Functional characterization of the plant centromeric histone variant CENH3 as a prerequisite to understand kinetochore formation

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Substitution of histone H3 by the variant CENH3 is a common feature of centromeric nucleosomes of all eukaryotes studied so far. Despite the evolutionary conservation of this process it is unknown what triggers CENH3 deposition and how it becomes targeted to the highly variable centromeric DNA sequences. Such knowledge forms the basis to understand centromere formation and to generate artificial centromeres. The aim of this proposal is to get insight into CENTO deposition in plants by studying consequences of depletion (T-DNA insertion mutants, RNAi) and overexpression of recombinant CENH3 in A. thaliana. Transgenic approaches applying fluorescently tagged CENH3 from species of different phylogenetic affiliation to wild-type and mutant A. thaliana plants should reveal CENH3 functionality across species borders. Reciprocally, the capability of AtCENH3 to target centromeric DNA sequences of other species with different degrees of similarity to A. thaliana centromeric repeats will be tested. Furthermore we will study i) whether in allopolyploids (A. suecica) both parental CENH3 genes are active, ii) the functional requirement of N- and C-terminal parts of CENH3 and iii) the hypotheisis of CENH3 expression during G2 when deposition occurs.