Meiosis-specific loading of the centromere-specific histone CENH3 in Arabidopsis thaliana.

Centromere behavior is specialized in meiosis I, so that sister chromatids of homologous chromosomes are pulled toward the same side of the spindle (through kinetochore mono-orientation) and chromosome number is reduced. Factors required for mono-orientation have been identified in yeast. However, comparatively little is known about how meiotic centromere behavior is specialized in animals and plants that typically have large tandem repeat centromeres. Kinetochores are nucleated by the centromere-specific histone CENH3. Unlike conventional histone H3s, CENH3 is rapidly evolving, particularly in its N-terminal tail domain. Here we describe chimeric variants of CENH3 with alterations in the N-terminal tail that are specifically defective in meiosis. Arabidopsis thaliana cenh3 mutants expressing a GFP-tagged chimeric protein containing the H3 N-terminal tail and the CENH3 C-terminus (termed GFP-tailswap) are sterile because of random meiotic chromosome segregation. These defects result from the specific depletion of GFP-tailswap protein from meiotic kinetochores, which contrasts with its normal localization in mitotic cells. Loss of the GFP-tailswap CENH3 variant in meiosis affects recruitment of the essential kinetochore protein MIS12. Our findings suggest that CENH3 loading dynamics might be regulated differently in mitosis and meiosis. As further support for our hypothesis, we show that GFP-tailswap protein is recruited back to centromeres in a subset of pollen grains in GFP-tailswap once they resume haploid mitosis. Meiotic recruitment of the GFP-tailswap CENH3 variant is not restored by removal of the meiosis-specific cohesin subunit REC8. Our results reveal the existence of a specialized loading pathway for CENH3 during meiosis that is likely to involve the hypervariable N-terminal tail. Meiosis-specific CENH3 dynamics may play a role in modulating meiotic centromere behavior.

Ravi M ，Shibata F ，Ramahi JS ，Nagaki K ，Chen C ，Murata M ，Chan SW ... -  《PLoS Genetics》

The rapidly evolving centromere-specific histone has stringent functional requirements in Arabidopsis thaliana.

Centromeres control chromosome inheritance in eukaryotes, yet their DNA structure and primary sequence are hypervariable. Most animals and plants have megabases of tandem repeats at their centromeres, unlike yeast with unique centromere sequences. Centromere function requires the centromere-specific histone CENH3 (CENP-A in human), which replaces histone H3 in centromeric nucleosomes. CENH3 evolves rapidly, particularly in its N-terminal tail domain. A portion of the CENH3 histone-fold domain, the CENP-A targeting domain (CATD), has been previously shown to confer kinetochore localization and centromere function when swapped into human H3. Furthermore, CENP-A in human cells can be functionally replaced by CENH3 from distantly related organisms including Saccharomyces cerevisiae. We have used cenh3-1 (a null mutant in Arabidopsis thaliana) to replace endogenous CENH3 with GFP-tagged variants. A H3.3 tail domain-CENH3 histone-fold domain chimera rescued viability of cenh3-1, but CENH3's lacking a tail domain were nonfunctional. In contrast to human results, H3 containing the A. thaliana CATD cannot complement cenh3-1. GFP-CENH3 from the sister species A. arenosa functionally replaces A. thaliana CENH3. GFP-CENH3 from the close relative Brassica rapa was targeted to centromeres, but did not complement cenh3-1, indicating that kinetochore localization and centromere function can be uncoupled. We conclude that CENH3 function in A. thaliana, an organism with large tandem repeat centromeres, has stringent requirements for functional complementation in mitosis.

Ravi M ，Kwong PN ，Menorca RM ，Valencia JT ，Ramahi JS ，Stewart JL ，Tran RK ，Sundaresan V ，Comai L ，Chan SW ... -  《-》

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

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. 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.

Lermontova I ，Koroleva O ，Rutten T ，Fuchs J ，Schubert V ，Moraes I ，Koszegi D ，Schubert I ... -  《-》

Arabidopsis kinetochore null2 is an upstream component for centromeric histone H3 variant cenH3 deposition at centromeres.

Lermontova I ，Kuhlmann M ，Friedel S ，Rutten T ，Heckmann S ，Sandmann M ，Demidov D ，Schubert V ，Schubert I ... -  《-》

The H3 histone chaperone NASP(SIM3) escorts CenH3 in Arabidopsis.

Centromeres define the chromosomal position where kinetochores form to link the chromosome to microtubules during mitosis and meiosis. Centromere identity is determined by incorporation of a specific histone H3 variant termed CenH3. As for other histones, escort and deposition of CenH3 must be ensured by histone chaperones, which handle the non-nucleosomal CenH3 pool and replenish CenH3 chromatin in dividing cells. Here, we show that the Arabidopsis orthologue of the mammalian NUCLEAR AUTOANTIGENIC SPERM PROTEIN (NASP) and Schizosaccharomyces pombe histone chaperone Sim3 is a soluble nuclear protein that binds the histone variant CenH3 and affects its abundance at the centromeres. NASPSIM3 is co-expressed with Arabidopsis CenH3 in dividing cells and binds directly to both the N-terminal tail and the histone fold domain of non-nucleosomal CenH3. Reduced NASPSIM3 expression negatively affects CenH3 deposition, identifying NASPSIM3 as a CenH3 histone chaperone.

Le Goff S ，Keçeli BN ，Jeřábková H ，Heckmann S ，Rutten T ，Cotterell S ，Schubert V ，Roitinger E ，Mechtler K ，Franklin FCH ，Tatout C ，Houben A ，Geelen D ，Probst AV ，Lermontova I ... -  《-》