Centromere Maintenance Via CENH3

The histone H3 variant (CENH3) of centromeric nucleosomes is essential for kinetochore assembly and thus for chromosome segregation in eukaryotes. The mechanism(s) that determine centromere identity, assembly and maintenance of kinetochores are still poorly understood, the more so as CENH3 is not loaded during chromatin replication in S phase but in G2, as we have shown for angiosperms and protozoans. Whereas the role of CENH3 during mitosis has been studied in several organisms, little is known about its meiotic function. We show that RNAi-mediated knock down of CENH3 led to reduced CENH3 expression, a dwarf phenotype, but not to mitotic abnormalities in root tip meristems of transformants. Flow cytometry revealed a significant increase of the 4C:2C ratio indicating suppression of mitosis in RNAi plants. In CENH3 RNAi transformants fertility was reduced because of frequently disturbed meiotic chromosome segregation. N-terminally truncated EYFP-CENH3(C) is deposited to and functional within Arabidopsis centromeres of mitotic chromosomes but cannot be loaded to centromeres of meiotic nuclei. Apparently the N-terminal part is required for CENH3 loading during meiosis via meiosis-specific chaperones. EYFP-CENH3(C) expression can reduce the amount of endogenous CENH3, thus mimicking the effect of RNAi. The consequences of reduced endogenous CENH3 and of lack of meiotic incorporation of EYFP-CENH3(C) are reduced fertility caused by insufficient CENH3 loading to the centromeres of meiotic chromosomes, subsequent lagging of chromosomes and formation of micronuclei.