CENPI

CENP-I is a constitutive inner kinetochore protein built around N-terminal HEAT repeats that directly binds AT-rich centromeric DNA to stabilize CENP-A nucleosomes and promote new CENP-A loading, thereby maintaining centromere identity across cell divisions (PMID:36888657, PMID:10864871, PMID:35970865). It occupies a central position in kinetochore hierarchy: CENP-I (together with CENP-H) is required for centromeric localization of CENP-C, and it recruits M18BP1 downstream of CENP-C to couple kinetochore structure to the CENP-A assembly pathway (PMID:11970896, PMID:26527398). CENP-I is also essential for spindle assembly checkpoint signaling, generating stable kinetochore association of the RZZ complex and Mad1/Mad2 while inhibiting their dynein-mediated stripping; together with Aurora B kinase activity, CENP-I forms a molecular switch that sustains checkpoint signaling at unattached kinetochores until stable microtubule attachment is achieved (PMID:12640463, PMID:24862574). Beyond mitosis, CENP-I promotes homologous-recombination-mediated DNA double-strand break repair by suppressing R-loop accumulation (PMID:34206916).

1. 2000 High

   The first functional link between a CENP-I ortholog and centromere chromatin was established when fission yeast Mis6 was shown to be required during G1-S for loading the CENP-A homolog, defining CENP-I as upstream of centromeric nucleosome specification.

   Evidence Temperature-sensitive mis6 mutant analysis with cell-cycle staging and chromosome segregation assays in S. pombe

   PMID:10864871

   Open questions at the time

   • Mechanism by which Mis6 promotes CENP-A incorporation was unknown
   • Whether the requirement was direct or indirect was unresolved
   • Applicability to vertebrate centromeres not yet tested
2. 2002 High

   Parallel work in budding yeast (Ctf3p) and chicken DT40 cells placed CENP-I as a constitutive centromere component required for CENP-C localization and proper chromosome segregation, revealing a conserved kinetochore assembly hierarchy in which CENP-I and CENP-H act upstream of CENP-C but downstream of CENP-A.

   Evidence Conditional knockout in DT40 cells with co-localization analysis; ChIP and genetic epistasis in budding yeast

   PMID:11782448 PMID:11970896

   Open questions at the time

   • Whether CENP-I contacts centromeric DNA directly was unknown
   • Relationship between CENP-I and outer kinetochore assembly undefined in vertebrates
3. 2003 High

   Human CENP-I was shown to be required for kinetochore recruitment of CENP-F, MAD1, and MAD2, establishing its role as a platform for spindle assembly checkpoint signaling in human cells.

   Evidence RNAi depletion of CENP-I in human cells with immunofluorescence and checkpoint analysis

   PMID:12640463

   Open questions at the time

   • Whether CENP-I directly interacts with checkpoint proteins or acts indirectly was unclear
   • Mechanism by which residual MAD2-dependent delay persists after CENP-I depletion not resolved
4. 2005 Medium

   Physical interaction between the Mis6 complex (CENP-I ortholog) and Mad2 was demonstrated in fission yeast, providing evidence that the inner kinetochore directly engages checkpoint machinery rather than simply serving as a structural scaffold.

   Evidence Co-immunoprecipitation and genetic analysis in S. pombe under checkpoint-activating conditions

   PMID:15930132

   Open questions at the time

   • Whether the interaction is direct or bridged by other kinetochore components was not determined
   • Co-IP performed under one condition; generality across checkpoint states untested
5. 2014 High

   The checkpoint role of CENP-I was refined to a dual function: generating stable RZZ/Mad1 association at kinetochores and inhibiting dynein-mediated stripping, working together with Aurora B as a molecular switch that sustains checkpoint signaling until mature microtubule attachment.

   Evidence Epistasis between CENP-I depletion and Aurora B inhibition in human cells with quantitative kinetochore localization analysis

   PMID:24862574

   Open questions at the time

   • Direct biochemical basis for CENP-I inhibition of dynein-mediated stripping was not identified
   • How attachment status feeds back to relieve CENP-I's inhibitory role remained unknown
6. 2015 Medium

   CENP-I was linked to centromere maintenance by its ability to recruit M18BP1 and induce de novo CENP-A assembly downstream of CENP-C, revealing it as a coupling factor between kinetochore structure and epigenetic centromere propagation.

   Evidence Ectopic tethering of CENP-I to synthetic alphoid arrays on human artificial chromosomes with immunofluorescence readout

   PMID:26527398

   Open questions at the time

   • Physiological relevance of ectopic tethering system to endogenous centromeres not fully validated
   • Whether CENP-I directly contacts M18BP1 or acts through intermediate factors was not resolved
7. 2020 Medium

   Structural dissection of a conserved intramolecular helix (α11) in CENP-I revealed that it mediates assembly with CENP-H and CENP-M through distinct binding interfaces, beginning to define the structural basis of the CENP-H/I/K/M sub-complex.

   Evidence Mutagenesis, in vitro aggregation assays, and co-immunoprecipitation in HeLa cells

   PMID:32017295

   Open questions at the time

   • No high-resolution structure of the CENP-H/I/K/M complex was available
   • In vivo consequences of helix mutations on kinetochore function not tested
8. 2021 High

   Two distinct non-kinetochore functions were uncovered: in budding yeast, a conserved surface of Ctf3/CENP-I docks the desumoylase Ulp2 to regulate inner kinetochore sumoylation; in human cells, CENP-I promotes homologous recombination DSB repair by suppressing R-loop accumulation.

   Evidence Structural analysis and mutagenesis with sumoylation/segregation assays in yeast; HR/NHEJ reporter assays and RNaseH1 rescue in human cells

   PMID:34081091 PMID:34206916

   Open questions at the time

   • Whether the Ulp2-docking function is conserved in human CENP-I is unknown
   • Mechanism by which CENP-I suppresses R-loops (direct RNA binding vs indirect) was not determined
   • Whether the HR function operates at centromeric or genome-wide loci is unclear
9. 2022 Medium

   Fission yeast Mis6 (CENP-I) was shown to retain pre-existing CENP-A during mitosis by blocking non-coding RNA transcription at centromeres, providing a mechanistic explanation for CENP-A stabilization distinct from direct DNA binding.

   Evidence Conditional depletion of Mis6 in mitotic S. pombe cells with RNA analysis and RNA Pol II inhibition rescue

   PMID:35970865

   Open questions at the time

   • Whether this transcription-blocking mechanism operates in human cells is untested
   • Relationship between this RNA-based mechanism and direct DNA-binding stabilization of CENP-A is unresolved
10. 2023 High

    The long-standing question of whether CENP-I contacts centromeric DNA directly was resolved: CENP-I binds AT-rich DNA through conserved charged residues at the end of N-terminal HEAT repeats, and this interaction is required for centromeric localization, chromosome alignment, and CENP-A loading — independent of CENP-H/K/M binding.

    Evidence In vitro DNA-binding assays, charge-reversal mutagenesis, co-immunoprecipitation, and CENP-A loading assays in human cells

    PMID:36888657

    Open questions at the time

    • No atomic-resolution structure of CENP-I bound to nucleosomal DNA exists
    • Whether DNA-binding and transcription-blocking are the same or separable mechanisms is unclear

• A high-resolution structure of CENP-I bound to centromeric nucleosomal DNA is lacking, and the molecular basis by which CENP-I inhibits dynein-mediated stripping of checkpoint proteins, the conservation of the Ulp2-docking function in vertebrates, and the mechanism linking CENP-I to R-loop suppression remain unresolved.
• No cryo-EM or crystal structure of CENP-I on nucleosomal DNA
• Dynein-stripping inhibition mechanism not biochemically defined
• R-loop suppression mechanism (direct vs indirect) uncharacterized