The Mechanism of Centromere and Kinoetochore Assembly in Higher Eukaryotes
Zusammenfassung der Projektergebnisse
Faithful chromosomes segregation is essential for genetic stability and development. During chromosome segregation, replicated chromosomes attach to the mitotic spindle via a single microtubule-binding site, called the kinetochore. The kinetochore is a multi-protein complex that assembles during mitosis on the centromere. The centromere is a specialized chromatin region characterized by the presence of centromere specific nucleosomes in which histone H3 is replaced by the histone H3 variant centromere protein A (CENP-A). CENP-A is essential for centromere and kinetochore formation in eukaryotes but the mechanism by which CENP-A directs centromere and kinetochore formation is not well understood. To analyze how CENP-A directs centromere and kinetochore formation, we reconstituted CENP-A chromatin from recombinant components and tested its ability to recapitulate essential steps in centromere and kinetochore assembly in Xenopus egg extracts. We found that CENP-A chromatin specifically recruits centromere and kinetochore proteins, when compared to H3 chromatin. Further, microtubules bind to and get stabilized by CENP-A chromatin. When microtubule detachment is mimicked by microtubule depolymerization, mitotic checkpoint protein recruitment to CENP-A chromatin increases and mitotic exit is delayed. Microtubule binding and mitotic checkpoint function are hallmarks of native kinetochores and thus our findings indicate that reconstituted CENP-A chromatin recapitulates essential kinetochore functions in vitro. Our in vitro centromere and kinetochore assembly system provides a distinct advantage over previously existing assays, because it allows us to directly assess how specific CENP-A domains participate in centromere and kinetochore assembly, even when the mutations we analyze would be lethal in vivo. We exploited this advantage and generated chromatin arrays containing chimeric CENP-A/H3 proteins. We find that the conserved C-terminus of CENP-A is necessary and sufficient for centromere and kinetochore assembly and function, but the CENP-A targeting domain (CATD) that had been shown to be required for new CENP-A histone assembly, is not. We propose that centromere and kinetochore assembly and function of CENP-A can be separated molecularly from the recognition mechanism for targeting CENP-A to the centromere. We anticipate that our unique cell-free system that allows complete control and manipulation of the synthetic chromatin templates will be a powerful tool to further dissect the mechanism of centromere and kinetochore assembly in higher eukaryotes and to be applicable to other research areas such as chromatin biology and epigenetics.
