Production of a synthetic repeat array system for artificial centromere formation in maize & epigenetic regulation of kinetochore size in plants
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Centromeres are specified by a histone H3 variant CENH3 and a DNA-binding protein CENPC, both of which are conserved components of the inner kinetochores. In animals, ectopically localized CENH3 or CENPC is sufficient to assemble de novo kinetochores. In this study, we tested whether similar observations can be achieved in maize to create artificial centromeres. To this end, we have developed a satellite repeat array, which is composed of five different binding modules and was designed to be used as a tethering site. We showed that it is possible to synthetically engineer and introduce megabase repeat arrays into the maize genome by biolistic transformation, and that the array was sufficient to efficiently tether fluorescent proteins to specific loci. We utilized the array system to specifically tether kinetochore protein CENH3 and CENPC to the chromosome arms. We showed that although these proteins may be transiently targeted to ectopic loci, they tended to be unstable and insufficient to induce neocentromere formation. Rather, transgenic CENH3 or CENPC fused with different tethering proteins were localized at endogenous centromeres and caused strong dominant-negative affects on native kinetochores. Our observations suggest that plant kinetochores are highly stable and rarely move. We also investigated centromere size variations in the grass species. We showed that the size of the CENH3 domains is correlated with genome size divided by chromosome number. We also observed that CENH3 domains are flexible and can change rapidly in new environments where two species are crossed. These observations lead to the speculation that the size of the CENH3 domain is a reflection of how much kinetochore area is required to stabilize the spindle microtubules and that cell size has a strong influence on the kinetochore stable state.