Knockdown of CENH3 in Arabidopsis reduces mitotic divisions and causes sterility by disturbed meiotic chromosome segregation

Abstract

SUMMARYThe 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. Although the role of CENH3 during mitosis has been studied in several organisms, little is known about its meiotic function. We show that RNAi-mediated CENH3 knockdown in Arabidopsis thaliana caused dwarfism as the result of a reduced number of mitotic divisions. The remaining mitotic divisions appeared to be error-free. CENH3 RNAi transformants had reduced fertility 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 onto centromeres of meiotic nuclei. Thus the N-terminal part is apparently required for CENH3 loading during meiosis. EYFP-CENH3(C) expression reduces the amount of endogenous CENH3, thus mimicking the effect of RNAi. The consequences of reduced endogenous CENH3 and 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.

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

“…We observed that in a population of knl2 mutant plants (;60 plants), ;30% of plants show reduced growth (Figure 4) and a phenotype similar to that of the cenH3 RNAi transformants (Lermontova et al, 2011a), while growth rate of another 70% of plants was only slightly reduced or did not differ from the wild type. Such phenotypic variation within a population of homozygous mutants was also observed for epigenetic mutants of Arabidopsis, such as met1-3 (Mathieu et al, 2007) and ddm1, the latter showing a gradual accumulation of developmental aberrations during inbreeding (Vongs et al, 1993;Kakutani et al, 1996).…”

“…Therefore, we analyzed the expression of the histone methyltransferase encoding genes SUVH4, SUVH2, and SUVH9 in 14-d-old seedlings of Arabidopsis wild type, knl2 mutants, and cenH3 RNAi transformants (Lermontova et al, 2011a) by real-time RT-PCR ( Figure 6A). We found that expression of SUVH4 and SUVH9 was reduced in the knl2 mutant to 50% of the wild-type level, but expression of SUVH2 was only slightly lower than in the wild type ( Figure 6A).…”

Osmotic stress induces phosphorylation of histone H3 at threonine 3 in pericentromeric regions of Arabidopsis thaliana

“…S8). In addition, the abundance of a centromerespecific histone H3 variant (CENH3), required for centromere function and chromosome segregation (34), was reduced in the mutant background (SI Appendix, Fig. S9F).…”

The AAA-ATPase molecular chaperone Cdc48/p97 disassembles sumoylated centromeres, decondenses heterochromatin, and activates ribosomal RNA genes

“…If left unchecked, neocentromeres result in the loss of chromosome integrity, which leads to diseases, including cancer, in humans (1,38). Below a certain threshold of cellular CenH3 levels, cells cease mitosis in Arabidopsis (39). Similarly, in human cells, the posttranslational reduction of CenH3 levels results in senescence, where cells lose the ability to divide, likely to prevent defective mitoses (7), which may be a process that requires p97.…”