Elucidation of the functional role of A. thaliana KNL2 in epigenetic regulation of kinetochore assembly in plants

在大多数真核生物中，在细胞分裂期间染色体的正确分离需要着丝粒组蛋白H3变体（cenH3）的存在。cenH3装载到着丝粒是一个多步骤的过程，它不依赖于特定的DNA序列，而是取决于着丝粒染色质的表观遗传状态和不同的组装因子。cenH3的不正常表达或其装载到着丝粒上的不当导致植物和动物的有丝分裂和减数分裂缺陷。虽然有许多关于动物中cenH3装载到着丝粒的调控的出版物（对植物的了解较少），但这一多步骤过程的机制必须更详细地阐明。之前我们已经鉴定并表征了第一个植物cenH3组装因子KNL2。我们发现敲除KNL2导致着丝粒cenH3水平降低，DNA甲基化减少，有丝分裂和减数分裂缺陷，随后生长速度和生育能力降低。在应用项目的框架内，我们旨在阐明以拟南芥为模型的cenH3装载和着丝粒组装的表观遗传调控，并解读KNL2蛋白在这一过程中的作用。我们将研究i)表观遗传突变体中DNA和H3K9甲基化水平的改变如何影响cenH3和其他着丝粒蛋白的着丝粒装载，ii)敲除cenH3组装因子KNL2如何影响全球拟南芥基因组的甲基化，iii) MET1和VIM1等表观遗传因子如何定向靶向着丝粒调节着丝粒在细胞周期不同阶段的组装和功能。这些知识对于了解着丝粒上着丝粒蛋白复合物的组装和功能机制，为染色体工程生成人工着丝粒，以及提高双单倍体的产生效率，为基于着丝粒操纵的植物育种过程提速至关重要。

In most eukaryotic organisms correct segregation of chromosomes during cell divisions requires presence of the centromeric histone H3 variant (cenH3). Loading of cenH3 to centromeres is a multi-step process which does not depend on specific DNA sequences, but depends on the epigenetic status of centromeric chromatin and on different assembly factors. Deregulated expression of cenH3 or its improper loading to centromeres results in mitotic and meiotic defects in plants and animals. Although numerous publications on the regulation of cenH3 loading to centromeres in animals (less is known from plants) exist, the mechanism of this multi-step process has to be elucidated more in detail. Previously we have identified and characterized the first plant cenH3 assembly factor KNL2. We showed that the knockout of KNL2 results in a reduced level of cenH3 at centromeres, reduced DNA methylation, defects in mitosis and meiosis followed by reduced growth rate and fertility. In the frame of the applied project we aim to elucidate the epigenetic regulation of cenH3 loading and kinetochore assembly using Arabidopsis thaliana as a model and to decipher the role of KNL2 proteins in this process. We will study i) how in epigenetic mutants an altered level of DNA and H3K9 methylation influences the centromere loading of cenH3 and other kinetochore proteins, ii) how a knockout of the cenH3 assembly factor KNL2 influences the methylation of the global Arabidopsis genome, iii) how the directed targeting of epigenetic factors like MET1 and VIM1 to centromeres regulate kinetochore assembly and function during different stages of the cell cycle. Such knowledge is essential i) to understand the mechanism of the assembly and function of the kinetochore protein complex at the centromeres, ii) to generate artificial centromeres for chromosome engineering and iii) to improve the efficiency of generating double haploids for an accelerated plant breeding process based on centromere manipulation.