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

组蛋白H3被CENH3变异体取代是目前研究的所有真核生物着丝粒核小体的共同特征。尽管这一过程在进化上是保守的，但尚不清楚是什么触发了CENH3的沉积，以及它如何成为高度可变的着丝粒DNA序列的靶标。这些知识形成了理解着丝粒形成和产生人工着丝粒的基础。这项建议的目的是通过研究枯竭(T-DNA插入突变体，RNAi)和重组CENH3在拟南芥中过表达的后果来深入了解CENTO在植物中的沉积。将来自不同亲缘关系物种的荧光标记的CENH3转基因方法应用于野生型和突变型拟南芥植物，应该可以跨物种边界揭示CENH3的功能。反过来，将测试AtCENH3针对其他物种的着丝粒DNA序列的能力，这些DNA序列与拟南芥着丝粒重复序列具有不同程度的相似性。此外，我们还将研究：(1)在异源多倍体(A.suecica)中，亲本CENH3基因是否都是活性的；(2)CENH3的N-末端和C-末端部分的功能需求；(3)CENH3在G2发生沉积时表达的假设。

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.