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

• Applicant: Dr. Inna Lermontova
• Subject Area: Plant Genetics and Genomics ; Plant Cell and Developmental Biology
• Term: since 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 516520480

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.

• DFG Programme: Research Grants