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

组蛋白H3被CENH3变体取代是迄今为止所研究的所有真核生物的着丝粒核小体的共同特征。尽管这一过程具有进化保守性，但尚不清楚是什么触发了CENH3沉积，以及它如何成为高度可变的着丝粒DNA序列的目标。这些知识是了解着丝粒形成和人工产生着丝粒的基础。本研究的目的是通过研究拟南芥中重组CENH3的耗尽（T-DNA插入突变，RNAi）和过表达的后果来深入了解CENTO在植物中的沉积。将来自不同系统发育亲缘关系物种的CENH3荧光标记转基因方法应用于野生型和突变型拟南芥植物，应该可以揭示出CENH3跨物种的功能。反过来，AtCENH3靶向与拟南螺旋体着丝粒重复序列具有不同程度相似性的其他物种的着丝粒DNA序列的能力将被测试。我们将进一步研究i)在异源多倍体（A. suecica）中，亲本CENH3基因是否都有活性，ii) CENH3 N端和c端部分的功能需求，iii)沉积发生时G2期间CENH3表达的假设。

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.