Centromeric transcripts and their role in the formation of centromeric chromatin and kinetochore assembly

有丝分裂和减数分裂期间染色体的分离是由称为着丝粒的离散区域控制的。细胞分裂是由附着在着丝粒上的一种叫做着丝点的蛋白质结构控制的，着丝点是纺锤体微管结合的主要部位。含有cenH3的着丝染色质通过染色质修饰在表观遗传上被标记。cenH3在着丝粒上的装载受着丝粒许可因子、cenH3伴侣和着丝粒转录本的调控。AtKNL2作为着丝粒许可因子在cenH3进入着丝粒核小体的过程中起着重要作用。我们之前的报道表明，KNL2可以结合着丝粒DNA和转录本，而且转录本能够通过与KNL2的相互作用来竞争DNA。然而，KNL2在调节着丝粒转录本中的功能作用尚未得到详细研究。通过对KNL2进行免疫沉淀、质谱（IP-MS）和酵母双杂交（Y2H）文库筛选，我们共鉴定出44个可能通过RNAi沉默机制参与着丝粒重复序列调控的蛋白。其中，NRPB2 （RNA聚合酶II）和EIF4AIII（剪接因子）在两个实验中都被鉴定出，可以认为是推测的直接KNL2相互作用物，而AGO1、AGO9和MET1通过IP-MS鉴定得到高分，可以间接连接到蛋白质复合物中的KNL2和cenH3。在这个项目中，我们的目的是阐明KNL2在调节着丝粒转录本中的作用，以及在cenH3结合和着丝粒蛋白向着丝粒募集过程中着丝粒转录本的功能。为了验证所选候选物与着丝粒蛋白KNL2和cenH3的相互作用，将应用BiFC， Y2H， FRET和Co-IP方法。选择的候选蛋白动力学将通过细胞周期和组织特异性定位分析进行研究。此外，将通过免疫- smfish技术检测cen180转录本在拟南螺旋体中的分布及其与候选蛋白和着丝粒蛋白的定位模式。我们将对nrpb2、eif4a-iii、ago1、ago9和met1进行功能表征，并详细研究它们的失调控表达如何影响着丝粒蛋白的稳定性和改变着丝粒的表观遗传状态。

The segregation of chromosomes during mitosis and meiosis is controlled by discrete regions called centromeres. Cell divisions are governed by a proteinaceous structure called kinetochores found attached to centromeres which act as a major site for spindle microtubule binding. The centromeric chromatin containing cenH3 has been marked epigenetically through chromatin-based modifications. The loading of cenH3 on centromeres is regulated by centromere licensing factors, cenH3 chaperones, and centromeric transcripts. AtKNL2 plays an important role as a centromere licensing factor in the incorporation of cenH3 into centromeric nucleosomes. Our previous reports demonstrated that KNL2 can bind centromeric DNA and transcripts, moreover, the transcripts are able to compete out DNA from interaction with KNL2. However, the functional role of KNL2 in the regulation of centromeric transcripts has not been studied in detail. By performing immunoprecipitation followed by mass spectrometry (IP-MS) and yeast two-hybrid (Y2H) library screening of KNL2, we identified a total of 44 proteins that might be involved in the regulation of centromeric repeats through the RNAi silencing mechanism. Among them, NRPB2 (RNA polymerase II) and EIF4AIII (splicing factor) were identified in both experiments and can be considered as putative direct KNL2 interactors, whereas AGO1, AGO9, and MET1 were identified by IP-MS with high score and can be indirectly linked to KNL2 and cenH3 in a protein complex. In this project, we aim to elucidate the role of KNL2 in the regulation of centromeric transcripts and the function of centromeric transcripts during the incorporation of cenH3 and recruitment of kinetochore proteins to centromeres. To validate the interaction of the selected candidates with the centromeric proteins KNL2 and cenH3, BiFC, Y2H, FRET and Co-IP approaches will be applied. The protein dynamics of selected candidates will be studied through both cell cycle and tissue-specific localization analysis. Further, the distribution of cen180 transcripts in A. thaliana and their localization pattern with the candidate and centromeric proteins will be detected through the Immuno-smFISH technique. Functional characterization of nrpb2, eif4a-iii, ago1, ago9, and met1 will be performed, and how their deregulated expression influences the stabilization of centromeric proteins and alters the epigenetic status of centromeres will be investigated in detail.