CENP-I Regulates Localization Of Newly Synthesized CENP-A Through Binding To Centromere DNA

During mitosis,the proper attachment of spindle microtubules to centromeres ensures correct alignment and faithful segregation of chromosomes,which is essential for propagation of genetic information and survival of daughter cells.The centromere is a specialized locus on chromosomes that provide a platform for the assembly of kinetochore and the attachment of microtubules.CENP-A,a histone H3 variant,serves as an epigenetic marker that specifically localizes to centromere and initiates the assembly of kinetochore.Kinetochore is a multi-subunit complex containing hundreds of centromeric proteins,the core complex CENP-H/I/K/M is responsible for maintaining the overall kinetochore assembly,but the mechanism of interaction with centromeres and CENP-A nucleosomes remains unclear.In this study,the CENP-H/I/K/M subunits of fungal and human were reconstituted in vitro and electrophoretic mobility shift assays(EMSA)were performed with centromeric DNA.The interaction between the N-terminal HEAT Repeat domain of CENP-I and centromeric DNA was found to be conserved in different species.In addition,binding of CENP-I to DNA shows base preference.CENP-I is more likely to bind to AT-rich DNA fragments which is a major feature of human centromeric DNA.CENP-I interacts with CENP-A or H3 nucleosome by binding to centromeric DNA in vitro,which could stabilize CENP-A nucleosome but loosen the H3 nucleosome.The key amino acid sites that mediate ctCENP-I binding to DNA have been identified in our laboratory previously:K189,K209,R216,R225,and R226,and ctCENP-INT-3M/5M mutants were designed.Here,we further introduced corresponding amino acid site mutants in human CENP-I referred as hsCENP-INT-3M/5M,surface plasmon resonance(SPR)and EMSA showed that both fungal and human CENP-INT-5M affect binding of CENP-I to centromeric DNA.Complex reconstitution in vitro and co-immunoprecipitence assays in cells indicated that ct/hsCENP-INT-3M/5M did not affect protein folding and assembly activity of CENP-I.The interaction between CENP-I and centromeric DNA has a significant effect on kinetochore assembly and localization in cells.CRISPR-Cas9 technology was used to generate endogenous CENP-I conditional knockout HeLa cell lines.After inducing endogenous CENP-I deletion and expressing hsCENP-I-WT/3M/5M,immunofluorescence assay showed that the centromeric localization of CENP-I was affected in different degrees in both interphase and metaphase cells,and severe chromosome misalignment occurred in metaphase.Since the denovo human CENP-A localizes to centromere in G1 phase,influence of hsCENP-I-WT/3M/5M on the localization of newly synthesized CENP-A was examined by SNAP-tag-mediated quench-pulse assay during interphase.The results showed that the loss of CENP-I interaction with DNA significantly reduced the centromeric loading efficiency of nascent CENP-A and caused abnormal kinetochore assembly.Therefore,we confirmed the conservation of interaction between CENP-I and centromeric DNA,and effect on the localization of CENP-I and newly synthesized CENPA were also unveiled.Moreover,we revealed different patterns of interaction between CENP-I and CENP-A/H3 nucleosomes,which provided new insight for understanding binding of CENP-I to centromere.These findings provide a molecular perspective for CENP-I regulating centromeric localization of CENP-A,which contribute to explore the dynamic assembly of kinetochore and the maintenance mechanism of centromere.