CENPI

CENP-I (Mis6/Ctf3) is a constitutive inner kinetochore protein that builds and maintains the centromeric chromatin platform required for faithful chromosome segregation (PMID:9230309, PMID:11970896). It directly binds AT-rich centromeric DNA through a consecutive surface formed by conserved charged residues at the end of its N-terminal HEAT repeats, and this DNA contact—rather than histone binding—drives its own centromeric localization, stabilizes CENP-A nucleosomes, and enables loading of newly synthesized CENP-A (PMID:36888657). CENP-I acts upstream of CENP-A deposition in fission yeast and of CENP-C localization in vertebrate cells, while being dispensable for CENP-A localization itself in vertebrates (PMID:10864871, PMID:11970896), and it promotes CENP-A assembly by recruiting CENP-C and M18BP1 to enable HJURP/Mis18-mediated loading (PMID:26527398). It also protects pre-existing CENP-A during mitosis by suppressing RNA Pol II-dependent non-coding transcription at the central core (PMID:35970865). Within the kinetochore, CENP-I assembles through conserved HEAT-repeat contacts with CENP-H, CENP-K, and CENP-M (PMID:32017295, PMID:36888657) and provides a docking site for the desumoylase Ulp2 that controls inner kinetochore sumoylation (PMID:34081091). Beyond architecture, CENP-I sustains spindle assembly checkpoint signaling: it is required for kinetochore association of MAD1, MAD2, CENP-F, and the RZZ complex, and—together with Aurora B—forms a molecular switch that retains checkpoint proteins by antagonizing their dynein-mediated stripping at unattached kinetochores (PMID:12640463, PMID:24862574). Independently of its kinetochore role, human CENP-I promotes homologous-recombination repair of DNA double-strand breaks by suppressing R-loop accumulation, as RNaseH1 expression restores HR in CENP-I-deficient cells (PMID:34206916).

1. 1997 High

   Established that the CENP-I ortholog Mis6 is a constitutive centromere component acting in G1/S to build specialized inner centromere chromatin, identifying it as a foundational determinant of centromere identity rather than a transient mitotic factor.

   Evidence Temperature-sensitive mutant analysis with minichromosome loss and micrococcal nuclease chromatin profiling in fission yeast

   PMID:9230309

   Open questions at the time

   • Molecular nature of the chromatin alteration undefined
   • No direct DNA- or protein-binding activity demonstrated
   • Relationship to CENP-A not yet tested
2. 1999 Medium

   Linked Mis6 function to mitotic spindle morphogenesis, showing centromere defects translate into aberrant metaphase spindle geometry.

   Evidence Fluorescence microscopy of spindle length and suppressor analysis in mis6 mutants

   PMID:10398680

   Open questions at the time

   • Spindle defect likely indirect via centromere structure
   • Mechanism connecting centromere to spindle length unresolved
3. 2000 High

   Placed Mis6 upstream of CENP-A deposition, defining its role in establishing the epigenetic centromere mark.

   Evidence Genetic epistasis and immunofluorescence of SpCENP-A in mis6 mutants

   PMID:10864871

   Open questions at the time

   • Did not distinguish establishment versus maintenance of CENP-A
   • Direct loading mechanism unknown
4. 2002 High

   Defined the vertebrate kinetochore hierarchy, showing human/chicken CENP-I is required for CENP-C but not CENP-A localization, revealing species differences in the CENP-A dependency relationship.

   Evidence Conditional knockout in DT40 cells with co-localization immunocytochemistry

   PMID:11970896

   Open questions at the time

   • Direct versus indirect requirement for CENP-C not separated
   • DNA-binding basis of localization not addressed
5. 2002 High

   Identified the budding yeast Ctf3 sub-complex (with Mcm22/Mcm16) and showed Cse4/CENP-A-dependent, Ctf19-dependent centromere binding, mapping CENP-I into the CCAN interaction network.

   Evidence Two-hybrid, ChIP, synthetic dosage lethality, and microscopy in budding yeast

   PMID:11782448

   Open questions at the time

   • Ctf3 dispensable for Cse4 loading, contrasting fission yeast — unifying mechanism unclear
   • Direct DNA contact versus indirect recruitment not resolved
6. 2003 High

   Connected CENP-I to spindle checkpoint competence, showing its depletion abolishes kinetochore MAD1/MAD2/CENP-F and impairs mitotic arrest at unattached kinetochores.

   Evidence Antibody depletion, immunofluorescence, time-lapse imaging and checkpoint assays in human cells

   PMID:12640463

   Open questions at the time

   • Whether checkpoint defect is architectural or signaling-specific not separated
   • Mechanism of MAD1/MAD2 recruitment unknown
7. 2005 Medium

   Provided physical evidence that the Mis6 complex engages Mad2 upon checkpoint activation and is required for Mad2 accumulation at unattached kinetochores.

   Evidence Reciprocal co-IP and Mad2/Bub1 localization in mis6 mutants, plus Mis6 fragment localization in fission yeast

   PMID:15930132

   Open questions at the time

   • Microtubule-binding by N-terminal fragments not functionally validated
   • Direct versus indirect Mad2 interaction unresolved
   • Single lab
8. 2014 High

   Resolved the CENP-I checkpoint mechanism, showing it retains RZZ/Mad1 by inhibiting dynein-mediated stripping while Aurora B regulates their association — a two-input molecular switch for robust SAC signaling.

   Evidence siRNA depletion with Aurora B inhibition epistasis, immunofluorescence and live imaging in human cells

   PMID:24862574

   Open questions at the time

   • Direct molecular target of CENP-I in blocking stripping unknown
   • Structural basis of the switch undefined
9. 2014 Medium

   Connected CENP-I/CCAN to the CENP-A loading machinery via Eic1/Mis19, showing physical bridging of Mis18/HJURP factors to the Mis6/Ctf19 complex enables temporally regulated CENP-A deposition.

   Evidence Co-IP, mass spectrometry, conditional depletion and localization in fission yeast

   PMID:24789708 PMID:25375240

   Open questions at the time

   • Direct CENP-I contact with Eic1 versus other CCAN subunits unmapped
   • Maintenance versus establishment role of the bridge unclear
10. 2015 Medium

    Demonstrated CENP-I can drive de novo CENP-A assembly by recruiting CENP-C and then M18BP1, defining a CENP-C-independent route to M18BP1 recruitment.

    Evidence Tethering to synthetic alphoid(tetO) HAC array with immunofluorescence and CENP-A ChIP

    PMID:26527398

    Open questions at the time

    • Ectopic tethering may not reflect endogenous order of events
    • Direct M18BP1 binding not shown
    • Single lab
11. 2020 Medium

    Mapped CENP-I intramolecular architecture, showing a conserved α11 helix folds against N-terminal HEAT repeats to maintain folding and CENP-H/CENP-M binding.

    Evidence In vitro aggregation assays, co-IP, mutagenesis and fungal structural analysis

    PMID:32017295

    Open questions at the time

    • Human full-length structure not solved
    • Functional consequence of α11 mutations in cells limited
12. 2021 Medium

    Identified a non-kinetochore function: CENP-I promotes homologous-recombination DSB repair by limiting R-loop formation, with RNaseH1 rescuing HR in its absence.

    Evidence siRNA, HR/NHEJ reporters, 53BP1 foci, IR survival and RNaseH1 rescue in human cells

    PMID:34206916

    Open questions at the time

    • Whether CENP-I acts directly at break sites or via centromere transcription unclear
    • Mechanism of R-loop suppression undefined
    • Single lab
13. 2021 High

    Revealed CENP-I as a desumoylase docking platform, with a conserved Ctf3 surface binding Ulp2 to control inner kinetochore sumoylation and segregation fidelity.

    Evidence Interaction mapping, surface mutagenesis, sumoylation and segregation assays in budding yeast

    PMID:34081091

    Open questions at the time

    • Conservation of the Ulp2-docking role in humans untested
    • Sumoylation substrates at the kinetochore not enumerated
14. 2022 Medium

    Showed CENP-I maintains pre-existing CENP-A during mitosis by suppressing RNA Pol II non-coding transcription, since Pol II inhibition rescues CENP-A loss in mis6 mutants.

    Evidence Conditional mitotic depletion, CENP-A localization, ncRNA quantification and Pol II inhibition rescue in fission yeast

    PMID:35970865

    Open questions at the time

    • Direct mechanism of transcriptional suppression by CENP-I unknown
    • Link to its DNA-binding activity not tested
15. 2023 High

    Established the central biochemical activity: CENP-I directly binds AT-rich centromeric DNA via charged residues at its N-terminal HEAT repeats, and this DNA contact—not histone binding—drives localization, CENP-A nucleosome stabilization, CENP-A loading and chromosome alignment.

    Evidence In vitro DNA-binding assays, DNA-surface mutagenesis, co-IP, CENP-A loading and chromosome alignment assays in human cells

    PMID:36888657

    Open questions at the time

    • Structure of the CENP-I–DNA complex not solved
    • How DNA binding integrates with CCAN assembly and transcription suppression unresolved
16. 2025 Low

    Placed CENPI under FOXM1 transcriptional control with links to proline/arginine metabolism in glioblastoma, extending it into a cancer regulatory context.

    Evidence JASPAR prediction, dual luciferase reporter, ChIP, siRNA and metabolic assays in glioblastoma cells

    PMID:41206587

    Open questions at the time

    • No direct protein-level mechanism linking CENPI to metabolism
    • Single lab, correlative
    • Generality beyond glioblastoma untested

• How CENP-I's direct DNA-binding activity is mechanistically integrated with CCAN assembly, transcriptional suppression of the centromere core, checkpoint protein retention, and its R-loop-suppressing DNA-repair role remains unresolved.
• No high-resolution structure of human CENP-I bound to centromeric DNA or nucleosome
• Mechanistic basis of dynein-stripping antagonism unknown
• Whether the HR/R-loop function is separable from the kinetochore role untested