Detecting Co-Evolution in Centromere Proteins

The kinetochore is a multiprotein complex that assembles on centromeres to direct the chromosome segregation during mitosis and meiosis. The inner kinetochore is directly associated with DNA and a key component is the centromere-specific histone CENH3 (or CENP-A) which replaces the canonical histone H3 in centromeric nucleosomes. CENH3 is an unusual histone because it is both rapidly evolving and critical for centromere function. CENP-C is a direct interaction partner of CENH3 and it acts as bridge between the inner and outer kinetochore. Surprisingly, CENP-C is also a rapidly evolving protein. In this study we will perform genetic assays to test whether CENH3 and CENP-C are co-evolving. A previous study found that the despite extensive sequence divergence CENH3 from Brassica rapa and Lepidium oleraceum were able to complement the essential functions of A.thaliana cenh3 null mutation. In contrast, while Lepidium oleraceum CENP-C could complement an A. thaliana cenpc null mutation, B.rapa CENP-C complemented plants were sterile, suggesting different evolutionary constraints on the two proteins. Moreover, centromeres built on divergent CENH3 showed extensive mis-segregation in crosses to wild type, producing high frequency of haploid and aneuploid progeny. We will generate a background where we combine the A. thaliana cenh3 and cenpc complementation lines so that both divergent CENH3 and CENPC proteins are from the same species and hence have co-evolved with one another. We predict two outcomes: one, centromere function is restored, from which we can conclude that the centromere dysfunction in the cenh3 complemented lines is due to a weakened interaction with the A. thaliana CENP-C. Two, an additive effect where A. thaliana centromere with two non-native proteins is further weakened leading to even higher frequency of mis-segregation events.