The impact of novel and confirmed cenH3 assembly factors on plant kinetochore formation

组蛋白H3被变体CENH 3取代是迄今为止研究的所有真核生物的着丝粒核小体的共同特征。尽管这一过程在进化上是保守的，但尚不清楚是什么触发了CENH 3沉积，以及它如何靶向高度可变的着丝粒DNA序列。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。在确定了有丝分裂周期中的加载时间后，本提案的目的是通过找出相互作用的因素及其模式来指导CENH 3到着丝粒进行动粒组装，从而深入了解CENH 3在植物中的沉积。将通过体内表达EYFP融合构建体和原位表达特异性抗体来分析推定的CENH 3组装因子（Sim 3、KNL 2和Mis 12）的动力学和亚细胞定位。CENH 3组装因子和CENH 3之间的潜在相互作用将通过BiFC、酵母双杂交和IP方法进行测试。将在T-DNA插入突变体和/或相应RNAi构建体的转基因植物中研究CENH 3组装因子表达的敲除/敲低的后果。为了了解CENH 3的N端和C端部件的功能要求，将分别测试它们与CENH 3组装因子的相互作用。

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. After having established the loading time during the mitotic cycle, the aim of this proposal is to get insight into CENH3 deposition in plants by finding out interacting factors and their mode to guide CENH3 to centromeres for kinetochore assembly. Kinetics and subcellular localization of putative CENH3 assembly factors (Sim3, KNL2 and Mis12) will be analysed via expression of EYFP fusion constructs in vivo and with specific antibodies in situ. Potential interactions between CENH3 assembly factors and CENH3 will be tested by BiFC, yeast two-hybrid and IP approaches. Consequences of knock out/knock down of CENH3 assembly factor expression will be studied in T-DNA insertion mutants and/or in plants transgenic for the corresponding RNAi constructs. In order to understand functional requirement of N and C-terminal parts of CENH3, they will be tested separately for interaction with CENH3 assembly factors.