{"gene":"CENPT","run_date":"2026-04-28T17:28:52","timeline":{"discoveries":[{"year":2012,"finding":"CENP-T-W and CENP-S-X complexes coassemble to form a stable CENP-T-W-S-X heterotetramer with histone-fold domains that structurally resembles a nucleosome; the heterotetramer binds DNA and introduces supercoiling, and mutations disrupting heterotetramerization or DNA-protein contacts reduce DNA binding/supercoiling in vitro and compromise kinetochore assembly in vivo.","method":"Crystal structure, in vitro DNA binding and supercoiling assays, active-site/interface mutagenesis, in vivo kinetochore assembly assays","journal":"Cell","confidence":"High","confidence_rationale":"Tier 1 — high-resolution structure combined with reconstitution, mutagenesis, and in vivo validation in a single highly-cited study","pmids":["22304917"],"is_preprint":false},{"year":2013,"finding":"The N-terminal disordered region of vertebrate CENP-T directly interacts with the RWD domain of Spc24/Spc25 (Ndc80 complex); CDK phosphorylation of CENP-T strengthens a cryptic hydrophobic interaction with Spc25 in a phospho-regulated manner that does not require direct recognition of the phosphorylated residue. The CENP-T–Ndc80 and Mis12–Ndc80 interactions are mutually exclusive, defining two parallel pathways for Ndc80 recruitment.","method":"X-ray crystal structure, ITC, phospho-mutant analysis, co-immunoprecipitation, in vivo kinetochore recruitment assays","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 1 — crystal structure plus mutagenesis and in vivo functional validation, replicated concept across multiple papers","pmids":["23334297"],"is_preprint":false},{"year":2012,"finding":"The histone-fold protein Cnn1 (yeast CENP-T ortholog) is a direct centromere receptor of the Ndc80 complex; its conserved N-terminal peptide motif mediates stoichiometric binding to the Spc24-Spc25 domain; artificial tethering of Ndc80 through Cnn1 supports mini-chromosome segregation without a natural centromere.","method":"Biochemical reconstitution, pulldown, in vivo genetic complementation assay, mini-chromosome segregation assay","journal":"Nature cell biology","confidence":"High","confidence_rationale":"Tier 1-2 — biochemical reconstitution plus genetic epistasis, highly cited, independently confirmed","pmids":["22561346"],"is_preprint":false},{"year":2013,"finding":"The CENP-T-W-S-X complex preferentially binds ~100 bp of linker DNA (not nucleosome-bound DNA), primarily as a (CENP-T-W-S-X)₂ dimer of tetramers, and unlike canonical nucleosomes induces positive rather than negative DNA supercoils; DNA-binding regions in CENP-T and CENP-W (but not CENP-S or CENP-X) are required for positive supercoiling and kinetochore targeting.","method":"In vitro DNA binding assays, supercoiling assays, domain mutagenesis, in vivo localization assays","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 1 — in vitro reconstitution with mutagenesis identifying specific subunits required for activity","pmids":["24234442"],"is_preprint":false},{"year":2015,"finding":"CENP-T and CENP-C act in parallel but distinct pathways to recruit the KMN network: CENP-C recruits Ndc80 via KNL1 and Mis12, whereas CENP-T directly interacts with Ndc80, which in turn recruits KNL1/Mis12. Aurora B kinase promotes KMN recruitment to CENP-C, while CDK regulates KMN recruitment to CENP-T.","method":"Ectopic chromosomal locus targeting, co-immunoprecipitation, kinase inhibitor treatment, quantitative fluorescence imaging","journal":"Current biology : CB","confidence":"High","confidence_rationale":"Tier 2 — independent pathway separation by ectopic targeting with multiple orthogonal readouts, replicated across labs","pmids":["25660545"],"is_preprint":false},{"year":2016,"finding":"CDK1:Cyclin B phosphorylates CENP-T at three distinct N-terminal sites, enabling CENP-T to bind one MIS12:NDC80 complex and two additional NDC80 complexes; CENP-C and CENP-T together can recruit two MIS12 and up to four NDC80 complexes, explaining stoichiometry of kinetochore components.","method":"In vitro reconstitution, phospho-mutant analysis, electron microscopy visualization of reconstituted complexes, quantitative binding assays","journal":"eLife","confidence":"High","confidence_rationale":"Tier 1 — reconstitution with phospho-mutants and EM visualization, highly cited","pmids":["28012276"],"is_preprint":false},{"year":2016,"finding":"The histone chaperone FACT (subunits Spt16/SSRP1) binds CENP-T/W; the C-terminal domain of Spt16 specifically binds the histone fold region of CENP-T/W. Depletion of Spt16 impairs CENP-T and CENP-W deposition at centromeres, and site-directed targeting of Spt16 alone is sufficient to drive de novo CENP-T accumulation at centromeres.","method":"Proteomic screen, co-immunoprecipitation, domain mapping, RNAi depletion, ectopic targeting assay, immunofluorescence","journal":"Genes & development","confidence":"High","confidence_rationale":"Tier 2 — reciprocal Co-IP, domain mapping, and functional depletion/targeting experiments with multiple readouts","pmids":["27284163"],"is_preprint":false},{"year":2008,"finding":"In living human cells, CENP-T directly associates with CENP-A and CENP-B (detected by FRET); CENP-T exchange at centromeres is restricted to S-phase (shown by FRAP), indicating a co-replicational loading mechanism.","method":"Acceptor-bleaching FRET, FRAP in live cells","journal":"Journal of biophotonics","confidence":"Medium","confidence_rationale":"Tier 2 — live-cell FRET and FRAP with functional interpretation, single lab study","pmids":["19412974"],"is_preprint":false},{"year":2013,"finding":"CSN5/JAB1 directly interacts with both CENP-T and CENP-W (yeast two-hybrid and co-immunoprecipitation) and promotes ubiquitin- and proteasome-dependent degradation of CENP-T and CENP-W; formation of the CENP-T/W complex enhances protein stability by blocking CSN5-mediated degradation; CSN5 dysregulation impairs CENP-T/W recruitment to kinetochores during prophase.","method":"Yeast two-hybrid, co-immunoprecipitation, proteasome inhibitor experiments, in vivo localization assays","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2-3 — yeast two-hybrid confirmed by Co-IP with functional degradation assay, single lab","pmids":["23926101"],"is_preprint":false},{"year":2015,"finding":"In budding yeast, Cnn1 (CENP-T) harbors two kinetochore-localization activities: a C-terminal histone-fold domain associating with the centromere region, and an N-terminal Spc24/Spc25 interaction sequence (residues 25-91) mediating linkage to the Ndc80 complex; Mps1 kinase phosphorylates Cnn1-S74, regulating its interaction with Ndc80 and modulating kinetochore accumulation from G1 through metaphase.","method":"In vivo localization by fluorescence microscopy, domain deletion/mutation analysis, kinase in vitro phosphorylation assay, genetic epistasis","journal":"Genetics","confidence":"Medium","confidence_rationale":"Tier 2 — multiple domain mutants with in vivo functional readouts, single lab","pmids":["25716979"],"is_preprint":false},{"year":2018,"finding":"CENP-T directly binds HJURP (a CENP-A chaperone) via the C-terminus of CENP-T; HJURP knockout minimizes CENP-T recruitment to centromeres; a HJURP-binding-deficient CENP-T mutant fails to localize to centromeres; HJURP recruits CENP-T in S/G2 phase.","method":"Co-immunoprecipitation, CRISPR knockout, domain mapping mutagenesis, immunofluorescence cell cycle staging","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 — CRISPR KO plus domain-mapping Co-IP and localization assay, single lab","pmids":["30459232"],"is_preprint":false},{"year":2015,"finding":"In fission yeast, the CENP-A (Cnp1) N-tail specifically promotes localization of CENP-T (Cnp20) (but not CENP-C) at centromeres; overexpression of CENP-T suppresses centromere inactivation defects caused by N-tail mutations, placing CENP-T downstream of the CENP-A N-tail in a pathway that maintains epigenetic centromere stability.","method":"Genetic epistasis (suppressor overexpression), fluorescence microscopy localization, synthetic lethality analysis","journal":"Current biology : CB","confidence":"Medium","confidence_rationale":"Tier 2 — genetic epistasis with suppressor rescue, fission yeast ortholog","pmids":["25619765"],"is_preprint":false},{"year":2016,"finding":"By in vivo FRET in human cells, the CENP-T C-terminus is specifically proximal to histone H3.1 (but not H3.2, H3.3, or CENP-A), suggesting CENP-T bridges a CENP-A-containing and an H3.1-containing nucleosome at centromeres.","method":"In vivo acceptor-bleaching FRET in live human cells","journal":"International journal of molecular sciences","confidence":"Medium","confidence_rationale":"Tier 2 — direct FRET measurement in live cells with multiple histone variants tested as controls, single lab","pmids":["25775162"],"is_preprint":false},{"year":2016,"finding":"ChIP-seq and sequential ChIP in human cells show that CENPT is centered over the CENPB box between two well-positioned CENPA nucleosomes on α-satellite dimers and physically interacts with the CENPB/CENPC complex; cross-linking captures the entire CENPA/CENPB/CENPC/CENPT complex over an α-satellite dimer.","method":"ChIP-seq, sequential ChIP, base-pair resolution genomic readout","journal":"Genome research","confidence":"Medium","confidence_rationale":"Tier 2 — genome-wide base-pair resolution mapping with sequential ChIP validation, single lab","pmids":["27384170"],"is_preprint":false},{"year":2018,"finding":"In budding yeast, de novo kinetochore assembly assay demonstrates that when the Mis12 pathway is crippled (Dsn1 phosphorylation defect), the CENP-T pathway becomes essential for viability and Ndc80 complex recruitment, establishing functional redundancy and epistatic relationship between the two Ndc80 recruitment pathways.","method":"De novo kinetochore assembly in yeast extracts, genetic epistasis, microtubule-binding assay","journal":"eLife","confidence":"Medium","confidence_rationale":"Tier 2 — novel in vitro assembly assay combined with genetic epistasis in budding yeast ortholog","pmids":["30117803"],"is_preprint":false},{"year":2020,"finding":"Crystal structure of the Ctf3 complex (yeast CENP-I module) bound to the Cnn1-Wip1 (CENP-T/W) heterodimer reveals the structural basis for Ctf3c and Cnn1-Wip1 co-recruitment to the kinetochore; live-cell imaging provides a feedback regulation mechanism for Ctf19c assembly.","method":"High-resolution crystal structure, live-cell imaging","journal":"Current biology : CB","confidence":"Medium","confidence_rationale":"Tier 1 — crystal structure with live-cell imaging in yeast ortholog, single study","pmids":["32679099"],"is_preprint":false},{"year":2021,"finding":"In fission yeast, CDK1-mediated phosphorylation of the Ccp1-interaction motif (CIM) at the N-terminus of CENP-T disrupts Ccp1 binding, enabling competitive displacement of Ccp1 by Ndc80; the phospho-null CIM mutant retains Ccp1 at centromeres during mitosis and mispositions the Ndc80 complex, causing chromosome missegregation.","method":"Phospho-mutant analysis, co-immunoprecipitation, live-cell imaging, chromosome segregation assays in fission yeast","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"Medium","confidence_rationale":"Tier 2 — phospho-mutant genetic and biochemical analysis with in vivo functional readout, fission yeast ortholog","pmids":["34810257"],"is_preprint":false},{"year":2022,"finding":"In chicken DT40 cells, two copies of Ndc80 complex (N-N) on CENP-T (one via direct binding, one via Mis12C) are required for proper kinetochore-microtubule interactions; artificial direct attachment of two Ndc80 complexes to CENP-T can substitute for the native Mis12C-mediated linkage, demonstrating N-N functionality is independent of direct Mis12C-Ndc80 binding.","method":"DT40 cell genetic engineering (interaction mutants), artificial kinetochore tethering, chromosome segregation and spindle checkpoint assays","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal genetic and functional experiments with defined molecular mutants in vertebrate cells","pmids":["35165266"],"is_preprint":false},{"year":2024,"finding":"The CENP-T–Mis12 complex interaction occurs via three binding surfaces (identified by AlphaFold2 combined with biochemical validation); this interaction is cooperatively regulated by dual phosphorylation of Dsn1 (Mis12C component) and CENP-T, ensuring robust Mis12C recruitment to CENP-T during mitosis.","method":"AlphaFold2 structure prediction combined with cell biological and biochemical validation, phospho-mutant analysis in DT40 cells","journal":"iScience","confidence":"Medium","confidence_rationale":"Tier 2 — structural prediction validated by biochemical and cell biological assays with phospho-mutants, single lab","pmids":["39628583"],"is_preprint":false},{"year":2024,"finding":"Aurora B phosphorylates CENP-W at threonine 60, which enhances the CENP-W–CENP-T interaction (via the histone fold domain and an uncharacterized N-terminal region of CENP-T) to ensure robust metaphase chromosome alignment and accurate chromosome segregation.","method":"In vitro kinase assay, co-immunoprecipitation, phospho-mutant analysis, live-cell imaging, chromosome segregation assays","journal":"Journal of molecular cell biology","confidence":"Medium","confidence_rationale":"Tier 2 — kinase assay with phospho-mutant Co-IP and in vivo segregation readout, single lab","pmids":["38200711"],"is_preprint":false},{"year":2024,"finding":"Ndc80 binding to CENP-T is a two-step process: rapid initial association/dissociation at disordered N-terminal sites followed by a slower 'maturation' transition to stronger retention; this maturation kinetic barrier is markedly accelerated when CENP-T is clustered at high molecular density, explaining why clustered CENP-T recruits more Ndc80 than monomeric CENP-T, and the two Ndc80-binding sites on CENP-T exhibit distinct maturation rates.","method":"Quantitative in vitro binding assays with clustered vs. monomeric CENP-T, single-molecule imaging, live dividing human cell analysis","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 — in vitro reconstitution with quantitative kinetic analysis plus live-cell validation, published in peer-reviewed journal","pmids":["39700145"],"is_preprint":false},{"year":2020,"finding":"Depletion of CENP-T by siRNA in mouse oocytes increases CDH1/FZR1 levels, elevating APC-CDH1 activity and decreasing CCNB1, thereby attenuating MPF and severely compromising meiotic resumption (G2/M transition); these defects are rescued by CCNB1 overexpression or CDH1 knockdown, placing CENP-T upstream of CDH1 in regulating meiotic progression.","method":"siRNA knockdown, overexpression rescue, western blot for CDH1 and CCNB1, MPF activity assay, genetic epistasis in mouse oocytes","journal":"Journal of cell science","confidence":"Medium","confidence_rationale":"Tier 2 — multiple orthogonal rescue experiments establishing pathway position, single lab in mouse oocytes","pmids":["31964702"],"is_preprint":false},{"year":2016,"finding":"In Xenopus egg extracts, CENP-T centromeric recruitment occurs in late interphase independently of DNA synthesis and precedes CENP-W recruitment (which occurs in mitosis); unlike CENP-C, CENP-T does not participate in CENP-A deposition; depletion of CENP-C reduces CENP-T at centromeres, but kinetochores can still assemble with reduced Ndc80/Mis12, supporting the existence of two parallel assembly pathways.","method":"Xenopus egg cell-free extract immunodepletion, cell cycle staging, immunofluorescence, western blot","journal":"Nucleus (Austin, Tex.)","confidence":"Medium","confidence_rationale":"Tier 2 — cell-free reconstitution with immunodepletion and temporal staging, Xenopus ortholog study","pmids":["25569378"],"is_preprint":false},{"year":2025,"finding":"CENPT interacts with GCLC (γ-glutamyl-cysteine ligase catalytic subunit) by binding to residues 213-424 of GCLC competitively with GCLM (the modifier subunit), increasing GCLC catalytic activity and glutathione synthesis, thereby reducing ROS and inhibiting ferroptosis in renal cell carcinoma cells.","method":"Co-immunoprecipitation, domain mapping, ROS measurements, ferroptosis assays, GSH quantification","journal":"Cell death & disease","confidence":"Low","confidence_rationale":"Tier 3 — Co-IP with functional assays, single lab, non-canonical CENPT function outside kinetochore biology","pmids":["40651948"],"is_preprint":false},{"year":2025,"finding":"Adaptive evolution of the CENP-T histone fold domain (DNA-binding region) in mice reduced centromere binding; introducing the histone fold domain from closely related species into mouse CENP-T (chimeric variants) increased centromere binding in oocytes and somatic cells; reduced binding by mouse CENP-T supports robust female gametogenesis, and the adaptation is independent of centromeric DNA sequence.","method":"Transgenic mouse models with chimeric CENP-T variants, oocyte microinjection, quantitative centromere binding assays, gametogenesis phenotype analysis","journal":"Current biology : CB","confidence":"Medium","confidence_rationale":"Tier 2 — transgenic mouse model with chimeric proteins and quantitative binding assays","pmids":["39947176"],"is_preprint":false}],"current_model":"CENP-T is an inner kinetochore histone-fold protein that, as part of the CENP-T-W-S-X heterotetramer, forms a nucleosome-like structure that wraps and positively supercoils linker DNA at centromeres; its disordered N-terminal region serves as a phospho-regulated (CDK1) scaffold that directly recruits up to two Ndc80 complexes and one Mis12:Ndc80 complex to the outer kinetochore, acting in parallel with CENP-C to collectively recruit two MIS12 and up to four NDC80 complexes per kinetochore, while FACT chaperone mediates its cell-cycle-coupled deposition at centromeres and Aurora B/CDK phosphorylation events fine-tune assembly and stability of these interactions."},"narrative":{"teleology":[{"year":2008,"claim":"Before the molecular function of CENP-T was known, live-cell FRET and FRAP established that CENP-T physically associates with CENP-A chromatin and is deposited at centromeres specifically during S phase, framing it as a replication-coupled centromere component.","evidence":"Acceptor-bleaching FRET and FRAP in live human cells","pmids":["19412974"],"confidence":"Medium","gaps":["Single-lab FRET study without biochemical confirmation of direct CENP-A contact","Mechanism of S-phase coupling unknown"]},{"year":2012,"claim":"Structural and biochemical work revealed that CENP-T forms a nucleosome-like CENP-T-W-S-X heterotetramer with histone-fold domains that binds and positively supercoils DNA, establishing the molecular basis for how CENP-T integrates into centromeric chromatin, while yeast reconstitution showed the conserved N-terminal peptide directly recruits the Ndc80 complex via Spc24-Spc25.","evidence":"Crystal structure, in vitro DNA-binding/supercoiling assays, mutagenesis, in vivo kinetochore assembly (human); biochemical reconstitution and mini-chromosome segregation assay (yeast)","pmids":["22304917","22561346"],"confidence":"High","gaps":["How the heterotetramer is positioned relative to CENP-A nucleosomes in vivo","Stoichiometry of DNA wrapping at native centromeres"]},{"year":2013,"claim":"Crystal structures of the CENP-T N-terminus bound to Ndc80 RWD domains revealed that CDK phosphorylation strengthens this interaction through a cryptic hydrophobic mechanism, and demonstrated that CENP-T–Ndc80 and Mis12–Ndc80 binding are mutually exclusive, defining two independent Ndc80 recruitment routes.","evidence":"X-ray crystallography, ITC, phospho-mutant analysis, co-immunoprecipitation, in vivo recruitment assays (human); DNA preference and supercoiling domain analysis","pmids":["23334297","24234442"],"confidence":"High","gaps":["How the two pathways are coordinated temporally in mitosis","Whether CDK phosphorylation is switch-like or graded in vivo"]},{"year":2015,"claim":"Ectopic targeting experiments separated the CENP-T and CENP-C pathways: Aurora B promotes KMN recruitment to CENP-C while CDK regulates KMN recruitment to CENP-T, establishing distinct kinase-dependent regulatory logic for each parallel pathway.","evidence":"Ectopic chromosomal locus targeting, kinase inhibitor treatment, quantitative fluorescence imaging in human cells; genetic epistasis and Mps1 regulation in budding yeast","pmids":["25660545","25716979"],"confidence":"High","gaps":["Quantitative contribution of each pathway to total kinetochore-microtubule attachment strength","Cross-talk between Aurora B and CDK at CENP-T"]},{"year":2016,"claim":"Reconstitution of the full phospho-regulated CENP-T scaffold showed that CDK1-dependent triple phosphorylation enables recruitment of one Mis12:Ndc80 complex and two additional Ndc80 complexes, and together with CENP-C accounts for the measured kinetochore stoichiometry of two MIS12 and up to four NDC80 complexes.","evidence":"In vitro reconstitution with phospho-mutants, electron microscopy, quantitative binding assays","pmids":["28012276"],"confidence":"High","gaps":["Whether this stoichiometry is invariant across cell types","How stoichiometry is maintained during metaphase oscillations"]},{"year":2016,"claim":"Identification of the FACT chaperone as the loading machine for CENP-T/W answered how CENP-T is deposited at centromeres: FACT binds the histone-fold domain of CENP-T/W and is sufficient for de novo centromeric accumulation, while genomic mapping placed CENP-T over CENP-B boxes between flanking CENP-A nucleosomes.","evidence":"Proteomic screen, reciprocal Co-IP, domain mapping, RNAi depletion, ectopic targeting (human cells); ChIP-seq and sequential ChIP at base-pair resolution","pmids":["27284163","27384170"],"confidence":"High","gaps":["Whether FACT and HJURP act sequentially or in parallel for CENP-T loading","Structural basis of FACT–CENP-T interaction"]},{"year":2018,"claim":"HJURP was identified as a second chaperone that directly binds the CENP-T C-terminus and is required for S/G2-phase CENP-T recruitment, while yeast de novo assembly assays proved the CENP-T and Mis12 pathways are functionally redundant for Ndc80 recruitment and viability.","evidence":"CRISPR knockout, domain mapping, Co-IP, cell-cycle staged immunofluorescence (human); in vitro kinetochore assembly and genetic epistasis (yeast)","pmids":["30459232","30117803"],"confidence":"Medium","gaps":["Whether HJURP–CENP-T interaction occurs on or off chromatin","How FACT and HJURP coordinate during the loading cycle"]},{"year":2020,"claim":"Structural resolution of the yeast CENP-I module (Ctf3c) bound to CENP-T/W revealed the architectural basis for inner kinetochore co-recruitment, while oocyte studies uncovered an unexpected role for CENP-T upstream of APC-CDH1 in meiotic resumption.","evidence":"Crystal structure and live-cell imaging (yeast); siRNA knockdown with overexpression rescue in mouse oocytes","pmids":["32679099","31964702"],"confidence":"Medium","gaps":["Whether the CDH1 regulatory role is kinetochore-dependent or represents a moonlighting function","Whether Ctf3c–CENP-T/W structural interface is conserved in vertebrates"]},{"year":2021,"claim":"CDK1 phosphorylation of the CENP-T N-terminal CIM motif was shown to act as a molecular switch that displaces the interphase partner Ccp1 to allow mitotic Ndc80 binding, providing a mechanism for the temporal transition from centromere maintenance to kinetochore assembly.","evidence":"Phospho-mutant analysis, Co-IP, live-cell imaging, chromosome segregation assays in fission yeast","pmids":["34810257"],"confidence":"Medium","gaps":["Whether an analogous switching mechanism operates in vertebrates","Identity of a vertebrate Ccp1 equivalent"]},{"year":2022,"claim":"Genetic engineering in vertebrate DT40 cells demonstrated that two Ndc80 complexes on a single CENP-T molecule are functionally required for proper kinetochore–microtubule interactions, and that artificial direct tethering of two Ndc80 copies can bypass the need for Mis12-mediated linkage.","evidence":"Interaction-specific mutations, artificial tethering constructs, chromosome segregation and spindle checkpoint assays in chicken DT40 cells","pmids":["35165266"],"confidence":"High","gaps":["Whether this design principle holds for human kinetochores in vivo","How two Ndc80 copies on one CENP-T engage a single microtubule"]},{"year":2024,"claim":"Three complementary studies resolved outstanding questions: the CENP-T–Mis12 interface involves three binding surfaces cooperatively regulated by dual phosphorylation; Aurora B phosphorylation of CENP-W T60 stabilizes the CENP-W–CENP-T interaction for accurate segregation; and Ndc80 binding to CENP-T proceeds through a two-step maturation mechanism that is dramatically accelerated by molecular clustering of CENP-T.","evidence":"AlphaFold2 prediction with biochemical validation and phospho-mutants (DT40); kinase assay, Co-IP, and segregation assays; quantitative in vitro kinetics with single-molecule imaging and live-cell validation (human)","pmids":["39628583","38200711","39700145"],"confidence":"High","gaps":["In vivo clustering stoichiometry at native centromeres","Whether maturation kinetics differ across cell types or species"]},{"year":2025,"claim":"Adaptive evolution of the CENP-T histone-fold domain in mice reduced centromere binding in a manner that supports female gametogenesis, revealing that CENP-T centromere affinity is under evolutionary selection independent of centromeric DNA sequence.","evidence":"Transgenic mouse models with chimeric CENP-T variants, quantitative centromere binding assays, gametogenesis phenotyping","pmids":["39947176"],"confidence":"Medium","gaps":["Whether this adaptation impacts kinetochore strength or meiotic drive","Molecular basis of the histone-fold changes that alter binding"]},{"year":null,"claim":"Key unresolved questions include the precise structural organization of the CENP-T-W-S-X complex on native centromeric chromatin in situ, the coordination between FACT and HJURP chaperones during CENP-T deposition, and whether the Ndc80 maturation kinetics observed in vitro govern error correction dynamics in vivo.","evidence":"","pmids":[],"confidence":"Low","gaps":["No in situ structural view of CENP-T on centromeric chromatin","FACT vs. HJURP temporal coordination unresolved","Physiological relevance of Ndc80 binding maturation kinetics for error correction"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0003677","term_label":"DNA binding","supporting_discovery_ids":[0,3]},{"term_id":"GO:0005198","term_label":"structural molecule activity","supporting_discovery_ids":[0,1,5,17]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[1,2,5,17]}],"localization":[{"term_id":"GO:0005694","term_label":"chromosome","supporting_discovery_ids":[0,3,7,13]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[7,12]}],"pathway":[{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[4,5,16,17]},{"term_id":"R-HSA-4839726","term_label":"Chromatin organization","supporting_discovery_ids":[0,3,13]}],"complexes":["CENP-T-W-S-X heterotetramer","CCAN (constitutive centromere-associated network)"],"partners":["CENPW","CENPS","CENPX","SPC24","SPC25","DSN1","SUPT16H","HJURP"],"other_free_text":[]},"mechanistic_narrative":"CENP-T is an inner kinetochore histone-fold protein that nucleates a parallel pathway for outer kinetochore assembly at centromeres. Together with CENP-W, CENP-S, and CENP-X, it forms a nucleosome-like heterotetramer that binds centromeric linker DNA and introduces positive supercoils, with DNA contacts in CENP-T and CENP-W essential for this activity and for kinetochore targeting [PMID:22304917, PMID:24234442]. The intrinsically disordered N-terminal region of CENP-T functions as a CDK1-phosphorylated scaffold that directly recruits up to two Ndc80 complexes and one Mis12-associated Ndc80 complex, operating in parallel with the CENP-C pathway to collectively assemble two MIS12 and up to four NDC80 complexes per kinetochore unit [PMID:23334297, PMID:28012276, PMID:35165266]. Cell-cycle-coupled deposition of CENP-T at centromeres is mediated by the FACT chaperone complex and HJURP, while Aurora B and CDK1 phosphorylation events regulate CENP-T–CENP-W stability and the phospho-dependent switching from interphase binding partners to mitotic Ndc80 engagement [PMID:27284163, PMID:30459232, PMID:34810257, PMID:38200711]."},"prefetch_data":{"uniprot":{"accession":"Q96BT3","full_name":"Centromere protein T","aliases":["Interphase centromere complex protein 22"],"length_aa":561,"mass_kda":60.4,"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. Part of a nucleosome-associated complex that binds specifically to histone H3-containing nucleosomes at the centromere, as opposed to nucleosomes containing CENPA. Component of the heterotetrameric CENP-T-W-S-X complex that binds and supercoils DNA, and plays an important role in kinetochore assembly. CENPT has a fundamental role in kinetochore assembly and function. It is one of the inner kinetochore proteins, with most further proteins binding downstream. Required for normal chromosome organization and normal progress through mitosis","subcellular_location":"Nucleus; Chromosome, centromere; Chromosome, centromere, kinetochore","url":"https://www.uniprot.org/uniprotkb/Q96BT3/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":true,"resolved_as":"","url":"https://depmap.org/portal/gene/CENPT","classification":"Common Essential","n_dependent_lines":1064,"n_total_lines":1208,"dependency_fraction":0.8807947019867549},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/CENPT","total_profiled":1310},"omim":[{"mim_id":"618702","title":"SHORT STATURE AND MICROCEPHALY WITH GENITAL ANOMALIES; SSMGA","url":"https://www.omim.org/entry/618702"},{"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"},{"mim_id":"611264","title":"CENTROMERIC PROTEIN W; CENPW","url":"https://www.omim.org/entry/611264"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Nuclear bodies","reliability":"Supported"},{"location":"Nucleoplasm","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in 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\"Crystal structure, in vitro DNA binding and supercoiling assays, active-site/interface mutagenesis, in vivo kinetochore assembly assays\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — high-resolution structure combined with reconstitution, mutagenesis, and in vivo validation in a single highly-cited study\",\n      \"pmids\": [\"22304917\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"The N-terminal disordered region of vertebrate CENP-T directly interacts with the RWD domain of Spc24/Spc25 (Ndc80 complex); CDK phosphorylation of CENP-T strengthens a cryptic hydrophobic interaction with Spc25 in a phospho-regulated manner that does not require direct recognition of the phosphorylated residue. The CENP-T–Ndc80 and Mis12–Ndc80 interactions are mutually exclusive, defining two parallel pathways for Ndc80 recruitment.\",\n      \"method\": \"X-ray crystal structure, ITC, phospho-mutant analysis, co-immunoprecipitation, in vivo kinetochore recruitment assays\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — crystal structure plus mutagenesis and in vivo functional validation, replicated concept across multiple papers\",\n      \"pmids\": [\"23334297\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"The histone-fold protein Cnn1 (yeast CENP-T ortholog) is a direct centromere receptor of the Ndc80 complex; its conserved N-terminal peptide motif mediates stoichiometric binding to the Spc24-Spc25 domain; artificial tethering of Ndc80 through Cnn1 supports mini-chromosome segregation without a natural centromere.\",\n      \"method\": \"Biochemical reconstitution, pulldown, in vivo genetic complementation assay, mini-chromosome segregation assay\",\n      \"journal\": \"Nature cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — biochemical reconstitution plus genetic epistasis, highly cited, independently confirmed\",\n      \"pmids\": [\"22561346\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"The CENP-T-W-S-X complex preferentially binds ~100 bp of linker DNA (not nucleosome-bound DNA), primarily as a (CENP-T-W-S-X)₂ dimer of tetramers, and unlike canonical nucleosomes induces positive rather than negative DNA supercoils; DNA-binding regions in CENP-T and CENP-W (but not CENP-S or CENP-X) are required for positive supercoiling and kinetochore targeting.\",\n      \"method\": \"In vitro DNA binding assays, supercoiling assays, domain mutagenesis, in vivo localization assays\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro reconstitution with mutagenesis identifying specific subunits required for activity\",\n      \"pmids\": [\"24234442\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"CENP-T and CENP-C act in parallel but distinct pathways to recruit the KMN network: CENP-C recruits Ndc80 via KNL1 and Mis12, whereas CENP-T directly interacts with Ndc80, which in turn recruits KNL1/Mis12. Aurora B kinase promotes KMN recruitment to CENP-C, while CDK regulates KMN recruitment to CENP-T.\",\n      \"method\": \"Ectopic chromosomal locus targeting, co-immunoprecipitation, kinase inhibitor treatment, quantitative fluorescence imaging\",\n      \"journal\": \"Current biology : CB\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — independent pathway separation by ectopic targeting with multiple orthogonal readouts, replicated across labs\",\n      \"pmids\": [\"25660545\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"CDK1:Cyclin B phosphorylates CENP-T at three distinct N-terminal sites, enabling CENP-T to bind one MIS12:NDC80 complex and two additional NDC80 complexes; CENP-C and CENP-T together can recruit two MIS12 and up to four NDC80 complexes, explaining stoichiometry of kinetochore components.\",\n      \"method\": \"In vitro reconstitution, phospho-mutant analysis, electron microscopy visualization of reconstituted complexes, quantitative binding assays\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — reconstitution with phospho-mutants and EM visualization, highly cited\",\n      \"pmids\": [\"28012276\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"The histone chaperone FACT (subunits Spt16/SSRP1) binds CENP-T/W; the C-terminal domain of Spt16 specifically binds the histone fold region of CENP-T/W. Depletion of Spt16 impairs CENP-T and CENP-W deposition at centromeres, and site-directed targeting of Spt16 alone is sufficient to drive de novo CENP-T accumulation at centromeres.\",\n      \"method\": \"Proteomic screen, co-immunoprecipitation, domain mapping, RNAi depletion, ectopic targeting assay, immunofluorescence\",\n      \"journal\": \"Genes & development\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal Co-IP, domain mapping, and functional depletion/targeting experiments with multiple readouts\",\n      \"pmids\": [\"27284163\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"In living human cells, CENP-T directly associates with CENP-A and CENP-B (detected by FRET); CENP-T exchange at centromeres is restricted to S-phase (shown by FRAP), indicating a co-replicational loading mechanism.\",\n      \"method\": \"Acceptor-bleaching FRET, FRAP in live cells\",\n      \"journal\": \"Journal of biophotonics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — live-cell FRET and FRAP with functional interpretation, single lab study\",\n      \"pmids\": [\"19412974\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"CSN5/JAB1 directly interacts with both CENP-T and CENP-W (yeast two-hybrid and co-immunoprecipitation) and promotes ubiquitin- and proteasome-dependent degradation of CENP-T and CENP-W; formation of the CENP-T/W complex enhances protein stability by blocking CSN5-mediated degradation; CSN5 dysregulation impairs CENP-T/W recruitment to kinetochores during prophase.\",\n      \"method\": \"Yeast two-hybrid, co-immunoprecipitation, proteasome inhibitor experiments, in vivo localization assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — yeast two-hybrid confirmed by Co-IP with functional degradation assay, single lab\",\n      \"pmids\": [\"23926101\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"In budding yeast, Cnn1 (CENP-T) harbors two kinetochore-localization activities: a C-terminal histone-fold domain associating with the centromere region, and an N-terminal Spc24/Spc25 interaction sequence (residues 25-91) mediating linkage to the Ndc80 complex; Mps1 kinase phosphorylates Cnn1-S74, regulating its interaction with Ndc80 and modulating kinetochore accumulation from G1 through metaphase.\",\n      \"method\": \"In vivo localization by fluorescence microscopy, domain deletion/mutation analysis, kinase in vitro phosphorylation assay, genetic epistasis\",\n      \"journal\": \"Genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple domain mutants with in vivo functional readouts, single lab\",\n      \"pmids\": [\"25716979\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"CENP-T directly binds HJURP (a CENP-A chaperone) via the C-terminus of CENP-T; HJURP knockout minimizes CENP-T recruitment to centromeres; a HJURP-binding-deficient CENP-T mutant fails to localize to centromeres; HJURP recruits CENP-T in S/G2 phase.\",\n      \"method\": \"Co-immunoprecipitation, CRISPR knockout, domain mapping mutagenesis, immunofluorescence cell cycle staging\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — CRISPR KO plus domain-mapping Co-IP and localization assay, single lab\",\n      \"pmids\": [\"30459232\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"In fission yeast, the CENP-A (Cnp1) N-tail specifically promotes localization of CENP-T (Cnp20) (but not CENP-C) at centromeres; overexpression of CENP-T suppresses centromere inactivation defects caused by N-tail mutations, placing CENP-T downstream of the CENP-A N-tail in a pathway that maintains epigenetic centromere stability.\",\n      \"method\": \"Genetic epistasis (suppressor overexpression), fluorescence microscopy localization, synthetic lethality analysis\",\n      \"journal\": \"Current biology : CB\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — genetic epistasis with suppressor rescue, fission yeast ortholog\",\n      \"pmids\": [\"25619765\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"By in vivo FRET in human cells, the CENP-T C-terminus is specifically proximal to histone H3.1 (but not H3.2, H3.3, or CENP-A), suggesting CENP-T bridges a CENP-A-containing and an H3.1-containing nucleosome at centromeres.\",\n      \"method\": \"In vivo acceptor-bleaching FRET in live human cells\",\n      \"journal\": \"International journal of molecular sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — direct FRET measurement in live cells with multiple histone variants tested as controls, single lab\",\n      \"pmids\": [\"25775162\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"ChIP-seq and sequential ChIP in human cells show that CENPT is centered over the CENPB box between two well-positioned CENPA nucleosomes on α-satellite dimers and physically interacts with the CENPB/CENPC complex; cross-linking captures the entire CENPA/CENPB/CENPC/CENPT complex over an α-satellite dimer.\",\n      \"method\": \"ChIP-seq, sequential ChIP, base-pair resolution genomic readout\",\n      \"journal\": \"Genome research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — genome-wide base-pair resolution mapping with sequential ChIP validation, single lab\",\n      \"pmids\": [\"27384170\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"In budding yeast, de novo kinetochore assembly assay demonstrates that when the Mis12 pathway is crippled (Dsn1 phosphorylation defect), the CENP-T pathway becomes essential for viability and Ndc80 complex recruitment, establishing functional redundancy and epistatic relationship between the two Ndc80 recruitment pathways.\",\n      \"method\": \"De novo kinetochore assembly in yeast extracts, genetic epistasis, microtubule-binding assay\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — novel in vitro assembly assay combined with genetic epistasis in budding yeast ortholog\",\n      \"pmids\": [\"30117803\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Crystal structure of the Ctf3 complex (yeast CENP-I module) bound to the Cnn1-Wip1 (CENP-T/W) heterodimer reveals the structural basis for Ctf3c and Cnn1-Wip1 co-recruitment to the kinetochore; live-cell imaging provides a feedback regulation mechanism for Ctf19c assembly.\",\n      \"method\": \"High-resolution crystal structure, live-cell imaging\",\n      \"journal\": \"Current biology : CB\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 — crystal structure with live-cell imaging in yeast ortholog, single study\",\n      \"pmids\": [\"32679099\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"In fission yeast, CDK1-mediated phosphorylation of the Ccp1-interaction motif (CIM) at the N-terminus of CENP-T disrupts Ccp1 binding, enabling competitive displacement of Ccp1 by Ndc80; the phospho-null CIM mutant retains Ccp1 at centromeres during mitosis and mispositions the Ndc80 complex, causing chromosome missegregation.\",\n      \"method\": \"Phospho-mutant analysis, co-immunoprecipitation, live-cell imaging, chromosome segregation assays in fission yeast\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — phospho-mutant genetic and biochemical analysis with in vivo functional readout, fission yeast ortholog\",\n      \"pmids\": [\"34810257\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"In chicken DT40 cells, two copies of Ndc80 complex (N-N) on CENP-T (one via direct binding, one via Mis12C) are required for proper kinetochore-microtubule interactions; artificial direct attachment of two Ndc80 complexes to CENP-T can substitute for the native Mis12C-mediated linkage, demonstrating N-N functionality is independent of direct Mis12C-Ndc80 binding.\",\n      \"method\": \"DT40 cell genetic engineering (interaction mutants), artificial kinetochore tethering, chromosome segregation and spindle checkpoint assays\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal genetic and functional experiments with defined molecular mutants in vertebrate cells\",\n      \"pmids\": [\"35165266\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"The CENP-T–Mis12 complex interaction occurs via three binding surfaces (identified by AlphaFold2 combined with biochemical validation); this interaction is cooperatively regulated by dual phosphorylation of Dsn1 (Mis12C component) and CENP-T, ensuring robust Mis12C recruitment to CENP-T during mitosis.\",\n      \"method\": \"AlphaFold2 structure prediction combined with cell biological and biochemical validation, phospho-mutant analysis in DT40 cells\",\n      \"journal\": \"iScience\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — structural prediction validated by biochemical and cell biological assays with phospho-mutants, single lab\",\n      \"pmids\": [\"39628583\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Aurora B phosphorylates CENP-W at threonine 60, which enhances the CENP-W–CENP-T interaction (via the histone fold domain and an uncharacterized N-terminal region of CENP-T) to ensure robust metaphase chromosome alignment and accurate chromosome segregation.\",\n      \"method\": \"In vitro kinase assay, co-immunoprecipitation, phospho-mutant analysis, live-cell imaging, chromosome segregation assays\",\n      \"journal\": \"Journal of molecular cell biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — kinase assay with phospho-mutant Co-IP and in vivo segregation readout, single lab\",\n      \"pmids\": [\"38200711\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Ndc80 binding to CENP-T is a two-step process: rapid initial association/dissociation at disordered N-terminal sites followed by a slower 'maturation' transition to stronger retention; this maturation kinetic barrier is markedly accelerated when CENP-T is clustered at high molecular density, explaining why clustered CENP-T recruits more Ndc80 than monomeric CENP-T, and the two Ndc80-binding sites on CENP-T exhibit distinct maturation rates.\",\n      \"method\": \"Quantitative in vitro binding assays with clustered vs. monomeric CENP-T, single-molecule imaging, live dividing human cell analysis\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro reconstitution with quantitative kinetic analysis plus live-cell validation, published in peer-reviewed journal\",\n      \"pmids\": [\"39700145\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Depletion of CENP-T by siRNA in mouse oocytes increases CDH1/FZR1 levels, elevating APC-CDH1 activity and decreasing CCNB1, thereby attenuating MPF and severely compromising meiotic resumption (G2/M transition); these defects are rescued by CCNB1 overexpression or CDH1 knockdown, placing CENP-T upstream of CDH1 in regulating meiotic progression.\",\n      \"method\": \"siRNA knockdown, overexpression rescue, western blot for CDH1 and CCNB1, MPF activity assay, genetic epistasis in mouse oocytes\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal rescue experiments establishing pathway position, single lab in mouse oocytes\",\n      \"pmids\": [\"31964702\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"In Xenopus egg extracts, CENP-T centromeric recruitment occurs in late interphase independently of DNA synthesis and precedes CENP-W recruitment (which occurs in mitosis); unlike CENP-C, CENP-T does not participate in CENP-A deposition; depletion of CENP-C reduces CENP-T at centromeres, but kinetochores can still assemble with reduced Ndc80/Mis12, supporting the existence of two parallel assembly pathways.\",\n      \"method\": \"Xenopus egg cell-free extract immunodepletion, cell cycle staging, immunofluorescence, western blot\",\n      \"journal\": \"Nucleus (Austin, Tex.)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — cell-free reconstitution with immunodepletion and temporal staging, Xenopus ortholog study\",\n      \"pmids\": [\"25569378\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"CENPT interacts with GCLC (γ-glutamyl-cysteine ligase catalytic subunit) by binding to residues 213-424 of GCLC competitively with GCLM (the modifier subunit), increasing GCLC catalytic activity and glutathione synthesis, thereby reducing ROS and inhibiting ferroptosis in renal cell carcinoma cells.\",\n      \"method\": \"Co-immunoprecipitation, domain mapping, ROS measurements, ferroptosis assays, GSH quantification\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — Co-IP with functional assays, single lab, non-canonical CENPT function outside kinetochore biology\",\n      \"pmids\": [\"40651948\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Adaptive evolution of the CENP-T histone fold domain (DNA-binding region) in mice reduced centromere binding; introducing the histone fold domain from closely related species into mouse CENP-T (chimeric variants) increased centromere binding in oocytes and somatic cells; reduced binding by mouse CENP-T supports robust female gametogenesis, and the adaptation is independent of centromeric DNA sequence.\",\n      \"method\": \"Transgenic mouse models with chimeric CENP-T variants, oocyte microinjection, quantitative centromere binding assays, gametogenesis phenotype analysis\",\n      \"journal\": \"Current biology : CB\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — transgenic mouse model with chimeric proteins and quantitative binding assays\",\n      \"pmids\": [\"39947176\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"CENP-T is an inner kinetochore histone-fold protein that, as part of the CENP-T-W-S-X heterotetramer, forms a nucleosome-like structure that wraps and positively supercoils linker DNA at centromeres; its disordered N-terminal region serves as a phospho-regulated (CDK1) scaffold that directly recruits up to two Ndc80 complexes and one Mis12:Ndc80 complex to the outer kinetochore, acting in parallel with CENP-C to collectively recruit two MIS12 and up to four NDC80 complexes per kinetochore, while FACT chaperone mediates its cell-cycle-coupled deposition at centromeres and Aurora B/CDK phosphorylation events fine-tune assembly and stability of these interactions.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"CENP-T is an inner kinetochore histone-fold protein that nucleates a parallel pathway for outer kinetochore assembly at centromeres. Together with CENP-W, CENP-S, and CENP-X, it forms a nucleosome-like heterotetramer that binds centromeric linker DNA and introduces positive supercoils, with DNA contacts in CENP-T and CENP-W essential for this activity and for kinetochore targeting [PMID:22304917, PMID:24234442]. The intrinsically disordered N-terminal region of CENP-T functions as a CDK1-phosphorylated scaffold that directly recruits up to two Ndc80 complexes and one Mis12-associated Ndc80 complex, operating in parallel with the CENP-C pathway to collectively assemble two MIS12 and up to four NDC80 complexes per kinetochore unit [PMID:23334297, PMID:28012276, PMID:35165266]. Cell-cycle-coupled deposition of CENP-T at centromeres is mediated by the FACT chaperone complex and HJURP, while Aurora B and CDK1 phosphorylation events regulate CENP-T–CENP-W stability and the phospho-dependent switching from interphase binding partners to mitotic Ndc80 engagement [PMID:27284163, PMID:30459232, PMID:34810257, PMID:38200711].\",\n  \"teleology\": [\n    {\n      \"year\": 2008,\n      \"claim\": \"Before the molecular function of CENP-T was known, live-cell FRET and FRAP established that CENP-T physically associates with CENP-A chromatin and is deposited at centromeres specifically during S phase, framing it as a replication-coupled centromere component.\",\n      \"evidence\": \"Acceptor-bleaching FRET and FRAP in live human cells\",\n      \"pmids\": [\"19412974\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-lab FRET study without biochemical confirmation of direct CENP-A contact\", \"Mechanism of S-phase coupling unknown\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Structural and biochemical work revealed that CENP-T forms a nucleosome-like CENP-T-W-S-X heterotetramer with histone-fold domains that binds and positively supercoils DNA, establishing the molecular basis for how CENP-T integrates into centromeric chromatin, while yeast reconstitution showed the conserved N-terminal peptide directly recruits the Ndc80 complex via Spc24-Spc25.\",\n      \"evidence\": \"Crystal structure, in vitro DNA-binding/supercoiling assays, mutagenesis, in vivo kinetochore assembly (human); biochemical reconstitution and mini-chromosome segregation assay (yeast)\",\n      \"pmids\": [\"22304917\", \"22561346\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How the heterotetramer is positioned relative to CENP-A nucleosomes in vivo\", \"Stoichiometry of DNA wrapping at native centromeres\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Crystal structures of the CENP-T N-terminus bound to Ndc80 RWD domains revealed that CDK phosphorylation strengthens this interaction through a cryptic hydrophobic mechanism, and demonstrated that CENP-T–Ndc80 and Mis12–Ndc80 binding are mutually exclusive, defining two independent Ndc80 recruitment routes.\",\n      \"evidence\": \"X-ray crystallography, ITC, phospho-mutant analysis, co-immunoprecipitation, in vivo recruitment assays (human); DNA preference and supercoiling domain analysis\",\n      \"pmids\": [\"23334297\", \"24234442\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How the two pathways are coordinated temporally in mitosis\", \"Whether CDK phosphorylation is switch-like or graded in vivo\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Ectopic targeting experiments separated the CENP-T and CENP-C pathways: Aurora B promotes KMN recruitment to CENP-C while CDK regulates KMN recruitment to CENP-T, establishing distinct kinase-dependent regulatory logic for each parallel pathway.\",\n      \"evidence\": \"Ectopic chromosomal locus targeting, kinase inhibitor treatment, quantitative fluorescence imaging in human cells; genetic epistasis and Mps1 regulation in budding yeast\",\n      \"pmids\": [\"25660545\", \"25716979\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Quantitative contribution of each pathway to total kinetochore-microtubule attachment strength\", \"Cross-talk between Aurora B and CDK at CENP-T\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Reconstitution of the full phospho-regulated CENP-T scaffold showed that CDK1-dependent triple phosphorylation enables recruitment of one Mis12:Ndc80 complex and two additional Ndc80 complexes, and together with CENP-C accounts for the measured kinetochore stoichiometry of two MIS12 and up to four NDC80 complexes.\",\n      \"evidence\": \"In vitro reconstitution with phospho-mutants, electron microscopy, quantitative binding assays\",\n      \"pmids\": [\"28012276\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether this stoichiometry is invariant across cell types\", \"How stoichiometry is maintained during metaphase oscillations\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Identification of the FACT chaperone as the loading machine for CENP-T/W answered how CENP-T is deposited at centromeres: FACT binds the histone-fold domain of CENP-T/W and is sufficient for de novo centromeric accumulation, while genomic mapping placed CENP-T over CENP-B boxes between flanking CENP-A nucleosomes.\",\n      \"evidence\": \"Proteomic screen, reciprocal Co-IP, domain mapping, RNAi depletion, ectopic targeting (human cells); ChIP-seq and sequential ChIP at base-pair resolution\",\n      \"pmids\": [\"27284163\", \"27384170\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether FACT and HJURP act sequentially or in parallel for CENP-T loading\", \"Structural basis of FACT–CENP-T interaction\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"HJURP was identified as a second chaperone that directly binds the CENP-T C-terminus and is required for S/G2-phase CENP-T recruitment, while yeast de novo assembly assays proved the CENP-T and Mis12 pathways are functionally redundant for Ndc80 recruitment and viability.\",\n      \"evidence\": \"CRISPR knockout, domain mapping, Co-IP, cell-cycle staged immunofluorescence (human); in vitro kinetochore assembly and genetic epistasis (yeast)\",\n      \"pmids\": [\"30459232\", \"30117803\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether HJURP–CENP-T interaction occurs on or off chromatin\", \"How FACT and HJURP coordinate during the loading cycle\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Structural resolution of the yeast CENP-I module (Ctf3c) bound to CENP-T/W revealed the architectural basis for inner kinetochore co-recruitment, while oocyte studies uncovered an unexpected role for CENP-T upstream of APC-CDH1 in meiotic resumption.\",\n      \"evidence\": \"Crystal structure and live-cell imaging (yeast); siRNA knockdown with overexpression rescue in mouse oocytes\",\n      \"pmids\": [\"32679099\", \"31964702\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether the CDH1 regulatory role is kinetochore-dependent or represents a moonlighting function\", \"Whether Ctf3c–CENP-T/W structural interface is conserved in vertebrates\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"CDK1 phosphorylation of the CENP-T N-terminal CIM motif was shown to act as a molecular switch that displaces the interphase partner Ccp1 to allow mitotic Ndc80 binding, providing a mechanism for the temporal transition from centromere maintenance to kinetochore assembly.\",\n      \"evidence\": \"Phospho-mutant analysis, Co-IP, live-cell imaging, chromosome segregation assays in fission yeast\",\n      \"pmids\": [\"34810257\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether an analogous switching mechanism operates in vertebrates\", \"Identity of a vertebrate Ccp1 equivalent\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Genetic engineering in vertebrate DT40 cells demonstrated that two Ndc80 complexes on a single CENP-T molecule are functionally required for proper kinetochore–microtubule interactions, and that artificial direct tethering of two Ndc80 copies can bypass the need for Mis12-mediated linkage.\",\n      \"evidence\": \"Interaction-specific mutations, artificial tethering constructs, chromosome segregation and spindle checkpoint assays in chicken DT40 cells\",\n      \"pmids\": [\"35165266\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether this design principle holds for human kinetochores in vivo\", \"How two Ndc80 copies on one CENP-T engage a single microtubule\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Three complementary studies resolved outstanding questions: the CENP-T–Mis12 interface involves three binding surfaces cooperatively regulated by dual phosphorylation; Aurora B phosphorylation of CENP-W T60 stabilizes the CENP-W–CENP-T interaction for accurate segregation; and Ndc80 binding to CENP-T proceeds through a two-step maturation mechanism that is dramatically accelerated by molecular clustering of CENP-T.\",\n      \"evidence\": \"AlphaFold2 prediction with biochemical validation and phospho-mutants (DT40); kinase assay, Co-IP, and segregation assays; quantitative in vitro kinetics with single-molecule imaging and live-cell validation (human)\",\n      \"pmids\": [\"39628583\", \"38200711\", \"39700145\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo clustering stoichiometry at native centromeres\", \"Whether maturation kinetics differ across cell types or species\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Adaptive evolution of the CENP-T histone-fold domain in mice reduced centromere binding in a manner that supports female gametogenesis, revealing that CENP-T centromere affinity is under evolutionary selection independent of centromeric DNA sequence.\",\n      \"evidence\": \"Transgenic mouse models with chimeric CENP-T variants, quantitative centromere binding assays, gametogenesis phenotyping\",\n      \"pmids\": [\"39947176\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether this adaptation impacts kinetochore strength or meiotic drive\", \"Molecular basis of the histone-fold changes that alter binding\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Key unresolved questions include the precise structural organization of the CENP-T-W-S-X complex on native centromeric chromatin in situ, the coordination between FACT and HJURP chaperones during CENP-T deposition, and whether the Ndc80 maturation kinetics observed in vitro govern error correction dynamics in vivo.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No in situ structural view of CENP-T on centromeric chromatin\", \"FACT vs. HJURP temporal coordination unresolved\", \"Physiological relevance of Ndc80 binding maturation kinetics for error correction\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0003677\", \"supporting_discovery_ids\": [0, 3]},\n      {\"term_id\": \"GO:0005198\", \"supporting_discovery_ids\": [0, 1, 5, 17]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [1, 2, 5, 17]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005694\", \"supporting_discovery_ids\": [0, 3, 7, 13]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [7, 12]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [4, 5, 16, 17]},\n      {\"term_id\": \"R-HSA-4839726\", \"supporting_discovery_ids\": [0, 3, 13]}\n    ],\n    \"complexes\": [\n      \"CENP-T-W-S-X heterotetramer\",\n      \"CCAN (constitutive centromere-associated network)\"\n    ],\n    \"partners\": [\n      \"CENPW\",\n      \"CENPS\",\n      \"CENPX\",\n      \"SPC24\",\n      \"SPC25\",\n      \"DSN1\",\n      \"SUPT16H\",\n      \"HJURP\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}