Arabidopsis KINETOCHORE NULL2 Is an Upstream Component for Centromeric Histone H3 Variant cenH3 Deposition at Centromeres

Abstract

The centromeric histone H3 variant cenH3 is an essential centromeric protein required for assembly, maintenance, and proper function of kinetochores during mitosis and meiosis. We identified a KINETOCHORE NULL2 (KNL2) homolog in Arabidopsis thaliana and uncovered features of its role in cenH3 loading at centromeres. We show that Arabidopsis KNL2 colocalizes with cenH3 and is associated with centromeres during all stages of the mitotic cell cycle, except from metaphase to mid-anaphase. KNL2 is regulated by the proteasome degradation pathway. The KNL2 promoter is mainly active in meristematic tissues, similar to the cenH3 promoter. A knockout mutant for KNL2 shows a reduced level of cenH3 expression and reduced amount of cenH3 protein at chromocenters of meristematic nuclei, anaphase bridges during mitosis, micronuclei in pollen tetrads, and 30% seed abortion. Moreover, knl2 mutant plants display reduced expression of suppressor of variegation 3-9 homologs2, 4, and 9 and reduced DNA methylation, suggesting an impact of KNL2 on the epigenetic environment for centromere maintenance.

Nonrandom domain organization of the Arabidopsis genome at the nuclear periphery

“…A possible scenario accounting for this observation is that NPCs (or at least NUP1-containing NPCs) are not evenly distributed at the nuclear envelope, such that the NPC density is lower in regions where chromocenters are anchored. For instance, kinetochore proteins interact with Arabidopsis centromeres in almost all stages of the cell cycle (Lermontova et al 2013), and Gamma-tubulin Complex Protein 3-interacting proteins (GIPs) play essential roles in centromere assembly (Batzenschlager et al 2015); these interactions might sequester centromeres away from NPCs. We examined nuclei in transgenic plants coexpressing NUP1:GFP and mCherry:CENH3, in which the latter was exclusively loaded to centromeres (Lermontova et al 2006).…”

Targeting of Arabidopsis KNL2 to Centromeres Depends on the Conserved CENPC-k Motif in Its C Terminus

“…Therefore, we suggest that the CENPC-k motif of KNL2 is involved in binding centromeric nucleosomes, similar to the corresponding motif of the CENP-C protein. In view of the poor conservation of CENP-C and KNL2 proteins across eukaryotic kingdoms, outside of their CENPC-like motifs (Talbert et al, 2004;Maddox et al, 2007;Lermontova et al, 2013), it is possible that the CENPC-k motif of KNL2 or CENPC motif of CENP-C fused with any desired protein may lead to centromeric localization. Although this remains to be tested in further studies, this approach may provide an interesting tool for centromere research.…”

The Centromere: Epigenetic Control of Chromosome Segregation during Mitosis

“…All three proteins interact but it is not known whether Mis18a, Mis18b, and M18BP1 function in CENP-A assembly exclusively as a complex. A homologous M18BP1 protein, Kinetochore Null 2 (KNL2), was discovered in a screen for kinetochore defects in C. elegans and M18BP1 has been identified in humans, Xenopus, and Arabidopsis (Fujita et al 2007;Maddox et al 2007;Moree et al 2011;Lermontova et al 2013). The function of the Mis18 complex remains unclear, and more proteins are likely to be involved.…”