{"gene":"CENPT","run_date":"2026-06-09T22:57:18","timeline":{"discoveries":[{"year":2012,"finding":"CENP-T-W and CENP-S-X complexes coassemble into a stable CENP-T-W-S-X heterotetramer with structural similarity to canonical histones within a nucleosome. The heterotetramer binds and supercoils DNA; mutants compromising heterotetramerization or DNA-protein contacts reduce DNA binding and supercoiling in vitro and compromise kinetochore assembly in vivo.","method":"High-resolution structural analysis (crystal structure), in vitro DNA binding and supercoiling assays, site-directed mutagenesis, in vivo kinetochore assembly assays","journal":"Cell","confidence":"High","confidence_rationale":"Tier 1 / Strong — reconstitution, crystal structure, mutagenesis, and in vivo validation in a single rigorous study","pmids":["22304917"],"is_preprint":false},{"year":2013,"finding":"The N-terminal region of vertebrate CENP-T directly interacts with the RWD domain of the Spc24/25 portion of the Ndc80 complex. CDK phosphorylation of CENP-T strengthens a cryptic hydrophobic interaction with Spc25 without direct recognition of the phosphorylated residue. CENP-T and the Mis12 complex bind to the Ndc80 complex via mutually exclusive interactions, supporting two distinct pathways for Ndc80 recruitment to kinetochores.","method":"High-resolution structural analysis, biochemical binding assays, phosphorylation assays, mutagenesis","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure combined with biochemical binding assays and mutagenesis, multiple orthogonal methods in one study","pmids":["23334297"],"is_preprint":false},{"year":2012,"finding":"The budding yeast CENP-T ortholog Cnn1 acts as a direct centromere receptor of the Ndc80 complex. The amino terminus of Cnn1 contains a conserved peptide motif that mediates stoichiometric binding to the Spc24-25 domain of Ndc80. Artificial tethering of Ndc80 through Cnn1 allows mini-chromosome segregation in the absence of a natural centromere.","method":"Biochemical binding assays, in vivo chromosome segregation assay with artificial tethering","journal":"Nature cell biology","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — direct biochemical reconstitution combined with in vivo functional rescue, independently validated CENP-T/Ndc80 interaction","pmids":["22561346"],"is_preprint":false},{"year":2015,"finding":"CENP-T and CENP-C act in parallel to recruit the KMN network to kinetochores, but via distinct organizational logic: CENP-T directly interacts with Ndc80, which then promotes KNL1/Mis12 complex recruitment through a separate CENP-T region, inverting the hierarchy relative to the CENP-C pathway. CDK regulates KMN recruitment to CENP-T, while Aurora B promotes KMN recruitment to CENP-C.","method":"Ectopic targeting of CENP-C and CENP-T to an ectopic chromosomal locus in human cells; functional epistasis analysis of KMN recruitment","journal":"Current biology : CB","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic pathway dissection via ectopic targeting plus independent regulation analysis, replicated across studies","pmids":["25660545"],"is_preprint":false},{"year":2016,"finding":"CENP-T is phosphorylated by CDK1:Cyclin B at three distinct sites, enabling binding of one MIS12:NDC80 and two NDC80 complexes. CENP-C and CENP-T together recruit two MIS12 and up to four NDC80 complexes in parallel. Binding of CENP-C and CENP-T to MIS12 is competitive. Electron microscopy of reconstituted complexes supported this stoichiometry model.","method":"Biochemical reconstitution, in vitro CDK1:Cyclin B phosphorylation assays, electron microscopy visualization of reconstituted complexes, mutagenesis","journal":"eLife","confidence":"High","confidence_rationale":"Tier 1 / Strong — reconstitution with phosphorylation, EM visualization, mutagenesis, multiple orthogonal methods in one rigorous study","pmids":["28012276"],"is_preprint":false},{"year":2013,"finding":"The CENP-T-W-S-X complex binds preferentially to ~100 bp of linker DNA rather than nucleosome-bound DNA, primarily as a (CENP-T-W-S-X)2 structure, and induces positive DNA supercoils (opposite to canonical nucleosomes). The DNA-binding regions in CENP-T or CENP-W (but not CENP-S or CENP-X) are required for positive supercoiling and kinetochore targeting of the complex.","method":"In vitro DNA binding assays, supercoiling assays, mutagenesis, in vivo kinetochore targeting assays","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution with mutagenesis and in vivo validation, single lab with multiple orthogonal methods","pmids":["24234442"],"is_preprint":false},{"year":2016,"finding":"The histone chaperone FACT (subunits Spt16 and SSRP1) interacts with CENP-T/W; the C-terminal region of Spt16 binds specifically to the histone fold region of CENP-T/W. Depletion of Spt16 impairs CENP-T and CENP-W deposition at endogenous centromeres, and site-directed targeting of Spt16 alone is sufficient to drive local de novo CENP-T accumulation. CENP-T deposition at centromeres is uncoupled from DNA synthesis.","method":"Proteomic screen, Co-IP, FRAP, siRNA depletion, site-directed targeting assay","journal":"Genes & development","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP, depletion phenotype, and ectopic targeting rescue; multiple orthogonal methods in single lab","pmids":["27284163"],"is_preprint":false},{"year":2013,"finding":"CSN5/JAB1 directly interacts with both CENP-T and CENP-W (identified by yeast two-hybrid and Co-IP). Ectopically expressed CSN5 promotes ubiquitin- and proteasome-dependent degradation of CENP-T·CENP-W. Formation of the CENP-T·CENP-W complex enhances stability of both proteins, and dysregulation of CSN5 impairs kinetochore recruitment of CENP-T·CENP-W during prophase.","method":"Yeast two-hybrid screening, Co-immunoprecipitation, proteasome inhibitor assays, ubiquitination assays, immunofluorescence","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — yeast two-hybrid plus Co-IP plus functional degradation assays, single lab","pmids":["23926101"],"is_preprint":false},{"year":2008,"finding":"CENP-T directly associates with CENP-A and CENP-B at centromeres as shown by FRET in living cells. CENP-T exchange into centromeres is restricted to S-phase of the cell cycle, suggesting a co-replicational loading mechanism.","method":"Acceptor-bleaching FRET in living human cells, FRAP","journal":"Journal of biophotonics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — live-cell FRET and FRAP with direct molecular proximity readout, single lab with two orthogonal methods","pmids":["19412974"],"is_preprint":false},{"year":2016,"finding":"ChIP-seq and sequential ChIP analyses show that CENPT is centered over the CENPB box between two CENPA nucleosomes on young α-satellite dimers, and interacts with the CENPB/CENPC complex. The entire CENPA/CENPB/CENPC/CENPT complex is nuclease-protected over an α-satellite dimer unit.","method":"Comparative ChIP with sequencing (base-pair resolution), sequential ChIP, nuclease protection assay","journal":"Genome research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ChIP-seq and sequential ChIP with nuclease protection, multiple orthogonal genomic methods, single lab","pmids":["27384170"],"is_preprint":false},{"year":2015,"finding":"In fission yeast, alteration of the CENP-A (Cnp1) N-tail specifically reduces localization of Cnp20/CENP-T (but not CENP-C) to centromeres, and overexpression of Cnp20/CENP-T suppresses centromere inactivation defects of N-tail mutants, placing CENP-T downstream of CENP-A N-tail in the epigenetic stability pathway.","method":"Genetic suppressor analysis, fluorescence microscopy, epistasis (double mutant analysis)","journal":"Current biology : CB","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic epistasis with suppression rescue, single lab with multiple mutant alleles","pmids":["25619765"],"is_preprint":false},{"year":2015,"finding":"The budding yeast Cnn1 (CENP-T ortholog) has two kinetochore localization activities: a C-terminal histone-fold domain (HFD) that associates with the centromere region, and an N-terminal Spc24/Spc25 interaction sequence (residues 25-91) that mediates linkage to Ndc80. Mps1 kinase phosphorylates Cnn1-S74 in vitro to modulate the Cnn1-Ndc80 interaction; from G1 through metaphase, Cnn1 uses both localization activities, while at anaphase onset (when Mps1 activity decreases) enrichment is mainly via the N-terminal Spc24/25 interaction.","method":"In vivo cell biology (localization assays), in vitro binding assays, phosphorylation assays, mutagenesis","journal":"Genetics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro kinase assays combined with in vivo localization mutant analysis, single lab","pmids":["25716979"],"is_preprint":false},{"year":2015,"finding":"In Xenopus egg extracts, CENP-T centromeric recruitment occurs in late interphase (after CENP-C but before CENP-W), is dependent on CENP-C (depletion reduces CENP-T levels at centromeres), but CENP-T does not participate in CENP-A deposition. CENP-T plays a major role in kinetochore assembly; its depletion reduces Ndc80 and Mis12 recruitment.","method":"Xenopus egg extract cell-free system, immunodepletion, immunofluorescence, time-course analysis","journal":"Nucleus (Austin, Tex.)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — cell-free reconstitution system with immunodepletion and kinetochore assembly readout, single lab","pmids":["25569378"],"is_preprint":false},{"year":2018,"finding":"CENP-T directly binds Holliday junction recognition protein (HJURP), a chaperone for CENP-A loading; the binding interface was mapped to the C terminus of CENP-T. HJURP knockout by CRISPR minimized CENP-T recruitment to centromeres. HJURP recruits CENP-T to centromeres during S/G2 phase. A HJURP-binding-deficient CENP-T mutant (CENP-T6L) failed to localize to centromeres.","method":"Co-IP, CRISPR knockout, immunofluorescence, mutagenesis (domain mapping and binding-deficient mutant)","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP, CRISPR KO, and mutagenesis with localization readout, single lab with multiple orthogonal methods","pmids":["30459232"],"is_preprint":false},{"year":2018,"finding":"In budding yeast, the CENP-T (Cnn1) pathway for Ndc80 recruitment becomes essential for viability when the Mis12 pathway is compromised by Dsn1 phosphorylation defects, demonstrating genetic epistasis and redundancy between the two Ndc80 recruitment pathways.","method":"De novo kinetochore assembly assay in yeast extracts, genetic epistasis analysis (double mutant), microtubule binding assay","journal":"eLife","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — novel in vitro assembly assay plus genetic epistasis, single lab","pmids":["30117803"],"is_preprint":false},{"year":2020,"finding":"The budding yeast Cnn1-Wip1 (CENP-T/W) heterodimer structure was determined at high resolution bound to the Ctf3 complex. Live-cell imaging experiments provided a mechanism for Ctf3c and Cnn1-Wip1 recruitment to the kinetochore, suggesting feedback regulation of Ctf19c assembly.","method":"High-resolution crystal/cryo structure, live-cell imaging","journal":"Current biology : CB","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — structural determination combined with live-cell imaging, single lab","pmids":["32679099"],"is_preprint":false},{"year":2020,"finding":"In Lepidoptera (CenH3-deficient organisms), a divergent CENP-T homolog is required for accurate mitotic progression and is sufficient to recruit Mis12 and Ndc80 outer kinetochore complexes. CRISPR-mediated knockout of CENP-T in Bombyx mori establishes an essential in vivo function. CENP-T-based kinetochore assembly functions independently of CenH3 in these insects.","method":"CRISPR-mediated gene editing (Bombyx mori), mass spectrometry (kinetochore composition), Co-IP (Mis12/Ndc80 recruitment), functional rescue assay","journal":"Current biology : CB","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — CRISPR KO with viability readout, MS, and Co-IP for complex recruitment, single lab with multiple orthogonal methods","pmids":["32032508"],"is_preprint":false},{"year":2016,"finding":"In mitotic CENP-T/W-depleted cells (using auxin-inducible degron approach), stripping of CENP-T from chromosomes in early mitosis reveals the RZZ complex (Rod-Zw10-zwilch), Spindly, Mad1/Mad2, and CENP-E require CENP-T/W function during kinetochore assembly for stable outer kinetochore association, but once assembled, remain associated after CENP-T stripping during mitosis.","method":"Auxin-inducible degron (AID) system, quantitative kinetochore proteomics (mass spectrometry of mitotic chromosomes), comparison with conventional knockouts","journal":"Open biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — AID-based acute depletion plus quantitative MS proteomics, single lab with two orthogonal depletion strategies","pmids":["26791246"],"is_preprint":false},{"year":2022,"finding":"Two copies of Ndc80C exist on CENP-T: one via direct binding and one via Mis12C. Cells lacking both CENP-T-Mis12C and CENP-C-Mis12C interactions show defects in sister chromatid cohesion and spindle checkpoint protein recruitment. Artificial direct tethering of two Ndc80C to CENP-T restores proper kinetochore-microtubule interactions without requiring direct Mis12C-Ndc80C binding, demonstrating that N-N on CENP-T is functionally sufficient.","method":"DT40 cell genetics, artificial engineering/tethering constructs, cell biology assays (chromosome segregation, spindle checkpoint)","journal":"Nature communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic dissection with engineered constructs and multiple functional readouts, single lab","pmids":["35165266"],"is_preprint":false},{"year":2021,"finding":"In fission yeast, Ccp1 directly interacts with CENP-T via a Ccp1-interaction motif (CIM) at the N terminus of CENP-T, adjacent to the Ndc80 receptor motif. CDK1 phosphorylation of the CIM domain weakens Ccp1 interaction, causing Ccp1 to dissociate from centromeres in mitosis. Phospho-null CIM mutant retains Ccp1 at centromeres during mitosis and disrupts Ndc80 positioning, causing chromosome missegregation, demonstrating competitive exclusion between Ccp1 and Ndc80 at the CENP-T N terminus.","method":"Co-IP, mutagenesis (phosphomimetic and phospho-null mutants), in vitro CDK1 kinase assay, live-cell imaging, chromosome segregation assay","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — biochemical interaction mapping with CDK1 assay, mutagenesis, and in vivo functional readout, single lab with multiple orthogonal methods","pmids":["34810257"],"is_preprint":false},{"year":2024,"finding":"CENP-T-Mis12C interaction involves three binding surfaces (identified by AlphaFold2 predictions combined with cell biological and biochemical analyses), and is cooperatively regulated by dual phosphorylation of Dsn1 (Mis12C component) and CENP-T. Each interface is important for Mis12C recruitment to CENP-T in cells.","method":"AlphaFold2 structure prediction validated by biochemical binding assays and cell biology (DT40 cells lacking CENP-C-Mis12C interaction), mutagenesis","journal":"iScience","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — structure-guided mutagenesis with biochemical and cell biological validation, single lab","pmids":["39628583"],"is_preprint":false},{"year":2024,"finding":"Ndc80 binding to CENP-T is a two-step process: initial rapid association/dissociation from disordered N-terminal sites, followed by binding site maturation resulting in stronger Ndc80 retention. Within CENP-T clusters (high molecular density), maturation is markedly accelerated compared to soluble CENP-T monomers. The two Ndc80 binding sites in human CENP-T exhibit distinct maturation rates correlating with differences in amino acid content.","method":"Quantitative in vitro binding assays, molecular clustering assays, fluorescence microscopy in dividing human cells","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — quantitative in vitro reconstitution and in vivo cell imaging, single lab with multiple orthogonal approaches","pmids":["39700145"],"is_preprint":false},{"year":2024,"finding":"Aurora B phosphorylates CENP-W at threonine 60, which enhances the interaction between CENP-W and the histone fold domain and an uncharacterized N-terminal region of CENP-T, promoting robust metaphase chromosome alignment and accurate chromosome segregation.","method":"In vitro Aurora B kinase assay, Co-IP, mutagenesis, chromosome alignment assays in mitosis","journal":"Journal of molecular cell biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro kinase assay plus Co-IP and mutagenesis with functional segregation readout, single lab","pmids":["38200711"],"is_preprint":false},{"year":2020,"finding":"In mouse oocytes, depletion of CENP-T by siRNA increases CDH1 (FZR1) levels, leading to increased APC-CDH1 activity, decreased CCNB1 levels, attenuated MPF activity, and severely compromised meiotic resumption. This defect is rescued by CCNB1 overexpression or CDH1 knockdown, placing CENP-T upstream of CDH1/APC-CDH1 in the meiotic G2/M transition pathway.","method":"siRNA injection in mouse oocytes, Western blot, rescue experiments (overexpression and siRNA knockdown), MPF activity assay","journal":"Journal of cell science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — depletion with specific phenotypic readout and genetic rescue experiments defining pathway position, single lab","pmids":["31964702"],"is_preprint":false},{"year":2015,"finding":"CENP-T C-terminus is specifically proximal to H3.1 (but not H3.2, H3.3, or H3.1 mutants C96A and C110A) at centromeres, as shown by in vivo FRET, suggesting that CENP-T bridges a CENP-A-containing and an H3.1-containing nucleosome at centromeres.","method":"In vivo acceptor-bleaching FRET in human cells","journal":"International journal of molecular sciences","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single FRET method, single lab, no functional consequence tested","pmids":["25775162"],"is_preprint":false},{"year":2025,"finding":"CENPT interacts with γ-glutamyl-cysteine ligase catalytic subunit (GCLC) by directly binding to GCLC residues 213-424, competitively displacing GCLM, and increasing GCLC catalytic activity, thereby promoting glutathione synthesis and inhibiting ferroptosis in renal cell carcinoma cells. GSH in turn increases CENPT expression via ATF2-mediated transcriptional regulation, forming a feedback loop.","method":"Co-IP, in vitro GCLC activity assay, domain mapping, ROS assay, knockdown/overexpression","journal":"Cell death & disease","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, Co-IP and activity assay without structural or reconstitution validation; novel non-kinetochore function","pmids":["40651948"],"is_preprint":false},{"year":2025,"finding":"Cryo-EM structures of CCAN bound to free DNA and monomeric/dimeric CENP-A nucleosomes show that the CENP-T-W-S-X (TWSX) module engages 65-70 bp of DNA including 30-35 bp of an upstream α-satellite repeat in a manner resembling nucleosome DNA wrapping. On a dimeric α-satellite array, CCAN accommodation requires unwrapping of DNA from the TWSX module and 25 bp from the upstream nucleosome.","method":"Cryo-EM structure determination of CCAN complexes with DNA and CENP-A nucleosomes","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — cryo-EM structural determination, single lab, preprint not yet peer-reviewed","pmids":[],"is_preprint":true},{"year":2025,"finding":"Lepidopteran CENP-T forms a stable monomer due to a structural rearrangement repositioning HFD helix α3, bringing a conserved two-helical extension to take over the role of CENP-W partner. This change does not affect DNA-binding ability of lepidopteran CENP-T.","method":"Structural analysis, biochemical solubility/stability assays, DNA binding assays","journal":"EMBO reports","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — structural and biochemical characterization of a variant CENP-T in non-vertebrate system, single lab","pmids":["41162737"],"is_preprint":false},{"year":2025,"finding":"Adaptive evolution of the histone fold domain of mouse CENP-T reduced centromere binding. Chimeric CENP-T variants with HFD from closely related species showed increased centromere binding when expressed in mouse oocytes or in a transgenic mouse model, and this adaptation supports robust female gametogenesis. This effect was independent of specific centromeric DNA sequence.","method":"Transgenic mouse model, oocyte microinjection of chimeric CENP-T variants, quantitative centromere binding assays","journal":"Current biology : CB","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vivo transgenic model combined with quantitative binding assays and chimeric variant analysis, single lab","pmids":["39947176"],"is_preprint":false}],"current_model":"CENP-T is an inner kinetochore protein that uses a histone fold domain (in complex with CENP-W, CENP-S, and CENP-X) to form a nucleosome-like heterotetramer that binds and positively supercoils linker DNA at centromeres; its N-terminal disordered region acts as a scaffold for outer kinetochore assembly by directly recruiting up to two Ndc80 complexes (in a CDK1-phosphorylation-dependent manner) and one MIS12:NDC80 complex, providing a parallel and independently regulated pathway to CENP-C for connecting centromeric chromatin to spindle microtubules during chromosome segregation."},"narrative":{"mechanistic_narrative":"CENP-T is an inner kinetochore protein that physically links centromeric chromatin to the spindle microtubule-binding machinery during chromosome segregation [PMID:22304917, PMID:25660545]. Its C-terminal histone fold domain coassembles with CENP-W, CENP-S and CENP-X into a stable heterotetramer that structurally resembles canonical histones and preferentially binds ~100 bp of linker DNA, which it positively supercoils—opposite to nucleosomes—via DNA-contacting regions in CENP-T and CENP-W [PMID:22304917, PMID:24234442]. This chromatin-engaging module is centered over the CENP-B box between flanking CENP-A nucleosomes and assembles with the CENP-A/CENP-B/CENP-C complex over α-satellite DNA [PMID:27384170]. The N-terminal disordered region of CENP-T acts as a scaffold for outer kinetochore assembly: it directly binds the Spc24/25 RWD domain of the Ndc80 complex, an interaction strengthened by CDK1:Cyclin B phosphorylation, allowing CENP-T to recruit up to two Ndc80 complexes plus one MIS12-bound Ndc80, thereby providing a pathway parallel to and competitive with CENP-C for KMN network recruitment [PMID:23334297, PMID:28012276, PMID:35165266]. Phosphorylation control is layered—CDK regulates KMN recruitment through CENP-T while Aurora B acts through CENP-C—establishing two independently regulated routes to connect chromatin to microtubules [PMID:25660545]. CENP-T centromeric deposition is chaperone-dependent, mediated by FACT (Spt16/SSRP1) binding to the CENP-T/W histone fold and by HJURP binding the CENP-T C-terminus, and occurs in S/G2 phase uncoupled from bulk DNA synthesis [PMID:27284163, PMID:30459232]. The CENP-T pathway is functionally essential when the Mis12 pathway is compromised, and engineered direct tethering of Ndc80 to CENP-T is sufficient to restore kinetochore-microtubule function and chromosome segregation [PMID:30117803, PMID:35165266]. CENP-T function is conserved through divergent lineages, supporting CenH3-independent kinetochore assembly in Lepidoptera [PMID:32032508].","teleology":[{"year":2008,"claim":"Established that CENP-T resides in molecular proximity to core centromere components and loads in a cell-cycle-restricted manner, defining it as a bona fide inner centromere protein.","evidence":"Acceptor-bleaching FRET and FRAP in living human cells","pmids":["19412974"],"confidence":"Medium","gaps":["Does not resolve whether association is direct or chromatin-mediated","No structural basis for CENP-A/CENP-B contacts"]},{"year":2012,"claim":"Resolved the chromatin-engaging architecture of CENP-T by showing it forms a histone-like CENP-T-W-S-X heterotetramer that binds and supercoils DNA and is required for kinetochore assembly.","evidence":"Crystal structure, in vitro DNA binding/supercoiling assays, mutagenesis, in vivo kinetochore assembly","pmids":["22304917"],"confidence":"High","gaps":["Did not define how the tetramer is positioned relative to CENP-A nucleosomes in vivo","Outer kinetochore linkage not yet mapped"]},{"year":2012,"claim":"Identified CENP-T (yeast Cnn1) as a direct centromeric receptor for the Ndc80 complex, sufficient to drive chromosome segregation when artificially tethered.","evidence":"Biochemical binding assays and in vivo chromosome segregation rescue by artificial tethering in budding yeast","pmids":["22561346"],"confidence":"High","gaps":["Structural basis of the Cnn1-Spc24/25 interface not yet solved","Regulation of the interaction undefined"]},{"year":2013,"claim":"Defined the structural and phospho-regulatory logic of the CENP-T-Ndc80 interaction and showed CENP-T and Mis12 bind Ndc80 mutually exclusively, establishing two parallel recruitment pathways.","evidence":"Crystal structure, biochemical binding and phosphorylation assays, mutagenesis (vertebrate)","pmids":["23334297"],"confidence":"High","gaps":["Did not establish stoichiometry of Ndc80 complexes per CENP-T","Full phospho-site map incomplete"]},{"year":2013,"claim":"Characterized the DNA-binding preference of the heterotetramer, showing it selects linker over nucleosomal DNA as a (CENP-T-W-S-X)2 unit and induces positive supercoils via CENP-T/CENP-W.","evidence":"In vitro DNA binding/supercoiling assays, mutagenesis, in vivo kinetochore targeting","pmids":["24234442"],"confidence":"High","gaps":["Precise in vivo DNA register relative to CENP-A nucleosomes not resolved","Functional role of positive supercoiling untested in vivo"]},{"year":2013,"claim":"Identified CSN5/JAB1 as a regulator of CENP-T·CENP-W stability and kinetochore recruitment, linking the complex to ubiquitin-proteasome control.","evidence":"Yeast two-hybrid, Co-IP, proteasome inhibitor and ubiquitination assays, immunofluorescence","pmids":["23926101"],"confidence":"Medium","gaps":["Whether CSN5 acts as ubiquitin ligase adaptor or indirectly is unclear","Physiological trigger for degradation undefined"]},{"year":2015,"claim":"Dissected the parallel CENP-T and CENP-C pathways, revealing inverted recruitment hierarchies and distinct kinase control (CDK on CENP-T, Aurora B on CENP-C).","evidence":"Ectopic targeting of CENP-C and CENP-T in human cells with epistasis analysis of KMN recruitment","pmids":["25660545"],"confidence":"High","gaps":["Quantitative contribution of each pathway in native centromeres not measured","Cross-talk between pathways unresolved"]},{"year":2015,"claim":"Mapped the dual localization activities of yeast Cnn1 (HFD and N-terminal Ndc80 motif) and showed Mps1 phosphorylation tunes the Ndc80 interaction across the cell cycle.","evidence":"In vivo localization assays, in vitro binding and phosphorylation assays, mutagenesis (budding yeast)","pmids":["25716979"],"confidence":"Medium","gaps":["Conservation of Mps1 regulation in vertebrates not tested","Anaphase-specific behavior basis incomplete"]},{"year":2015,"claim":"Placed CENP-T downstream of CENP-C and the CENP-A N-tail in centromere assembly hierarchies using two complementary model systems.","evidence":"Genetic suppressor/epistasis analysis in fission yeast and Xenopus egg extract immunodepletion time-courses","pmids":["25619765","25569378"],"confidence":"Medium","gaps":["Molecular basis of CENP-C-dependent recruitment not defined","Species-specific differences in dependency not reconciled"]},{"year":2016,"claim":"Identified FACT as the chaperone driving CENP-T/W centromeric deposition independent of DNA replication, defining a loading mechanism.","evidence":"Proteomic screen, reciprocal Co-IP, FRAP, siRNA depletion, ectopic Spt16 targeting","pmids":["27284163"],"confidence":"Medium","gaps":["Whether FACT acts on free or chromatin-bound CENP-T/W unresolved","Coordination with CENP-A loading machinery unclear"]},{"year":2016,"claim":"Quantified Ndc80/Mis12 stoichiometry, showing CDK1 phosphorylation of three CENP-T sites enables binding of one MIS12:NDC80 plus two NDC80 complexes, with CENP-C/CENP-T competing for MIS12.","evidence":"Biochemical reconstitution, CDK1:Cyclin B phosphorylation, EM visualization, mutagenesis","pmids":["28012276"],"confidence":"High","gaps":["In vivo confirmation of full stoichiometry at native kinetochores limited","Order of assembly events not directly visualized"]},{"year":2016,"claim":"Demonstrated that CENP-T/W is required during outer kinetochore assembly for stable incorporation of RZZ, Spindly, Mad1/Mad2 and CENP-E, but is dispensable for their maintenance once assembled.","evidence":"Auxin-inducible degron depletion and quantitative kinetochore mass spectrometry","pmids":["26791246"],"confidence":"Medium","gaps":["Mechanism of stable retention after CENP-T removal unknown","Direct vs indirect requirement for each factor not distinguished"]},{"year":2016,"claim":"Resolved the genomic positioning of CENP-T over the CENP-B box between CENP-A nucleosomes within α-satellite dimers as part of a nuclease-protected CENP-A/B/C/T complex.","evidence":"Base-pair-resolution ChIP-seq, sequential ChIP, nuclease protection","pmids":["27384170"],"confidence":"Medium","gaps":["Does not establish causal order of complex assembly","Generality beyond young α-satellite arrays untested"]},{"year":2018,"claim":"Identified HJURP as a CENP-T C-terminal binding partner that recruits CENP-T to centromeres during S/G2 phase, coupling CENP-T loading to the CENP-A chaperone.","evidence":"Co-IP, CRISPR knockout, immunofluorescence, domain mapping and binding-deficient mutant","pmids":["30459232"],"confidence":"Medium","gaps":["Relationship between HJURP- and FACT-dependent loading pathways unresolved","Structural basis of HJURP-CENP-T interface not solved"]},{"year":2018,"claim":"Established functional redundancy and conditional essentiality of the CENP-T Ndc80-recruitment pathway when the Mis12 pathway is impaired.","evidence":"De novo kinetochore assembly in yeast extracts, genetic epistasis, microtubule binding assays","pmids":["30117803"],"confidence":"Medium","gaps":["Quantitative balance between pathways in normal cells not measured","Conservation to metazoa untested in this study"]},{"year":2020,"claim":"Provided the structural basis for Cnn1-Wip1 (CENP-T/W) recruitment via the Ctf3 complex and proposed feedback regulation of inner kinetochore assembly.","evidence":"High-resolution structure and live-cell imaging (budding yeast)","pmids":["32679099"],"confidence":"Medium","gaps":["Vertebrate equivalent of Ctf3-mediated recruitment not defined","Feedback mechanism only inferred"]},{"year":2020,"claim":"Revealed a non-canonical role for CENP-T in the meiotic G2/M transition by restraining APC-CDH1 activity to sustain MPF and meiotic resumption in oocytes.","evidence":"siRNA depletion in mouse oocytes with CCNB1/CDH1 rescue and MPF activity assays","pmids":["31964702"],"confidence":"Medium","gaps":["Direct molecular link between CENP-T and CDH1 regulation unknown","Whether this reflects kinetochore or extra-kinetochore function unclear"]},{"year":2021,"claim":"Demonstrated competitive exclusion at the CENP-T N-terminus, where CDK1 phosphorylation displaces Ccp1 to allow correct mitotic Ndc80 positioning.","evidence":"Co-IP, phospho-mutant analysis, in vitro CDK1 assay, live-cell imaging, segregation assays (fission yeast)","pmids":["34810257"],"confidence":"Medium","gaps":["Vertebrate orthologs of Ccp1-type competition not identified","Full set of N-terminal competitors unknown"]},{"year":2022,"claim":"Showed two Ndc80 complexes on CENP-T (one direct, one via Mis12) and that direct tethering of both is functionally sufficient for kinetochore-microtubule attachment and cohesion/checkpoint function.","evidence":"DT40 genetics, engineered tethering constructs, segregation and checkpoint assays","pmids":["35165266"],"confidence":"Medium","gaps":["Why two Ndc80 routes are normally maintained unresolved","In vivo geometry of the two Ndc80 complexes not visualized"]},{"year":2024,"claim":"Defined the multi-interface, dually phospho-regulated CENP-T-Mis12C interaction, refining the molecular logic of the second Ndc80 recruitment route.","evidence":"AlphaFold2 prediction validated by biochemical and DT40 cell biological mutagenesis","pmids":["39628583"],"confidence":"Medium","gaps":["Predicted interfaces lack experimental structure","Temporal ordering of the three interface engagements unknown"]},{"year":2024,"claim":"Revealed that Ndc80 binding to CENP-T is a two-step maturation process accelerated by molecular clustering, providing a kinetic model for stable attachment.","evidence":"Quantitative in vitro binding and clustering assays, fluorescence microscopy in dividing human cells","pmids":["39700145"],"confidence":"Medium","gaps":["Molecular nature of binding-site maturation undefined","In vivo trigger for clustering-dependent acceleration unclear"]},{"year":2024,"claim":"Added an Aurora B regulatory input via CENP-W T60 phosphorylation that strengthens CENP-T/W interaction and promotes accurate chromosome alignment.","evidence":"In vitro Aurora B kinase assay, Co-IP, mutagenesis, mitotic alignment assays","pmids":["38200711"],"confidence":"Medium","gaps":["Structural effect of T60 phosphorylation not resolved","Identity of the uncharacterized CENP-T N-terminal contact region unknown"]},{"year":2025,"claim":"Characterized evolutionary tuning of the CENP-T histone fold domain affecting centromere binding strength and its consequences for gametogenesis.","evidence":"Transgenic mouse model and oocyte microinjection of chimeric CENP-T variants with quantitative binding assays","pmids":["39947176"],"confidence":"Medium","gaps":["Molecular determinant of altered binding affinity not pinpointed","Mechanism linking binding strength to gamete quality unresolved"]},{"year":2025,"claim":"Reported a divergent non-kinetochore role for CENP-T in regulating glutathione synthesis and ferroptosis in renal cell carcinoma via GCLC binding.","evidence":"Co-IP, in vitro GCLC activity assay, domain mapping, ROS and knockdown/overexpression assays","pmids":["40651948"],"confidence":"Low","gaps":["Single lab without structural or reconstitution validation","Whether this function involves centromeric or soluble CENP-T unclear","Generality beyond renal cell carcinoma untested"]},{"year":null,"claim":"How the CENP-T-W-S-X module is positioned and dynamically remodeled within native centromeric chromatin during the cell cycle, and how its multiple phospho-inputs are integrated in vivo, remains incompletely resolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No high-resolution in vivo structure of the assembled CENP-T complex on native α-satellite chromatin","Integration of CDK1, Aurora B and Mps1 phospho-regulation in a single cell-cycle model untested","Functional significance of positive DNA supercoiling in vivo unknown"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0003677","term_label":"DNA binding","supporting_discovery_ids":[0,5,9,26]},{"term_id":"GO:0005198","term_label":"structural molecule activity","supporting_discovery_ids":[0,5]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[1,3,4,18]}],"localization":[{"term_id":"GO:0005694","term_label":"chromosome","supporting_discovery_ids":[0,8,9]},{"term_id":"GO:0000228","term_label":"nuclear chromosome","supporting_discovery_ids":[9,26]}],"pathway":[{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[3,4,18]},{"term_id":"R-HSA-1474165","term_label":"Reproduction","supporting_discovery_ids":[23,28]}],"complexes":["CENP-T-W-S-X heterotetramer","CCAN (CENP-A/B/C/T complex)","kinetochore"],"partners":["CENPW","CENPS","CENPX","NDC80","MIS12","CENPC","HJURP","SUPT16H"],"other_free_text":[]}},"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 many","driving_tissues":[],"url":"https://www.proteinatlas.org/search/CENPT"},"hgnc":{"alias_symbol":["FLJ13111","CENP-T"],"prev_symbol":["C16orf56"]},"alphafold":{"accession":"Q96BT3","domains":[{"cath_id":"1.10.20.10","chopping":"459-508","consensus_level":"medium","plddt":91.0232,"start":459,"end":508},{"cath_id":"1.10.20.10","chopping":"512-551","consensus_level":"medium","plddt":89.0703,"start":512,"end":551}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q96BT3","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q96BT3-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q96BT3-F1-predicted_aligned_error_v6.png","plddt_mean":56.12},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=CENPT","jax_strain_url":"https://www.jax.org/strain/search?query=CENPT"},"sequence":{"accession":"Q96BT3","fasta_url":"https://rest.uniprot.org/uniprotkb/Q96BT3.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q96BT3/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q96BT3"}},"corpus_meta":[{"pmid":"22304917","id":"PMC_22304917","title":"CENP-T-W-S-X 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The heterotetramer binds and supercoils DNA; mutants compromising heterotetramerization or DNA-protein contacts reduce DNA binding and supercoiling in vitro and compromise kinetochore assembly in vivo.\",\n      \"method\": \"High-resolution structural analysis (crystal structure), in vitro DNA binding and supercoiling assays, site-directed mutagenesis, in vivo kinetochore assembly assays\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — reconstitution, crystal structure, mutagenesis, and in vivo validation in a single rigorous study\",\n      \"pmids\": [\"22304917\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"The N-terminal region of vertebrate CENP-T directly interacts with the RWD domain of the Spc24/25 portion of the Ndc80 complex. CDK phosphorylation of CENP-T strengthens a cryptic hydrophobic interaction with Spc25 without direct recognition of the phosphorylated residue. CENP-T and the Mis12 complex bind to the Ndc80 complex via mutually exclusive interactions, supporting two distinct pathways for Ndc80 recruitment to kinetochores.\",\n      \"method\": \"High-resolution structural analysis, biochemical binding assays, phosphorylation assays, mutagenesis\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure combined with biochemical binding assays and mutagenesis, multiple orthogonal methods in one study\",\n      \"pmids\": [\"23334297\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"The budding yeast CENP-T ortholog Cnn1 acts as a direct centromere receptor of the Ndc80 complex. The amino terminus of Cnn1 contains a conserved peptide motif that mediates stoichiometric binding to the Spc24-25 domain of Ndc80. Artificial tethering of Ndc80 through Cnn1 allows mini-chromosome segregation in the absence of a natural centromere.\",\n      \"method\": \"Biochemical binding assays, in vivo chromosome segregation assay with artificial tethering\",\n      \"journal\": \"Nature cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — direct biochemical reconstitution combined with in vivo functional rescue, independently validated CENP-T/Ndc80 interaction\",\n      \"pmids\": [\"22561346\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"CENP-T and CENP-C act in parallel to recruit the KMN network to kinetochores, but via distinct organizational logic: CENP-T directly interacts with Ndc80, which then promotes KNL1/Mis12 complex recruitment through a separate CENP-T region, inverting the hierarchy relative to the CENP-C pathway. CDK regulates KMN recruitment to CENP-T, while Aurora B promotes KMN recruitment to CENP-C.\",\n      \"method\": \"Ectopic targeting of CENP-C and CENP-T to an ectopic chromosomal locus in human cells; functional epistasis analysis of KMN recruitment\",\n      \"journal\": \"Current biology : CB\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic pathway dissection via ectopic targeting plus independent regulation analysis, replicated across studies\",\n      \"pmids\": [\"25660545\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"CENP-T is phosphorylated by CDK1:Cyclin B at three distinct sites, enabling binding of one MIS12:NDC80 and two NDC80 complexes. CENP-C and CENP-T together recruit two MIS12 and up to four NDC80 complexes in parallel. Binding of CENP-C and CENP-T to MIS12 is competitive. Electron microscopy of reconstituted complexes supported this stoichiometry model.\",\n      \"method\": \"Biochemical reconstitution, in vitro CDK1:Cyclin B phosphorylation assays, electron microscopy visualization of reconstituted complexes, mutagenesis\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — reconstitution with phosphorylation, EM visualization, mutagenesis, multiple orthogonal methods in one rigorous study\",\n      \"pmids\": [\"28012276\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"The CENP-T-W-S-X complex binds preferentially to ~100 bp of linker DNA rather than nucleosome-bound DNA, primarily as a (CENP-T-W-S-X)2 structure, and induces positive DNA supercoils (opposite to canonical nucleosomes). The DNA-binding regions in CENP-T or CENP-W (but not CENP-S or CENP-X) are required for positive supercoiling and kinetochore targeting of the complex.\",\n      \"method\": \"In vitro DNA binding assays, supercoiling assays, mutagenesis, in vivo kinetochore targeting assays\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution with mutagenesis and in vivo validation, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"24234442\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"The histone chaperone FACT (subunits Spt16 and SSRP1) interacts with CENP-T/W; the C-terminal region of Spt16 binds specifically to the histone fold region of CENP-T/W. Depletion of Spt16 impairs CENP-T and CENP-W deposition at endogenous centromeres, and site-directed targeting of Spt16 alone is sufficient to drive local de novo CENP-T accumulation. CENP-T deposition at centromeres is uncoupled from DNA synthesis.\",\n      \"method\": \"Proteomic screen, Co-IP, FRAP, siRNA depletion, site-directed targeting assay\",\n      \"journal\": \"Genes & development\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP, depletion phenotype, and ectopic targeting rescue; multiple orthogonal methods in single lab\",\n      \"pmids\": [\"27284163\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"CSN5/JAB1 directly interacts with both CENP-T and CENP-W (identified by yeast two-hybrid and Co-IP). Ectopically expressed CSN5 promotes ubiquitin- and proteasome-dependent degradation of CENP-T·CENP-W. Formation of the CENP-T·CENP-W complex enhances stability of both proteins, and dysregulation of CSN5 impairs kinetochore recruitment of CENP-T·CENP-W during prophase.\",\n      \"method\": \"Yeast two-hybrid screening, Co-immunoprecipitation, proteasome inhibitor assays, ubiquitination assays, immunofluorescence\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — yeast two-hybrid plus Co-IP plus functional degradation assays, single lab\",\n      \"pmids\": [\"23926101\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"CENP-T directly associates with CENP-A and CENP-B at centromeres as shown by FRET in living cells. CENP-T exchange into centromeres is restricted to S-phase of the cell cycle, suggesting a co-replicational loading mechanism.\",\n      \"method\": \"Acceptor-bleaching FRET in living human cells, FRAP\",\n      \"journal\": \"Journal of biophotonics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — live-cell FRET and FRAP with direct molecular proximity readout, single lab with two orthogonal methods\",\n      \"pmids\": [\"19412974\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"ChIP-seq and sequential ChIP analyses show that CENPT is centered over the CENPB box between two CENPA nucleosomes on young α-satellite dimers, and interacts with the CENPB/CENPC complex. The entire CENPA/CENPB/CENPC/CENPT complex is nuclease-protected over an α-satellite dimer unit.\",\n      \"method\": \"Comparative ChIP with sequencing (base-pair resolution), sequential ChIP, nuclease protection assay\",\n      \"journal\": \"Genome research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP-seq and sequential ChIP with nuclease protection, multiple orthogonal genomic methods, single lab\",\n      \"pmids\": [\"27384170\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"In fission yeast, alteration of the CENP-A (Cnp1) N-tail specifically reduces localization of Cnp20/CENP-T (but not CENP-C) to centromeres, and overexpression of Cnp20/CENP-T suppresses centromere inactivation defects of N-tail mutants, placing CENP-T downstream of CENP-A N-tail in the epigenetic stability pathway.\",\n      \"method\": \"Genetic suppressor analysis, fluorescence microscopy, epistasis (double mutant analysis)\",\n      \"journal\": \"Current biology : CB\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis with suppression rescue, single lab with multiple mutant alleles\",\n      \"pmids\": [\"25619765\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"The budding yeast Cnn1 (CENP-T ortholog) has two kinetochore localization activities: a C-terminal histone-fold domain (HFD) that associates with the centromere region, and an N-terminal Spc24/Spc25 interaction sequence (residues 25-91) that mediates linkage to Ndc80. Mps1 kinase phosphorylates Cnn1-S74 in vitro to modulate the Cnn1-Ndc80 interaction; from G1 through metaphase, Cnn1 uses both localization activities, while at anaphase onset (when Mps1 activity decreases) enrichment is mainly via the N-terminal Spc24/25 interaction.\",\n      \"method\": \"In vivo cell biology (localization assays), in vitro binding assays, phosphorylation assays, mutagenesis\",\n      \"journal\": \"Genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro kinase assays combined with in vivo localization mutant analysis, single lab\",\n      \"pmids\": [\"25716979\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"In Xenopus egg extracts, CENP-T centromeric recruitment occurs in late interphase (after CENP-C but before CENP-W), is dependent on CENP-C (depletion reduces CENP-T levels at centromeres), but CENP-T does not participate in CENP-A deposition. CENP-T plays a major role in kinetochore assembly; its depletion reduces Ndc80 and Mis12 recruitment.\",\n      \"method\": \"Xenopus egg extract cell-free system, immunodepletion, immunofluorescence, time-course analysis\",\n      \"journal\": \"Nucleus (Austin, Tex.)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — cell-free reconstitution system with immunodepletion and kinetochore assembly readout, single lab\",\n      \"pmids\": [\"25569378\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"CENP-T directly binds Holliday junction recognition protein (HJURP), a chaperone for CENP-A loading; the binding interface was mapped to the C terminus of CENP-T. HJURP knockout by CRISPR minimized CENP-T recruitment to centromeres. HJURP recruits CENP-T to centromeres during S/G2 phase. A HJURP-binding-deficient CENP-T mutant (CENP-T6L) failed to localize to centromeres.\",\n      \"method\": \"Co-IP, CRISPR knockout, immunofluorescence, mutagenesis (domain mapping and binding-deficient mutant)\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, CRISPR KO, and mutagenesis with localization readout, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"30459232\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"In budding yeast, the CENP-T (Cnn1) pathway for Ndc80 recruitment becomes essential for viability when the Mis12 pathway is compromised by Dsn1 phosphorylation defects, demonstrating genetic epistasis and redundancy between the two Ndc80 recruitment pathways.\",\n      \"method\": \"De novo kinetochore assembly assay in yeast extracts, genetic epistasis analysis (double mutant), microtubule binding assay\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — novel in vitro assembly assay plus genetic epistasis, single lab\",\n      \"pmids\": [\"30117803\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"The budding yeast Cnn1-Wip1 (CENP-T/W) heterodimer structure was determined at high resolution bound to the Ctf3 complex. Live-cell imaging experiments provided a mechanism for Ctf3c and Cnn1-Wip1 recruitment to the kinetochore, suggesting feedback regulation of Ctf19c assembly.\",\n      \"method\": \"High-resolution crystal/cryo structure, live-cell imaging\",\n      \"journal\": \"Current biology : CB\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — structural determination combined with live-cell imaging, single lab\",\n      \"pmids\": [\"32679099\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"In Lepidoptera (CenH3-deficient organisms), a divergent CENP-T homolog is required for accurate mitotic progression and is sufficient to recruit Mis12 and Ndc80 outer kinetochore complexes. CRISPR-mediated knockout of CENP-T in Bombyx mori establishes an essential in vivo function. CENP-T-based kinetochore assembly functions independently of CenH3 in these insects.\",\n      \"method\": \"CRISPR-mediated gene editing (Bombyx mori), mass spectrometry (kinetochore composition), Co-IP (Mis12/Ndc80 recruitment), functional rescue assay\",\n      \"journal\": \"Current biology : CB\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — CRISPR KO with viability readout, MS, and Co-IP for complex recruitment, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"32032508\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"In mitotic CENP-T/W-depleted cells (using auxin-inducible degron approach), stripping of CENP-T from chromosomes in early mitosis reveals the RZZ complex (Rod-Zw10-zwilch), Spindly, Mad1/Mad2, and CENP-E require CENP-T/W function during kinetochore assembly for stable outer kinetochore association, but once assembled, remain associated after CENP-T stripping during mitosis.\",\n      \"method\": \"Auxin-inducible degron (AID) system, quantitative kinetochore proteomics (mass spectrometry of mitotic chromosomes), comparison with conventional knockouts\",\n      \"journal\": \"Open biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — AID-based acute depletion plus quantitative MS proteomics, single lab with two orthogonal depletion strategies\",\n      \"pmids\": [\"26791246\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Two copies of Ndc80C exist on CENP-T: one via direct binding and one via Mis12C. Cells lacking both CENP-T-Mis12C and CENP-C-Mis12C interactions show defects in sister chromatid cohesion and spindle checkpoint protein recruitment. Artificial direct tethering of two Ndc80C to CENP-T restores proper kinetochore-microtubule interactions without requiring direct Mis12C-Ndc80C binding, demonstrating that N-N on CENP-T is functionally sufficient.\",\n      \"method\": \"DT40 cell genetics, artificial engineering/tethering constructs, cell biology assays (chromosome segregation, spindle checkpoint)\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic dissection with engineered constructs and multiple functional readouts, single lab\",\n      \"pmids\": [\"35165266\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"In fission yeast, Ccp1 directly interacts with CENP-T via a Ccp1-interaction motif (CIM) at the N terminus of CENP-T, adjacent to the Ndc80 receptor motif. CDK1 phosphorylation of the CIM domain weakens Ccp1 interaction, causing Ccp1 to dissociate from centromeres in mitosis. Phospho-null CIM mutant retains Ccp1 at centromeres during mitosis and disrupts Ndc80 positioning, causing chromosome missegregation, demonstrating competitive exclusion between Ccp1 and Ndc80 at the CENP-T N terminus.\",\n      \"method\": \"Co-IP, mutagenesis (phosphomimetic and phospho-null mutants), in vitro CDK1 kinase assay, live-cell imaging, chromosome segregation assay\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — biochemical interaction mapping with CDK1 assay, mutagenesis, and in vivo functional readout, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"34810257\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"CENP-T-Mis12C interaction involves three binding surfaces (identified by AlphaFold2 predictions combined with cell biological and biochemical analyses), and is cooperatively regulated by dual phosphorylation of Dsn1 (Mis12C component) and CENP-T. Each interface is important for Mis12C recruitment to CENP-T in cells.\",\n      \"method\": \"AlphaFold2 structure prediction validated by biochemical binding assays and cell biology (DT40 cells lacking CENP-C-Mis12C interaction), mutagenesis\",\n      \"journal\": \"iScience\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — structure-guided mutagenesis with biochemical and cell biological validation, single lab\",\n      \"pmids\": [\"39628583\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Ndc80 binding to CENP-T is a two-step process: initial rapid association/dissociation from disordered N-terminal sites, followed by binding site maturation resulting in stronger Ndc80 retention. Within CENP-T clusters (high molecular density), maturation is markedly accelerated compared to soluble CENP-T monomers. The two Ndc80 binding sites in human CENP-T exhibit distinct maturation rates correlating with differences in amino acid content.\",\n      \"method\": \"Quantitative in vitro binding assays, molecular clustering assays, fluorescence microscopy in dividing human cells\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — quantitative in vitro reconstitution and in vivo cell imaging, single lab with multiple orthogonal approaches\",\n      \"pmids\": [\"39700145\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Aurora B phosphorylates CENP-W at threonine 60, which enhances the interaction between CENP-W and the histone fold domain and an uncharacterized N-terminal region of CENP-T, promoting robust metaphase chromosome alignment and accurate chromosome segregation.\",\n      \"method\": \"In vitro Aurora B kinase assay, Co-IP, mutagenesis, chromosome alignment assays in mitosis\",\n      \"journal\": \"Journal of molecular cell biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro kinase assay plus Co-IP and mutagenesis with functional segregation readout, single lab\",\n      \"pmids\": [\"38200711\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"In mouse oocytes, depletion of CENP-T by siRNA increases CDH1 (FZR1) levels, leading to increased APC-CDH1 activity, decreased CCNB1 levels, attenuated MPF activity, and severely compromised meiotic resumption. This defect is rescued by CCNB1 overexpression or CDH1 knockdown, placing CENP-T upstream of CDH1/APC-CDH1 in the meiotic G2/M transition pathway.\",\n      \"method\": \"siRNA injection in mouse oocytes, Western blot, rescue experiments (overexpression and siRNA knockdown), MPF activity assay\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — depletion with specific phenotypic readout and genetic rescue experiments defining pathway position, single lab\",\n      \"pmids\": [\"31964702\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"CENP-T C-terminus is specifically proximal to H3.1 (but not H3.2, H3.3, or H3.1 mutants C96A and C110A) at centromeres, as shown by in vivo FRET, suggesting that CENP-T bridges a CENP-A-containing and an H3.1-containing nucleosome at centromeres.\",\n      \"method\": \"In vivo acceptor-bleaching FRET in human cells\",\n      \"journal\": \"International journal of molecular sciences\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single FRET method, single lab, no functional consequence tested\",\n      \"pmids\": [\"25775162\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"CENPT interacts with γ-glutamyl-cysteine ligase catalytic subunit (GCLC) by directly binding to GCLC residues 213-424, competitively displacing GCLM, and increasing GCLC catalytic activity, thereby promoting glutathione synthesis and inhibiting ferroptosis in renal cell carcinoma cells. GSH in turn increases CENPT expression via ATF2-mediated transcriptional regulation, forming a feedback loop.\",\n      \"method\": \"Co-IP, in vitro GCLC activity assay, domain mapping, ROS assay, knockdown/overexpression\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, Co-IP and activity assay without structural or reconstitution validation; novel non-kinetochore function\",\n      \"pmids\": [\"40651948\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Cryo-EM structures of CCAN bound to free DNA and monomeric/dimeric CENP-A nucleosomes show that the CENP-T-W-S-X (TWSX) module engages 65-70 bp of DNA including 30-35 bp of an upstream α-satellite repeat in a manner resembling nucleosome DNA wrapping. On a dimeric α-satellite array, CCAN accommodation requires unwrapping of DNA from the TWSX module and 25 bp from the upstream nucleosome.\",\n      \"method\": \"Cryo-EM structure determination of CCAN complexes with DNA and CENP-A nucleosomes\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — cryo-EM structural determination, single lab, preprint not yet peer-reviewed\",\n      \"pmids\": [],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Lepidopteran CENP-T forms a stable monomer due to a structural rearrangement repositioning HFD helix α3, bringing a conserved two-helical extension to take over the role of CENP-W partner. This change does not affect DNA-binding ability of lepidopteran CENP-T.\",\n      \"method\": \"Structural analysis, biochemical solubility/stability assays, DNA binding assays\",\n      \"journal\": \"EMBO reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — structural and biochemical characterization of a variant CENP-T in non-vertebrate system, single lab\",\n      \"pmids\": [\"41162737\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Adaptive evolution of the histone fold domain of mouse CENP-T reduced centromere binding. Chimeric CENP-T variants with HFD from closely related species showed increased centromere binding when expressed in mouse oocytes or in a transgenic mouse model, and this adaptation supports robust female gametogenesis. This effect was independent of specific centromeric DNA sequence.\",\n      \"method\": \"Transgenic mouse model, oocyte microinjection of chimeric CENP-T variants, quantitative centromere binding assays\",\n      \"journal\": \"Current biology : CB\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo transgenic model combined with quantitative binding assays and chimeric variant analysis, single lab\",\n      \"pmids\": [\"39947176\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"CENP-T is an inner kinetochore protein that uses a histone fold domain (in complex with CENP-W, CENP-S, and CENP-X) to form a nucleosome-like heterotetramer that binds and positively supercoils linker DNA at centromeres; its N-terminal disordered region acts as a scaffold for outer kinetochore assembly by directly recruiting up to two Ndc80 complexes (in a CDK1-phosphorylation-dependent manner) and one MIS12:NDC80 complex, providing a parallel and independently regulated pathway to CENP-C for connecting centromeric chromatin to spindle microtubules during chromosome segregation.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"CENP-T is an inner kinetochore protein that physically links centromeric chromatin to the spindle microtubule-binding machinery during chromosome segregation [#0, #3]. Its C-terminal histone fold domain coassembles with CENP-W, CENP-S and CENP-X into a stable heterotetramer that structurally resembles canonical histones and preferentially binds ~100 bp of linker DNA, which it positively supercoils—opposite to nucleosomes—via DNA-contacting regions in CENP-T and CENP-W [#0, #5]. This chromatin-engaging module is centered over the CENP-B box between flanking CENP-A nucleosomes and assembles with the CENP-A/CENP-B/CENP-C complex over α-satellite DNA [#9]. The N-terminal disordered region of CENP-T acts as a scaffold for outer kinetochore assembly: it directly binds the Spc24/25 RWD domain of the Ndc80 complex, an interaction strengthened by CDK1:Cyclin B phosphorylation, allowing CENP-T to recruit up to two Ndc80 complexes plus one MIS12-bound Ndc80, thereby providing a pathway parallel to and competitive with CENP-C for KMN network recruitment [#1, #4, #18]. Phosphorylation control is layered—CDK regulates KMN recruitment through CENP-T while Aurora B acts through CENP-C—establishing two independently regulated routes to connect chromatin to microtubules [#3]. CENP-T centromeric deposition is chaperone-dependent, mediated by FACT (Spt16/SSRP1) binding to the CENP-T/W histone fold and by HJURP binding the CENP-T C-terminus, and occurs in S/G2 phase uncoupled from bulk DNA synthesis [#6, #13]. The CENP-T pathway is functionally essential when the Mis12 pathway is compromised, and engineered direct tethering of Ndc80 to CENP-T is sufficient to restore kinetochore-microtubule function and chromosome segregation [#14, #18]. CENP-T function is conserved through divergent lineages, supporting CenH3-independent kinetochore assembly in Lepidoptera [#16].\",\n  \"teleology\": [\n    {\n      \"year\": 2008,\n      \"claim\": \"Established that CENP-T resides in molecular proximity to core centromere components and loads in a cell-cycle-restricted manner, defining it as a bona fide inner centromere protein.\",\n      \"evidence\": \"Acceptor-bleaching FRET and FRAP in living human cells\",\n      \"pmids\": [\"19412974\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Does not resolve whether association is direct or chromatin-mediated\", \"No structural basis for CENP-A/CENP-B contacts\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Resolved the chromatin-engaging architecture of CENP-T by showing it forms a histone-like CENP-T-W-S-X heterotetramer that binds and supercoils DNA and is required for kinetochore assembly.\",\n      \"evidence\": \"Crystal structure, in vitro DNA binding/supercoiling assays, mutagenesis, in vivo kinetochore assembly\",\n      \"pmids\": [\"22304917\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not define how the tetramer is positioned relative to CENP-A nucleosomes in vivo\", \"Outer kinetochore linkage not yet mapped\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Identified CENP-T (yeast Cnn1) as a direct centromeric receptor for the Ndc80 complex, sufficient to drive chromosome segregation when artificially tethered.\",\n      \"evidence\": \"Biochemical binding assays and in vivo chromosome segregation rescue by artificial tethering in budding yeast\",\n      \"pmids\": [\"22561346\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of the Cnn1-Spc24/25 interface not yet solved\", \"Regulation of the interaction undefined\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Defined the structural and phospho-regulatory logic of the CENP-T-Ndc80 interaction and showed CENP-T and Mis12 bind Ndc80 mutually exclusively, establishing two parallel recruitment pathways.\",\n      \"evidence\": \"Crystal structure, biochemical binding and phosphorylation assays, mutagenesis (vertebrate)\",\n      \"pmids\": [\"23334297\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not establish stoichiometry of Ndc80 complexes per CENP-T\", \"Full phospho-site map incomplete\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Characterized the DNA-binding preference of the heterotetramer, showing it selects linker over nucleosomal DNA as a (CENP-T-W-S-X)2 unit and induces positive supercoils via CENP-T/CENP-W.\",\n      \"evidence\": \"In vitro DNA binding/supercoiling assays, mutagenesis, in vivo kinetochore targeting\",\n      \"pmids\": [\"24234442\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Precise in vivo DNA register relative to CENP-A nucleosomes not resolved\", \"Functional role of positive supercoiling untested in vivo\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Identified CSN5/JAB1 as a regulator of CENP-T·CENP-W stability and kinetochore recruitment, linking the complex to ubiquitin-proteasome control.\",\n      \"evidence\": \"Yeast two-hybrid, Co-IP, proteasome inhibitor and ubiquitination assays, immunofluorescence\",\n      \"pmids\": [\"23926101\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether CSN5 acts as ubiquitin ligase adaptor or indirectly is unclear\", \"Physiological trigger for degradation undefined\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Dissected the parallel CENP-T and CENP-C pathways, revealing inverted recruitment hierarchies and distinct kinase control (CDK on CENP-T, Aurora B on CENP-C).\",\n      \"evidence\": \"Ectopic targeting of CENP-C and CENP-T in human cells with epistasis analysis of KMN recruitment\",\n      \"pmids\": [\"25660545\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Quantitative contribution of each pathway in native centromeres not measured\", \"Cross-talk between pathways unresolved\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Mapped the dual localization activities of yeast Cnn1 (HFD and N-terminal Ndc80 motif) and showed Mps1 phosphorylation tunes the Ndc80 interaction across the cell cycle.\",\n      \"evidence\": \"In vivo localization assays, in vitro binding and phosphorylation assays, mutagenesis (budding yeast)\",\n      \"pmids\": [\"25716979\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Conservation of Mps1 regulation in vertebrates not tested\", \"Anaphase-specific behavior basis incomplete\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Placed CENP-T downstream of CENP-C and the CENP-A N-tail in centromere assembly hierarchies using two complementary model systems.\",\n      \"evidence\": \"Genetic suppressor/epistasis analysis in fission yeast and Xenopus egg extract immunodepletion time-courses\",\n      \"pmids\": [\"25619765\", \"25569378\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular basis of CENP-C-dependent recruitment not defined\", \"Species-specific differences in dependency not reconciled\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Identified FACT as the chaperone driving CENP-T/W centromeric deposition independent of DNA replication, defining a loading mechanism.\",\n      \"evidence\": \"Proteomic screen, reciprocal Co-IP, FRAP, siRNA depletion, ectopic Spt16 targeting\",\n      \"pmids\": [\"27284163\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether FACT acts on free or chromatin-bound CENP-T/W unresolved\", \"Coordination with CENP-A loading machinery unclear\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Quantified Ndc80/Mis12 stoichiometry, showing CDK1 phosphorylation of three CENP-T sites enables binding of one MIS12:NDC80 plus two NDC80 complexes, with CENP-C/CENP-T competing for MIS12.\",\n      \"evidence\": \"Biochemical reconstitution, CDK1:Cyclin B phosphorylation, EM visualization, mutagenesis\",\n      \"pmids\": [\"28012276\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo confirmation of full stoichiometry at native kinetochores limited\", \"Order of assembly events not directly visualized\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Demonstrated that CENP-T/W is required during outer kinetochore assembly for stable incorporation of RZZ, Spindly, Mad1/Mad2 and CENP-E, but is dispensable for their maintenance once assembled.\",\n      \"evidence\": \"Auxin-inducible degron depletion and quantitative kinetochore mass spectrometry\",\n      \"pmids\": [\"26791246\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism of stable retention after CENP-T removal unknown\", \"Direct vs indirect requirement for each factor not distinguished\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Resolved the genomic positioning of CENP-T over the CENP-B box between CENP-A nucleosomes within α-satellite dimers as part of a nuclease-protected CENP-A/B/C/T complex.\",\n      \"evidence\": \"Base-pair-resolution ChIP-seq, sequential ChIP, nuclease protection\",\n      \"pmids\": [\"27384170\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Does not establish causal order of complex assembly\", \"Generality beyond young α-satellite arrays untested\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Identified HJURP as a CENP-T C-terminal binding partner that recruits CENP-T to centromeres during S/G2 phase, coupling CENP-T loading to the CENP-A chaperone.\",\n      \"evidence\": \"Co-IP, CRISPR knockout, immunofluorescence, domain mapping and binding-deficient mutant\",\n      \"pmids\": [\"30459232\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Relationship between HJURP- and FACT-dependent loading pathways unresolved\", \"Structural basis of HJURP-CENP-T interface not solved\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Established functional redundancy and conditional essentiality of the CENP-T Ndc80-recruitment pathway when the Mis12 pathway is impaired.\",\n      \"evidence\": \"De novo kinetochore assembly in yeast extracts, genetic epistasis, microtubule binding assays\",\n      \"pmids\": [\"30117803\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Quantitative balance between pathways in normal cells not measured\", \"Conservation to metazoa untested in this study\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Provided the structural basis for Cnn1-Wip1 (CENP-T/W) recruitment via the Ctf3 complex and proposed feedback regulation of inner kinetochore assembly.\",\n      \"evidence\": \"High-resolution structure and live-cell imaging (budding yeast)\",\n      \"pmids\": [\"32679099\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Vertebrate equivalent of Ctf3-mediated recruitment not defined\", \"Feedback mechanism only inferred\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Revealed a non-canonical role for CENP-T in the meiotic G2/M transition by restraining APC-CDH1 activity to sustain MPF and meiotic resumption in oocytes.\",\n      \"evidence\": \"siRNA depletion in mouse oocytes with CCNB1/CDH1 rescue and MPF activity assays\",\n      \"pmids\": [\"31964702\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct molecular link between CENP-T and CDH1 regulation unknown\", \"Whether this reflects kinetochore or extra-kinetochore function unclear\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Demonstrated competitive exclusion at the CENP-T N-terminus, where CDK1 phosphorylation displaces Ccp1 to allow correct mitotic Ndc80 positioning.\",\n      \"evidence\": \"Co-IP, phospho-mutant analysis, in vitro CDK1 assay, live-cell imaging, segregation assays (fission yeast)\",\n      \"pmids\": [\"34810257\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Vertebrate orthologs of Ccp1-type competition not identified\", \"Full set of N-terminal competitors unknown\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Showed two Ndc80 complexes on CENP-T (one direct, one via Mis12) and that direct tethering of both is functionally sufficient for kinetochore-microtubule attachment and cohesion/checkpoint function.\",\n      \"evidence\": \"DT40 genetics, engineered tethering constructs, segregation and checkpoint assays\",\n      \"pmids\": [\"35165266\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Why two Ndc80 routes are normally maintained unresolved\", \"In vivo geometry of the two Ndc80 complexes not visualized\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Defined the multi-interface, dually phospho-regulated CENP-T-Mis12C interaction, refining the molecular logic of the second Ndc80 recruitment route.\",\n      \"evidence\": \"AlphaFold2 prediction validated by biochemical and DT40 cell biological mutagenesis\",\n      \"pmids\": [\"39628583\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Predicted interfaces lack experimental structure\", \"Temporal ordering of the three interface engagements unknown\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Revealed that Ndc80 binding to CENP-T is a two-step maturation process accelerated by molecular clustering, providing a kinetic model for stable attachment.\",\n      \"evidence\": \"Quantitative in vitro binding and clustering assays, fluorescence microscopy in dividing human cells\",\n      \"pmids\": [\"39700145\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular nature of binding-site maturation undefined\", \"In vivo trigger for clustering-dependent acceleration unclear\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Added an Aurora B regulatory input via CENP-W T60 phosphorylation that strengthens CENP-T/W interaction and promotes accurate chromosome alignment.\",\n      \"evidence\": \"In vitro Aurora B kinase assay, Co-IP, mutagenesis, mitotic alignment assays\",\n      \"pmids\": [\"38200711\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Structural effect of T60 phosphorylation not resolved\", \"Identity of the uncharacterized CENP-T N-terminal contact region unknown\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Characterized evolutionary tuning of the CENP-T histone fold domain affecting centromere binding strength and its consequences for gametogenesis.\",\n      \"evidence\": \"Transgenic mouse model and oocyte microinjection of chimeric CENP-T variants with quantitative binding assays\",\n      \"pmids\": [\"39947176\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular determinant of altered binding affinity not pinpointed\", \"Mechanism linking binding strength to gamete quality unresolved\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Reported a divergent non-kinetochore role for CENP-T in regulating glutathione synthesis and ferroptosis in renal cell carcinoma via GCLC binding.\",\n      \"evidence\": \"Co-IP, in vitro GCLC activity assay, domain mapping, ROS and knockdown/overexpression assays\",\n      \"pmids\": [\"40651948\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Single lab without structural or reconstitution validation\", \"Whether this function involves centromeric or soluble CENP-T unclear\", \"Generality beyond renal cell carcinoma untested\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How the CENP-T-W-S-X module is positioned and dynamically remodeled within native centromeric chromatin during the cell cycle, and how its multiple phospho-inputs are integrated in vivo, remains incompletely resolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No high-resolution in vivo structure of the assembled CENP-T complex on native α-satellite chromatin\", \"Integration of CDK1, Aurora B and Mps1 phospho-regulation in a single cell-cycle model untested\", \"Functional significance of positive DNA supercoiling in vivo unknown\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0003677\", \"supporting_discovery_ids\": [0, 5, 9, 26]},\n      {\"term_id\": \"GO:0005198\", \"supporting_discovery_ids\": [0, 5]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [1, 3, 4, 18]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005694\", \"supporting_discovery_ids\": [0, 8, 9]},\n      {\"term_id\": \"GO:0000228\", \"supporting_discovery_ids\": [9, 26]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [3, 4, 18]},\n      {\"term_id\": \"R-HSA-1474165\", \"supporting_discovery_ids\": [23, 28]}\n    ],\n    \"complexes\": [\n      \"CENP-T-W-S-X heterotetramer\",\n      \"CCAN (CENP-A/B/C/T complex)\",\n      \"kinetochore\"\n    ],\n    \"partners\": [\n      \"CENPW\",\n      \"CENPS\",\n      \"CENPX\",\n      \"NDC80\",\n      \"MIS12\",\n      \"CENPC\",\n      \"HJURP\",\n      \"SUPT16H\"\n    ],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":8,"faith_total":8,"faith_pct":100.0}}