{"gene":"DSN1","run_date":"2026-04-28T17:46:03","timeline":{"discoveries":[{"year":2004,"finding":"DSN1 (KNL3 in C. elegans) was identified as a subunit of a conserved outer kinetochore protein network (KMN). Depletion of the C. elegans ortholog (KNL-3) caused a 'kinetochore null' phenotype, abolishing kinetochore assembly and chromosome segregation, placing DSN1 as a core component of the MIS/KMN outer kinetochore module.","method":"Protein co-purification from C. elegans embryos, RNAi depletion with live-imaging phenotypic readout, homolog identification in human cells","journal":"Genes & development","confidence":"High","confidence_rationale":"Tier 2 — reciprocal co-purification and RNAi epistasis replicated across organisms","pmids":["15371340"],"is_preprint":false},{"year":2004,"finding":"Human DSN1 (c20orf172) was identified as a subunit of the conserved hMis12 core complex. The complex also contained hNnf1(PMF1), hNsl1(DC8), and hMis12, and interacted with centromeric heterochromatin components HP1α and HP1γ; RNAi of any subunit disrupted chromosome segregation in HeLa cells.","method":"Tandem-affinity purification of hMis12 complex, RNAi knockdown, immunofluorescence localization in HeLa cells","journal":"Nature cell biology","confidence":"High","confidence_rationale":"Tier 2 — reciprocal co-purification plus RNAi functional validation","pmids":["15502821"],"is_preprint":false},{"year":2006,"finding":"Human DSN1 (hDsn1/Q9H410), together with hNnf1(PMF1) and hNsl1(DC31), forms a stable four-subunit complex with hMis12 demonstrable by coexpression in bacteria and fractionation of mitotic extracts. Depletion of any Mis12 complex subunit caused mitotic delay, chromosome misalignment, reduced centromere stretch, and severely diminished outer kinetochore protein Ndc80/HEC1 localization, establishing the Mis12 complex as essential for outer kinetochore assembly.","method":"Bacterial coexpression, mitotic extract fractionation, RNAi depletion in human and chicken DT40 cells, immunofluorescence","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 1–2 — bacterial reconstitution plus RNAi with multiple phenotypic readouts","pmids":["16585270"],"is_preprint":false},{"year":2006,"finding":"DSN1 was detected as a phosphorylated protein in purified human mitotic spindles, identifying it as a substrate of mitotic phosphorylation events.","method":"Mass spectrometry-based phosphoproteomics of purified human mitotic spindles","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"Medium","confidence_rationale":"Tier 2 — in vivo phosphosite detection by MS, single study","pmids":["16565220"],"is_preprint":false},{"year":2007,"finding":"DSN1-containing hMis12 complex subunits (c20orf172/hMis13 and DC8/hMis14) were shown to associate with the C-terminal domain of blinkin (KNL1/AF15q14), placing DSN1 at the interface between the inner kinetochore and the KNL1 scaffold that recruits spindle-checkpoint proteins Bub1 and BubR1.","method":"Co-immunoprecipitation, domain-mapping pulldown assays, RNAi in HeLa cells","journal":"Developmental cell","confidence":"Medium","confidence_rationale":"Tier 2–3 — co-IP and domain mapping, single lab","pmids":["17981135"],"is_preprint":false},{"year":2010,"finding":"Biochemical analysis and negative-stain electron microscopy of the human MIS12 complex revealed an elongated ~22 nm structure. NSL1 (not DSN1) acts as the scaffold, but DSN1 occupies a defined position in the complex head and makes contacts required for interaction with the NDC80 and KNL1 complexes within the KMN network.","method":"Biochemical fractionation, chemical cross-linking, negative-stain electron microscopy, pulldown assays","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 1 — structural EM plus biochemical interaction mapping","pmids":["20819937"],"is_preprint":false},{"year":2010,"finding":"High-throughput tandem-affinity purification and mass spectrometry (MitoCheck) confirmed DSN1 as a stable component of the human Mis12 complex and localized it to kinetochores during mitosis.","method":"BAC-based gene tagging, TAP-MS, immunofluorescence localization","journal":"Science","confidence":"Medium","confidence_rationale":"Tier 2 — systematic AP-MS with localization, large consortium study","pmids":["20360068"],"is_preprint":false},{"year":2013,"finding":"In budding yeast, the Mub1/Ubr2 ubiquitin ligase complex associates with kinetochore particles via CENP-C (Mif2) and ubiquitylates Dsn1 to regulate its protein levels. Deletion of Mub1/Ubr2 restores levels and viability of a mutant Dsn1, suggesting Mub1/Ubr2 act as a quality control system that degrades aberrant Dsn1 to maintain kinetochore integrity.","method":"Kinetochore particle purification, co-immunoprecipitation, genetic deletion analysis, ubiquitylation assay, viability rescue experiments in S. cerevisiae","journal":"PLoS genetics","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods (purification, genetic epistasis, ubiquitylation assay) in single rigorous study","pmids":["23408894"],"is_preprint":false},{"year":2013,"finding":"CENP-T interacts with the Mis12 complex (containing DSN1) and with the Ndc80 complex via mutually exclusive binding; CENP-T and Mis12C compete for Ndc80 binding, defining two distinct pathways that independently recruit the Ndc80 complex to kinetochores.","method":"Structural analysis, biochemical reconstitution, pulldown competition assays, DT40 cell depletion experiments","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 1 — structural analysis plus biochemical reconstitution and genetic validation","pmids":["23334297"],"is_preprint":false},{"year":2016,"finding":"Crystal structures of the human MIS12 complex (containing DSN1) bound to a CENP-C fragment revealed the structural basis of MIS12C's bridging function between the outer kinetochore (KMN) and inner kinetochore. Aurora B kinase phosphorylation of the DSN1 N-terminal region (specifically Ser100 and Ser109) releases auto-inhibition and dramatically strengthens CENP-C binding, revealing a phospho-regulatory switch controlling kinetochore assembly.","method":"X-ray crystallography of MIS12C:CENP-C complex, biochemical binding assays with phosphomimetic/phosphodead mutants, Aurora B kinase assay","journal":"Cell","confidence":"High","confidence_rationale":"Tier 1 — crystal structure plus mutagenesis and kinase assay, strong mechanistic validation","pmids":["27881301"],"is_preprint":false},{"year":2016,"finding":"DSN1 protein levels were sequentially up-regulated during colorectal adenoma-to-carcinoma progression; DSN1 knockdown in CRC cells induced G2/M arrest and decreased migration, invasion, and anchorage-independent growth, implicating DSN1 in chromosome 20q amplification-associated malignant transformation.","method":"SNP genotyping, RNA sequencing of patient tri-part samples, siRNA knockdown, cell growth/apoptosis/migration/invasion assays","journal":"Oncotarget","confidence":"Medium","confidence_rationale":"Tier 3 — loss-of-function with defined phenotypic readouts but limited pathway mechanistic detail","pmids":["27329586"],"is_preprint":false},{"year":2021,"finding":"ESRRA was shown to transcriptionally regulate DSN1 expression in gastric cancer. ChIP and dual-luciferase assays confirmed ESRRA directly binds the DSN1 promoter. ESRRA silencing caused G2/M arrest via the CDC25C–CDK1–Cyclin B1 pathway downstream of DSN1 reduction, placing DSN1 in an ESRRA→DSN1→CDC25C/CDK1/CyclinB1 signaling axis.","method":"RNA-seq, ChIP assay, dual-luciferase reporter assay, Western blot, flow cytometry (cell cycle), siRNA knockdown","journal":"International journal of biological sciences","confidence":"Medium","confidence_rationale":"Tier 2 — ChIP and luciferase directly demonstrate transcriptional regulation; pathway placement via Western blot, single lab","pmids":["34131395"],"is_preprint":false},{"year":2022,"finding":"SRSF9 stabilizes DSN1 mRNA in an m6A-dependent manner in colorectal cancer cells. SRSF9 acts as an m6A reader, binding to two m6A modification sites in the DSN1 mRNA 3′ region; METTL3 knockdown impaired both SRSF9 binding and DSN1 mRNA stabilization. DSN1 knockdown partially reversed SRSF9 overexpression-induced CRC cell phenotypes.","method":"Methylated RNA affinity assay, gene-specific m6A qRT-PCR, dual-luciferase reporter, RNA stability assay, siRNA knockdown, xenograft model","journal":"Journal of translational medicine","confidence":"Medium","confidence_rationale":"Tier 2 — multiple orthogonal biochemical methods demonstrating m6A-mediated mRNA stabilization, single lab","pmids":["35509101"],"is_preprint":false},{"year":2024,"finding":"Mammalian germ cells express a germline-specific alternative splice isoform of DSN1 that lacks a key N-terminal regulatory region required for Aurora kinase phosphorylation-dependent centromere localization. This isoform displays constitutive centromere localization independent of Aurora B phosphorylation. Expression of the germline isoform in somatic cells causes constitutive kinetochore localization, chromosome segregation errors, and growth defects. Selective elimination of the germline isoform in mouse models disrupts oocyte maturation and early embryonic divisions, reducing fertility.","method":"Alternative splice isoform cloning, immunofluorescence in oocytes and somatic cells, Aurora kinase inhibitor treatment, CRISPR mouse models with isoform-specific deletion, live-cell imaging, fertility assays","journal":"Current biology : CB","confidence":"High","confidence_rationale":"Tier 1–2 — multiple orthogonal methods including mouse genetic models, cell imaging, and pharmacological perturbation; peer-reviewed","pmids":["39178843"],"is_preprint":false},{"year":2024,"finding":"DSN1 directly interacts with Centromere Protein T (CENP-T) in hepatocellular carcinoma cells; elevated DSN1 expression led to overproduction of cell cycle-related proteins through this interaction, contributing to chromosomal instability and aberrant cell cycle progression. siRNA/shRNA knockdown of DSN1 reduced xenograft tumor growth.","method":"Co-immunoprecipitation, siRNA/shRNA knockdown, overexpression vectors, xenograft tumor model, TCGA database analysis","journal":"Molecular carcinogenesis","confidence":"Medium","confidence_rationale":"Tier 3 — co-IP identifies interaction, functional consequences shown by KD/OE, single lab","pmids":["39560395"],"is_preprint":false},{"year":2024,"finding":"AlphaFold2-based predictions and cell biological experiments in chicken DT40 cells identified two binding surfaces for the CENP-T–Mis12C (DSN1-containing) interaction. Dual phosphorylation of Dsn1 and CENP-T cooperatively regulates this interaction to ensure robust CENP-T–Mis12C binding and proper mitotic progression.","method":"AlphaFold2 structure prediction, mutagenesis of binding surfaces, DT40 cell conditional depletion, co-immunoprecipitation, cell viability assays","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 2 — structure-guided mutagenesis with cell biological validation, preprint","pmids":["bio_10.1101_2024.06.20.599825"],"is_preprint":true},{"year":2025,"finding":"Cryo-EM structures of the budding yeast KMN complex revealed that the N-terminal auto-inhibitory segment of Dsn1 (Dsn1-AI) folds into two α-helices that engage its head domain, occluding binding sites for inner kinetochore subunits CENP-C (Mif2) and CENP-U (Ame1). Aurora B (Ipl1) phosphorylation of Dsn1-AI releases this auto-inhibition to strengthen inner–outer kinetochore connections. C-terminal α-helical motifs of Dsn1 and other Mis12c subunits mediate binding to Knl1c and Ndc80c.","method":"Cryo-EM structure determination, biochemical binding assays, genetic experiments in S. cerevisiae","journal":"bioRxiv","confidence":"High","confidence_rationale":"Tier 1 — cryo-EM structure plus biochemical and genetic validation, multiple orthogonal methods","pmids":["bio_10.1101_2025.06.03.657598"],"is_preprint":true},{"year":2026,"finding":"DSN1 stabilizes c-MYC protein in colorectal cancer by competing with the E3 ubiquitin ligase FZR1 for c-MYC binding, thereby attenuating FZR1-mediated ubiquitination and proteasomal degradation of c-MYC. DSN1 knockdown accelerated c-MYC degradation and reduced CRC invasion/metastasis in vitro and in vivo; c-MYC overexpression rescued the anti-metastatic effect of DSN1 silencing.","method":"Cycloheximide chase assay, proteasome inhibitor assay, co-immunoprecipitation (DSN1–FZR1, FZR1–c-MYC competition), siRNA knockdown, rescue overexpression, in vivo metastasis model","journal":"Experimental cell research","confidence":"Medium","confidence_rationale":"Tier 2 — multiple biochemical methods (CHX chase, proteasome inhibition, co-IP competition) with in vivo validation, single lab","pmids":["41713835"],"is_preprint":false}],"current_model":"DSN1 is a core subunit of the evolutionarily conserved four-subunit MIS12 complex at the outer kinetochore, where its N-terminal auto-inhibitory domain (released by Aurora B phosphorylation) controls binding to inner kinetochore proteins CENP-C and CENP-T, while its C-terminal region connects to KNL1 and NDC80 complexes to mediate chromosome–microtubule attachment; protein levels are regulated by Mub1/Ubr2-mediated ubiquitylation (quality control), its mRNA is stabilized by SRSF9 via m6A recognition, a germline-specific splice isoform bypasses Aurora B regulation to support oocyte and embryo development, and in cancer contexts DSN1 also stabilizes c-MYC by competing with the FZR1 E3 ligase."},"narrative":{"teleology":[{"year":2004,"claim":"Identification of DSN1 as a conserved outer kinetochore component resolved the molecular composition of the KMN network and established that loss of DSN1 abolishes kinetochore assembly and chromosome segregation.","evidence":"Co-purification from C. elegans embryos with RNAi phenotyping, and tandem-affinity purification of the human hMis12 complex with RNAi in HeLa cells","pmids":["15371340","15502821"],"confidence":"High","gaps":["Precise stoichiometry of DSN1 within the complex was unresolved","No structural information on DSN1 or its interaction interfaces"]},{"year":2006,"claim":"Reconstitution of the four-subunit MIS12 complex and demonstration that its depletion eliminates NDC80/HEC1 kinetochore recruitment established DSN1 as essential for outer kinetochore assembly in vertebrate cells.","evidence":"Bacterial coexpression of the four-subunit complex, mitotic extract fractionation, RNAi in HeLa and DT40 cells","pmids":["16585270"],"confidence":"High","gaps":["Which subunit directly contacts NDC80 and KNL1 was unknown","Regulation of DSN1 during the cell cycle was uncharacterized"]},{"year":2010,"claim":"Structural visualization of the elongated MIS12 complex by negative-stain EM and mapping of DSN1's position within the complex head defined the architecture that mediates bridging between KNL1/NDC80 and the inner kinetochore.","evidence":"Negative-stain EM, chemical cross-linking, and pulldown assays with recombinant MIS12 complex","pmids":["20819937"],"confidence":"High","gaps":["Atomic-resolution structure of DSN1 was still lacking","How DSN1 phosphorylation controls binding was unknown"]},{"year":2013,"claim":"Two parallel studies revealed that CENP-T binds the MIS12 complex independently of CENP-C, defining dual pathways for NDC80 recruitment, and that Dsn1 protein levels are controlled by Mub1/Ubr2-mediated ubiquitylation as a kinetochore quality-control mechanism.","evidence":"Structural and biochemical reconstitution of CENP-T–MIS12C interaction in DT40 cells; kinetochore particle purification, genetic epistasis, and ubiquitylation assays in S. cerevisiae","pmids":["23334297","23408894"],"confidence":"High","gaps":["The structural basis of DSN1's contacts with CENP-C and CENP-T was unresolved","Whether ubiquitin-mediated regulation of DSN1 is conserved in mammals was untested"]},{"year":2016,"claim":"Crystal structures of the MIS12 complex bound to CENP-C revealed that Aurora B phosphorylation of DSN1 Ser100/Ser109 releases an N-terminal auto-inhibitory segment that competes with CENP-C binding, establishing the phospho-regulatory switch governing inner–outer kinetochore coupling.","evidence":"X-ray crystallography of MIS12C:CENP-C complex, phosphomimetic/phosphodead mutagenesis, Aurora B kinase assays","pmids":["27881301"],"confidence":"High","gaps":["Whether the same switch controls CENP-T binding was unclear","The structural basis of auto-inhibition at atomic resolution in the context of the full KMN was unresolved"]},{"year":2022,"claim":"Post-transcriptional regulation of DSN1 was revealed: SRSF9 functions as an m6A reader that stabilizes DSN1 mRNA via METTL3-deposited m6A marks, linking epitranscriptomic regulation to DSN1 protein abundance in colorectal cancer.","evidence":"Methylated RNA affinity assay, gene-specific m6A qRT-PCR, RNA stability assays, and xenograft models in CRC cells","pmids":["35509101"],"confidence":"Medium","gaps":["Whether m6A-mediated DSN1 regulation occurs in normal (non-cancer) cells is unknown","The specific m6A writer–reader axis for DSN1 in other tissue contexts is uncharacterized"]},{"year":2024,"claim":"Discovery of a germline-specific DSN1 splice isoform that lacks the Aurora B-regulated N-terminal domain showed that germ cells bypass mitotic phospho-regulation to achieve constitutive kinetochore assembly, and that this isoform is required for oocyte maturation and fertility.","evidence":"Isoform cloning, CRISPR mouse models with isoform-specific deletion, live-cell imaging of oocytes, Aurora kinase inhibitor experiments","pmids":["39178843"],"confidence":"High","gaps":["Whether the germline isoform is conserved in non-mammalian vertebrates is untested","How meiotic chromosome segregation fidelity is maintained without the auto-inhibitory regulatory step is unclear"]},{"year":2025,"claim":"Cryo-EM structures of the budding yeast KMN complex provided atomic detail of the DSN1 auto-inhibitory helices engaging its head domain and the C-terminal helical motifs mediating KNL1 and NDC80 binding, unifying genetic and biochemical data into a complete structural framework.","evidence":"Cryo-EM structure determination with biochemical and genetic validation in S. cerevisiae (preprint)","pmids":["bio_10.1101_2025.06.03.657598"],"confidence":"High","gaps":["Full-length human KMN cryo-EM structure is still unavailable","Dynamics of auto-inhibition release in vivo are unresolved"]},{"year":2026,"claim":"A non-canonical cancer-associated function was identified: DSN1 stabilizes c-MYC by competing with the FZR1 E3 ligase for c-MYC binding, thereby attenuating ubiquitin-mediated c-MYC degradation and promoting colorectal cancer metastasis.","evidence":"Cycloheximide chase, co-IP competition between DSN1 and FZR1 for c-MYC, siRNA knockdown with c-MYC rescue, in vivo metastasis model","pmids":["41713835"],"confidence":"Medium","gaps":["Whether DSN1–c-MYC interaction occurs in normal proliferating cells is unknown","The structural basis of DSN1–c-MYC binding and FZR1 competition is uncharacterized","Independent replication in other cancer types is lacking"]},{"year":null,"claim":"Key unresolved questions include the full atomic structure of the human KMN super-complex in the kinetochore context, whether ubiquitin-mediated DSN1 quality control (Mub1/Ubr2) is conserved in mammals, and how the germline DSN1 isoform maintains segregation fidelity without Aurora B-dependent regulation.","evidence":"","pmids":[],"confidence":"Low","gaps":["No human KMN cryo-EM structure available","Mammalian DSN1 ubiquitylation pathway not characterized","Germline isoform segregation fidelity mechanism unknown"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0005198","term_label":"structural molecule activity","supporting_discovery_ids":[0,1,2,5,9]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[9,16,17]}],"localization":[{"term_id":"GO:0005694","term_label":"chromosome","supporting_discovery_ids":[0,1,2,6,9,13]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[1,6]}],"pathway":[{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[0,1,2,9,13]}],"complexes":["MIS12 complex","KMN network"],"partners":["MIS12","PMF1","NSL1","CENP-C","CENP-T","KNL1","NDC80"],"other_free_text":[]},"mechanistic_narrative":"DSN1 is a core subunit of the conserved four-subunit MIS12 complex (MIS12–PMF1–NSL1–DSN1) that serves as the central bridge between inner kinetochore receptors (CENP-C and CENP-T) and the outer kinetochore microtubule-binding complexes NDC80 and KNL1, and is therefore essential for chromosome segregation during mitosis [PMID:15371340, PMID:16585270, PMID:20819937]. Crystal and cryo-EM structures show that the DSN1 N-terminal auto-inhibitory segment occludes CENP-C and CENP-T binding sites on the MIS12 complex; Aurora B phosphorylation of DSN1 Ser100/Ser109 releases this auto-inhibition, acting as a phospho-switch that controls kinetochore assembly [PMID:27881301, PMID:39178843]. Protein levels of DSN1 are regulated by Mub1/Ubr2-mediated ubiquitylation in yeast as a quality-control mechanism, and its mRNA is stabilized by the m6A reader SRSF9 in human colorectal cancer cells [PMID:23408894, PMID:35509101]. A germline-specific splice isoform that lacks the Aurora B-regulated N-terminal domain enables constitutive centromere localization and is required for oocyte maturation and early embryonic divisions in mice [PMID:39178843]."},"prefetch_data":{"uniprot":{"accession":"Q9H410","full_name":"Kinetochore-associated protein DSN1 homolog","aliases":[],"length_aa":356,"mass_kda":40.1,"function":"Part of the MIS12 complex which is required for normal chromosome alignment and segregation and kinetochore formation during mitosis","subcellular_location":"Nucleus; Chromosome, centromere, kinetochore","url":"https://www.uniprot.org/uniprotkb/Q9H410/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":true,"resolved_as":"","url":"https://depmap.org/portal/gene/DSN1","classification":"Common Essential","n_dependent_lines":1106,"n_total_lines":1208,"dependency_fraction":0.9155629139072847},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"MIS12","stoichiometry":10.0},{"gene":"CBX1","stoichiometry":0.2},{"gene":"DNAJC7","stoichiometry":0.2},{"gene":"HSPA4","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/DSN1","total_profiled":1310},"omim":[{"mim_id":"609175","title":"DSN1, MIS12 KINETOCHORE COMPLEX COMPONENT; DSN1","url":"https://www.omim.org/entry/609175"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Nucleoplasm","reliability":"Supported"},{"location":"Nucleoli fibrillar center","reliability":"Additional"},{"location":"Nuclear bodies","reliability":"Additional"},{"location":"Kinetochore","reliability":"Additional"},{"location":"Cytosol","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/DSN1"},"hgnc":{"alias_symbol":["dJ469A13.2","MIS13","KNL3","hKNL-3"],"prev_symbol":["C20orf172"]},"alphafold":{"accession":"Q9H410","domains":[{"cath_id":"-","chopping":"118-194","consensus_level":"high","plddt":88.5304,"start":118,"end":194}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9H410","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9H410-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9H410-F1-predicted_aligned_error_v6.png","plddt_mean":67.75},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=DSN1","jax_strain_url":"https://www.jax.org/strain/search?query=DSN1"},"sequence":{"accession":"Q9H410","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9H410.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9H410/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9H410"}},"corpus_meta":[{"pmid":"27329586","id":"PMC_27329586","title":"Over-expression 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Sensitivity.","date":"2018","source":"Cell","url":"https://pubmed.ncbi.nlm.nih.gov/29656893","citation_count":379,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"15371340","id":"PMC_15371340","title":"A conserved protein network controls assembly of the outer kinetochore and its ability to sustain tension.","date":"2004","source":"Genes & development","url":"https://pubmed.ncbi.nlm.nih.gov/15371340","citation_count":356,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"34079125","id":"PMC_34079125","title":"A proximity-dependent biotinylation map of a human cell.","date":"2021","source":"Nature","url":"https://pubmed.ncbi.nlm.nih.gov/34079125","citation_count":339,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"16565220","id":"PMC_16565220","title":"Phosphoproteome analysis of the human mitotic spindle.","date":"2006","source":"Proceedings of the National Academy of Sciences of the United States of 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biology","url":"https://pubmed.ncbi.nlm.nih.gov/15502821","citation_count":222,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"17207965","id":"PMC_17207965","title":"hORFeome v3.1: a resource of human open reading frames representing over 10,000 human genes.","date":"2007","source":"Genomics","url":"https://pubmed.ncbi.nlm.nih.gov/17207965","citation_count":222,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"20562864","id":"PMC_20562864","title":"Human POGZ modulates dissociation of HP1alpha from mitotic chromosome arms through Aurora B activation.","date":"2010","source":"Nature cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/20562864","citation_count":217,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"27705803","id":"PMC_27705803","title":"A High-Density Map for Navigating the Human Polycomb Complexome.","date":"2016","source":"Cell reports","url":"https://pubmed.ncbi.nlm.nih.gov/27705803","citation_count":216,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"15824131","id":"PMC_15824131","title":"ZW10 links mitotic checkpoint signaling to the structural kinetochore.","date":"2005","source":"The Journal of cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/15824131","citation_count":214,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"20819937","id":"PMC_20819937","title":"The MIS12 complex is a protein interaction hub for outer kinetochore assembly.","date":"2010","source":"The Journal of cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/20819937","citation_count":190,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"16585270","id":"PMC_16585270","title":"The human Mis12 complex is required for kinetochore assembly and proper chromosome segregation.","date":"2006","source":"The Journal of cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/16585270","citation_count":178,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"23334297","id":"PMC_23334297","title":"CENP-T provides a structural platform for outer kinetochore assembly.","date":"2013","source":"The EMBO journal","url":"https://pubmed.ncbi.nlm.nih.gov/23334297","citation_count":170,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"11780052","id":"PMC_11780052","title":"The DNA sequence and comparative analysis of human chromosome 20.","date":"2001","source":"Nature","url":"https://pubmed.ncbi.nlm.nih.gov/11780052","citation_count":168,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"27881301","id":"PMC_27881301","title":"Structure of the MIS12 Complex and Molecular Basis of Its Interaction with CENP-C at Human Kinetochores.","date":"2016","source":"Cell","url":"https://pubmed.ncbi.nlm.nih.gov/27881301","citation_count":132,"is_preprint":false,"source_track":"gene2pubmed"}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":8338,"output_tokens":2373,"usd":0.030305},"stage2":{"model":"claude-opus-4-6","input_tokens":5671,"output_tokens":2339,"usd":0.130245},"total_usd":0.395912,"stage1_batch_id":"msgbatch_0118RScg3WGaGkaZRtWZbtce","stage2_batch_id":"msgbatch_01Cpfzvtbj42aqp5oYZ5Te2s","note":"batch pricing = 50% of standard","round2_stage1":{"model":"claude-sonnet-4-6","input_tokens":19628,"output_tokens":5665,"usd":0.071929},"round2_rules_fired":"R3","round2_stage2":{"model":"claude-opus-4-6","input_tokens":8296,"output_tokens":2699,"usd":0.163433}},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2013,\n      \"finding\": \"The Mub1/Ubr2 ubiquitin ligase complex associates with kinetochore particles through CENP-C (Mif2) and ubiquitylates the conserved outer kinetochore protein Dsn1, regulating its protein levels as part of a quality control mechanism that monitors kinetochore integrity.\",\n      \"method\": \"Kinetochore particle purification, Co-IP, ubiquitylation assays, genetic deletion/rescue experiments in budding yeast\",\n      \"journal\": \"PLoS genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal Co-IP, biochemical ubiquitylation assay, and genetic epistasis with functional rescue, multiple orthogonal methods\",\n      \"pmids\": [\"23408894\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Mammalian germ cells express a germline-specific alternative splice isoform of DSN1 that lacks a key regulatory region, bypassing the requirement for Aurora kinase phosphorylation for centromere localization and enabling persistent centromere localization. This isoform is required for oocyte maturation and early embryonic divisions; expression in somatic cells causes constitutive kinetochore localization, chromosome segregation errors, and growth defects.\",\n      \"method\": \"Mouse genetic models (germline-specific isoform knockout), live cell imaging, kinetochore localization assays, Aurora kinase phosphorylation assays, somatic cell expression experiments\",\n      \"journal\": \"Current biology : CB\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — multiple orthogonal methods including mouse knockouts, live imaging, and phospho-regulation experiments, replicated in peer-reviewed and preprint versions\",\n      \"pmids\": [\"39178843\", \"38659852\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"The N-terminal auto-inhibitory segment of Dsn1 (Dsn1 AI) folds into two α-helices that engage its head 1 domain, occluding binding sites for inner kinetochore subunits CENP-C (Mif2) and CENP-U (Ame1), thereby reducing their affinity for the Mis12 complex. Aurora B (Ipl1) phosphorylation of Dsn1 AI releases this auto-inhibition, strengthening connections between the inner and outer kinetochore. C-terminal α-helical motifs of Mis12c subunits Dsn1, Mis12, and Pmf1 bind Knl1c and Ndc80c to assemble the KMN complex.\",\n      \"method\": \"Cryo-EM structure determination, biochemical binding assays, genetic experiments in budding yeast\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — cryo-EM structure with biochemical and genetic validation, multiple orthogonal methods in single study\",\n      \"pmids\": [\"bio_10.1101_2025.06.03.657598\"],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"The Mis12 complex (Mis12C) is recruited to kinetochores via dual phospho-regulation: DSN1 phosphorylation and CENP-T phosphorylation cooperatively regulate two interaction surfaces between CENP-T and Mis12C, ensuring robust Mis12C recruitment and proper mitotic progression.\",\n      \"method\": \"AlphaFold2 structural prediction, site-directed mutagenesis of interaction surfaces, functional rescue experiments in chicken DT40 cells lacking CENP-C-Mis12C interaction\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — epistatic cell line system with mutagenesis and functional readout, but single lab preprint\",\n      \"pmids\": [\"bio_10.1101_2024.06.20.599825\"],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"DSN1 directly interacts with Centromere Protein T (CENP-T) in hepatocellular carcinoma cells, and elevated DSN1 expression through this interaction leads to overproduction of cell cycle-related proteins and chromosomal instability.\",\n      \"method\": \"Co-IP, siRNA/shRNA knockdown, overexpression vectors in hepatoma cell lines and xenograft models\",\n      \"journal\": \"Molecular carcinogenesis\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — Co-IP interaction with functional knockdown/overexpression phenotypes, single lab study\",\n      \"pmids\": [\"39560395\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"SRSF9 binds to m6A-modified DSN1 mRNA in an m6A motif- and dose-dependent manner, stabilizing DSN1 mRNA and preventing its degradation; this stabilization is dependent on the methyltransferase METTL3 and two specific m6A modification sites in the SRSF9-binding region of DSN1 mRNA.\",\n      \"method\": \"Methylated single-stranded RNA affinity assays, gene-specific m6A quantitative RT-PCR, METTL3 knockdown, RNA stability assays, dual-luciferase reporter assays\",\n      \"journal\": \"Journal of translational medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal biochemical methods establishing m6A-dependent mRNA stabilization mechanism, single lab\",\n      \"pmids\": [\"35509101\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"ESRRA transcriptionally activates DSN1 expression (validated by dual-luciferase assay and ChIP), and ESRRA silencing causes G2/M arrest via the CDC25C-CDK1-CyclinB1 pathway downstream of DSN1.\",\n      \"method\": \"RNA-seq, dual-luciferase assay, ChIP assay, western blot, flow cytometry, siRNA knockdown\",\n      \"journal\": \"International journal of biological sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — ChIP and luciferase assays establishing direct transcriptional regulation, with downstream pathway validation, single lab\",\n      \"pmids\": [\"34131395\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"DSN1 competes with c-MYC for binding to the E3 ubiquitin ligase FZR1, thereby attenuating FZR1-mediated ubiquitination and proteasomal degradation of c-MYC, stabilizing c-MYC protein to promote CRC invasion and metastasis.\",\n      \"method\": \"Co-IP (DSN1-FZR1 and FZR1-c-MYC interactions), cycloheximide chase assay, proteasome inhibition assay, competitive binding assay, rescue experiments with c-MYC overexpression, in vivo metastasis model\",\n      \"journal\": \"Experimental cell research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal Co-IP, competition assay, and cycloheximide/proteasome inhibitor experiments with rescue, single lab\",\n      \"pmids\": [\"41713835\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"DSN1 is a conserved and essential subunit of the Mis12/MIND kinetochore complex; depletion of Dsn1 (KNL3) in C. elegans results in a 'kinetochore null' phenotype, and in human cells DSN1 is required to shape the interface between the Mis12 complex and Blinkin (Spc105 counterpart).\",\n      \"method\": \"RNAi depletion in C. elegans, functional studies in human cells, sequence comparisons and proteomics\",\n      \"journal\": \"Cell cycle (Georgetown, Tex.)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — loss-of-function with defined phenotype in multiple organisms, but summary/review nature limits confidence\",\n      \"pmids\": [\"19270503\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"DSN1 is an essential subunit of the MIS12 kinetochore complex that bridges centromeric chromatin (via CENP-C/CENP-T interactions) to microtubule-binding outer kinetochore proteins (Knl1c/Ndc80c); its centromere recruitment is phospho-regulated by Aurora B kinase through relief of N-terminal auto-inhibition, its protein levels are controlled by Mub1/Ubr2-mediated ubiquitylation as a quality control mechanism, and a germline-specific splice isoform bypasses Aurora kinase regulation to support meiosis and early embryogenesis, while in cancer contexts DSN1 additionally stabilizes c-MYC by competing with the FZR1 E3 ligase.\"\n}\n```\n\n--- ROUND 2 (R2/R3, augmented corpus) ---\n\nI'll classify each paper and extract discoveries only from KEEP papers.\n\n**Classification:**\n\nRetrieved papers:\n1. AURKA/SKA3/DSN1 in CRC — KEEP (DSN1 protein, functional assays)\n2. ESRRA/DSN1/CDC25C in gastric cancer — KEEP (DSN1 as transcriptional target with downstream pathway)\n3. SRSF9/DSN1 mRNA stability — KEEP (DSN1 mRNA regulation mechanism)\n4. Mub1/Ubr2 ubiquitin ligase regulates Dsn1 — KEEP (yeast ortholog, direct mechanism)\n5. CDCA8/DSN1/BIRC5 in osteosarcoma — KEEP (DSN1 cell cycle role, limited mechanism)\n6. Drosophila Dsn1 — KEEP (ortholog discussion, limited mechanism)\n7. Germline DSN1 splice isoform (CB 2024) — KEEP (canonical DSN1 protein, strong mechanism)\n8. Germline DSN1 splice isoform (bioRxiv preprint) — KEEP but duplicate of #7; mark as preprint\n9. DSN1 in breast cancer — KEEP (cell cycle mechanism)\n10. DSN1/CENP-T in HCC — KEEP (direct interaction mechanism)\n11. DSN1/FZR1/c-MYC in CRC — KEEP (direct mechanism)\n12. KMN complex cryo-EM (preprint) — KEEP (Dsn1 structural mechanism, yeast ortholog)\n13. Bioinformatics endometrial cancer — EXCLUDE (pure bioinformatics/expression, no direct mechanism)\n14. CENP-T-Mis12 interaction (preprint) — KEEP (Dsn1 phospho-regulation mechanism)\n\nGene2pubmed papers:\n- PMID:17081983 (phosphoproteome) — KEEP (DSN1 phosphorylation detected)\n- PMID:22658674 (mRNA interactome) — EXCLUDE (no DSN1-specific finding)\n- PMID:12477932, 14702039, 15489334, 17207965 (cDNA collections) — EXCLUDE\n- PMID:26186194, 28514442, 33961781, 32296183 (BioPlex/interactome) — KEEP (DSN1 interactions mapped)\n- PMID:26496610 (HeLa interactome) — KEEP\n- PMID:21873635 (GO annotation) — EXCLUDE\n- PMID:20360068 (MitoCheck) — KEEP (DSN1 complex characterization)\n- PMID:29656893 (Shieldin) — EXCLUDE (no DSN1-specific finding)\n- PMID:34079125, 35271311 (BioID/OpenCell maps) — KEEP (localization)\n- PMID:16565220 (mitotic spindle phosphoproteome) — KEEP (DSN1 phosphorylation)\n- PMID:21900206 — EXCLUDE (no DSN1 specific)\n- PMID:17981135 (Blinkin/hMis12) — KEEP (DSN1 complex context)\n- PMID:15502821 (Mis12 complex) — KEEP (DSN1 as complex member)\n- PMID:20562864 (POGZ/HP1) — EXCLUDE (no DSN1 specific)\n- PMID:27705803 (PcG complexome) — EXCLUDE\n- PMID:15824131 (ZW10/Zwint) — KEEP (Mis12 complex context)\n- PMID:15371340 (C. elegans KMN) — KEEP (KNL3/DSN1 ortholog)\n- PMID:20819937 (MIS12 complex hub) — KEEP (DSN1/NSL1 interactions)\n- PMID:16585270 (human Mis12 complex) — KEEP (hDsn1 direct characterization)\n- PMID:23334297 (CENP-T/Ndc80) — KEEP (Mis12C/DSN1 interaction context)\n- PMID:27881301 (MIS12C structure) — KEEP (structural basis including DSN1)\n- PMID:11780052 (chr20 sequence) — EXCLUDE\n- PMID:25416956 (not in list) — N/A\n\n```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2004,\n      \"finding\": \"DSN1 (KNL3 in C. elegans) was identified as a subunit of a conserved outer kinetochore protein network (KMN). Depletion of the C. elegans ortholog (KNL-3) caused a 'kinetochore null' phenotype, abolishing kinetochore assembly and chromosome segregation, placing DSN1 as a core component of the MIS/KMN outer kinetochore module.\",\n      \"method\": \"Protein co-purification from C. elegans embryos, RNAi depletion with live-imaging phenotypic readout, homolog identification in human cells\",\n      \"journal\": \"Genes & development\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal co-purification and RNAi epistasis replicated across organisms\",\n      \"pmids\": [\"15371340\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Human DSN1 (c20orf172) was identified as a subunit of the conserved hMis12 core complex. The complex also contained hNnf1(PMF1), hNsl1(DC8), and hMis12, and interacted with centromeric heterochromatin components HP1α and HP1γ; RNAi of any subunit disrupted chromosome segregation in HeLa cells.\",\n      \"method\": \"Tandem-affinity purification of hMis12 complex, RNAi knockdown, immunofluorescence localization in HeLa cells\",\n      \"journal\": \"Nature cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal co-purification plus RNAi functional validation\",\n      \"pmids\": [\"15502821\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"Human DSN1 (hDsn1/Q9H410), together with hNnf1(PMF1) and hNsl1(DC31), forms a stable four-subunit complex with hMis12 demonstrable by coexpression in bacteria and fractionation of mitotic extracts. Depletion of any Mis12 complex subunit caused mitotic delay, chromosome misalignment, reduced centromere stretch, and severely diminished outer kinetochore protein Ndc80/HEC1 localization, establishing the Mis12 complex as essential for outer kinetochore assembly.\",\n      \"method\": \"Bacterial coexpression, mitotic extract fractionation, RNAi depletion in human and chicken DT40 cells, immunofluorescence\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — bacterial reconstitution plus RNAi with multiple phenotypic readouts\",\n      \"pmids\": [\"16585270\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"DSN1 was detected as a phosphorylated protein in purified human mitotic spindles, identifying it as a substrate of mitotic phosphorylation events.\",\n      \"method\": \"Mass spectrometry-based phosphoproteomics of purified human mitotic spindles\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — in vivo phosphosite detection by MS, single study\",\n      \"pmids\": [\"16565220\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"DSN1-containing hMis12 complex subunits (c20orf172/hMis13 and DC8/hMis14) were shown to associate with the C-terminal domain of blinkin (KNL1/AF15q14), placing DSN1 at the interface between the inner kinetochore and the KNL1 scaffold that recruits spindle-checkpoint proteins Bub1 and BubR1.\",\n      \"method\": \"Co-immunoprecipitation, domain-mapping pulldown assays, RNAi in HeLa cells\",\n      \"journal\": \"Developmental cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 — co-IP and domain mapping, single lab\",\n      \"pmids\": [\"17981135\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Biochemical analysis and negative-stain electron microscopy of the human MIS12 complex revealed an elongated ~22 nm structure. NSL1 (not DSN1) acts as the scaffold, but DSN1 occupies a defined position in the complex head and makes contacts required for interaction with the NDC80 and KNL1 complexes within the KMN network.\",\n      \"method\": \"Biochemical fractionation, chemical cross-linking, negative-stain electron microscopy, pulldown assays\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — structural EM plus biochemical interaction mapping\",\n      \"pmids\": [\"20819937\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"High-throughput tandem-affinity purification and mass spectrometry (MitoCheck) confirmed DSN1 as a stable component of the human Mis12 complex and localized it to kinetochores during mitosis.\",\n      \"method\": \"BAC-based gene tagging, TAP-MS, immunofluorescence localization\",\n      \"journal\": \"Science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — systematic AP-MS with localization, large consortium study\",\n      \"pmids\": [\"20360068\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"In budding yeast, the Mub1/Ubr2 ubiquitin ligase complex associates with kinetochore particles via CENP-C (Mif2) and ubiquitylates Dsn1 to regulate its protein levels. Deletion of Mub1/Ubr2 restores levels and viability of a mutant Dsn1, suggesting Mub1/Ubr2 act as a quality control system that degrades aberrant Dsn1 to maintain kinetochore integrity.\",\n      \"method\": \"Kinetochore particle purification, co-immunoprecipitation, genetic deletion analysis, ubiquitylation assay, viability rescue experiments in S. cerevisiae\",\n      \"journal\": \"PLoS genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods (purification, genetic epistasis, ubiquitylation assay) in single rigorous study\",\n      \"pmids\": [\"23408894\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"CENP-T interacts with the Mis12 complex (containing DSN1) and with the Ndc80 complex via mutually exclusive binding; CENP-T and Mis12C compete for Ndc80 binding, defining two distinct pathways that independently recruit the Ndc80 complex to kinetochores.\",\n      \"method\": \"Structural analysis, biochemical reconstitution, pulldown competition assays, DT40 cell depletion experiments\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — structural analysis plus biochemical reconstitution and genetic validation\",\n      \"pmids\": [\"23334297\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Crystal structures of the human MIS12 complex (containing DSN1) bound to a CENP-C fragment revealed the structural basis of MIS12C's bridging function between the outer kinetochore (KMN) and inner kinetochore. Aurora B kinase phosphorylation of the DSN1 N-terminal region (specifically Ser100 and Ser109) releases auto-inhibition and dramatically strengthens CENP-C binding, revealing a phospho-regulatory switch controlling kinetochore assembly.\",\n      \"method\": \"X-ray crystallography of MIS12C:CENP-C complex, biochemical binding assays with phosphomimetic/phosphodead mutants, Aurora B kinase assay\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — crystal structure plus mutagenesis and kinase assay, strong mechanistic validation\",\n      \"pmids\": [\"27881301\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"DSN1 protein levels were sequentially up-regulated during colorectal adenoma-to-carcinoma progression; DSN1 knockdown in CRC cells induced G2/M arrest and decreased migration, invasion, and anchorage-independent growth, implicating DSN1 in chromosome 20q amplification-associated malignant transformation.\",\n      \"method\": \"SNP genotyping, RNA sequencing of patient tri-part samples, siRNA knockdown, cell growth/apoptosis/migration/invasion assays\",\n      \"journal\": \"Oncotarget\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — loss-of-function with defined phenotypic readouts but limited pathway mechanistic detail\",\n      \"pmids\": [\"27329586\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"ESRRA was shown to transcriptionally regulate DSN1 expression in gastric cancer. ChIP and dual-luciferase assays confirmed ESRRA directly binds the DSN1 promoter. ESRRA silencing caused G2/M arrest via the CDC25C–CDK1–Cyclin B1 pathway downstream of DSN1 reduction, placing DSN1 in an ESRRA→DSN1→CDC25C/CDK1/CyclinB1 signaling axis.\",\n      \"method\": \"RNA-seq, ChIP assay, dual-luciferase reporter assay, Western blot, flow cytometry (cell cycle), siRNA knockdown\",\n      \"journal\": \"International journal of biological sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — ChIP and luciferase directly demonstrate transcriptional regulation; pathway placement via Western blot, single lab\",\n      \"pmids\": [\"34131395\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"SRSF9 stabilizes DSN1 mRNA in an m6A-dependent manner in colorectal cancer cells. SRSF9 acts as an m6A reader, binding to two m6A modification sites in the DSN1 mRNA 3′ region; METTL3 knockdown impaired both SRSF9 binding and DSN1 mRNA stabilization. DSN1 knockdown partially reversed SRSF9 overexpression-induced CRC cell phenotypes.\",\n      \"method\": \"Methylated RNA affinity assay, gene-specific m6A qRT-PCR, dual-luciferase reporter, RNA stability assay, siRNA knockdown, xenograft model\",\n      \"journal\": \"Journal of translational medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal biochemical methods demonstrating m6A-mediated mRNA stabilization, single lab\",\n      \"pmids\": [\"35509101\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Mammalian germ cells express a germline-specific alternative splice isoform of DSN1 that lacks a key N-terminal regulatory region required for Aurora kinase phosphorylation-dependent centromere localization. This isoform displays constitutive centromere localization independent of Aurora B phosphorylation. Expression of the germline isoform in somatic cells causes constitutive kinetochore localization, chromosome segregation errors, and growth defects. Selective elimination of the germline isoform in mouse models disrupts oocyte maturation and early embryonic divisions, reducing fertility.\",\n      \"method\": \"Alternative splice isoform cloning, immunofluorescence in oocytes and somatic cells, Aurora kinase inhibitor treatment, CRISPR mouse models with isoform-specific deletion, live-cell imaging, fertility assays\",\n      \"journal\": \"Current biology : CB\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — multiple orthogonal methods including mouse genetic models, cell imaging, and pharmacological perturbation; peer-reviewed\",\n      \"pmids\": [\"39178843\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"DSN1 directly interacts with Centromere Protein T (CENP-T) in hepatocellular carcinoma cells; elevated DSN1 expression led to overproduction of cell cycle-related proteins through this interaction, contributing to chromosomal instability and aberrant cell cycle progression. siRNA/shRNA knockdown of DSN1 reduced xenograft tumor growth.\",\n      \"method\": \"Co-immunoprecipitation, siRNA/shRNA knockdown, overexpression vectors, xenograft tumor model, TCGA database analysis\",\n      \"journal\": \"Molecular carcinogenesis\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — co-IP identifies interaction, functional consequences shown by KD/OE, single lab\",\n      \"pmids\": [\"39560395\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"AlphaFold2-based predictions and cell biological experiments in chicken DT40 cells identified two binding surfaces for the CENP-T–Mis12C (DSN1-containing) interaction. Dual phosphorylation of Dsn1 and CENP-T cooperatively regulates this interaction to ensure robust CENP-T–Mis12C binding and proper mitotic progression.\",\n      \"method\": \"AlphaFold2 structure prediction, mutagenesis of binding surfaces, DT40 cell conditional depletion, co-immunoprecipitation, cell viability assays\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — structure-guided mutagenesis with cell biological validation, preprint\",\n      \"pmids\": [\"bio_10.1101_2024.06.20.599825\"],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Cryo-EM structures of the budding yeast KMN complex revealed that the N-terminal auto-inhibitory segment of Dsn1 (Dsn1-AI) folds into two α-helices that engage its head domain, occluding binding sites for inner kinetochore subunits CENP-C (Mif2) and CENP-U (Ame1). Aurora B (Ipl1) phosphorylation of Dsn1-AI releases this auto-inhibition to strengthen inner–outer kinetochore connections. C-terminal α-helical motifs of Dsn1 and other Mis12c subunits mediate binding to Knl1c and Ndc80c.\",\n      \"method\": \"Cryo-EM structure determination, biochemical binding assays, genetic experiments in S. cerevisiae\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — cryo-EM structure plus biochemical and genetic validation, multiple orthogonal methods\",\n      \"pmids\": [\"bio_10.1101_2025.06.03.657598\"],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"DSN1 stabilizes c-MYC protein in colorectal cancer by competing with the E3 ubiquitin ligase FZR1 for c-MYC binding, thereby attenuating FZR1-mediated ubiquitination and proteasomal degradation of c-MYC. DSN1 knockdown accelerated c-MYC degradation and reduced CRC invasion/metastasis in vitro and in vivo; c-MYC overexpression rescued the anti-metastatic effect of DSN1 silencing.\",\n      \"method\": \"Cycloheximide chase assay, proteasome inhibitor assay, co-immunoprecipitation (DSN1–FZR1, FZR1–c-MYC competition), siRNA knockdown, rescue overexpression, in vivo metastasis model\",\n      \"journal\": \"Experimental cell research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple biochemical methods (CHX chase, proteasome inhibition, co-IP competition) with in vivo validation, single lab\",\n      \"pmids\": [\"41713835\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"DSN1 is a core subunit of the evolutionarily conserved four-subunit MIS12 complex at the outer kinetochore, where its N-terminal auto-inhibitory domain (released by Aurora B phosphorylation) controls binding to inner kinetochore proteins CENP-C and CENP-T, while its C-terminal region connects to KNL1 and NDC80 complexes to mediate chromosome–microtubule attachment; protein levels are regulated by Mub1/Ubr2-mediated ubiquitylation (quality control), its mRNA is stabilized by SRSF9 via m6A recognition, a germline-specific splice isoform bypasses Aurora B regulation to support oocyte and embryo development, and in cancer contexts DSN1 also stabilizes c-MYC by competing with the FZR1 E3 ligase.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"DSN1 is an essential subunit of the MIS12/MIND kinetochore complex that bridges inner centromeric chromatin to outer microtubule-binding components, serving as a critical regulatory node for chromosome segregation. Its N-terminal auto-inhibitory segment occludes binding sites for inner kinetochore proteins CENP-C and CENP-U; Aurora B kinase phosphorylation relieves this auto-inhibition, strengthening inner–outer kinetochore connections, while C-terminal helical motifs of DSN1 and other MIS12 complex subunits recruit the KMN network (Knl1c/Ndc80c) [PMID:19270503, PMID:39178843]. DSN1 protein levels are controlled by Mub1/Ubr2-mediated ubiquitylation acting through CENP-C as a kinetochore integrity quality-control mechanism, and germ cells express an alternative splice isoform lacking the Aurora kinase–regulated region, enabling constitutive centromere localization essential for oocyte maturation and early embryogenesis [PMID:23408894, PMID:39178843]. In cancer contexts, DSN1 overexpression promotes chromosomal instability through its CENP-T interaction and stabilizes c-MYC by competing with the FZR1 E3 ligase for binding, thereby attenuating c-MYC ubiquitination [PMID:39560395, PMID:41713835].\",\n  \"teleology\": [\n    {\n      \"year\": 2009,\n      \"claim\": \"Establishing DSN1 as a conserved, essential MIS12/MIND complex subunit resolved the identity of the bridge connecting inner and outer kinetochore layers across metazoans.\",\n      \"evidence\": \"RNAi depletion in C. elegans producing kinetochore-null phenotype; functional studies in human cells\",\n      \"pmids\": [\"19270503\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Structural basis for DSN1 integration into the MIS12 complex was unknown\",\n        \"Regulatory mechanisms controlling DSN1 recruitment to centromeres were uncharacterized\"\n      ]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Discovery that the Mub1/Ubr2 ubiquitin ligase targets Dsn1 through CENP-C revealed a quality-control pathway that monitors and adjusts outer kinetochore protein levels.\",\n      \"evidence\": \"Kinetochore particle purification, Co-IP, ubiquitylation assays, and genetic epistasis in budding yeast\",\n      \"pmids\": [\"23408894\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Whether analogous ubiquitin-dependent quality control operates on mammalian DSN1 was not tested\",\n        \"The signal distinguishing defective from functional kinetochores in this pathway remained unclear\"\n      ]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Identification of ESRRA as a direct transcriptional activator of DSN1 linked nuclear receptor signaling to mitotic gene expression and G2/M progression.\",\n      \"evidence\": \"ChIP assay and dual-luciferase reporter in cancer cells; siRNA knockdown causing G2/M arrest via CDC25C-CDK1-CyclinB1\",\n      \"pmids\": [\"34131395\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Whether ESRRA-driven DSN1 transcription operates in normal proliferating tissues was not assessed\",\n        \"Other transcription factors regulating DSN1 remain uncharacterized\"\n      ]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Demonstration that SRSF9 stabilizes DSN1 mRNA in an m6A-dependent manner established a post-transcriptional regulatory layer controlling DSN1 protein abundance.\",\n      \"evidence\": \"m6A-RNA affinity assays, METTL3 knockdown, RNA stability and dual-luciferase assays\",\n      \"pmids\": [\"35509101\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Physiological contexts where m6A-mediated DSN1 regulation is rate-limiting were not defined\",\n        \"Whether DSN1 mRNA destabilization feeds back on kinetochore integrity was not tested\"\n      ]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Discovery of a germline-specific DSN1 splice isoform that bypasses Aurora kinase phospho-regulation explained how meiotic and early embryonic kinetochores achieve constitutive centromere localization without mitotic phospho-switches.\",\n      \"evidence\": \"Mouse germline-specific isoform knockout, live imaging, Aurora kinase phosphorylation assays, somatic misexpression causing segregation errors\",\n      \"pmids\": [\"39178843\", \"38659852\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"How the germline isoform is spliced and what controls its tissue-specific expression remain unknown\",\n        \"Whether this isoform alters affinity for specific outer kinetochore partners was not measured\"\n      ]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Functional evidence that DSN1 interacts with CENP-T in hepatocellular carcinoma cells and that its overexpression drives chromosomal instability connected DSN1 dosage to oncogenic karyotype evolution.\",\n      \"evidence\": \"Co-IP, siRNA/shRNA knockdown, overexpression in hepatoma cell lines and xenograft models\",\n      \"pmids\": [\"39560395\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Whether DSN1-CENP-T stoichiometric imbalance alone suffices for CIN or requires additional factors is unclear\",\n        \"No structural detail of the DSN1-CENP-T interface in mammalian cells\"\n      ]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Cryo-EM resolution of the Dsn1 auto-inhibitory segment revealed the structural mechanism by which Aurora B phosphorylation toggles inner kinetochore binding and how C-terminal helices assemble the KMN network.\",\n      \"evidence\": \"Cryo-EM structure of budding yeast MIS12 complex, biochemical binding assays, genetic validation (preprint)\",\n      \"pmids\": [\"bio_10.1101_2025.06.03.657598\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Mammalian cryo-EM structure of the DSN1 auto-inhibitory switch has not been determined\",\n        \"Dynamic conformational changes during mitotic phosphorylation in vivo remain uncharacterized\"\n      ]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Identification of a non-kinetochore function in which DSN1 competes with c-MYC for FZR1 binding expanded its role to oncoprotein stabilization and metastasis promotion.\",\n      \"evidence\": \"Reciprocal Co-IP, competitive binding assay, cycloheximide chase, proteasome inhibition, and in vivo metastasis model in CRC\",\n      \"pmids\": [\"41713835\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Whether this FZR1-competitive mechanism operates outside colorectal cancer contexts is unknown\",\n        \"The subcellular compartment where DSN1-FZR1 competition occurs was not defined\",\n        \"Stoichiometric thresholds of DSN1 needed to stabilize c-MYC were not determined\"\n      ]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How the multiple regulatory inputs on DSN1 — phosphorylation, ubiquitylation, mRNA stabilization, alternative splicing, and protein–protein competition — are integrated in different cell types and cell-cycle stages to achieve precise kinetochore assembly remains an open question.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"A unified quantitative model of DSN1 regulation across mitosis, meiosis, and cancer is lacking\",\n        \"Whether DSN1's non-kinetochore oncogenic functions are independent of its kinetochore role is unresolved\"\n      ]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0005198\", \"supporting_discovery_ids\": [0, 2, 8]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [7]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005694\", \"supporting_discovery_ids\": [1, 2, 8]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [1, 2, 6, 8]}\n    ],\n    \"complexes\": [\n      \"MIS12/MIND complex\",\n      \"KMN network\"\n    ],\n    \"partners\": [\n      \"MIS12\",\n      \"CENP-C\",\n      \"CENP-T\",\n      \"CENP-U\",\n      \"FZR1\",\n      \"UBR2\",\n      \"SRSF9\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```\n\n--- ROUND 2 (R2/R3, augmented corpus) ---\n\n```json\n{\n  \"mechanistic_narrative\": \"DSN1 is a core subunit of the conserved four-subunit MIS12 complex (MIS12–PMF1–NSL1–DSN1) that serves as the central bridge between inner kinetochore receptors (CENP-C and CENP-T) and the outer kinetochore microtubule-binding complexes NDC80 and KNL1, and is therefore essential for chromosome segregation during mitosis [PMID:15371340, PMID:16585270, PMID:20819937]. Crystal and cryo-EM structures show that the DSN1 N-terminal auto-inhibitory segment occludes CENP-C and CENP-T binding sites on the MIS12 complex; Aurora B phosphorylation of DSN1 Ser100/Ser109 releases this auto-inhibition, acting as a phospho-switch that controls kinetochore assembly [PMID:27881301, PMID:39178843]. Protein levels of DSN1 are regulated by Mub1/Ubr2-mediated ubiquitylation in yeast as a quality-control mechanism, and its mRNA is stabilized by the m6A reader SRSF9 in human colorectal cancer cells [PMID:23408894, PMID:35509101]. A germline-specific splice isoform that lacks the Aurora B-regulated N-terminal domain enables constitutive centromere localization and is required for oocyte maturation and early embryonic divisions in mice [PMID:39178843].\",\n  \"teleology\": [\n    {\n      \"year\": 2004,\n      \"claim\": \"Identification of DSN1 as a conserved outer kinetochore component resolved the molecular composition of the KMN network and established that loss of DSN1 abolishes kinetochore assembly and chromosome segregation.\",\n      \"evidence\": \"Co-purification from C. elegans embryos with RNAi phenotyping, and tandem-affinity purification of the human hMis12 complex with RNAi in HeLa cells\",\n      \"pmids\": [\"15371340\", \"15502821\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Precise stoichiometry of DSN1 within the complex was unresolved\", \"No structural information on DSN1 or its interaction interfaces\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Reconstitution of the four-subunit MIS12 complex and demonstration that its depletion eliminates NDC80/HEC1 kinetochore recruitment established DSN1 as essential for outer kinetochore assembly in vertebrate cells.\",\n      \"evidence\": \"Bacterial coexpression of the four-subunit complex, mitotic extract fractionation, RNAi in HeLa and DT40 cells\",\n      \"pmids\": [\"16585270\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Which subunit directly contacts NDC80 and KNL1 was unknown\", \"Regulation of DSN1 during the cell cycle was uncharacterized\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Structural visualization of the elongated MIS12 complex by negative-stain EM and mapping of DSN1's position within the complex head defined the architecture that mediates bridging between KNL1/NDC80 and the inner kinetochore.\",\n      \"evidence\": \"Negative-stain EM, chemical cross-linking, and pulldown assays with recombinant MIS12 complex\",\n      \"pmids\": [\"20819937\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Atomic-resolution structure of DSN1 was still lacking\", \"How DSN1 phosphorylation controls binding was unknown\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Two parallel studies revealed that CENP-T binds the MIS12 complex independently of CENP-C, defining dual pathways for NDC80 recruitment, and that Dsn1 protein levels are controlled by Mub1/Ubr2-mediated ubiquitylation as a kinetochore quality-control mechanism.\",\n      \"evidence\": \"Structural and biochemical reconstitution of CENP-T–MIS12C interaction in DT40 cells; kinetochore particle purification, genetic epistasis, and ubiquitylation assays in S. cerevisiae\",\n      \"pmids\": [\"23334297\", \"23408894\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"The structural basis of DSN1's contacts with CENP-C and CENP-T was unresolved\", \"Whether ubiquitin-mediated regulation of DSN1 is conserved in mammals was untested\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Crystal structures of the MIS12 complex bound to CENP-C revealed that Aurora B phosphorylation of DSN1 Ser100/Ser109 releases an N-terminal auto-inhibitory segment that competes with CENP-C binding, establishing the phospho-regulatory switch governing inner–outer kinetochore coupling.\",\n      \"evidence\": \"X-ray crystallography of MIS12C:CENP-C complex, phosphomimetic/phosphodead mutagenesis, Aurora B kinase assays\",\n      \"pmids\": [\"27881301\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether the same switch controls CENP-T binding was unclear\", \"The structural basis of auto-inhibition at atomic resolution in the context of the full KMN was unresolved\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Post-transcriptional regulation of DSN1 was revealed: SRSF9 functions as an m6A reader that stabilizes DSN1 mRNA via METTL3-deposited m6A marks, linking epitranscriptomic regulation to DSN1 protein abundance in colorectal cancer.\",\n      \"evidence\": \"Methylated RNA affinity assay, gene-specific m6A qRT-PCR, RNA stability assays, and xenograft models in CRC cells\",\n      \"pmids\": [\"35509101\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether m6A-mediated DSN1 regulation occurs in normal (non-cancer) cells is unknown\", \"The specific m6A writer–reader axis for DSN1 in other tissue contexts is uncharacterized\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Discovery of a germline-specific DSN1 splice isoform that lacks the Aurora B-regulated N-terminal domain showed that germ cells bypass mitotic phospho-regulation to achieve constitutive kinetochore assembly, and that this isoform is required for oocyte maturation and fertility.\",\n      \"evidence\": \"Isoform cloning, CRISPR mouse models with isoform-specific deletion, live-cell imaging of oocytes, Aurora kinase inhibitor experiments\",\n      \"pmids\": [\"39178843\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether the germline isoform is conserved in non-mammalian vertebrates is untested\", \"How meiotic chromosome segregation fidelity is maintained without the auto-inhibitory regulatory step is unclear\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Cryo-EM structures of the budding yeast KMN complex provided atomic detail of the DSN1 auto-inhibitory helices engaging its head domain and the C-terminal helical motifs mediating KNL1 and NDC80 binding, unifying genetic and biochemical data into a complete structural framework.\",\n      \"evidence\": \"Cryo-EM structure determination with biochemical and genetic validation in S. cerevisiae (preprint)\",\n      \"pmids\": [\"bio_10.1101_2025.06.03.657598\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Full-length human KMN cryo-EM structure is still unavailable\", \"Dynamics of auto-inhibition release in vivo are unresolved\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"A non-canonical cancer-associated function was identified: DSN1 stabilizes c-MYC by competing with the FZR1 E3 ligase for c-MYC binding, thereby attenuating ubiquitin-mediated c-MYC degradation and promoting colorectal cancer metastasis.\",\n      \"evidence\": \"Cycloheximide chase, co-IP competition between DSN1 and FZR1 for c-MYC, siRNA knockdown with c-MYC rescue, in vivo metastasis model\",\n      \"pmids\": [\"41713835\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether DSN1–c-MYC interaction occurs in normal proliferating cells is unknown\", \"The structural basis of DSN1–c-MYC binding and FZR1 competition is uncharacterized\", \"Independent replication in other cancer types is lacking\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Key unresolved questions include the full atomic structure of the human KMN super-complex in the kinetochore context, whether ubiquitin-mediated DSN1 quality control (Mub1/Ubr2) is conserved in mammals, and how the germline DSN1 isoform maintains segregation fidelity without Aurora B-dependent regulation.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No human KMN cryo-EM structure available\", \"Mammalian DSN1 ubiquitylation pathway not characterized\", \"Germline isoform segregation fidelity mechanism unknown\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0005198\", \"supporting_discovery_ids\": [0, 1, 2, 5, 9]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [9, 16, 17]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005694\", \"supporting_discovery_ids\": [0, 1, 2, 6, 9, 13]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [1, 6]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [0, 1, 2, 9, 13]}\n    ],\n    \"complexes\": [\n      \"MIS12 complex\",\n      \"KMN network\"\n    ],\n    \"partners\": [\n      \"MIS12\",\n      \"PMF1\",\n      \"NSL1\",\n      \"CENP-C\",\n      \"CENP-T\",\n      \"KNL1\",\n      \"NDC80\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}