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

• Applicant: Dr. Inna Lermontova
• Subject Area: Plant Genetics and Genomics ; Plant Cell and Developmental Biology
• Term: from 2011 to 2018
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 192140482

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.

• DFG Programme: Research Grants