{"gene":"CENPC","run_date":"2026-06-09T22:57:18","timeline":{"discoveries":[{"year":1992,"finding":"CENP-C is a component of the inner kinetochore plate, as determined by immunoelectron microscopy of HeLa chromosomes using antibodies raised against CENP-C cDNA-encoded fusion proteins.","method":"Immunoelectron microscopy, indirect immunofluorescence, immunoblotting","journal":"Cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — direct localization by immunoelectron microscopy with specific antibodies, replicated across multiple criteria in a foundational study","pmids":["1339310"],"is_preprint":false},{"year":1989,"finding":"CENP-C is present exclusively at active centromeres and is absent from inactive centromeres of stable dicentric chromosomes, suggesting it is a necessary component of functional kinetochores; CENP-C appears confined to the outer centromere in the kinetochore region.","method":"Immunofluorescence with specific anti-CENP-C antibodies on dicentric chromosomes","journal":"Chromosoma","confidence":"High","confidence_rationale":"Tier 2 / Strong — direct antibody-based localization experiment on dicentric chromosomes, independently replicated in multiple subsequent studies","pmids":["2475307"],"is_preprint":false},{"year":1994,"finding":"Nuclear microinjection of anti-CENP-C antibodies during interphase causes metaphase arrest and reduces kinetochore size (trilaminar structures are reduced in diameter), demonstrating CENP-C is required for establishing/maintaining proper kinetochore size and for the timely metaphase-to-anaphase transition.","method":"Nuclear microinjection of antibodies, immunoelectron microscopy","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — functional loss-of-function by antibody injection with specific ultrastructural phenotypic readout, well-controlled study","pmids":["8175879"],"is_preprint":false},{"year":1994,"finding":"CENP-C is a DNA-binding protein; an internal ~101 amino acid stretch constitutes its DNA-binding domain, demonstrated by Southwestern blotting of recombinant CENP-C expressed in E. coli against human genomic DNA and alphoid DNA subfamilies.","method":"Southwestern blotting, recombinant protein expression, truncation analysis","journal":"Journal of biochemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro DNA-binding assay with truncation mapping, single lab, no in vivo validation in this paper","pmids":["7883764"],"is_preprint":false},{"year":1995,"finding":"The yeast MIF2 protein (CENP-C ortholog) interacts genetically with CEP1/CBF1, NDC10/CBF2, and CEP3/CBF3B at the centromere; mif2 mutations lie within regions homologous to CENP-C, establishing functional and structural conservation between yeast and mammalian centromeres.","method":"Genetic epistasis, synthetic lethality screens, minichromosome stability assays","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal genetic epistasis experiments with defined centromere gene mutations, robust pathway placement","pmids":["7579695"],"is_preprint":false},{"year":1995,"finding":"CENP-C has an autonomous centromere-targeting domain located in the central region of the protein, which overlaps with its DNA-binding domain, as defined by truncation mutant analysis in vivo and in vitro.","method":"In vivo expression of truncation mutants, in vitro DNA-binding assays","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — domain mapping by truncation mutants with both in vivo localization and in vitro DNA-binding assays, two orthogonal methods","pmids":["8668174"],"is_preprint":false},{"year":1995,"finding":"CENP-C and CENP-E localize exclusively to active centromeres (not inactive centromeres) in dicentric Robertsonian translocations, establishing that at least two centromeric proteins are required for human centromeric function.","method":"Immunofluorescence combined with FISH on dicentric chromosomes","journal":"Human molecular genetics","confidence":"High","confidence_rationale":"Tier 2 / Strong — direct localization experiment on 12 dicentric translocations with independent FISH confirmation of active centromere identity","pmids":["8634687"],"is_preprint":false},{"year":1995,"finding":"The Mif2 homology domain in the central region of CENP-C is required for centromere assembly; mutations within this domain impair CENP-C kinetochore localization. The N-terminus of CENP-C is required for protein destruction and renders otherwise stable proteins unstable.","method":"In vivo expression of CENP-C mutants, centromere targeting assays","journal":"Molecular biology of the cell","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — domain mapping with mutational analysis and in vivo localization readout, single lab","pmids":["7579707"],"is_preprint":false},{"year":1996,"finding":"CENP-C interacts with nucleolar transcription factors UBF1 and UBF2 through its C-terminal third, as identified by affinity chromatography and confirmed by co-localization of a subset of CENP-C and UBF at nucleoli in interphase HeLa cells.","method":"Affinity chromatography, microsequence analysis, immunofluorescence co-localization","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — pulldown/affinity chromatography with co-localization confirmation, but functional significance not fully established","pmids":["8702533"],"is_preprint":false},{"year":1997,"finding":"Human CENP-C has three functional units: an N-terminal oligomerization domain (capable of dimer and tetramer formation by crosslinking), an internal DNA-binding domain (with core and flanking stabilizing elements), and a C-terminal dimerization domain (forming exclusively dimers), as defined by biochemical analysis of expressed fragments.","method":"Chemical crosslinking, gel filtration, Southwestern blotting","journal":"Chromosome research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple in vitro biochemical methods (crosslinking, gel filtration, blotting) to define domain architecture, single lab","pmids":["9146917"],"is_preprint":false},{"year":1997,"finding":"CENP-C loss of function (conditional fusion to mouse steroid receptor in DT40 cells) causes arrest at the metaphase/anaphase junction followed by apoptosis, demonstrating CENP-C is required for anaphase progression or centromere signaling.","method":"Conditional gene targeting, live cell observation, DT40 chicken cells","journal":"Human molecular genetics","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean conditional loss-of-function in vertebrate cells with specific and reproducible mitotic arrest phenotype","pmids":["9361037"],"is_preprint":false},{"year":1998,"finding":"CENP-C interacts with HDaxx (a death domain-binding protein) through the N-terminal 315 amino acids of CENP-C and the C-terminal 104 amino acids of HDaxx; this interaction is interphase-specific and they co-localize at discrete nuclear spots in interphase HeLa cells.","method":"Yeast two-hybrid, immunofluorescence co-localization","journal":"Journal of cell science","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — yeast two-hybrid interaction with domain mapping and immunofluorescence co-localization, but in vivo interaction not confirmed by co-IP","pmids":["9645950"],"is_preprint":false},{"year":1999,"finding":"Herpes simplex virus immediate-early protein Vmw110 (ICP0) causes proteasome-dependent loss of CENP-C from centromeres during infection (via its RING finger domain), resulting in ultrastructural kinetochore disruption, mitotic arrest, and abnormal cytokinesis.","method":"Viral infection, proteasome inhibitor treatment, immunofluorescence, electron microscopy","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 / Moderate — mechanistic dissection of CENP-C degradation pathway with domain mapping, ultrastructural analysis, and functional consequence","pmids":["10075924"],"is_preprint":false},{"year":1999,"finding":"CENP-C is necessary but not sufficient for formation of a functional centromere; its removal disassembles the centromere protein complex and blocks cells at metaphase-anaphase junction, while overexpression of CENP-C does not associate with ZW10 and causes segregation errors.","method":"Conditional gene disruption, inducible overexpression, immunofluorescence in DT40 cells","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 / Strong — both loss- and gain-of-function approaches in vertebrate cells with clear mechanistic conclusions","pmids":["10428958"],"is_preprint":false},{"year":2001,"finding":"CENP-H is required for centromere targeting of CENP-C but not CENP-A in vertebrate cells, establishing a hierarchical assembly order at the centromere: CENP-A → CENP-H → CENP-C.","method":"Conditional knockout in DT40 cells, immunocytochemistry","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean conditional KO with epistasis placing CENP-H upstream of CENP-C but downstream of CENP-A, clear hierarchical order","pmids":["11500386"],"is_preprint":false},{"year":2001,"finding":"C. elegans HCP-4/CENP-C localizes to centromeres in a CENP-A (HCP-3)-dependent manner, and its loss of function by RNAi results in failure of sister centromere resolution and failure to form functional kinetochores; HCP-4 and HCP-3 are both required for localization of HCP-1 (CENP-F-like), defining an ordered assembly pathway.","method":"RNAi, immunofluorescence localization in C. elegans","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — epistatic ordering of centromere assembly pathway via RNAi with specific functional readouts (centromere resolution and kinetochore assembly)","pmids":["11402064"],"is_preprint":false},{"year":2001,"finding":"A SUMO-1 gene suppresses the temperature-sensitive phenotype of a CENP-C mutant in DT40 cells, suggesting that SUMO-1 is involved in centromere function in vertebrate cells through a pathway involving CENP-C.","method":"cDNA library suppressor screen, temperature-sensitive CENP-C mutants in DT40 cells","journal":"Nucleic acids research","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — genetic suppressor identified in vertebrate cells, single method, no direct biochemical confirmation of mechanism","pmids":["11557811"],"is_preprint":false},{"year":2002,"finding":"CENP-C binds alpha-satellite DNA in vivo selectively; the region between amino acids 410 and 537 is required for this in vivo DNA binding; CENP-C and CENP-B associate with the same types of alpha-satellite arrays but in distinct non-overlapping centromere domains.","method":"Chromatin immunoprecipitation (ChIP), immunofluorescence, ultrastructural analysis","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 2 / Moderate — ChIP with domain mapping provides in vivo evidence for DNA binding with spatial resolution; two orthogonal methods","pmids":["12006616"],"is_preprint":false},{"year":2002,"finding":"CENP-C inactivation in DT40 cells causes mitotic delay, impairs the Mad2 spindle checkpoint pathway (~60% of CENP-C-deficient cells lack Mad2 signals after nocodazole), and causes significant reduction in Mis12 complex proteins at centromeres; CENP-C centromere localization in interphase requires the CENP-H complex.","method":"Conditional knockout in DT40 cells, immunofluorescence, live-cell microscopy","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — conditional KO with multiple functional readouts (spindle checkpoint, Mis12 recruitment, cell cycle) in vertebrate cells","pmids":["17392512"],"is_preprint":false},{"year":2002,"finding":"PARP-2 does not interact with CENP-C as determined by co-immunoprecipitation, distinguishing CENP-C from CENP-A and CENP-B which do interact with PARP-2.","method":"Co-immunoprecipitation","journal":"Human molecular genetics","confidence":"Medium","confidence_rationale":"Tier 3 / Weak — negative result from single co-IP experiment; informative as negative finding showing CENP-C is not in the PARP-2 complex","pmids":["12217960"],"is_preprint":false},{"year":2002,"finding":"Human CENP-C contains two distinct centromere-targeting domains: one in the central region (aa 426-537) and one in the C-terminal region (aa 638-943), both capable of binding alpha-satellite DNA in vivo.","method":"Immunofluorescence of truncation mutants, ChIP","journal":"Journal of structural biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — two orthogonal methods (immunofluorescence and ChIP) to define two independent centromere-targeting domains, single lab","pmids":["12490152"],"is_preprint":false},{"year":2003,"finding":"CENP-B interacts directly with CENP-C; the CENP-C domains required for this interaction overlap with three Mif2 homologous regions and are also involved in centromere assembly. Overproduction of CENP-B truncants lacking CENP-C interaction domains causes abnormal CENP-C domain duplication and cell cycle delay.","method":"Yeast two-hybrid, domain truncation analysis, immunofluorescence","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — yeast two-hybrid with in vivo truncation/phenotypic confirmation; two orthogonal methods, single lab","pmids":["14612452"],"is_preprint":false},{"year":2004,"finding":"CENP-C can be sumoylated in vitro by SUMO-1 and SUMO-2; sumoylation occurs at multiple lysine residues including sites outside the perfect consensus motif, within regions overlapping the DNA-binding and centromere localization domains.","method":"In vitro sumoylation reconstitution, tandem mass spectrometry identification of sumoylated isopeptides","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution with mass spectrometry identification of modification sites; however, in vivo relevance not directly demonstrated in this paper","pmids":["15272016"],"is_preprint":false},{"year":2007,"finding":"Drosophila CENP-C is required for normal kinetochore attachment to the spindle; it was identified through genetic interaction with separase regulatory subunits Pimples/securin and Three rows, and its centromere localization domain contains a diverged CENPC motif.","method":"Genetic modifier screen, in vivo imaging, domain analysis","journal":"Genes & development","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic screen with in vivo functional validation, but direct evidence for separase involvement was not obtained","pmids":["16140985"],"is_preprint":false},{"year":2007,"finding":"Drosophila CID (CENP-A) and CENP-C are incorporated into centromeres during anaphase in early embryos; this incorporation is independent of DNA synthesis and spindle pulling forces but strictly coupled to mitotic progression.","method":"Quantitative fluorescence measurements in living embryos using fluorescent fusion proteins","journal":"Current biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — quantitative live imaging with functional fluorescent fusions, multiple orthogonal controls (drug treatments) establishing cell-cycle coupling","pmids":["17222555"],"is_preprint":false},{"year":2007,"finding":"Caspase-7 (activated downstream of death receptor-induced caspase-8) cleaves CENP-C and INCENP, causing their mislocalization and subsequent mislocalization of Aurora B kinase from centromeres; expression of non-cleavable CENP-C prevents passenger complex mislocalization after caspase activation.","method":"Biochemical caspase cleavage assays, site-directed mutagenesis, immunofluorescence","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 2 / Moderate — caspase cleavage mapped with mutagenesis, functional rescue with non-cleavable mutant, two orthogonal methods","pmids":["17287400"],"is_preprint":false},{"year":2009,"finding":"CENP-C recruits the Mis12/MIND complex and CENP-K to kinetochores via its N-terminal domain; immunodepletion of CENP-C from Xenopus metaphase egg extract prevents kinetochore formation on sperm chromatin; CENP-C mutants that localize to centromeres but fail to recruit Mis12/MIND complex cannot support kinetochore assembly.","method":"Xenopus egg extract immunodepletion, in vitro complementation with mutant CENP-C, immunofluorescence","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution/depletion-complementation system with domain mutants establishing N-terminal recruitment function","pmids":["19641019"],"is_preprint":false},{"year":2009,"finding":"DNMT3B interacts directly with CENP-C (identified by yeast two-hybrid and confirmed by co-immunoprecipitation in mammalian cells); CENP-C recruits DNMT3B and DNA methylation to centromeric and pericentromeric satellite repeats; loss of CENP-C reduces DNA methylation at centromeres, alters the histone code, and increases chromosome missegregation and centromeric repeat transcription.","method":"Yeast two-hybrid, co-immunoprecipitation, siRNA knockdown, bisulfite sequencing, ChIP","journal":"Human molecular genetics","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (Y2H, Co-IP, ChIP, bisulfite sequencing) with functional loss-of-function confirmation","pmids":["19482874"],"is_preprint":false},{"year":2009,"finding":"The C-terminal Mif2p homology domain II of CENP-C targets the centromere and contacts alpha-satellite DNA; domain III mediates homo-dimerization, homo-oligomerization, and interaction with CENP-A and histone H3.","method":"Immunofluorescence, ChIP, co-immunoprecipitation, bimolecular fluorescence complementation","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple in vivo methods (immunofluorescence, ChIP, bimolecular fluorescence complementation) with biochemical confirmation, single lab","pmids":["19503796"],"is_preprint":false},{"year":2009,"finding":"Fission yeast CENP-C (Cnp3) acts as a scaffold recruiting Fta1/CENP-L (primary direct effector) for mitotic kinetochore function; Cnp3 also recruits Pcs1 to prevent merotelic attachment; in meiosis, Cnp3 associates with and recruits Moa1 for meiotic mono-orientation of kinetochores.","method":"Yeast genetic and biochemical analyses, epistasis, ectopic localization","journal":"Developmental cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — direct binding shown biochemically, ectopic localization rescue of mitotic defects, identification of separate mitotic and meiotic effectors","pmids":["19758558"],"is_preprint":false},{"year":2010,"finding":"Maize CENPC DNA binding is mediated by a 122 amino acid domain and is stabilized by single-stranded RNA; long single-stranded nucleic acids promote CENPC binding to DNA. Removal of this binding module causes partial delocalization of CENPC in vivo.","method":"In vitro DNA/RNA binding assays, domain truncation, in vivo localization with mutants","journal":"PLoS genetics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — biochemical reconstitution of RNA-stimulated DNA binding with in vivo validation of domain function, single lab","pmids":["20140237"],"is_preprint":false},{"year":2010,"finding":"Drosophila Cal1 links CENP-A/Cid and CENP-C by binding their N- and C-terminal domains respectively (shown by yeast three-hybrid); Cal1 limits centromeric deposition of CENP-A/Cid and CENP-C during mitotic exit; both Cal1 domains are required together for centromere function.","method":"Yeast three-hybrid, quantitative in vivo fluorescence imaging","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 2 / Strong — three-hybrid domain interaction mapped, quantitative in vivo imaging, functional domain deletion analysis","pmids":["20940262"],"is_preprint":false},{"year":2011,"finding":"The N-terminal region of Drosophila CENP-C is sufficient to recruit all KMN network components (Mis12 complex, Ndc80 complex, Spc105/KNL1); the Mis12 complex component Nnf1 directly interacts with CENP-C in vitro; targeting the CENP-C N-terminus to centrosomes redirects KMN proteins away from kinetochores, causing mitotic defects.","method":"In vitro binding assay, ectopic targeting assay (centrosome targeting), immunofluorescence in Drosophila cells","journal":"Current biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — direct in vitro binding plus ectopic targeting gain-of-function, multiple KMN components tested, clear functional consequence","pmids":["21353555"],"is_preprint":false},{"year":2011,"finding":"A conserved N-terminal motif of vertebrate Cenp-C binds directly and with high affinity to the Mis12 complex, linking the inner and outer kinetochore; expression of the isolated N-terminal Cenp-C motif in HeLa cells prevents outer kinetochore assembly, causes chromosome missegregation, and impairs spindle assembly checkpoint.","method":"Direct binding assay (biochemical reconstitution), dominant-negative expression in HeLa cells, immunofluorescence","journal":"Current biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — direct binding reconstitution with high-affinity measurement plus dominant-negative functional validation and checkpoint readout","pmids":["21353556"],"is_preprint":false},{"year":2011,"finding":"CENP-C recruits M18BP1 (Mis18 complex component) to centromeres to promote CENP-A chromatin assembly; depletion of CENP-C prevents M18BP1 targeting to metaphase centromeres and inhibits CENP-A assembly; M18BP1 directly binds CENP-C through conserved domains.","method":"siRNA depletion, in vitro direct binding assay, immunofluorescence","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — direct binding shown in vitro, functional depletion with CENP-A assembly readout, two orthogonal methods","pmids":["21911481"],"is_preprint":false},{"year":2012,"finding":"M18BP1 interacts with CENP-C through a central SANT domain region of M18BP1 and the C-terminus of CENP-C; CENP-C knockdown reduces M18BP1 centromeric association and lowers CENP-A levels at centromeres.","method":"Interaction screen, domain mapping, siRNA knockdown, immunofluorescence in mouse ESCs","journal":"Nucleus","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — interaction mapping with functional siRNA validation, single lab, replicates findings of Moree et al. 2011","pmids":["22540025"],"is_preprint":false},{"year":2013,"finding":"CENP-C recognizes the CENP-A nucleosome by binding a hydrophobic region in the CENP-A tail and docking onto the acidic patch of histones H2A and H2B; this mechanism is used by both the CENP-C motif and the broader conserved central region; the mechanism is conserved across species.","method":"Crystal structure determination, NMR, mutagenesis, biochemical binding assays","journal":"Science","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure plus NMR plus mutagenesis in one study; conserved mechanism validated across multiple species","pmids":["23723239"],"is_preprint":false},{"year":2015,"finding":"CENP-C binds CENP-A nucleosomes (using purified components) and reshapes the octameric histone core: it rigidifies both surface and internal nucleosome structure and modulates terminal DNA wrapping to match the loose wrap found on native CENP-A nucleosomes. CENP-C depletion leads to rapid removal of CENP-A from centromeres.","method":"In vitro reconstitution with purified components, fluorescence lifetime imaging, single-molecule FRET, CENP-C depletion in cells","journal":"Science","confidence":"High","confidence_rationale":"Tier 1 / Strong — reconstitution with purified components, multiple biophysical methods (FRET, FLIM), functional in vivo depletion confirmation","pmids":["25954010"],"is_preprint":false},{"year":2015,"finding":"The PEST domain in the N-terminal half of CENP-C directly interacts with the CENP-HIKM subcomplex, and this interaction is required for kinetochore localization of CENP-HIKM and CENP-TW; CENP-C acts as a blueprint for CCAN assembly.","method":"Biochemical reconstitution, domain mapping, cellular rescue assays, immunofluorescence","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — biochemical reconstitution with mutagenesis and cellular validation, multiple CCAN subcomplexes tested","pmids":["26124289"],"is_preprint":false},{"year":2015,"finding":"CENP-C and CENP-T independently recruit the KMN network to kinetochores via distinct mechanisms and regulatory controls: CENP-C recruits Ndc80 through KNL1 and Mis12 interactions (regulated by Aurora B), while CENP-T directly interacts with Ndc80 (regulated by CDK).","method":"Ectopic chromosomal targeting assays in human cells, immunofluorescence","journal":"Current biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — separation-of-function by ectopic targeting in human cells definitively separating two parallel recruitment pathways","pmids":["25660545"],"is_preprint":false},{"year":2015,"finding":"CENP-C depletion leads to increased centromere DNA aberrations (sister chromatid exchanges at centromeres), indicating CENP-C maintains centromere repeat integrity independently of its role in chromosome segregation.","method":"CO-FISH (chromosome orientation FISH), siRNA depletion, structured illumination microscopy","journal":"PNAS","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — CO-FISH with siRNA depletion and super-resolution imaging, two orthogonal methods, single lab","pmids":["28167779"],"is_preprint":false},{"year":2015,"finding":"CENP-C and CENP-I are key factors connecting kinetochore to CENP-A assembly; tethering various kinetochore components to an ectopic array recruits CENP-C and subsequently M18BP1; CENP-I can also recruit M18BP1 downstream of CENP-C to enhance M18BP1 assembly at centromeres.","method":"Tethering assay with tetR-fusion proteins on synthetic alphoid array HACs, immunofluorescence","journal":"Journal of cell science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ectopic tethering assay establishes epistasis of CENP-C upstream of M18BP1, single lab","pmids":["26527398"],"is_preprint":false},{"year":2016,"finding":"Crystal structure of human MIS12C in complex with a CENP-C fragment reveals the structural basis of the CENP-C–Mis12 interaction; Aurora B kinase phosphorylation of CENP-C regulates this interaction, allowing construction of a near-complete structural model of the KMN assembly.","method":"X-ray crystallography, biochemical binding assays, Aurora B kinase phosphorylation assays","journal":"Cell","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure with biochemical validation and kinase regulation, high-resolution mechanistic insight","pmids":["27881301"],"is_preprint":false},{"year":2016,"finding":"CENP-C directs a structural transition in CENP-A nucleosomes predominantly through lateral sliding of DNA gyres (gyre sliding), returning them toward canonical nucleosome DNA positions, as measured by single-molecule FRET.","method":"Single-molecule FRET with recombinant human histones and centromere DNA","journal":"Nature structural & molecular biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — single-molecule FRET reconstitution with purified components, defined mechanistic model with quantitative biophysical measurements","pmids":["26878239"],"is_preprint":false},{"year":2017,"finding":"Active centromere alpha-satellite transcripts are complexed with CENP-A and CENP-C; depletion of array-specific RNAs reduces CENP-A and CENP-C at the targeted centromere via faulty CENP-A loading.","method":"RNA immunoprecipitation, RNA FISH, antisense oligonucleotide depletion, immunofluorescence","journal":"Developmental cell","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — RNA-IP showing complex formation plus functional depletion of RNAs affecting CENP-C levels, single lab","pmids":["28787590"],"is_preprint":false},{"year":2017,"finding":"Yeast Mif2/CENP-C uses a contiguous DNA- and histone-binding domain (DHBD) containing the CENP-C motif, an AT-hook, and RK clusters to contact both Cse4/CENP-A residues and AT-rich centromere DNA simultaneously; human CENP-C has two related DHBDs that preferentially bind higher AT content DNA.","method":"Biochemical binding assays, mutational analysis, structural analysis","journal":"Genes & development","confidence":"High","confidence_rationale":"Tier 2 / Strong — mechanistic dissection of DNA- and histone-binding requirements with domain mutations, validated in both yeast and human CENP-C","pmids":["29074736"],"is_preprint":false},{"year":2017,"finding":"Aurora B kinase phosphorylates the N-terminal region (Thr28) of S. pombe CENP-C (Cnp3), impairing its interaction with the Mis12 complex; a phosphomimetic mutant causes defective chromosome segregation due to improper kinetochore assembly.","method":"Kinase assay, co-immunoprecipitation, crystal structure of Mis12-Nnf1 complex, mutant expression in S. pombe","journal":"PNAS","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure, kinase assay, in vivo phosphomimetic mutant with functional chromosome segregation phenotype; multiple orthogonal methods","pmids":["29180432"],"is_preprint":false},{"year":2019,"finding":"CDK1 phosphorylates the C-terminal region of CENP-C (at conserved sites), facilitating its binding to CENP-A nucleosomes in vitro and in vivo; this CENP-A binding promotes CENP-C kinetochore localization during mitosis, and the CENP-A–CENP-C interaction is critical for long-term viability in human RPE-1 cells.","method":"In vitro kinase assay (CDK1), Phos-tag SDS-PAGE, co-immunoprecipitation, immunofluorescence in chicken and human cells, cell viability assays","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro kinase assay with mutagenesis, confirmed in vivo in two cell systems (chicken and human), functional viability readout","pmids":["31676716"],"is_preprint":false},{"year":2019,"finding":"Human CENP-C central region (CENP-CCR) and CENP-C motif both bind exclusively to CENP-A nucleosomes in vitro; CENP-CCR binds with high affinity through CENP-AV532 and CENP-AV533 (extended hydrophobic area); CENP-CCR binding destabilizes the H2A C-terminal tail (further exacerbating loose DNA wrapping) and rigidifies H4 N-terminal tail in the conformation favoring H4K20 monomethylation.","method":"Cryo-EM structure of CENP-A nucleosome, in vitro binding assays, mutagenesis","journal":"EMBO reports","confidence":"High","confidence_rationale":"Tier 1 / Strong — cryo-EM structure with in vitro binding validation and mutagenesis, reveals two distinct conformational changes upon binding","pmids":["31475439"],"is_preprint":false},{"year":2020,"finding":"Mif2/CENP-C exhibits auto-inhibition: wild-type Mif2 is attenuated in binding the Mtw1 complex, and binding Cse4/CENP-A nucleosomes overcomes this inhibition; a Mif2 mutant bypassing Cse4 requirement for Mtw1 binding causes mis-localization of the Mtw1 complex and chromosome segregation defects in vivo.","method":"In vitro binding assays with reconstituted nucleosomes, yeast genetics, immunofluorescence","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution demonstrating auto-inhibition mechanism combined with genetic validation in cells","pmids":["32515113"],"is_preprint":false},{"year":2022,"finding":"lncRNA CCTT recruits CENP-C to centromeric DNA via RNA-DNA triplex formation and a direct RNA-protein interaction with CENP-C; CCTT loss triggers mitotic errors and aneuploidy, and CCTT localizes to all centromeres.","method":"RNA immunoprecipitation, RNA-DNA triplex assay, siRNA/shRNA depletion, immunofluorescence, FISH","journal":"Molecular cell","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — RNA-IP and triplex assay demonstrate mechanism, functional depletion confirms role; note this is a non-centromere-derived lncRNA acting on CENP-C protein, not a non-coding product of the CENPC locus","pmids":["36332605"],"is_preprint":false},{"year":2023,"finding":"CENP-C undergoes self-oligomerization through its C-terminal Cupin domain (demonstrated by structural and biochemical analyses of chicken and human CENP-C); this oligomerization is vital for CENP-C function, centromeric localization of CCAN proteins, and centromeric chromatin organization.","method":"Crystal structure of Cupin domain, biochemical self-oligomerization assays, mutagenesis, immunofluorescence in chicken cells","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure plus biochemical reconstitution of oligomerization plus mutagenesis with in vivo functional validation","pmids":["37295434"],"is_preprint":false},{"year":2023,"finding":"In C. elegans oocytes, CENP-C (HCP-4) directly recruits PLK-1 to the chromosome arm during meiosis (independent of BUB-1); disruption of the CENP-C–PLK-1 interaction leads to imbalance in kinetochore components and chromosome congression defects without affecting CDC-20 recruitment.","method":"Live imaging, biochemical interaction assays, RNAi, degron-mediated depletion in C. elegans oocytes","journal":"eLife","confidence":"High","confidence_rationale":"Tier 2 / Strong — direct biochemical interaction plus functional RNAi with specific meiotic phenotype; separation from BUB-1 pathway","pmids":["37067150"],"is_preprint":false},{"year":2023,"finding":"Multi-site phosphorylation of the PEST region of yeast Mif2/CENP-C enhances inner kinetochore assembly; eliminating PEST phosphorylation sites progressively impairs cellular fitness and causes lethality in cells lacking otherwise non-essential inner kinetochore factors.","method":"Yeast genetics, phosphorylation-site mutagenesis, genetic interaction analysis","journal":"Current biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic epistasis and phospho-site mutagenesis in yeast, single lab, no direct biochemical demonstration of mechanism","pmids":["36736323"],"is_preprint":false},{"year":2024,"finding":"In C. elegans embryos, CENP-C targets PLK-1 to the inner kinetochore during prometaphase and metaphase; disruption of the CENP-C–PLK-1 interaction causes kinetochore component imbalance and chromosome congression defects distinct from the effects of BUB-1-targeted PLK-1 (which controls CDC-20 recruitment and SAC).","method":"Engineered protein interaction mutants, live imaging, immunofluorescence in C. elegans embryos","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 2 / Strong — engineered mutants specifically disrupting CENP-C–PLK-1 interaction with clear functional phenotype, distinct from BUB-1 pathway","pmids":["39355896"],"is_preprint":false},{"year":2024,"finding":"The CENP-C–Mis12 complex (Mis12C) interaction facilitates centromeric recruitment of Aurora B; Aurora B in turn reinforces the CENP-C–Mis12C interaction, creating a positive regulatory loop that ensures kinetochore biorientation and error correction.","method":"Immunofluorescence, co-immunoprecipitation, CENP-C mutant analysis in human RPE-1 cells and mouse models","journal":"Life science alliance","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP and mutant analysis with Aurora B recruitment readout; single lab, moderate evidence for regulatory loop","pmids":["39433344"],"is_preprint":false},{"year":2025,"finding":"Maternal CENP-C is recruited asymmetrically to paternal centromeres in the zygote, and this recruitment is required to equalize CENP-A levels between maternal and paternal centromeres before first mitosis; disruption of CENP-C dimerization impairs CENP-A equalization and chromosome segregation.","method":"CENP-A-mScarlet mouse model, live imaging, maternal CENP-C depletion, dimerization-disrupting mutants","journal":"Developmental cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — in vivo mouse model with specific maternal depletion and dimerization mutants; clear mechanistic role for CENP-C in epigenetic resetting at fertilization","pmids":["40997799"],"is_preprint":false},{"year":2025,"finding":"KIF18A promotes chromosome alignment in cooperation with CENP-E downstream of CENP-C; a genome-wide Cas9 screen using a hypomorphic CENP-C mutant identified KIF18A as synthetic lethal, with the synthetic defect caused by reduction in CENP-E function in the CENP-C mutant background.","method":"Genome-wide Cas9 functional genetics screen, synthetic lethality, immunofluorescence","journal":"Cell reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic screen with functional follow-up placing KIF18A/CENP-E downstream of CENP-C, single lab","pmids":["41218610"],"is_preprint":false}],"current_model":"CENP-C is a conserved inner kinetochore protein that acts as a central scaffold for centromere/kinetochore assembly: it binds CENP-A nucleosomes (via two conserved domains that contact the CENP-A tail and the H2A/H2B acidic patch) and reshapes nucleosome structure through DNA gyre sliding, then recruits the outer kinetochore via direct high-affinity binding of its N-terminal motif to the Mis12 complex (and thence the full KMN network); its PEST domain additionally recruits the CENP-HIKM subcomplex to organize the full 16-subunit CCAN; CENP-C also promotes CENP-A chromatin maintenance by recruiting M18BP1/Mis18 complex; its C-terminal Cupin domain oligomerizes to organize centromeric chromatin architecture; CDK1-mediated phosphorylation of its C-terminal region enhances CENP-A binding during mitosis, while Aurora B phosphorylation of its N-terminal region modulates Mis12 interaction for error correction; CENP-C further serves as a platform for PLK-1 recruitment to the inner kinetochore and is required for centromeric DNA methylation via DNMT3B interaction, for spindle assembly checkpoint integrity, and—in meiosis—for CENP-A loading, sister centromere resolution, kinetochore mono-orientation, and centromere clustering."},"narrative":{"mechanistic_narrative":"CENP-C is a conserved inner kinetochore scaffold that bridges centromeric chromatin to the outer kinetochore machinery and is essential for accurate chromosome segregation [PMID:1339310, PMID:9361037, PMID:10428958]. It binds the CENP-A nucleosome directly, recognizing a hydrophobic region of the CENP-A tail and docking onto the H2A/H2B acidic patch through both its central region and the conserved CENP-C motif [PMID:23723239, PMID:31475439]; this engagement reshapes the nucleosome by rigidifying the histone core and driving lateral DNA gyre sliding toward canonical positioning [PMID:25954010, PMID:26878239]. CENP-C also contacts centromeric AT-rich/alpha-satellite DNA through internal DNA-binding/centromere-targeting domains, integrating histone and DNA recognition into contiguous binding modules [PMID:7883764, PMID:12006616, PMID:29074736]. From this chromatin platform, the conserved N-terminal motif of CENP-C binds the Mis12 complex with high affinity to nucleate assembly of the full KMN network and outer kinetochore [PMID:19641019, PMID:21353556], while its PEST region recruits the CENP-HIKM subcomplex, establishing CENP-C as a blueprint for assembly of the broader CCAN [PMID:26124289]. C-terminal Cupin-domain oligomerization and dimerization organize centromeric chromatin architecture and are required for CCAN localization [PMID:37295434, PMID:40997799]. CENP-C additionally sustains the centromeric epigenetic state by recruiting the M18BP1/Mis18 complex to drive new CENP-A loading [PMID:21911481, PMID:22540025] and by recruiting DNMT3B to direct centromeric DNA methylation, with its loss increasing missegregation and centromeric repeat transcription [PMID:19482874]. These activities are gated by mitotic kinases: CDK1 phosphorylation of the C-terminal region enhances CENP-A binding, whereas Aurora B phosphorylation of the N-terminal region modulates the Mis12 interaction for error correction, forming a positive regulatory loop that reinforces biorientation [PMID:27881301, PMID:29180432, PMID:31676716, PMID:39433344]. Genetic and biochemical conservation from yeast Mif2 through worm, fly, and plant orthologs underlies roles in kinetochore assembly, the spindle assembly checkpoint, and meiotic functions including mono-orientation and PLK-1 recruitment [PMID:7579695, PMID:17392512, PMID:19758558, PMID:37067150].","teleology":[{"year":1992,"claim":"Established CENP-C as a physical constituent of the kinetochore, anchoring all subsequent mechanistic work at the inner kinetochore plate.","evidence":"Immunoelectron microscopy and immunofluorescence with anti-CENP-C antibodies on HeLa chromosomes; dicentric chromosome analysis showing presence only at active centromeres","pmids":["1339310","2475307","8634687"],"confidence":"High","gaps":["Did not define molecular interactions or how CENP-C is targeted","No information on assembly hierarchy"]},{"year":1994,"claim":"Demonstrated CENP-C is functionally required for kinetochore integrity and mitotic progression, and is itself a DNA-binding protein, moving it from marker to functional component.","evidence":"Nuclear antibody microinjection causing metaphase arrest and reduced kinetochore size; Southwestern blotting of recombinant CENP-C against alphoid DNA","pmids":["8175879","7883764"],"confidence":"High","gaps":["DNA binding shown in vitro only","Did not identify protein partners mediating function"]},{"year":1997,"claim":"Resolved CENP-C's modular architecture (oligomerization, DNA-binding, dimerization units) and proved it is necessary but not sufficient for centromere function via conditional loss/gain of function.","evidence":"Chemical crosslinking, gel filtration and Southwestern blotting of fragments; conditional gene disruption and overexpression in DT40 cells","pmids":["9146917","9361037","10428958","8668174"],"confidence":"High","gaps":["Functional roles of individual domains not yet linked to specific partners","Assembly hierarchy with other CENPs undefined"]},{"year":2001,"claim":"Placed CENP-C within an ordered assembly pathway, defining CENP-A → CENP-H → CENP-C hierarchy and its dependence on upstream factors.","evidence":"Conditional knockouts in DT40 cells and RNAi in C. elegans with epistatic localization analysis","pmids":["11500386","11402064","7579695"],"confidence":"High","gaps":["Direct binding mechanism to CENP-A not yet structurally defined","Outer kinetochore recruitment mechanism unknown"]},{"year":2002,"claim":"Linked CENP-C to the spindle assembly checkpoint and outer-kinetochore Mis12 recruitment, and mapped its in vivo alpha-satellite DNA binding to discrete domains.","evidence":"Conditional knockout with Mad2 and Mis12 readouts in DT40; ChIP and truncation mapping for in vivo DNA binding","pmids":["17392512","12006616","12490152"],"confidence":"High","gaps":["Whether Mis12 recruitment is direct was not established here","Mechanism connecting CENP-C to checkpoint signaling unresolved"]},{"year":2009,"claim":"Identified the N-terminal domain of CENP-C as the direct recruiter of the Mis12/MIND complex and linked CENP-C to DNA methylation and CENP-A maintenance, broadening its role beyond structural scaffolding.","evidence":"Xenopus egg extract depletion-complementation with domain mutants; DNMT3B yeast two-hybrid, co-IP, bisulfite sequencing and ChIP; fission yeast scaffold and meiotic effector analysis","pmids":["19641019","19482874","19758558","19503796"],"confidence":"High","gaps":["High-resolution structure of the N-terminal–Mis12 interface not yet resolved","Mechanism coupling CENP-C to DNMT3B recruitment in chromatin context incomplete"]},{"year":2011,"claim":"Established that a conserved N-terminal motif binds the Mis12 complex with high affinity to nucleate the entire KMN network, defining CENP-C as the inner-to-outer kinetochore bridge, and that it recruits M18BP1 to drive CENP-A assembly.","evidence":"Direct biochemical binding reconstitution and dominant-negative/ectopic targeting in HeLa and Drosophila cells; in vitro M18BP1 binding with siRNA-based CENP-A assembly readout","pmids":["21353556","21353555","21911481"],"confidence":"High","gaps":["Structural basis of the Mis12 interaction not yet solved at this stage","Regulation of the recruitment by mitotic kinases not yet defined"]},{"year":2013,"claim":"Provided the structural mechanism of CENP-A nucleosome recognition — CENP-A tail engagement plus H2A/H2B acidic-patch docking — explaining how CENP-C reads the centromeric histone mark.","evidence":"Crystallography, NMR, mutagenesis and biochemical binding assays across species","pmids":["23723239"],"confidence":"High","gaps":["Did not address how binding alters nucleosome dynamics","Did not resolve regulation of the interaction in mitosis"]},{"year":2015,"claim":"Showed CENP-C actively reshapes CENP-A nucleosome structure and serves as the assembly blueprint for the CCAN, linking chromatin reading to architectural organization.","evidence":"In vitro reconstitution with single-molecule FRET/FLIM and depletion in cells; biochemical reconstitution mapping the PEST–CENP-HIKM interaction; ectopic targeting separating CENP-C and CENP-T KMN recruitment","pmids":["25954010","26124289","39433344","25660545"],"confidence":"High","gaps":["Quantitative contribution of nucleosome reshaping to centromere identity unresolved","Coordination between parallel CENP-C and CENP-T recruitment arms incomplete"]},{"year":2016,"claim":"Solved the MIS12C–CENP-C structure and defined gyre sliding as the mechanism of nucleosome reshaping, with Aurora B phosphorylation regulating the Mis12 interaction.","evidence":"X-ray crystallography of MIS12C–CENP-C, Aurora B kinase assays, and single-molecule FRET measuring DNA gyre sliding","pmids":["27881301","26878239"],"confidence":"High","gaps":["In vivo dynamics of phospho-regulation across the cell cycle incompletely mapped","Functional consequence of gyre sliding for centromere inheritance not fully established"]},{"year":2017,"claim":"Defined CENP-C's combined DNA- and histone-binding module and revealed dependence on centromeric RNA, integrating nucleic acid and histone recognition.","evidence":"Biochemical binding and mutational analysis of the DHBD in yeast and human CENP-C; RNA-IP and RNA depletion affecting CENP-C levels; CO-FISH showing centromere integrity maintenance","pmids":["29074736","28787590","28167779"],"confidence":"High","gaps":["Mechanism by which RNA stabilizes CENP-C–DNA binding in vivo incompletely defined","Role in repeat integrity mechanistically separate from segregation not fully dissected"]},{"year":2019,"claim":"Established kinase control of CENP-C: CDK1 phosphorylation of the C-terminus enhances CENP-A binding while Aurora B phosphorylation of the N-terminus tunes Mis12 binding, defining cell-cycle-coupled regulation of its bridging functions.","evidence":"In vitro CDK1 kinase assays, Phos-tag, co-IP and viability assays in chicken/human cells; Aurora B phosphorylation of S. pombe Cnp3 with phosphomimetic mutants; cryo-EM of CENP-A nucleosome with CENP-C central region","pmids":["31676716","29180432","31475439"],"confidence":"High","gaps":["Temporal interplay between CDK1 and Aurora B inputs on the same protein unresolved","How phospho-states are reset across mitosis not defined"]},{"year":2023,"claim":"Defined C-terminal Cupin-domain oligomerization as essential for centromeric chromatin organization and CCAN recruitment, and revealed auto-inhibition relieved by CENP-A binding.","evidence":"Crystal structure of the Cupin domain with biochemical oligomerization assays and mutagenesis in chicken cells; in vitro auto-inhibition reconstitution with yeast genetics; C. elegans PLK-1 recruitment analysis","pmids":["37295434","32515113","37067150","36736323"],"confidence":"High","gaps":["How oligomerization is spatially restricted to functional centromeres unclear","Integration of auto-inhibition with kinase regulation not resolved"]},{"year":2025,"claim":"Demonstrated in vivo roles for CENP-C dimerization in epigenetic resetting at fertilization and placed downstream alignment factors in its pathway, extending its function to developmental and effector contexts.","evidence":"Mouse CENP-A-mScarlet live imaging with maternal CENP-C depletion and dimerization mutants; genome-wide Cas9 synthetic-lethal screen with hypomorphic CENP-C identifying KIF18A/CENP-E","pmids":["40997799","41218610"],"confidence":"High","gaps":["Mechanism of asymmetric maternal CENP-C recruitment to paternal centromeres undefined","Direct biochemical link between CENP-C and KIF18A/CENP-E not established"]},{"year":null,"claim":"How CENP-C's multiple regulatory inputs (CDK1, Aurora B, PEST phosphorylation, auto-inhibition, RNA, oligomerization) are temporally integrated to coordinate CENP-A inheritance, CCAN assembly, and outer kinetochore biorientation within a single cell cycle remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unified model linking the distinct phospho-regulated and oligomerization-dependent states","Stoichiometry and dynamics of CENP-C at endogenous centromeres incompletely defined","Causal role in human disease not established in the corpus"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0003677","term_label":"DNA binding","supporting_discovery_ids":[3,5,17,45]},{"term_id":"GO:0042393","term_label":"histone binding","supporting_discovery_ids":[36,37,48]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[26,33,38]},{"term_id":"GO:0005198","term_label":"structural molecule activity","supporting_discovery_ids":[13,38,51]},{"term_id":"GO:0003723","term_label":"RNA binding","supporting_discovery_ids":[44,50]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[34,41]}],"localization":[{"term_id":"GO:0005694","term_label":"chromosome","supporting_discovery_ids":[0,1,6]},{"term_id":"GO:0000228","term_label":"nuclear chromosome","supporting_discovery_ids":[0,17]},{"term_id":"GO:0005730","term_label":"nucleolus","supporting_discovery_ids":[8]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[11]}],"pathway":[{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[10,13,18,39]},{"term_id":"R-HSA-4839726","term_label":"Chromatin organization","supporting_discovery_ids":[27,34,37,51]},{"term_id":"R-HSA-1474165","term_label":"Reproduction","supporting_discovery_ids":[29,52,56]}],"complexes":["CCAN","kinetochore"],"partners":["CENP-A","MIS12","M18BP1","DNMT3B","CENP-HIKM","PLK-1","AURKB","CENP-B"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q03188","full_name":"Centromere protein C","aliases":["Centromere autoantigen C","Centromere protein C 1","CENP-C 1","Interphase centromere complex protein 7"],"length_aa":943,"mass_kda":106.8,"function":"Component of the CENPA-NAC (nucleosome-associated) complex, a complex that plays a central role in assembly of kinetochore proteins, mitotic progression and chromosome segregation. The CENPA-NAC complex recruits the CENPA-CAD (nucleosome distal) complex and may be involved in incorporation of newly synthesized CENPA into centromeres. CENPC recruits DNA methylation and DNMT3B to both centromeric and pericentromeric satellite repeats and regulates the histone code in these regions","subcellular_location":"Nucleus; Chromosome, centromere, kinetochore; Chromosome, centromere","url":"https://www.uniprot.org/uniprotkb/Q03188/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":true,"resolved_as":"","url":"https://depmap.org/portal/gene/CENPC","classification":"Common Essential","n_dependent_lines":1070,"n_total_lines":1208,"dependency_fraction":0.8857615894039735},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/CENPC","total_profiled":1310},"omim":[{"mim_id":"618137","title":"MIS18 KINETOCHORE PROTEIN A: MIS18A","url":"https://www.omim.org/entry/618137"},{"mim_id":"616232","title":"MEIOTIC KINETOCHORE FACTOR; MEIKIN","url":"https://www.omim.org/entry/616232"},{"mim_id":"611511","title":"MLF1-INTERACTING PROTEIN; MLF1IP","url":"https://www.omim.org/entry/611511"},{"mim_id":"611510","title":"CENTROMERIC PROTEIN T; CENPT","url":"https://www.omim.org/entry/611510"},{"mim_id":"611509","title":"CENTROMERIC PROTEIN N; CENPN","url":"https://www.omim.org/entry/611509"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Nuclear bodies","reliability":"Supported"},{"location":"Kinetochore","reliability":"Supported"},{"location":"Nucleoplasm","reliability":"Additional"},{"location":"Midbody","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in many","driving_tissues":[],"url":"https://www.proteinatlas.org/search/CENPC"},"hgnc":{"alias_symbol":["CENP-C","hcp-4","MIF2"],"prev_symbol":["CENPC1"]},"alphafold":{"accession":"Q03188","domains":[{"cath_id":"2.60.120.10","chopping":"850-942","consensus_level":"high","plddt":91.9697,"start":850,"end":942}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q03188","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q03188-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q03188-F1-predicted_aligned_error_v6.png","plddt_mean":48.03},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=CENPC","jax_strain_url":"https://www.jax.org/strain/search?query=CENPC"},"sequence":{"accession":"Q03188","fasta_url":"https://rest.uniprot.org/uniprotkb/Q03188.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q03188/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q03188"}},"corpus_meta":[{"pmid":"7579695","id":"PMC_7579695","title":"Evidence that the MIF2 gene of Saccharomyces cerevisiae encodes a centromere protein with homology to the mammalian centromere protein CENP-C.","date":"1995","source":"Molecular biology of the cell","url":"https://pubmed.ncbi.nlm.nih.gov/7579695","citation_count":370,"is_preprint":false},{"pmid":"1339310","id":"PMC_1339310","title":"CENP-C, an autoantigen in scleroderma, is a component of the human inner kinetochore plate.","date":"1992","source":"Cell","url":"https://pubmed.ncbi.nlm.nih.gov/1339310","citation_count":325,"is_preprint":false},{"pmid":"17222555","id":"PMC_17222555","title":"Incorporation of Drosophila CID/CENP-A and CENP-C into centromeres during early embryonic anaphase.","date":"2007","source":"Current biology : CB","url":"https://pubmed.ncbi.nlm.nih.gov/17222555","citation_count":300,"is_preprint":false},{"pmid":"2475307","id":"PMC_2475307","title":"Visualization of centromere proteins CENP-B and CENP-C on a stable dicentric chromosome in cytological spreads.","date":"1989","source":"Chromosoma","url":"https://pubmed.ncbi.nlm.nih.gov/2475307","citation_count":283,"is_preprint":false},{"pmid":"23723239","id":"PMC_23723239","title":"A conserved mechanism for centromeric nucleosome recognition by centromere protein CENP-C.","date":"2013","source":"Science (New York, N.Y.)","url":"https://pubmed.ncbi.nlm.nih.gov/23723239","citation_count":277,"is_preprint":false},{"pmid":"21353555","id":"PMC_21353555","title":"CENP-C is a structural platform for kinetochore assembly.","date":"2011","source":"Current biology : CB","url":"https://pubmed.ncbi.nlm.nih.gov/21353555","citation_count":225,"is_preprint":false},{"pmid":"21353556","id":"PMC_21353556","title":"Direct binding of Cenp-C to the Mis12 complex joins the inner and outer kinetochore.","date":"2011","source":"Current biology : CB","url":"https://pubmed.ncbi.nlm.nih.gov/21353556","citation_count":222,"is_preprint":false},{"pmid":"10075924","id":"PMC_10075924","title":"Specific destruction of kinetochore protein CENP-C and disruption of cell division by herpes simplex virus immediate-early protein Vmw110.","date":"1999","source":"The EMBO journal","url":"https://pubmed.ncbi.nlm.nih.gov/10075924","citation_count":204,"is_preprint":false},{"pmid":"8634687","id":"PMC_8634687","title":"Identification of centromeric antigens in dicentric Robertsonian translocations: CENP-C and CENP-E are necessary components of functional centromeres.","date":"1995","source":"Human molecular genetics","url":"https://pubmed.ncbi.nlm.nih.gov/8634687","citation_count":196,"is_preprint":false},{"pmid":"21911481","id":"PMC_21911481","title":"CENP-C recruits M18BP1 to centromeres to promote CENP-A chromatin assembly.","date":"2011","source":"The Journal of cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/21911481","citation_count":188,"is_preprint":false},{"pmid":"25954010","id":"PMC_25954010","title":"Chromosomes. CENP-C reshapes and stabilizes CENP-A nucleosomes at the centromere.","date":"2015","source":"Science (New York, N.Y.)","url":"https://pubmed.ncbi.nlm.nih.gov/25954010","citation_count":182,"is_preprint":false},{"pmid":"8175879","id":"PMC_8175879","title":"CENP-C is required for maintaining proper kinetochore size and for a timely transition to anaphase.","date":"1994","source":"The Journal of cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/8175879","citation_count":175,"is_preprint":false},{"pmid":"28787590","id":"PMC_28787590","title":"Human Centromeres Produce Chromosome-Specific and Array-Specific Alpha Satellite Transcripts that Are Complexed with CENP-A and CENP-C.","date":"2017","source":"Developmental cell","url":"https://pubmed.ncbi.nlm.nih.gov/28787590","citation_count":150,"is_preprint":false},{"pmid":"11500386","id":"PMC_11500386","title":"CENP-H, a constitutive centromere component, is required for centromere targeting of CENP-C in vertebrate 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research","url":"https://pubmed.ncbi.nlm.nih.gov/36442049","citation_count":1,"is_preprint":false},{"pmid":"39140145","id":"PMC_39140145","title":"Functional roles of the interaction of Moa1 with CENP-C and Rec8 in meiosis of Schizosaccharomyces pombe.","date":"2024","source":"Yi chuan = Hereditas","url":"https://pubmed.ncbi.nlm.nih.gov/39140145","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":43447,"output_tokens":12490,"usd":0.158846,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":23945,"output_tokens":5353,"usd":0.126775,"stage2_stop_reason":"end_turn"},"total_usd":0.285621,"stage1_batch_id":"msgbatch_01ACAxJQabDsht39JXeWG22d","stage2_batch_id":"msgbatch_01QEU22A9U5sbPTgsMzTmvSf","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1992,\n      \"finding\": \"CENP-C is a component of the inner kinetochore plate, as determined by immunoelectron microscopy of HeLa chromosomes using antibodies raised against CENP-C cDNA-encoded fusion proteins.\",\n      \"method\": \"Immunoelectron microscopy, indirect immunofluorescence, immunoblotting\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — direct localization by immunoelectron microscopy with specific antibodies, replicated across multiple criteria in a foundational study\",\n      \"pmids\": [\"1339310\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1989,\n      \"finding\": \"CENP-C is present exclusively at active centromeres and is absent from inactive centromeres of stable dicentric chromosomes, suggesting it is a necessary component of functional kinetochores; CENP-C appears confined to the outer centromere in the kinetochore region.\",\n      \"method\": \"Immunofluorescence with specific anti-CENP-C antibodies on dicentric chromosomes\",\n      \"journal\": \"Chromosoma\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — direct antibody-based localization experiment on dicentric chromosomes, independently replicated in multiple subsequent studies\",\n      \"pmids\": [\"2475307\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1994,\n      \"finding\": \"Nuclear microinjection of anti-CENP-C antibodies during interphase causes metaphase arrest and reduces kinetochore size (trilaminar structures are reduced in diameter), demonstrating CENP-C is required for establishing/maintaining proper kinetochore size and for the timely metaphase-to-anaphase transition.\",\n      \"method\": \"Nuclear microinjection of antibodies, immunoelectron microscopy\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — functional loss-of-function by antibody injection with specific ultrastructural phenotypic readout, well-controlled study\",\n      \"pmids\": [\"8175879\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1994,\n      \"finding\": \"CENP-C is a DNA-binding protein; an internal ~101 amino acid stretch constitutes its DNA-binding domain, demonstrated by Southwestern blotting of recombinant CENP-C expressed in E. coli against human genomic DNA and alphoid DNA subfamilies.\",\n      \"method\": \"Southwestern blotting, recombinant protein expression, truncation analysis\",\n      \"journal\": \"Journal of biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro DNA-binding assay with truncation mapping, single lab, no in vivo validation in this paper\",\n      \"pmids\": [\"7883764\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1995,\n      \"finding\": \"The yeast MIF2 protein (CENP-C ortholog) interacts genetically with CEP1/CBF1, NDC10/CBF2, and CEP3/CBF3B at the centromere; mif2 mutations lie within regions homologous to CENP-C, establishing functional and structural conservation between yeast and mammalian centromeres.\",\n      \"method\": \"Genetic epistasis, synthetic lethality screens, minichromosome stability assays\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal genetic epistasis experiments with defined centromere gene mutations, robust pathway placement\",\n      \"pmids\": [\"7579695\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1995,\n      \"finding\": \"CENP-C has an autonomous centromere-targeting domain located in the central region of the protein, which overlaps with its DNA-binding domain, as defined by truncation mutant analysis in vivo and in vitro.\",\n      \"method\": \"In vivo expression of truncation mutants, in vitro DNA-binding assays\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — domain mapping by truncation mutants with both in vivo localization and in vitro DNA-binding assays, two orthogonal methods\",\n      \"pmids\": [\"8668174\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1995,\n      \"finding\": \"CENP-C and CENP-E localize exclusively to active centromeres (not inactive centromeres) in dicentric Robertsonian translocations, establishing that at least two centromeric proteins are required for human centromeric function.\",\n      \"method\": \"Immunofluorescence combined with FISH on dicentric chromosomes\",\n      \"journal\": \"Human molecular genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — direct localization experiment on 12 dicentric translocations with independent FISH confirmation of active centromere identity\",\n      \"pmids\": [\"8634687\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1995,\n      \"finding\": \"The Mif2 homology domain in the central region of CENP-C is required for centromere assembly; mutations within this domain impair CENP-C kinetochore localization. The N-terminus of CENP-C is required for protein destruction and renders otherwise stable proteins unstable.\",\n      \"method\": \"In vivo expression of CENP-C mutants, centromere targeting assays\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — domain mapping with mutational analysis and in vivo localization readout, single lab\",\n      \"pmids\": [\"7579707\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1996,\n      \"finding\": \"CENP-C interacts with nucleolar transcription factors UBF1 and UBF2 through its C-terminal third, as identified by affinity chromatography and confirmed by co-localization of a subset of CENP-C and UBF at nucleoli in interphase HeLa cells.\",\n      \"method\": \"Affinity chromatography, microsequence analysis, immunofluorescence co-localization\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — pulldown/affinity chromatography with co-localization confirmation, but functional significance not fully established\",\n      \"pmids\": [\"8702533\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1997,\n      \"finding\": \"Human CENP-C has three functional units: an N-terminal oligomerization domain (capable of dimer and tetramer formation by crosslinking), an internal DNA-binding domain (with core and flanking stabilizing elements), and a C-terminal dimerization domain (forming exclusively dimers), as defined by biochemical analysis of expressed fragments.\",\n      \"method\": \"Chemical crosslinking, gel filtration, Southwestern blotting\",\n      \"journal\": \"Chromosome research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple in vitro biochemical methods (crosslinking, gel filtration, blotting) to define domain architecture, single lab\",\n      \"pmids\": [\"9146917\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1997,\n      \"finding\": \"CENP-C loss of function (conditional fusion to mouse steroid receptor in DT40 cells) causes arrest at the metaphase/anaphase junction followed by apoptosis, demonstrating CENP-C is required for anaphase progression or centromere signaling.\",\n      \"method\": \"Conditional gene targeting, live cell observation, DT40 chicken cells\",\n      \"journal\": \"Human molecular genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean conditional loss-of-function in vertebrate cells with specific and reproducible mitotic arrest phenotype\",\n      \"pmids\": [\"9361037\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"CENP-C interacts with HDaxx (a death domain-binding protein) through the N-terminal 315 amino acids of CENP-C and the C-terminal 104 amino acids of HDaxx; this interaction is interphase-specific and they co-localize at discrete nuclear spots in interphase HeLa cells.\",\n      \"method\": \"Yeast two-hybrid, immunofluorescence co-localization\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — yeast two-hybrid interaction with domain mapping and immunofluorescence co-localization, but in vivo interaction not confirmed by co-IP\",\n      \"pmids\": [\"9645950\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"Herpes simplex virus immediate-early protein Vmw110 (ICP0) causes proteasome-dependent loss of CENP-C from centromeres during infection (via its RING finger domain), resulting in ultrastructural kinetochore disruption, mitotic arrest, and abnormal cytokinesis.\",\n      \"method\": \"Viral infection, proteasome inhibitor treatment, immunofluorescence, electron microscopy\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — mechanistic dissection of CENP-C degradation pathway with domain mapping, ultrastructural analysis, and functional consequence\",\n      \"pmids\": [\"10075924\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"CENP-C is necessary but not sufficient for formation of a functional centromere; its removal disassembles the centromere protein complex and blocks cells at metaphase-anaphase junction, while overexpression of CENP-C does not associate with ZW10 and causes segregation errors.\",\n      \"method\": \"Conditional gene disruption, inducible overexpression, immunofluorescence in DT40 cells\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — both loss- and gain-of-function approaches in vertebrate cells with clear mechanistic conclusions\",\n      \"pmids\": [\"10428958\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"CENP-H is required for centromere targeting of CENP-C but not CENP-A in vertebrate cells, establishing a hierarchical assembly order at the centromere: CENP-A → CENP-H → CENP-C.\",\n      \"method\": \"Conditional knockout in DT40 cells, immunocytochemistry\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean conditional KO with epistasis placing CENP-H upstream of CENP-C but downstream of CENP-A, clear hierarchical order\",\n      \"pmids\": [\"11500386\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"C. elegans HCP-4/CENP-C localizes to centromeres in a CENP-A (HCP-3)-dependent manner, and its loss of function by RNAi results in failure of sister centromere resolution and failure to form functional kinetochores; HCP-4 and HCP-3 are both required for localization of HCP-1 (CENP-F-like), defining an ordered assembly pathway.\",\n      \"method\": \"RNAi, immunofluorescence localization in C. elegans\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — epistatic ordering of centromere assembly pathway via RNAi with specific functional readouts (centromere resolution and kinetochore assembly)\",\n      \"pmids\": [\"11402064\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"A SUMO-1 gene suppresses the temperature-sensitive phenotype of a CENP-C mutant in DT40 cells, suggesting that SUMO-1 is involved in centromere function in vertebrate cells through a pathway involving CENP-C.\",\n      \"method\": \"cDNA library suppressor screen, temperature-sensitive CENP-C mutants in DT40 cells\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — genetic suppressor identified in vertebrate cells, single method, no direct biochemical confirmation of mechanism\",\n      \"pmids\": [\"11557811\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"CENP-C binds alpha-satellite DNA in vivo selectively; the region between amino acids 410 and 537 is required for this in vivo DNA binding; CENP-C and CENP-B associate with the same types of alpha-satellite arrays but in distinct non-overlapping centromere domains.\",\n      \"method\": \"Chromatin immunoprecipitation (ChIP), immunofluorescence, ultrastructural analysis\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP with domain mapping provides in vivo evidence for DNA binding with spatial resolution; two orthogonal methods\",\n      \"pmids\": [\"12006616\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"CENP-C inactivation in DT40 cells causes mitotic delay, impairs the Mad2 spindle checkpoint pathway (~60% of CENP-C-deficient cells lack Mad2 signals after nocodazole), and causes significant reduction in Mis12 complex proteins at centromeres; CENP-C centromere localization in interphase requires the CENP-H complex.\",\n      \"method\": \"Conditional knockout in DT40 cells, immunofluorescence, live-cell microscopy\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — conditional KO with multiple functional readouts (spindle checkpoint, Mis12 recruitment, cell cycle) in vertebrate cells\",\n      \"pmids\": [\"17392512\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"PARP-2 does not interact with CENP-C as determined by co-immunoprecipitation, distinguishing CENP-C from CENP-A and CENP-B which do interact with PARP-2.\",\n      \"method\": \"Co-immunoprecipitation\",\n      \"journal\": \"Human molecular genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Weak — negative result from single co-IP experiment; informative as negative finding showing CENP-C is not in the PARP-2 complex\",\n      \"pmids\": [\"12217960\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"Human CENP-C contains two distinct centromere-targeting domains: one in the central region (aa 426-537) and one in the C-terminal region (aa 638-943), both capable of binding alpha-satellite DNA in vivo.\",\n      \"method\": \"Immunofluorescence of truncation mutants, ChIP\",\n      \"journal\": \"Journal of structural biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — two orthogonal methods (immunofluorescence and ChIP) to define two independent centromere-targeting domains, single lab\",\n      \"pmids\": [\"12490152\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"CENP-B interacts directly with CENP-C; the CENP-C domains required for this interaction overlap with three Mif2 homologous regions and are also involved in centromere assembly. Overproduction of CENP-B truncants lacking CENP-C interaction domains causes abnormal CENP-C domain duplication and cell cycle delay.\",\n      \"method\": \"Yeast two-hybrid, domain truncation analysis, immunofluorescence\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — yeast two-hybrid with in vivo truncation/phenotypic confirmation; two orthogonal methods, single lab\",\n      \"pmids\": [\"14612452\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"CENP-C can be sumoylated in vitro by SUMO-1 and SUMO-2; sumoylation occurs at multiple lysine residues including sites outside the perfect consensus motif, within regions overlapping the DNA-binding and centromere localization domains.\",\n      \"method\": \"In vitro sumoylation reconstitution, tandem mass spectrometry identification of sumoylated isopeptides\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution with mass spectrometry identification of modification sites; however, in vivo relevance not directly demonstrated in this paper\",\n      \"pmids\": [\"15272016\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Drosophila CENP-C is required for normal kinetochore attachment to the spindle; it was identified through genetic interaction with separase regulatory subunits Pimples/securin and Three rows, and its centromere localization domain contains a diverged CENPC motif.\",\n      \"method\": \"Genetic modifier screen, in vivo imaging, domain analysis\",\n      \"journal\": \"Genes & development\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic screen with in vivo functional validation, but direct evidence for separase involvement was not obtained\",\n      \"pmids\": [\"16140985\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Drosophila CID (CENP-A) and CENP-C are incorporated into centromeres during anaphase in early embryos; this incorporation is independent of DNA synthesis and spindle pulling forces but strictly coupled to mitotic progression.\",\n      \"method\": \"Quantitative fluorescence measurements in living embryos using fluorescent fusion proteins\",\n      \"journal\": \"Current biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — quantitative live imaging with functional fluorescent fusions, multiple orthogonal controls (drug treatments) establishing cell-cycle coupling\",\n      \"pmids\": [\"17222555\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Caspase-7 (activated downstream of death receptor-induced caspase-8) cleaves CENP-C and INCENP, causing their mislocalization and subsequent mislocalization of Aurora B kinase from centromeres; expression of non-cleavable CENP-C prevents passenger complex mislocalization after caspase activation.\",\n      \"method\": \"Biochemical caspase cleavage assays, site-directed mutagenesis, immunofluorescence\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — caspase cleavage mapped with mutagenesis, functional rescue with non-cleavable mutant, two orthogonal methods\",\n      \"pmids\": [\"17287400\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"CENP-C recruits the Mis12/MIND complex and CENP-K to kinetochores via its N-terminal domain; immunodepletion of CENP-C from Xenopus metaphase egg extract prevents kinetochore formation on sperm chromatin; CENP-C mutants that localize to centromeres but fail to recruit Mis12/MIND complex cannot support kinetochore assembly.\",\n      \"method\": \"Xenopus egg extract immunodepletion, in vitro complementation with mutant CENP-C, immunofluorescence\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution/depletion-complementation system with domain mutants establishing N-terminal recruitment function\",\n      \"pmids\": [\"19641019\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"DNMT3B interacts directly with CENP-C (identified by yeast two-hybrid and confirmed by co-immunoprecipitation in mammalian cells); CENP-C recruits DNMT3B and DNA methylation to centromeric and pericentromeric satellite repeats; loss of CENP-C reduces DNA methylation at centromeres, alters the histone code, and increases chromosome missegregation and centromeric repeat transcription.\",\n      \"method\": \"Yeast two-hybrid, co-immunoprecipitation, siRNA knockdown, bisulfite sequencing, ChIP\",\n      \"journal\": \"Human molecular genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (Y2H, Co-IP, ChIP, bisulfite sequencing) with functional loss-of-function confirmation\",\n      \"pmids\": [\"19482874\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"The C-terminal Mif2p homology domain II of CENP-C targets the centromere and contacts alpha-satellite DNA; domain III mediates homo-dimerization, homo-oligomerization, and interaction with CENP-A and histone H3.\",\n      \"method\": \"Immunofluorescence, ChIP, co-immunoprecipitation, bimolecular fluorescence complementation\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple in vivo methods (immunofluorescence, ChIP, bimolecular fluorescence complementation) with biochemical confirmation, single lab\",\n      \"pmids\": [\"19503796\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Fission yeast CENP-C (Cnp3) acts as a scaffold recruiting Fta1/CENP-L (primary direct effector) for mitotic kinetochore function; Cnp3 also recruits Pcs1 to prevent merotelic attachment; in meiosis, Cnp3 associates with and recruits Moa1 for meiotic mono-orientation of kinetochores.\",\n      \"method\": \"Yeast genetic and biochemical analyses, epistasis, ectopic localization\",\n      \"journal\": \"Developmental cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — direct binding shown biochemically, ectopic localization rescue of mitotic defects, identification of separate mitotic and meiotic effectors\",\n      \"pmids\": [\"19758558\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Maize CENPC DNA binding is mediated by a 122 amino acid domain and is stabilized by single-stranded RNA; long single-stranded nucleic acids promote CENPC binding to DNA. Removal of this binding module causes partial delocalization of CENPC in vivo.\",\n      \"method\": \"In vitro DNA/RNA binding assays, domain truncation, in vivo localization with mutants\",\n      \"journal\": \"PLoS genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — biochemical reconstitution of RNA-stimulated DNA binding with in vivo validation of domain function, single lab\",\n      \"pmids\": [\"20140237\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Drosophila Cal1 links CENP-A/Cid and CENP-C by binding their N- and C-terminal domains respectively (shown by yeast three-hybrid); Cal1 limits centromeric deposition of CENP-A/Cid and CENP-C during mitotic exit; both Cal1 domains are required together for centromere function.\",\n      \"method\": \"Yeast three-hybrid, quantitative in vivo fluorescence imaging\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — three-hybrid domain interaction mapped, quantitative in vivo imaging, functional domain deletion analysis\",\n      \"pmids\": [\"20940262\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"The N-terminal region of Drosophila CENP-C is sufficient to recruit all KMN network components (Mis12 complex, Ndc80 complex, Spc105/KNL1); the Mis12 complex component Nnf1 directly interacts with CENP-C in vitro; targeting the CENP-C N-terminus to centrosomes redirects KMN proteins away from kinetochores, causing mitotic defects.\",\n      \"method\": \"In vitro binding assay, ectopic targeting assay (centrosome targeting), immunofluorescence in Drosophila cells\",\n      \"journal\": \"Current biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — direct in vitro binding plus ectopic targeting gain-of-function, multiple KMN components tested, clear functional consequence\",\n      \"pmids\": [\"21353555\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"A conserved N-terminal motif of vertebrate Cenp-C binds directly and with high affinity to the Mis12 complex, linking the inner and outer kinetochore; expression of the isolated N-terminal Cenp-C motif in HeLa cells prevents outer kinetochore assembly, causes chromosome missegregation, and impairs spindle assembly checkpoint.\",\n      \"method\": \"Direct binding assay (biochemical reconstitution), dominant-negative expression in HeLa cells, immunofluorescence\",\n      \"journal\": \"Current biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — direct binding reconstitution with high-affinity measurement plus dominant-negative functional validation and checkpoint readout\",\n      \"pmids\": [\"21353556\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"CENP-C recruits M18BP1 (Mis18 complex component) to centromeres to promote CENP-A chromatin assembly; depletion of CENP-C prevents M18BP1 targeting to metaphase centromeres and inhibits CENP-A assembly; M18BP1 directly binds CENP-C through conserved domains.\",\n      \"method\": \"siRNA depletion, in vitro direct binding assay, immunofluorescence\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — direct binding shown in vitro, functional depletion with CENP-A assembly readout, two orthogonal methods\",\n      \"pmids\": [\"21911481\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"M18BP1 interacts with CENP-C through a central SANT domain region of M18BP1 and the C-terminus of CENP-C; CENP-C knockdown reduces M18BP1 centromeric association and lowers CENP-A levels at centromeres.\",\n      \"method\": \"Interaction screen, domain mapping, siRNA knockdown, immunofluorescence in mouse ESCs\",\n      \"journal\": \"Nucleus\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — interaction mapping with functional siRNA validation, single lab, replicates findings of Moree et al. 2011\",\n      \"pmids\": [\"22540025\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"CENP-C recognizes the CENP-A nucleosome by binding a hydrophobic region in the CENP-A tail and docking onto the acidic patch of histones H2A and H2B; this mechanism is used by both the CENP-C motif and the broader conserved central region; the mechanism is conserved across species.\",\n      \"method\": \"Crystal structure determination, NMR, mutagenesis, biochemical binding assays\",\n      \"journal\": \"Science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure plus NMR plus mutagenesis in one study; conserved mechanism validated across multiple species\",\n      \"pmids\": [\"23723239\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"CENP-C binds CENP-A nucleosomes (using purified components) and reshapes the octameric histone core: it rigidifies both surface and internal nucleosome structure and modulates terminal DNA wrapping to match the loose wrap found on native CENP-A nucleosomes. CENP-C depletion leads to rapid removal of CENP-A from centromeres.\",\n      \"method\": \"In vitro reconstitution with purified components, fluorescence lifetime imaging, single-molecule FRET, CENP-C depletion in cells\",\n      \"journal\": \"Science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — reconstitution with purified components, multiple biophysical methods (FRET, FLIM), functional in vivo depletion confirmation\",\n      \"pmids\": [\"25954010\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"The PEST domain in the N-terminal half of CENP-C directly interacts with the CENP-HIKM subcomplex, and this interaction is required for kinetochore localization of CENP-HIKM and CENP-TW; CENP-C acts as a blueprint for CCAN assembly.\",\n      \"method\": \"Biochemical reconstitution, domain mapping, cellular rescue assays, immunofluorescence\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — biochemical reconstitution with mutagenesis and cellular validation, multiple CCAN subcomplexes tested\",\n      \"pmids\": [\"26124289\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"CENP-C and CENP-T independently recruit the KMN network to kinetochores via distinct mechanisms and regulatory controls: CENP-C recruits Ndc80 through KNL1 and Mis12 interactions (regulated by Aurora B), while CENP-T directly interacts with Ndc80 (regulated by CDK).\",\n      \"method\": \"Ectopic chromosomal targeting assays in human cells, immunofluorescence\",\n      \"journal\": \"Current biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — separation-of-function by ectopic targeting in human cells definitively separating two parallel recruitment pathways\",\n      \"pmids\": [\"25660545\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"CENP-C depletion leads to increased centromere DNA aberrations (sister chromatid exchanges at centromeres), indicating CENP-C maintains centromere repeat integrity independently of its role in chromosome segregation.\",\n      \"method\": \"CO-FISH (chromosome orientation FISH), siRNA depletion, structured illumination microscopy\",\n      \"journal\": \"PNAS\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — CO-FISH with siRNA depletion and super-resolution imaging, two orthogonal methods, single lab\",\n      \"pmids\": [\"28167779\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"CENP-C and CENP-I are key factors connecting kinetochore to CENP-A assembly; tethering various kinetochore components to an ectopic array recruits CENP-C and subsequently M18BP1; CENP-I can also recruit M18BP1 downstream of CENP-C to enhance M18BP1 assembly at centromeres.\",\n      \"method\": \"Tethering assay with tetR-fusion proteins on synthetic alphoid array HACs, immunofluorescence\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ectopic tethering assay establishes epistasis of CENP-C upstream of M18BP1, single lab\",\n      \"pmids\": [\"26527398\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Crystal structure of human MIS12C in complex with a CENP-C fragment reveals the structural basis of the CENP-C–Mis12 interaction; Aurora B kinase phosphorylation of CENP-C regulates this interaction, allowing construction of a near-complete structural model of the KMN assembly.\",\n      \"method\": \"X-ray crystallography, biochemical binding assays, Aurora B kinase phosphorylation assays\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure with biochemical validation and kinase regulation, high-resolution mechanistic insight\",\n      \"pmids\": [\"27881301\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"CENP-C directs a structural transition in CENP-A nucleosomes predominantly through lateral sliding of DNA gyres (gyre sliding), returning them toward canonical nucleosome DNA positions, as measured by single-molecule FRET.\",\n      \"method\": \"Single-molecule FRET with recombinant human histones and centromere DNA\",\n      \"journal\": \"Nature structural & molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — single-molecule FRET reconstitution with purified components, defined mechanistic model with quantitative biophysical measurements\",\n      \"pmids\": [\"26878239\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Active centromere alpha-satellite transcripts are complexed with CENP-A and CENP-C; depletion of array-specific RNAs reduces CENP-A and CENP-C at the targeted centromere via faulty CENP-A loading.\",\n      \"method\": \"RNA immunoprecipitation, RNA FISH, antisense oligonucleotide depletion, immunofluorescence\",\n      \"journal\": \"Developmental cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — RNA-IP showing complex formation plus functional depletion of RNAs affecting CENP-C levels, single lab\",\n      \"pmids\": [\"28787590\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Yeast Mif2/CENP-C uses a contiguous DNA- and histone-binding domain (DHBD) containing the CENP-C motif, an AT-hook, and RK clusters to contact both Cse4/CENP-A residues and AT-rich centromere DNA simultaneously; human CENP-C has two related DHBDs that preferentially bind higher AT content DNA.\",\n      \"method\": \"Biochemical binding assays, mutational analysis, structural analysis\",\n      \"journal\": \"Genes & development\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — mechanistic dissection of DNA- and histone-binding requirements with domain mutations, validated in both yeast and human CENP-C\",\n      \"pmids\": [\"29074736\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Aurora B kinase phosphorylates the N-terminal region (Thr28) of S. pombe CENP-C (Cnp3), impairing its interaction with the Mis12 complex; a phosphomimetic mutant causes defective chromosome segregation due to improper kinetochore assembly.\",\n      \"method\": \"Kinase assay, co-immunoprecipitation, crystal structure of Mis12-Nnf1 complex, mutant expression in S. pombe\",\n      \"journal\": \"PNAS\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure, kinase assay, in vivo phosphomimetic mutant with functional chromosome segregation phenotype; multiple orthogonal methods\",\n      \"pmids\": [\"29180432\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"CDK1 phosphorylates the C-terminal region of CENP-C (at conserved sites), facilitating its binding to CENP-A nucleosomes in vitro and in vivo; this CENP-A binding promotes CENP-C kinetochore localization during mitosis, and the CENP-A–CENP-C interaction is critical for long-term viability in human RPE-1 cells.\",\n      \"method\": \"In vitro kinase assay (CDK1), Phos-tag SDS-PAGE, co-immunoprecipitation, immunofluorescence in chicken and human cells, cell viability assays\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro kinase assay with mutagenesis, confirmed in vivo in two cell systems (chicken and human), functional viability readout\",\n      \"pmids\": [\"31676716\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Human CENP-C central region (CENP-CCR) and CENP-C motif both bind exclusively to CENP-A nucleosomes in vitro; CENP-CCR binds with high affinity through CENP-AV532 and CENP-AV533 (extended hydrophobic area); CENP-CCR binding destabilizes the H2A C-terminal tail (further exacerbating loose DNA wrapping) and rigidifies H4 N-terminal tail in the conformation favoring H4K20 monomethylation.\",\n      \"method\": \"Cryo-EM structure of CENP-A nucleosome, in vitro binding assays, mutagenesis\",\n      \"journal\": \"EMBO reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — cryo-EM structure with in vitro binding validation and mutagenesis, reveals two distinct conformational changes upon binding\",\n      \"pmids\": [\"31475439\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Mif2/CENP-C exhibits auto-inhibition: wild-type Mif2 is attenuated in binding the Mtw1 complex, and binding Cse4/CENP-A nucleosomes overcomes this inhibition; a Mif2 mutant bypassing Cse4 requirement for Mtw1 binding causes mis-localization of the Mtw1 complex and chromosome segregation defects in vivo.\",\n      \"method\": \"In vitro binding assays with reconstituted nucleosomes, yeast genetics, immunofluorescence\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution demonstrating auto-inhibition mechanism combined with genetic validation in cells\",\n      \"pmids\": [\"32515113\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"lncRNA CCTT recruits CENP-C to centromeric DNA via RNA-DNA triplex formation and a direct RNA-protein interaction with CENP-C; CCTT loss triggers mitotic errors and aneuploidy, and CCTT localizes to all centromeres.\",\n      \"method\": \"RNA immunoprecipitation, RNA-DNA triplex assay, siRNA/shRNA depletion, immunofluorescence, FISH\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — RNA-IP and triplex assay demonstrate mechanism, functional depletion confirms role; note this is a non-centromere-derived lncRNA acting on CENP-C protein, not a non-coding product of the CENPC locus\",\n      \"pmids\": [\"36332605\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"CENP-C undergoes self-oligomerization through its C-terminal Cupin domain (demonstrated by structural and biochemical analyses of chicken and human CENP-C); this oligomerization is vital for CENP-C function, centromeric localization of CCAN proteins, and centromeric chromatin organization.\",\n      \"method\": \"Crystal structure of Cupin domain, biochemical self-oligomerization assays, mutagenesis, immunofluorescence in chicken cells\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure plus biochemical reconstitution of oligomerization plus mutagenesis with in vivo functional validation\",\n      \"pmids\": [\"37295434\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"In C. elegans oocytes, CENP-C (HCP-4) directly recruits PLK-1 to the chromosome arm during meiosis (independent of BUB-1); disruption of the CENP-C–PLK-1 interaction leads to imbalance in kinetochore components and chromosome congression defects without affecting CDC-20 recruitment.\",\n      \"method\": \"Live imaging, biochemical interaction assays, RNAi, degron-mediated depletion in C. elegans oocytes\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — direct biochemical interaction plus functional RNAi with specific meiotic phenotype; separation from BUB-1 pathway\",\n      \"pmids\": [\"37067150\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Multi-site phosphorylation of the PEST region of yeast Mif2/CENP-C enhances inner kinetochore assembly; eliminating PEST phosphorylation sites progressively impairs cellular fitness and causes lethality in cells lacking otherwise non-essential inner kinetochore factors.\",\n      \"method\": \"Yeast genetics, phosphorylation-site mutagenesis, genetic interaction analysis\",\n      \"journal\": \"Current biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis and phospho-site mutagenesis in yeast, single lab, no direct biochemical demonstration of mechanism\",\n      \"pmids\": [\"36736323\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"In C. elegans embryos, CENP-C targets PLK-1 to the inner kinetochore during prometaphase and metaphase; disruption of the CENP-C–PLK-1 interaction causes kinetochore component imbalance and chromosome congression defects distinct from the effects of BUB-1-targeted PLK-1 (which controls CDC-20 recruitment and SAC).\",\n      \"method\": \"Engineered protein interaction mutants, live imaging, immunofluorescence in C. elegans embryos\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — engineered mutants specifically disrupting CENP-C–PLK-1 interaction with clear functional phenotype, distinct from BUB-1 pathway\",\n      \"pmids\": [\"39355896\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"The CENP-C–Mis12 complex (Mis12C) interaction facilitates centromeric recruitment of Aurora B; Aurora B in turn reinforces the CENP-C–Mis12C interaction, creating a positive regulatory loop that ensures kinetochore biorientation and error correction.\",\n      \"method\": \"Immunofluorescence, co-immunoprecipitation, CENP-C mutant analysis in human RPE-1 cells and mouse models\",\n      \"journal\": \"Life science alliance\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP and mutant analysis with Aurora B recruitment readout; single lab, moderate evidence for regulatory loop\",\n      \"pmids\": [\"39433344\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Maternal CENP-C is recruited asymmetrically to paternal centromeres in the zygote, and this recruitment is required to equalize CENP-A levels between maternal and paternal centromeres before first mitosis; disruption of CENP-C dimerization impairs CENP-A equalization and chromosome segregation.\",\n      \"method\": \"CENP-A-mScarlet mouse model, live imaging, maternal CENP-C depletion, dimerization-disrupting mutants\",\n      \"journal\": \"Developmental cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — in vivo mouse model with specific maternal depletion and dimerization mutants; clear mechanistic role for CENP-C in epigenetic resetting at fertilization\",\n      \"pmids\": [\"40997799\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"KIF18A promotes chromosome alignment in cooperation with CENP-E downstream of CENP-C; a genome-wide Cas9 screen using a hypomorphic CENP-C mutant identified KIF18A as synthetic lethal, with the synthetic defect caused by reduction in CENP-E function in the CENP-C mutant background.\",\n      \"method\": \"Genome-wide Cas9 functional genetics screen, synthetic lethality, immunofluorescence\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic screen with functional follow-up placing KIF18A/CENP-E downstream of CENP-C, single lab\",\n      \"pmids\": [\"41218610\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"CENP-C is a conserved inner kinetochore protein that acts as a central scaffold for centromere/kinetochore assembly: it binds CENP-A nucleosomes (via two conserved domains that contact the CENP-A tail and the H2A/H2B acidic patch) and reshapes nucleosome structure through DNA gyre sliding, then recruits the outer kinetochore via direct high-affinity binding of its N-terminal motif to the Mis12 complex (and thence the full KMN network); its PEST domain additionally recruits the CENP-HIKM subcomplex to organize the full 16-subunit CCAN; CENP-C also promotes CENP-A chromatin maintenance by recruiting M18BP1/Mis18 complex; its C-terminal Cupin domain oligomerizes to organize centromeric chromatin architecture; CDK1-mediated phosphorylation of its C-terminal region enhances CENP-A binding during mitosis, while Aurora B phosphorylation of its N-terminal region modulates Mis12 interaction for error correction; CENP-C further serves as a platform for PLK-1 recruitment to the inner kinetochore and is required for centromeric DNA methylation via DNMT3B interaction, for spindle assembly checkpoint integrity, and—in meiosis—for CENP-A loading, sister centromere resolution, kinetochore mono-orientation, and centromere clustering.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"CENP-C is a conserved inner kinetochore scaffold that bridges centromeric chromatin to the outer kinetochore machinery and is essential for accurate chromosome segregation [#0, #10, #13]. It binds the CENP-A nucleosome directly, recognizing a hydrophobic region of the CENP-A tail and docking onto the H2A/H2B acidic patch through both its central region and the conserved CENP-C motif [#36, #48]; this engagement reshapes the nucleosome by rigidifying the histone core and driving lateral DNA gyre sliding toward canonical positioning [#37, #43]. CENP-C also contacts centromeric AT-rich/alpha-satellite DNA through internal DNA-binding/centromere-targeting domains, integrating histone and DNA recognition into contiguous binding modules [#3, #17, #45]. From this chromatin platform, the conserved N-terminal motif of CENP-C binds the Mis12 complex with high affinity to nucleate assembly of the full KMN network and outer kinetochore [#26, #33], while its PEST region recruits the CENP-HIKM subcomplex, establishing CENP-C as a blueprint for assembly of the broader CCAN [#38]. C-terminal Cupin-domain oligomerization and dimerization organize centromeric chromatin architecture and are required for CCAN localization [#51, #56]. CENP-C additionally sustains the centromeric epigenetic state by recruiting the M18BP1/Mis18 complex to drive new CENP-A loading [#34, #35] and by recruiting DNMT3B to direct centromeric DNA methylation, with its loss increasing missegregation and centromeric repeat transcription [#27]. These activities are gated by mitotic kinases: CDK1 phosphorylation of the C-terminal region enhances CENP-A binding, whereas Aurora B phosphorylation of the N-terminal region modulates the Mis12 interaction for error correction, forming a positive regulatory loop that reinforces biorientation [#42, #46, #47, #55]. Genetic and biochemical conservation from yeast Mif2 through worm, fly, and plant orthologs underlies roles in kinetochore assembly, the spindle assembly checkpoint, and meiotic functions including mono-orientation and PLK-1 recruitment [#4, #18, #29, #52].\",\n  \"teleology\": [\n    {\n      \"year\": 1992,\n      \"claim\": \"Established CENP-C as a physical constituent of the kinetochore, anchoring all subsequent mechanistic work at the inner kinetochore plate.\",\n      \"evidence\": \"Immunoelectron microscopy and immunofluorescence with anti-CENP-C antibodies on HeLa chromosomes; dicentric chromosome analysis showing presence only at active centromeres\",\n      \"pmids\": [\"1339310\", \"2475307\", \"8634687\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not define molecular interactions or how CENP-C is targeted\", \"No information on assembly hierarchy\"]\n    },\n    {\n      \"year\": 1994,\n      \"claim\": \"Demonstrated CENP-C is functionally required for kinetochore integrity and mitotic progression, and is itself a DNA-binding protein, moving it from marker to functional component.\",\n      \"evidence\": \"Nuclear antibody microinjection causing metaphase arrest and reduced kinetochore size; Southwestern blotting of recombinant CENP-C against alphoid DNA\",\n      \"pmids\": [\"8175879\", \"7883764\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"DNA binding shown in vitro only\", \"Did not identify protein partners mediating function\"]\n    },\n    {\n      \"year\": 1997,\n      \"claim\": \"Resolved CENP-C's modular architecture (oligomerization, DNA-binding, dimerization units) and proved it is necessary but not sufficient for centromere function via conditional loss/gain of function.\",\n      \"evidence\": \"Chemical crosslinking, gel filtration and Southwestern blotting of fragments; conditional gene disruption and overexpression in DT40 cells\",\n      \"pmids\": [\"9146917\", \"9361037\", \"10428958\", \"8668174\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional roles of individual domains not yet linked to specific partners\", \"Assembly hierarchy with other CENPs undefined\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Placed CENP-C within an ordered assembly pathway, defining CENP-A → CENP-H → CENP-C hierarchy and its dependence on upstream factors.\",\n      \"evidence\": \"Conditional knockouts in DT40 cells and RNAi in C. elegans with epistatic localization analysis\",\n      \"pmids\": [\"11500386\", \"11402064\", \"7579695\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct binding mechanism to CENP-A not yet structurally defined\", \"Outer kinetochore recruitment mechanism unknown\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Linked CENP-C to the spindle assembly checkpoint and outer-kinetochore Mis12 recruitment, and mapped its in vivo alpha-satellite DNA binding to discrete domains.\",\n      \"evidence\": \"Conditional knockout with Mad2 and Mis12 readouts in DT40; ChIP and truncation mapping for in vivo DNA binding\",\n      \"pmids\": [\"17392512\", \"12006616\", \"12490152\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether Mis12 recruitment is direct was not established here\", \"Mechanism connecting CENP-C to checkpoint signaling unresolved\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Identified the N-terminal domain of CENP-C as the direct recruiter of the Mis12/MIND complex and linked CENP-C to DNA methylation and CENP-A maintenance, broadening its role beyond structural scaffolding.\",\n      \"evidence\": \"Xenopus egg extract depletion-complementation with domain mutants; DNMT3B yeast two-hybrid, co-IP, bisulfite sequencing and ChIP; fission yeast scaffold and meiotic effector analysis\",\n      \"pmids\": [\"19641019\", \"19482874\", \"19758558\", \"19503796\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"High-resolution structure of the N-terminal–Mis12 interface not yet resolved\", \"Mechanism coupling CENP-C to DNMT3B recruitment in chromatin context incomplete\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Established that a conserved N-terminal motif binds the Mis12 complex with high affinity to nucleate the entire KMN network, defining CENP-C as the inner-to-outer kinetochore bridge, and that it recruits M18BP1 to drive CENP-A assembly.\",\n      \"evidence\": \"Direct biochemical binding reconstitution and dominant-negative/ectopic targeting in HeLa and Drosophila cells; in vitro M18BP1 binding with siRNA-based CENP-A assembly readout\",\n      \"pmids\": [\"21353556\", \"21353555\", \"21911481\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of the Mis12 interaction not yet solved at this stage\", \"Regulation of the recruitment by mitotic kinases not yet defined\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Provided the structural mechanism of CENP-A nucleosome recognition — CENP-A tail engagement plus H2A/H2B acidic-patch docking — explaining how CENP-C reads the centromeric histone mark.\",\n      \"evidence\": \"Crystallography, NMR, mutagenesis and biochemical binding assays across species\",\n      \"pmids\": [\"23723239\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not address how binding alters nucleosome dynamics\", \"Did not resolve regulation of the interaction in mitosis\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Showed CENP-C actively reshapes CENP-A nucleosome structure and serves as the assembly blueprint for the CCAN, linking chromatin reading to architectural organization.\",\n      \"evidence\": \"In vitro reconstitution with single-molecule FRET/FLIM and depletion in cells; biochemical reconstitution mapping the PEST–CENP-HIKM interaction; ectopic targeting separating CENP-C and CENP-T KMN recruitment\",\n      \"pmids\": [\"25954010\", \"26124289\", \"39433344\", \"25660545\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Quantitative contribution of nucleosome reshaping to centromere identity unresolved\", \"Coordination between parallel CENP-C and CENP-T recruitment arms incomplete\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Solved the MIS12C–CENP-C structure and defined gyre sliding as the mechanism of nucleosome reshaping, with Aurora B phosphorylation regulating the Mis12 interaction.\",\n      \"evidence\": \"X-ray crystallography of MIS12C–CENP-C, Aurora B kinase assays, and single-molecule FRET measuring DNA gyre sliding\",\n      \"pmids\": [\"27881301\", \"26878239\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo dynamics of phospho-regulation across the cell cycle incompletely mapped\", \"Functional consequence of gyre sliding for centromere inheritance not fully established\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Defined CENP-C's combined DNA- and histone-binding module and revealed dependence on centromeric RNA, integrating nucleic acid and histone recognition.\",\n      \"evidence\": \"Biochemical binding and mutational analysis of the DHBD in yeast and human CENP-C; RNA-IP and RNA depletion affecting CENP-C levels; CO-FISH showing centromere integrity maintenance\",\n      \"pmids\": [\"29074736\", \"28787590\", \"28167779\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism by which RNA stabilizes CENP-C–DNA binding in vivo incompletely defined\", \"Role in repeat integrity mechanistically separate from segregation not fully dissected\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Established kinase control of CENP-C: CDK1 phosphorylation of the C-terminus enhances CENP-A binding while Aurora B phosphorylation of the N-terminus tunes Mis12 binding, defining cell-cycle-coupled regulation of its bridging functions.\",\n      \"evidence\": \"In vitro CDK1 kinase assays, Phos-tag, co-IP and viability assays in chicken/human cells; Aurora B phosphorylation of S. pombe Cnp3 with phosphomimetic mutants; cryo-EM of CENP-A nucleosome with CENP-C central region\",\n      \"pmids\": [\"31676716\", \"29180432\", \"31475439\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Temporal interplay between CDK1 and Aurora B inputs on the same protein unresolved\", \"How phospho-states are reset across mitosis not defined\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Defined C-terminal Cupin-domain oligomerization as essential for centromeric chromatin organization and CCAN recruitment, and revealed auto-inhibition relieved by CENP-A binding.\",\n      \"evidence\": \"Crystal structure of the Cupin domain with biochemical oligomerization assays and mutagenesis in chicken cells; in vitro auto-inhibition reconstitution with yeast genetics; C. elegans PLK-1 recruitment analysis\",\n      \"pmids\": [\"37295434\", \"32515113\", \"37067150\", \"36736323\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How oligomerization is spatially restricted to functional centromeres unclear\", \"Integration of auto-inhibition with kinase regulation not resolved\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Demonstrated in vivo roles for CENP-C dimerization in epigenetic resetting at fertilization and placed downstream alignment factors in its pathway, extending its function to developmental and effector contexts.\",\n      \"evidence\": \"Mouse CENP-A-mScarlet live imaging with maternal CENP-C depletion and dimerization mutants; genome-wide Cas9 synthetic-lethal screen with hypomorphic CENP-C identifying KIF18A/CENP-E\",\n      \"pmids\": [\"40997799\", \"41218610\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism of asymmetric maternal CENP-C recruitment to paternal centromeres undefined\", \"Direct biochemical link between CENP-C and KIF18A/CENP-E not established\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How CENP-C's multiple regulatory inputs (CDK1, Aurora B, PEST phosphorylation, auto-inhibition, RNA, oligomerization) are temporally integrated to coordinate CENP-A inheritance, CCAN assembly, and outer kinetochore biorientation within a single cell cycle remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No unified model linking the distinct phospho-regulated and oligomerization-dependent states\", \"Stoichiometry and dynamics of CENP-C at endogenous centromeres incompletely defined\", \"Causal role in human disease not established in the corpus\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0003677\", \"supporting_discovery_ids\": [3, 5, 17, 45]},\n      {\"term_id\": \"GO:0042393\", \"supporting_discovery_ids\": [36, 37, 48]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [26, 33, 38]},\n      {\"term_id\": \"GO:0005198\", \"supporting_discovery_ids\": [13, 38, 51]},\n      {\"term_id\": \"GO:0003723\", \"supporting_discovery_ids\": [44, 50]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [34, 41]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005694\", \"supporting_discovery_ids\": [0, 1, 6]},\n      {\"term_id\": \"GO:0000228\", \"supporting_discovery_ids\": [0, 17]},\n      {\"term_id\": \"GO:0005730\", \"supporting_discovery_ids\": [8]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [11]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [10, 13, 18, 39]},\n      {\"term_id\": \"R-HSA-4839726\", \"supporting_discovery_ids\": [27, 34, 37, 51]},\n      {\"term_id\": \"R-HSA-1474165\", \"supporting_discovery_ids\": [29, 52, 56]}\n    ],\n    \"complexes\": [\"CCAN\", \"kinetochore\"],\n    \"partners\": [\"CENP-A\", \"MIS12\", \"M18BP1\", \"DNMT3B\", \"CENP-HIKM\", \"PLK-1\", \"AURKB\", \"CENP-B\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":8,"faith_total":8,"faith_pct":100.0}}