Arabidopsis MZT1 homologs GIP1 and GIP2 are essential for centromere architecture

Centromeres play a pivotal role in maintaining genome integrity by facilitating the recruitment of kinetochore and sister-chromatid cohesion proteins, both required for correct chromosome segregation. Centromeres are epigenetically specified by the presence of the histone H3 variant (CENH3). In this study, we investigate the role of the highly conserved γ-tubulin complex protein 3-interacting proteins (GIPs) in Arabidopsis centromere regulation. We show that GIPs form a complex with CENH3 in cycling cells. GIP depletion in the gip1gip2 knockdown mutant leads to a decreased CENH3 level at centromeres, despite a higher level of Mis18BP1/KNL2 present at both centromeric and ectopic sites. We thus postulate that GIPs are required to ensure CENH3 deposition and/or maintenance at centromeres. In addition, the recruitment at the centromere of other proteins such as the CENP-C kinetochore component and the cohesin subunit SMC3 is impaired in gip1gip2. These defects in centromere architecture result in aneuploidy due to severely altered centromeric cohesion. Altogether, we ascribe a central function to GIPs for the proper recruitment and/or stabilization of centromeric proteins essential in the specification of the centromere identity, as well as for centromeric cohesion in somatic cells.In eukaryotes, centromeres play a critical role in accurate chromosome segregation and in the maintenance of genome integrity through their regulated assembly and the maintenance of their cohesion until anaphase. Centromeres consist each of a central core (1) characterized epigenetically by the recruitment of the histone H3 variant CENH3 (CENP-A in animals). Extensive studies are still ongoing to identify the regulatory factors for loading and maintenance of CENH3 at centromeres. In yeast, suppressor of chromosome missegregation protein 3 was identified as a specific chaperone for CENP-A loading (2). In animals, the Mis18 complex, including the CENH3 assembly factor Kinetochore Null 2 (KNL2; also called Mis18BP1), recruits the cell cycle-dependent maintenance and deposition factor of CENP-A, HJURP (Holliday junction recognition protein), to centromeres (3). Recently, two Mis18-complex components, Eic1 and Eic2, were identified in fission yeast (4). Whereas Eic1 promotes CENH3 loading and maintenance, Eic2 is recruited at centromeres independently of its association with Mis18. Together with CENH3, the conserved kinetochore assembly protein CENP-C participates in pericentromeric cohesin recruitment (5). The CENH3 loading machinery changed rapidly during evolution, and a CENH3 chaperone has not been identified in plants thus far. Moreover, nothing is known about a possibly conserved interplay between CENH3 loading and sister chromatid cohesion at centromeres. Recently, the plant homolog of KNL2 was proposed as an upstream component for CENH3 deposition at centromeres (6). Finally, the regulation of centromeric complex positioning at the nuclear envelope environment is still elusive in plants.

Table S1.

Gene accession numbers

Gene	Accession number
GIP1	At4G09550
GIP2	At1g73790
CENH3	At1g01370
CENP-C	At1g15660
KNL2	At5G02520
CTF7	At4g31400
MAD3-1	At2g33560
MAD3-2	At5g05510

Open in a separate windowPreviously, we characterized the γ-tubulin complex protein 3-interacting proteins (GIPs), GIP1 and GIP2 (7, 8). This function seems conserved in the human and Schizosaccharomyces pombe GIP homologs named mitotic spindle organizing protein 1 (MZT1) (9–11). More recently, we localized GIPs at the nucleoplasm periphery, close to chromocenters, where they modulate the nuclear architecture (12, 13). Here, we exploit the various phenotype gradations of knockdown gip1gip2 mutants to investigate the role of GIPs at centromeres. We demonstrate that GIPs are required for CENH3 stabilization and centromere cohesion in Arabidopsis. We further show that these nuclear functions are not related to mitotic checkpoint controls and occur in addition to the previously established role of GIPs/MZT1 in spindle microtubule robustness. Our results highlight a previously unidentified aspect of centromere regulation mediated by GIPs/MZT1 to maintain genomic and ploidy stability.