The impact of novel and confirmed cenH3 assembly factors on plant kinetochore formation
Plant Cell and Developmental Biology
Final Report Abstract
Centromeres contain specialized nucleosomes at which histone H3 is replaced by the centromeric histone H3 variant cenH3 that is required for the assembly, maintenance, and proper function of kinetochores during mitotic and meiotic divisions. In higher eukaryotes, positions of cenH3 incorporation and kinetochore assembly do not depend on specific DNA sequences, but are rather determined epigenetically. We have identified a KNL2 (Kinetochore Null 2) of A. thaliana that is involved in the recognition of centromeres and centromeric localization of cenH3 similar to its homologues of C. elegans and mammals. KNL2 homologues identified so far contain a conserved SANTA domain, possibly involved in protein-protein interactions, at the N-terminus, but only some of them contain SANT/Myb (DNA binding) domain at the C-terminus. Our recent study revealed a cenH3 nucleosome-binding CENPC-k motif at the C-terminus of A. thaliana KNL2, which is conserved among a wide spectrum of eukaryotes. Centromeric localization of KNL2 is abolished by deletion of the CENPC-k motif and by mutating single conserved amino acids, but can be restored by insertion of the corresponding motif of A. thaliana CENP-C. We showed by electromobility shift assay (EMSA) that the C-terminus of KNL2 binds DNA sequence-independently and interacts with the centromeric pAL1 transcripts in vitro. Chromatin immunoprecipitation with anti-KNL2 antibodies indicated that in vivo KNL2 is preferentially associated with the centromeric repeat pAL1. Complete deletion of the CENPC-k motif did not influence its ability to interact with DNA in vitro. Therefore, we suggest that KNL2 recognizes centromeric nucleosomes, similar to CENP-C, via the CENPC-k motif and binds adjoining DNA. On the base of project results the patent application proposing the KNL2-based alternative mechanism of double haploid generation in plants was submitted in October 2015. Our unpublished results will be used as the base for new research projects.
Publications
- (2015) Arabidopsis MZT1 homologs GIP1 and GIP2 are essential for centromere architecture. Proc Natl Acad Sci USA, 112, 8656-8660
Batzenschlager, M., Lermontova, I., Schubert, V., Fuchs, J., Berr, A., Koini, M.A., Houlne, G., Herzog, E., Rutten, T., Alioua, A., Fransz, P., Schmit, A.C. and Chaboute, M.E.
(See online at https://doi.org/10.1073/pnas.1506351112) - (2015) Centromeric hromatin and its dynamics in plants. Plant J, 83, 4-17
Lermontova, I., Sandmann, M., Mascher, M., Schmit, A.C. and Chaboute, M.E.
(See online at https://doi.org/10.1111/tpj.12875) - (2016) Aurora Kinases Throughout Plant Development. Trends Plant Sci, 21, 69-79
Weimer, A.K., Demidov, D., Lermontova, I., Beeckman, T. and Van Damme, D.
(See online at https://doi.org/10.1016/j.tplants.2015.10.001) - (2016) Immunolabeling of Nuclei/Chromosomes in Arabidopsis thaliana. Methods Mol Biol, 1370, 127-135
Sandmann, M., Fuchs, J. and Lermontova, I.
(See online at https://doi.org/10.1007/978-1-4939-3142-2_10) - (2017) Targeting of A. thaliana KNL2 to centromeres depends on the conserved CENPC-k motif of its C-terminus. Plant Cell, 27(1), 144-155
Sandmann, M., Talbert, P., Demidov, D., Kuhlmann, M., Rutten, T., Conrad, U., Lermontova, I.
(See online at https://doi.org/10.1105/tpc.16.00720) - (2017). Generation of haploid plants based on KNL2. EP 15191078.3
Lermontova, I.
