium","gaps":["Endogenous AR target genes not mapped","Link between AR coregulation and enzymatic activity unresolved"]},{"year":2003,"claim":"Identified NSD1 as a histone methyltransferase and demonstrated its essentiality, answering what its catalytic function is and whether it is required for life.","evidence":"In vitro methyltransferase assay with recombinant SET domain plus NSD1-null knockout mice","pmids":["12805229"],"confidence":"High","gaps":["H3K36 vs H4K20 specificity later refined","Mechanism of developmental requirement not defined","Genomic targets unknown"]},{"year":2004,"claim":"Mapped the structural basis of NSD1's corepressor interaction by showing Nizp1 docks to the C5HCH domain via a novel C2HR zinc finger required for repression.","evidence":"Co-IP, GST pulldown, zinc-dependence and mutagenesis with transcriptional repression reporters","pmids":["15169884"],"confidence":"High","gaps":["Genome-wide Nizp1/NSD1 co-target genes not identified","Relationship to methyltransferase activity unclear"]},{"year":2007,"claim":"Established the oncogenic mechanism of NUP98-NSD1, showing it drives AML through SET-domain-dependent Hox-A activation and EZH2 antagonism.","evidence":"Murine bone marrow transplant AML model, colony assays, ChIP, SET-domain and FG-repeat mutagenesis","pmids":["17589499"],"confidence":"High","gaps":["Direct chromatin recruitment mechanism of the fusion not fully resolved","Cooperating mutations not addressed"]},{"year":2009,"claim":"Probed whether NSD1 acts as a tumor suppressor by showing promoter hypermethylation silences it in neuroblastoma/glioma, reducing H3K36/H4K20 methylation and growth.","evidence":"Bisulfite sequencing, ChIP, re-expression and colony formation assays in cell lines","pmids":["20018718"],"confidence":"Medium","gaps":["Context-dependent tumor-suppressor vs oncogenic role unreconciled","MEIS1 regulation mechanism limited to ChIP"]},{"year":2009,"claim":"Extended NSD1 substrate scope beyond histones by reporting methylation of NF-kB p65 at K218/K221 to activate NF-kB signaling.","evidence":"Overexpression/knockdown, MS site identification, reporter and MEF gene-expression assays","pmids":["20080798"],"confidence":"Medium","gaps":["p65 methylation not reproduced in a later systematic substrate assay (#11)","Single-lab finding"]},{"year":2010,"claim":"Linked NSD1-dependent H3K36 methylation to transcriptional elongation by showing it controls RNA Pol II CTD phospho-state at target promoters.","evidence":"ChIP-seq, ChIP-qPCR, siRNA, and Western blot for RNAP II Ser5/Ser2 phospho-forms","pmids":["20837538"],"confidence":"Medium","gaps":["Direct vs indirect effect on Pol II phosphorylation unresolved","Limited number of target genes examined"]},{"year":2010,"claim":"Provided the structural explanation for NSD1's substrate gating, revealing an autoinhibitory loop that occludes the H3K36 channel and is relieved by the nucleosome.","evidence":"1.7 A crystal structure of the catalytic domain plus molecular dynamics and docking","pmids":["21196496"],"confidence":"High","gaps":["Active-state conformation inferred computationally, not crystallized with nucleosome","Mechanism of loop opening on chromatin not directly observed"]},{"year":2011,"claim":"Defined the molecular consequences of Sotos-syndrome PHD mutations, showing they disrupt methyl-H3 reading and Nizp1 recruitment.","evidence":"Histone peptide pulldowns, PHD mutagenesis, and Nizp1 co-IP","pmids":["21972110"],"confidence":"Medium","gaps":["In vivo consequence of disrupted reading not tested","Functional readout of each PHD finger incomplete"]},{"year":2014,"claim":"Systematically resolved NSD1 substrate specificity, identifying preferred sequence context and ruling out H4K20 and p65 as substrates under these conditions.","evidence":"SPOT peptide arrays, in vitro methyltransferase assays, and SET-domain mutagenesis","pmids":["24412544"],"confidence":"High","gaps":["In vivo relevance of non-histone substrates (H1.5 K168, H4K44) untested","Conflict with earlier p65/H4K20 reports unresolved"]},{"year":2013,"claim":"Suggested an inflammasome-regulatory role by showing NSD1 dampens caspase-1 activation and pyroptosis in macrophages.","evidence":"siRNA knockdown with caspase-1 activity, cytokine ELISA, and cell death assays","pmids":["24058709"],"confidence":"Low","gaps":["Single knockdown approach without rescue","No direct biochemical mechanism for inflammasome modulation","Not independently confirmed"]},{"year":2020,"claim":"Established the H3K36me2-DNMT3A axis in vivo, showing germline NSD1 deposits broad H3K36me2 required for de novo DNA methylation and imprinting.","evidence":"Conditional KO mice with whole-genome bisulfite sequencing, ChIP-seq, RNA-seq in prospermatogonia and oocytes","pmids":["32929285"],"confidence":"High","gaps":["Sex-specific division of labor with SETD2 mechanism not fully resolved","Direct DNMT3A recruitment biochemistry not shown here"]},{"year":2020,"claim":"Demonstrated druggability of the NSD1 SET domain, with a covalent inhibitor that opens the autoinhibitory loop and downregulates NUP98-NSD1 target genes.","evidence":"Fragment screening, X-ray crystallography of BT5 complex, cell-based H3K36me2 and colony assays in patient AML cells","pmids":["32868895"],"confidence":"High","gaps":["Selectivity over other NSD family members not detailed here","In vivo efficacy untested in this study"]},{"year":2021,"claim":"Defined a skeletal developmental role, showing NSD1 in mesenchymal progenitors regulates Sox9 and HIF1a via H3K36 methylation to drive chondrogenesis.","evidence":"Prx1-Cre and Col2-Cre conditional KO mice, RNA-seq, ChIP-seq, in vitro differentiation","pmids":["34099628"],"confidence":"High","gaps":["Direct vs indirect HIF1a activation mechanism unresolved","Relevance to Sotos overgrowth phenotype not directly linked"]},{"year":2022,"claim":"Mechanistically connected NSD1/H3K36me2 to PRC2 antagonism and SWI/SNF cooperation, explaining EZH2-inhibitor resistance in NSD1-null tumors.","evidence":"CRISPR screen and KO, KDM2A inhibitor rescue, ChIP-seq in SMARCB1-mutant rhabdoid cells","pmids":["35537449"],"confidence":"High","gaps":["Precise biochemical link between H3K36me2 and PRC2 exclusion not fully reconstituted","Generalizability beyond SWI/SNF-deficient context unclear"]},{"year":2022,"claim":"Characterized NUP98-NSD1 condensate biology, identifying SMARCA5/BPTF as core interactors required for transformation.","evidence":"AP-MS, FRAP, proximity ligation, inducible knockdown, and SMARCA5 inhibition with colony assays","pmids":["35073946"],"confidence":"Medium","gaps":["Causal role of condensate formation vs SMARCA5 activity disentangled only partially","Single-lab interactome"]},{"year":2023,"claim":"Separated NSD1's catalytic and coactivator functions, revealing a qPHD-PWWP module that reads H3K18ac to drive enhancer activity and Pol II pause release independent of methyltransferase activity.","evidence":"Auxin-inducible degron, ChIP-seq/ATAC-seq/GRO-seq, domain binding assays, catalytic-dead rescue, ESC differentiation","pmids":["37402365"],"confidence":"High","gaps":["Mechanism by which non-catalytic NSD1 promotes pause release undefined","Relative contribution of catalytic vs non-catalytic functions context-dependent"]},{"year":2023,"claim":"Established the H3K36me2-DNMT3A-non-CG methylation pathway in neurons, linking NSD1 loss to DNMT3A-disorder-convergent gene dysregulation.","evidence":"Brain-specific conditional KO, ChIP-seq, whole-genome bisulfite sequencing, RNA-seq","pmids":["37098340"],"confidence":"High","gaps":["Behavioral/disease phenotype consequences not addressed here","Direct DNMT3A recruitment biochemistry not shown"]},{"year":2023,"claim":"Showed NSD1-deposited H3K36me2 establishes and maintains cortical neuron areal and laminar identity, defining a postmitotic developmental function.","evidence":"Conditional KO mice, RNA-seq, ChIP-seq, DNA methylation profiling, electrophysiology, axonal tracing","pmids":["37995181"],"confidence":"High","gaps":["Causal chain from H3K36me2 loss to wiring defects incompletely mapped","Reversibility not tested"]},{"year":2023,"claim":"Linked NSD1 loss to tumor immune evasion, showing reduced H3K36me2/elevated H3K27me3 silences T-cell chemokines CXCL9/10 in HNSCC.","evidence":"NSD1-mutant HNSCC models, ChIP-seq, KDM2A inhibition, syngeneic tumor models, flow cytometry","pmids":["37311054"],"confidence":"Medium","gaps":["Direct chromatin mechanism at chemokine loci vs indirect effects not fully separated","Clinical translatability of KDM2A inhibition untested"]},{"year":2024,"claim":"Ranked NSD1 atop the H3K36 methyltransferase hierarchy, establishing it as the predominant depositor of broad intergenic H3K36me2.","evidence":"CRISPR KO of five K36MTs with genome-wide ChIP-seq for H3K36me1/2/3 and RNA-seq in mouse mesenchymal stem cells","pmids":["39390582"],"confidence":"High","gaps":["Cell-type dependence of the hierarchy not exhaustively tested","Mechanism of intergenic targeting specificity unresolved"]},{"year":null,"claim":"How NSD1 is recruited to specific genomic loci and how it physically hands off H3K36me2 to DNMT3A and excludes PRC2 remain incompletely defined at the biochemical level.","evidence":"","pmids":[],"confidence":"High","gaps":["No reconstituted NSD1-DNMT3A handoff","Locus-targeting determinants for intergenic H3K36me2 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NSD1 also contains separate repression and activation domains, establishing it as a bifunctional transcriptional intermediary factor.\",\n      \"method\": \"Yeast two-hybrid, GST pulldown, LBD mutagenesis, co-transfection reporter assays\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — multiple orthogonal binding assays combined with domain mutagenesis in a single rigorous study; foundational characterization paper\",\n      \"pmids\": [\"9628876\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"NSD1's SET domain possesses intrinsic histone methyltransferase activity with specificity for H3K36 and H4K20. NSD1-null embryos die during gastrulation, demonstrating it is essential for early post-implantation development.\",\n      \"method\": \"In vitro histone methyltransferase assay with recombinant SET domain; gene-targeted knockout mice\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro enzymatic reconstitution combined with genetic loss-of-function in mice; replicated by subsequent studies\",\n      \"pmids\": [\"12805229\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"NSD1 is fused in-frame to NUP98 by the t(5;11)(q35;p15.5) translocation in childhood AML, producing a chimeric NUP98-NSD1 mRNA and the reciprocal NSD1-NUP98 transcript. This was the first implication of NSD1 in human malignancy.\",\n      \"method\": \"3'-RACE PCR, RT-PCR, FISH\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — molecular cloning of fusion transcript confirmed by multiple PCR-based methods in a single study; no functional assay in this paper\",\n      \"pmids\": [\"11493482\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"NSD1 (also known as ARA267-alpha) functions as an androgen receptor (AR) coregulator: both its N-terminal and C-terminal regions interact with the AR DNA- and ligand-binding domains, and it enhances AR transactivation in a dihydrotestosterone-dependent manner in prostate cancer cells.\",\n      \"method\": \"Yeast two-hybrid, GST pulldown, luciferase and CAT reporter assays in PC-3 and H1299 cells\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal binding assays plus functional reporter assays in two cell lines; single lab, two orthogonal methods\",\n      \"pmids\": [\"11509567\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"The NUP98-NSD1 fusion induces AML in vivo, sustains myeloid stem cell self-renewal in vitro, and enforces expression of HoxA7, HoxA9, HoxA10, and Meis1. Mechanistically, NUP98-NSD1 binds genomic elements at the Hox-A locus, maintains H3K36 methylation and histone acetylation there, and prevents EZH2-mediated transcriptional repression. Deletion of the NUP98 FG-repeat domain or inactivating mutations of the NSD1 SET domain (H3K36 methyltransferase activity) abolished Hox-A activation and progenitor immortalization.\",\n      \"method\": \"Retroviral transduction + murine bone marrow transplantation (in vivo AML), methylcellulose colony assay, ChIP, SET-domain point mutagenesis, gene-expression analysis\",\n      \"journal\": \"Nature cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — in vivo leukemia model plus SET-domain mutagenesis plus ChIP; multiple orthogonal methods establishing mechanism\",\n      \"pmids\": [\"17589499\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"NSD1 methylates p65 (NF-κB subunit) at K218 and K221, activating NF-κB target gene expression. The lysine demethylase FBXL11 reverses this modification. Overexpression of NSD1 activates NF-κB and reverses FBXL11-mediated inhibition; knockdown of NSD1 decreases NF-κB activation. NF-κB-dependent gene expression in mouse embryo fibroblasts relies on K218/K221 methylation.\",\n      \"method\": \"Overexpression/knockdown in cells, MS-based identification of methylation sites, luciferase reporter assays, MEF gene-expression analysis\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — MS identification of modification sites plus functional reporter assays; single lab, two orthogonal methods\",\n      \"pmids\": [\"20080798\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Epigenetic inactivation of NSD1 by CpG island promoter hypermethylation in neuroblastoma and glioma cells causes specifically diminished H4K20 and H3K36 methylation. Restored NSD1 expression reduces colony formation and inhibits cell growth (tumor-suppressor-like activity). ChIP analysis identified MEIS1 as a direct NSD1 target in neuroblastoma.\",\n      \"method\": \"Bisulfite sequencing, ChIP, expression microarray, colony formation assay, re-expression experiments in cell lines\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP plus functional rescue; single lab, two orthogonal methods\",\n      \"pmids\": [\"20018718\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"The 1.7 Å crystal structure of the NSD1 catalytic domain reveals that a regulatory (autoinhibitory) loop occludes the H3K36 substrate access channel to the bound SAM cofactor. Molecular dynamics and docking show this loop can adopt an active conformation, and that the nucleosome likely stabilizes the active state, explaining NSD1's preference for nucleosomal substrate and its dimethyl-H3K36 product specificity.\",\n      \"method\": \"X-ray crystallography (1.7 Å), molecular dynamics simulation, computational docking\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — high-resolution crystal structure plus MD simulation; single lab but Tier-1 structural method\",\n      \"pmids\": [\"21196496\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"NSD1 binds near promoter elements of target genes (e.g., BMP4, ZFP36L1), regulates H3K36 methylation primarily in the promoter-proximal region, reduces RNA Pol II recruitment to the BMP4 promoter, and causes inappropriate persistence of Ser5-phosphorylated RNAP II with reduced Ser2 phosphorylation within the CTD, linking NSD1-dependent H3K36 methylation to RNAP II elongation control.\",\n      \"method\": \"ChIP-seq, ChIP-qPCR, siRNA knockdown, Western blot for RNAP II CTD phospho-forms\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP-seq plus RNAP II phospho-state analysis; single lab, two orthogonal methods\",\n      \"pmids\": [\"20837538\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"NSD1 PHD domains 1, 4, 5, and 6 bind histone H3 methylated at K4 or K9. Eleven of twelve Sotos-syndrome-associated missense mutations in PHD4, PHD5, and PHD6 disrupt binding to these methylated lysines, and 8 of 9 mutations in PHD4 and PHD6 severely impair binding to the transcription cofactor Nizp1.\",\n      \"method\": \"Histone peptide binding assays (peptide pulldown), mutagenesis of PHD domains, co-immunoprecipitation with Nizp1\",\n      \"journal\": \"Human mutation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — peptide binding assays plus mutagenesis; single lab, two orthogonal methods\",\n      \"pmids\": [\"21972110\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Nizp1 interacts with NSD1 through a novel C2HR zinc-finger motif that docks to the cysteine-rich (C5HCH) domain of NSD1 in a Zn(II)-dependent manner. This interaction is required for Nizp1-mediated transcriptional repression at RNA Pol II promoters. Mutations of the C2HR cysteine/histidine residues or conversion to a canonical C2H2 zinc finger abolish NSD1 binding and transcriptional repression.\",\n      \"method\": \"Co-immunoprecipitation, GST pulldown, zinc-dependence assay, transcriptional repression assay, domain mutagenesis\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — reciprocal Co-IP plus GST pulldown plus mutagenesis plus functional reporter assay in a single study\",\n      \"pmids\": [\"15169884\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"NSD1 preferentially methylates substrates with aromatic/hydrophobic residues at the −2/−1 positions and basic residues at +1/+2 of the target peptide. NSD1 methylates 25 non-histone peptide substrates in vitro; the best protein substrate identified is H1.5 K168. H4K44 is also methylated. Methylation of H4K20 and p65 was NOT observed in this assay. Sotos-syndrome missense mutations in the SET domain inactivate enzymatic activity.\",\n      \"method\": \"SPOT peptide array assay, in vitro methyltransferase assay with recombinant NSD1, SET-domain mutagenesis\",\n      \"journal\": \"Chemistry & biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro enzymatic reconstitution with systematic substrate-specificity profiling plus mutagenesis; single lab, multiple orthogonal methods\",\n      \"pmids\": [\"24412544\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"CRISPR/Cas9-mediated NSD1 knockout in hepatocellular carcinoma cells increases H3K27me3 and reduces H3K36me2 at the Wnt10b promoter, suppressing Wnt10b expression and inactivating Wnt/β-catenin signaling, thereby inhibiting cell proliferation, migration, and invasion in vitro and tumor growth in vivo.\",\n      \"method\": \"CRISPR/Cas9 knockout, ChIP, Western blot, proliferation/migration/invasion assays, xenograft model\",\n      \"journal\": \"Journal of experimental & clinical cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP plus functional KO phenotype in vitro and in vivo; single lab, two orthogonal methods\",\n      \"pmids\": [\"31727171\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"In mouse prospermatogonia (male germline), NSD1 deposits broad H3K36me2 in euchromatic regions and is required for de novo DNA methylation by DNMT3A/DNMT3L, including at imprinted genes. Males with germline NSD1 deficiency show more severe spermatogenesis defects than Dnmt3l-/- males. NSD1 also safeguards a subset of genes against H3K27me3-associated silencing. In oocytes, H3K36me2 is predominantly dependent on SETD2, and NSD1-deficient oocytes support normal female fertility.\",\n      \"method\": \"Conditional knockout mice, whole-genome bisulfite sequencing, ChIP-seq, RNA-seq\",\n      \"journal\": \"Nature genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — genetic KO with genome-wide bisulfite sequencing and ChIP-seq; multiple orthogonal methods\",\n      \"pmids\": [\"32929285\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Crystal structure of NSD1 SET domain in complex with a covalently bound inhibitor (BT5) reveals a conformational change in the autoinhibitory loop, exposing a channel-like pocket. The covalent inhibitor inhibits H3K36 dimethylation and downregulates NUP98-NSD1 target genes and impairs colony formation in NUP98-NSD1 patient-derived AML cells.\",\n      \"method\": \"Fragment-based screening, chemical synthesis, X-ray crystallography, cell-based H3K36me2 assay, colony formation assay\",\n      \"journal\": \"Nature chemical biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — crystal structure plus functional cell-based validation; single lab, multiple orthogonal methods\",\n      \"pmids\": [\"32868895\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"NSD1 loss in skeletal mesenchymal progenitors (Prx1+) impairs chondrogenic differentiation, skeletal growth, and fracture healing. NSD1 regulates Sox9 expression by modulating H3K36me1 and H3K36me2 at the Sox9 promoter; NSD1 also directly activates HIF1α, which regulates Sox9. Conditional KO in Col2+ chondrocytes does not recapitulate the phenotype, placing NSD1 function upstream in the progenitor compartment.\",\n      \"method\": \"Conditional knockout mice (Prx1-Cre; Col2-Cre), RNA-seq, ChIP-seq, in vitro differentiation assays\",\n      \"journal\": \"Bone research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — conditional KO with ChIP-seq and RNA-seq; two tissue-specific Cre lines providing epistasis; multiple orthogonal methods\",\n      \"pmids\": [\"34099628\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"NSD1 antagonizes Polycomb repressor complex (PRC2/EZH2) activity via cooperation with SWI/SNF chromatin remodelers. Loss of NSD1 causes resistance to EZH2 inhibition in SMARCB1-mutant rhabdoid tumor cells; H3K36me2 itself is essential for activation of polycomb target genes. Inhibition of the H3K36me2 demethylase KDM2A restores EZH2 inhibitor efficacy in SWI/SNF-deficient NSD1-null cells.\",\n      \"method\": \"CRISPR screen, CRISPR KO, KDM2A inhibitor treatment, ChIP-seq, gene expression analysis\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — genome-wide CRISPR screen plus targeted KO plus pharmacological rescue plus ChIP-seq; multiple orthogonal methods\",\n      \"pmids\": [\"35537449\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"NUP98-NSD1 fusion protein forms liquid-like phase-separated nuclear condensates dependent on the NUP98 FG-repeat domains. These condensates co-interact with the ISWI-family chromatin remodeler SMARCA5 and BPTF (NURF complex members), which are identified as core interactome partners by AP-MS. SMARCA5 is functionally required for NUP98-NSD1/FLT3-ITD-mediated hematopoietic cell transformation; inhibition of SMARCA5 activity (not condensate formation per se) abrogates transformation.\",\n      \"method\": \"Affinity purification–mass spectrometry (AP-MS), inducible shRNA knockdown, pharmacological SMARCA5 inhibition, FRAP, proximity ligation assay, methylcellulose colony assay\",\n      \"journal\": \"Journal of experimental & clinical cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — AP-MS interactome plus functional genetic/pharmacological validation; single lab, multiple orthogonal methods\",\n      \"pmids\": [\"35073946\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"NSD1 and H3K36me2 are enriched at cis-regulatory elements, particularly enhancers. NSD1's enhancer association is mediated by a tandem quadruple PHD-PWWP (qPHD-PWWP) module that recognizes p300-catalyzed H3K18ac. Acute NSD1 depletion reduces enhancer activity and impairs RNA Pol II pause release at target genes. Importantly, NSD1 can act as a transcriptional coactivator independently of its catalytic (H3K36 methyltransferase) activity. NSD1 also controls embryonic stem cell multilineage differentiation.\",\n      \"method\": \"Auxin-inducible degron (acute depletion), ChIP-seq, ATAC-seq, GRO-seq, domain binding assays (PHD-PWWP module), catalytic-dead mutant rescue experiments, ESC differentiation assays\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — acute depletion system with time-resolved multi-omics plus domain binding assays plus catalytic-dead mutagenesis; multiple orthogonal methods in a single rigorous study\",\n      \"pmids\": [\"37402365\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"In mouse neurons, NSD1 deposits megabase-scale H3K36me2 that recruits DNMT3A to pattern non-CG DNA methylation. Brain-specific NSD1 deletion causes altered DNA methylation overlapping with DNMT3A-disorder models, driving convergent dysregulation of key neuronal genes. Loss of H3K36me2 (via NSD1 deletion) disrupts the H3K36me2–DNMT3A–non-CG-methylation pathway required for neuronal gene regulation.\",\n      \"method\": \"Brain-specific conditional KO, ChIP-seq, whole-genome bisulfite sequencing (non-CG methylation), RNA-seq\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — conditional KO with ChIP-seq and bisulfite sequencing; multiple orthogonal genome-wide methods\",\n      \"pmids\": [\"37098340\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"NSD1-mediated H3K36me2 in postmitotic neocortical neurons establishes and maintains area- and layer-specific pyramidal neuron identities. Nsd1 conditional KO causes area-shift of all four primary cortical regions, aberrant cortico-thalamic wiring, and progressive mis-expression of deep-layer markers in superficial neurons; neurons remain morphologically and electrophysiologically immature. Loss of Nsd1 causes genome-wide H3K36me2 loss and redistribution of DNA methylation.\",\n      \"method\": \"Conditional knockout mice, RNA-seq, ChIP-seq, DNA methylation profiling, electrophysiology, axonal tracing\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — conditional KO with ChIP-seq, transcriptomics, methylomics, and electrophysiology; multiple orthogonal methods\",\n      \"pmids\": [\"37995181\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"NSD1 inactivation in HNSCC reduces H3K36me2 and elevates H3K27me3 at promoters of T-cell chemokines CXCL9 and CXCL10, suppressing their expression and excluding T cells from the tumor microenvironment. Inhibition of KDM2A (the primary H3K36 demethylase) reverses these altered histone marks, restores CXCL9/10 expression, and rescues T-cell infiltration. KDM2A suppression reduces NSD1-deficient tumor growth in immunocompetent but not immunodeficient mice.\",\n      \"method\": \"NSD1-mutant HNSCC cell lines/patient samples, ChIP-seq, KDM2A inhibitor treatment, in vivo syngeneic tumor models, flow cytometry for T-cell infiltration\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP-seq plus pharmacological rescue plus in vivo model; single lab, multiple methods\",\n      \"pmids\": [\"37311054\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Among H3K36 methyltransferases, NSD1 is the predominant depositor of intergenic H3K36me2 (with NSD2 contributing partially), while SETD2 deposits most H3K36me3 within gene bodies. Systematic perturbation in mouse mesenchymal stem cells places NSD1 at the top of a K36MT hierarchy: NSD1 > NSD2 > NSD3 > ASH1L for broad H3K36me2 deposition.\",\n      \"method\": \"CRISPR KO of individual K36MTs, ChIP-seq for H3K36me1/2/3, RNA-seq in mouse mesenchymal stem cells\",\n      \"journal\": \"Genome biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — systematic genetic perturbation of five enzymes with genome-wide ChIP-seq; rigorous comparative approach establishing hierarchy\",\n      \"pmids\": [\"39390582\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Murine NSD1 diminishes caspase-1 activity and downstream IL-1β/IL-18 maturation during macrophage stimulation with listeriolysin O (LLO). Silencing Nsd1 enhances caspase-1 activation, cytokine maturation, and pyroptosis. NSD1 does not affect NF-κB signaling or NLRP3 gene expression at the chromatin or transcriptional level during LLO stimulation.\",\n      \"method\": \"siRNA knockdown, caspase-1 activity assay, ELISA for cytokines, cell death assay in macrophages\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single knockdown approach, single lab, no direct biochemical mechanism established for how NSD1 modulates the inflammasome\",\n      \"pmids\": [\"24058709\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"NSD1 is a bifunctional nuclear protein that primarily functions as a histone H3K36 mono- and di-methyltransferase (via its SET domain, whose autoinhibitory loop opens upon nucleosome engagement), depositing broad intergenic H3K36me2 that recruits DNMT3A to pattern DNA methylation and antagonizes PRC2-dependent H3K27me3; it also acts as a transcriptional coactivator at enhancers through a PHD-PWWP module that reads p300-catalyzed H3K18ac and facilitates RNA Pol II pause release—in part independent of catalytic activity—while interacting with nuclear receptors (via distinct ligand-dependent and -independent NR-box motifs), the transcriptional co-repressor Nizp1 (via its C5HCH domain), and the androgen receptor, with disease-associated SET-domain mutations abrogating enzymatic activity and PHD-domain mutations disrupting methyl-histone reading and Nizp1 recruitment.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"NSD1 is a chromatin-modifying enzyme that functions as the predominant depositor of broad intergenic and enhancer-associated histone H3K36 mono- and di-methylation, organizing the epigenomic landscape during development and differentiation [#13, #22]. Its SET domain carries intrinsic H3K36 methyltransferase activity gated by a regulatory autoinhibitory loop that occludes the substrate channel in the resting state and opens upon nucleosome engagement, explaining its preference for nucleosomal substrate and its dimethyl product specificity [#7, #14]; this enzymatic activity is essential for early post-implantation development [#1]. The H3K36me2 NSD1 deposits acts as a node that recruits DNMT3A to pattern (including non-CG) DNA methylation and antagonizes PRC2/EZH2-dependent H3K27me3 deposition, in cooperation with SWI/SNF remodelers [#13, #19, #16]. Beyond catalysis, NSD1 reads chromatin and protein partners through dedicated modules: a tandem quadruple PHD-PWWP module recognizes p300-catalyzed H3K18ac to localize NSD1 to enhancers and promote RNA Pol II pause release—a coactivator function that operates partly independent of catalytic activity [#18], while its PHD fingers bind methylated H3K4/H3K9 and its cysteine-rich C5HCH domain docks the corepressor Nizp1 via a C2HR zinc-finger motif [#9, #10]. NSD1 also serves as a bifunctional nuclear-receptor intermediary, contacting both unliganded and liganded receptor LBDs through distinct NR-box motifs and acting as an androgen receptor coregulator [#0, #3]. These activities translate into tissue-specific roles in spermatogenesis and imprinting, skeletal progenitor differentiation, and the establishment of cortical neuron identity [#13, #15, #20]. NSD1 is recurrently disrupted in cancer: it is fused to NUP98 in childhood AML, where the chimera maintains Hox-A locus activation in a SET-domain-dependent manner [#2, #4], and Sotos-syndrome-associated SET- and PHD-domain mutations abrogate catalytic activity and methyl-histone/Nizp1 reading, respectively [#11, #9].\",\n  \"teleology\": [\n    {\n      \"year\": 1998,\n      \"claim\": \"Established NSD1 as a bifunctional nuclear-receptor intermediary, answering whether it acts as activator or repressor by showing it does both depending on ligand state.\",\n      \"evidence\": \"Yeast two-hybrid, GST pulldown, and reporter assays with NID-L and NID+L domains against RAR/TR/RXR/ER LBDs\",\n      \"pmids\": [\"9628876\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not establish enzymatic activity\", \"No endogenous target genes identified\", \"In vivo relevance of NR interactions untested\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Implicated NSD1 in human malignancy by identifying its in-frame fusion to NUP98 in childhood AML.\",\n      \"evidence\": \"3'-RACE, RT-PCR, and FISH on patient AML material\",\n      \"pmids\": [\"11493482\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No functional assay of the fusion in this study\", \"Mechanism of transformation unknown\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Defined NSD1 (ARA267-alpha) as an androgen receptor coregulator, extending its nuclear-receptor role to prostate cancer signaling.\",\n      \"evidence\": \"Yeast two-hybrid, GST pulldown, and reporter assays in PC-3 and H1299 cells\",\n      \"pmids\": [\"11509567\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Endogenous AR target genes not mapped\", \"Link between AR coregulation and enzymatic activity unresolved\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Identified NSD1 as a histone methyltransferase and demonstrated its essentiality, answering what its catalytic function is and whether it is required for life.\",\n      \"evidence\": \"In vitro methyltransferase assay with recombinant SET domain plus NSD1-null knockout mice\",\n      \"pmids\": [\"12805229\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"H3K36 vs H4K20 specificity later refined\", \"Mechanism of developmental requirement not defined\", \"Genomic targets unknown\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Mapped the structural basis of NSD1's corepressor interaction by showing Nizp1 docks to the C5HCH domain via a novel C2HR zinc finger required for repression.\",\n      \"evidence\": \"Co-IP, GST pulldown, zinc-dependence and mutagenesis with transcriptional repression reporters\",\n      \"pmids\": [\"15169884\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Genome-wide Nizp1/NSD1 co-target genes not identified\", \"Relationship to methyltransferase activity unclear\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Established the oncogenic mechanism of NUP98-NSD1, showing it drives AML through SET-domain-dependent Hox-A activation and EZH2 antagonism.\",\n      \"evidence\": \"Murine bone marrow transplant AML model, colony assays, ChIP, SET-domain and FG-repeat mutagenesis\",\n      \"pmids\": [\"17589499\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct chromatin recruitment mechanism of the fusion not fully resolved\", \"Cooperating mutations not addressed\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Probed whether NSD1 acts as a tumor suppressor by showing promoter hypermethylation silences it in neuroblastoma/glioma, reducing H3K36/H4K20 methylation and growth.\",\n      \"evidence\": \"Bisulfite sequencing, ChIP, re-expression and colony formation assays in cell lines\",\n      \"pmids\": [\"20018718\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Context-dependent tumor-suppressor vs oncogenic role unreconciled\", \"MEIS1 regulation mechanism limited to ChIP\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Extended NSD1 substrate scope beyond histones by reporting methylation of NF-kB p65 at K218/K221 to activate NF-kB signaling.\",\n      \"evidence\": \"Overexpression/knockdown, MS site identification, reporter and MEF gene-expression assays\",\n      \"pmids\": [\"20080798\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"p65 methylation not reproduced in a later systematic substrate assay (#11)\", \"Single-lab finding\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Linked NSD1-dependent H3K36 methylation to transcriptional elongation by showing it controls RNA Pol II CTD phospho-state at target promoters.\",\n      \"evidence\": \"ChIP-seq, ChIP-qPCR, siRNA, and Western blot for RNAP II Ser5/Ser2 phospho-forms\",\n      \"pmids\": [\"20837538\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct vs indirect effect on Pol II phosphorylation unresolved\", \"Limited number of target genes examined\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Provided the structural explanation for NSD1's substrate gating, revealing an autoinhibitory loop that occludes the H3K36 channel and is relieved by the nucleosome.\",\n      \"evidence\": \"1.7 A crystal structure of the catalytic domain plus molecular dynamics and docking\",\n      \"pmids\": [\"21196496\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Active-state conformation inferred computationally, not crystallized with nucleosome\", \"Mechanism of loop opening on chromatin not directly observed\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Defined the molecular consequences of Sotos-syndrome PHD mutations, showing they disrupt methyl-H3 reading and Nizp1 recruitment.\",\n      \"evidence\": \"Histone peptide pulldowns, PHD mutagenesis, and Nizp1 co-IP\",\n      \"pmids\": [\"21972110\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"In vivo consequence of disrupted reading not tested\", \"Functional readout of each PHD finger incomplete\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Systematically resolved NSD1 substrate specificity, identifying preferred sequence context and ruling out H4K20 and p65 as substrates under these conditions.\",\n      \"evidence\": \"SPOT peptide arrays, in vitro methyltransferase assays, and SET-domain mutagenesis\",\n      \"pmids\": [\"24412544\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo relevance of non-histone substrates (H1.5 K168, H4K44) untested\", \"Conflict with earlier p65/H4K20 reports unresolved\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Suggested an inflammasome-regulatory role by showing NSD1 dampens caspase-1 activation and pyroptosis in macrophages.\",\n      \"evidence\": \"siRNA knockdown with caspase-1 activity, cytokine ELISA, and cell death assays\",\n      \"pmids\": [\"24058709\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Single knockdown approach without rescue\", \"No direct biochemical mechanism for inflammasome modulation\", \"Not independently confirmed\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Established the H3K36me2-DNMT3A axis in vivo, showing germline NSD1 deposits broad H3K36me2 required for de novo DNA methylation and imprinting.\",\n      \"evidence\": \"Conditional KO mice with whole-genome bisulfite sequencing, ChIP-seq, RNA-seq in prospermatogonia and oocytes\",\n      \"pmids\": [\"32929285\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Sex-specific division of labor with SETD2 mechanism not fully resolved\", \"Direct DNMT3A recruitment biochemistry not shown here\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Demonstrated druggability of the NSD1 SET domain, with a covalent inhibitor that opens the autoinhibitory loop and downregulates NUP98-NSD1 target genes.\",\n      \"evidence\": \"Fragment screening, X-ray crystallography of BT5 complex, cell-based H3K36me2 and colony assays in patient AML cells\",\n      \"pmids\": [\"32868895\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Selectivity over other NSD family members not detailed here\", \"In vivo efficacy untested in this study\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Defined a skeletal developmental role, showing NSD1 in mesenchymal progenitors regulates Sox9 and HIF1a via H3K36 methylation to drive chondrogenesis.\",\n      \"evidence\": \"Prx1-Cre and Col2-Cre conditional KO mice, RNA-seq, ChIP-seq, in vitro differentiation\",\n      \"pmids\": [\"34099628\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct vs indirect HIF1a activation mechanism unresolved\", \"Relevance to Sotos overgrowth phenotype not directly linked\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Mechanistically connected NSD1/H3K36me2 to PRC2 antagonism and SWI/SNF cooperation, explaining EZH2-inhibitor resistance in NSD1-null tumors.\",\n      \"evidence\": \"CRISPR screen and KO, KDM2A inhibitor rescue, ChIP-seq in SMARCB1-mutant rhabdoid cells\",\n      \"pmids\": [\"35537449\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Precise biochemical link between H3K36me2 and PRC2 exclusion not fully reconstituted\", \"Generalizability beyond SWI/SNF-deficient context unclear\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Characterized NUP98-NSD1 condensate biology, identifying SMARCA5/BPTF as core interactors required for transformation.\",\n      \"evidence\": \"AP-MS, FRAP, proximity ligation, inducible knockdown, and SMARCA5 inhibition with colony assays\",\n      \"pmids\": [\"35073946\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Causal role of condensate formation vs SMARCA5 activity disentangled only partially\", \"Single-lab interactome\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Separated NSD1's catalytic and coactivator functions, revealing a qPHD-PWWP module that reads H3K18ac to drive enhancer activity and Pol II pause release independent of methyltransferase activity.\",\n      \"evidence\": \"Auxin-inducible degron, ChIP-seq/ATAC-seq/GRO-seq, domain binding assays, catalytic-dead rescue, ESC differentiation\",\n      \"pmids\": [\"37402365\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism by which non-catalytic NSD1 promotes pause release undefined\", \"Relative contribution of catalytic vs non-catalytic functions context-dependent\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Established the H3K36me2-DNMT3A-non-CG methylation pathway in neurons, linking NSD1 loss to DNMT3A-disorder-convergent gene dysregulation.\",\n      \"evidence\": \"Brain-specific conditional KO, ChIP-seq, whole-genome bisulfite sequencing, RNA-seq\",\n      \"pmids\": [\"37098340\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Behavioral/disease phenotype consequences not addressed here\", \"Direct DNMT3A recruitment biochemistry not shown\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Showed NSD1-deposited H3K36me2 establishes and maintains cortical neuron areal and laminar identity, defining a postmitotic developmental function.\",\n      \"evidence\": \"Conditional KO mice, RNA-seq, ChIP-seq, DNA methylation profiling, electrophysiology, axonal tracing\",\n      \"pmids\": [\"37995181\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Causal chain from H3K36me2 loss to wiring defects incompletely mapped\", \"Reversibility not tested\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Linked NSD1 loss to tumor immune evasion, showing reduced H3K36me2/elevated H3K27me3 silences T-cell chemokines CXCL9/10 in HNSCC.\",\n      \"evidence\": \"NSD1-mutant HNSCC models, ChIP-seq, KDM2A inhibition, syngeneic tumor models, flow cytometry\",\n      \"pmids\": [\"37311054\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct chromatin mechanism at chemokine loci vs indirect effects not fully separated\", \"Clinical translatability of KDM2A inhibition untested\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Ranked NSD1 atop the H3K36 methyltransferase hierarchy, establishing it as the predominant depositor of broad intergenic H3K36me2.\",\n      \"evidence\": \"CRISPR KO of five K36MTs with genome-wide ChIP-seq for H3K36me1/2/3 and RNA-seq in mouse mesenchymal stem cells\",\n      \"pmids\": [\"39390582\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Cell-type dependence of the hierarchy not exhaustively tested\", \"Mechanism of intergenic targeting specificity unresolved\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How NSD1 is recruited to specific genomic loci and how it physically hands off H3K36me2 to DNMT3A and excludes PRC2 remain incompletely defined at the biochemical level.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No reconstituted NSD1-DNMT3A handoff\", \"Locus-targeting determinants for intergenic H3K36me2 unknown\", \"Non-catalytic coactivator mechanism for Pol II pause release undefined\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0016740\", \"supporting_discovery_ids\": [1, 11, 13, 22]},\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [1, 11, 5]},\n      {\"term_id\": \"GO:0042393\", \"supporting_discovery_ids\": [9, 18]},\n      {\"term_id\": \"GO:0140110\", \"supporting_discovery_ids\": [0, 18, 10]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [0, 3, 10]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [0, 8, 18]},\n      {\"term_id\": \"GO:0000228\", \"supporting_discovery_ids\": [4, 8, 13, 22]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-4839726\", \"supporting_discovery_ids\": [13, 16, 18, 22]},\n      {\"term_id\": \"R-HSA-74160\", \"supporting_discovery_ids\": [0, 8, 18]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [13, 15, 20]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [2, 4, 12, 21]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"NUP98\", \"DNMT3A\", \"Nizp1\", \"AR\", \"EZH2\", \"SMARCA5\", \"BPTF\", \"EP300\"]\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}