In vitro centromere and kinetochore assembly on defined chromatin templates

Abstract

Centromeres are specialized chromatin domains that are essential for chromosome segregation and cell division, but it is unclear how they are established and assembled to generate microtubule-binding sites, or kinetochores, during mitosis. Here, Aaron Straight and colleagues have reconstituted vertebrate centromeric chromatin in vitro and show that, when added to cell-free extracts, this reconstituted CENP-A chromatin is sufficient for the assembly of centromere and kinetochore proteins, binding of microtubules and mitotic checkpoint function. This cell-free system is used to dissect various molecular features of CENP-A, and should be a valuable approach to study the complex centromeres of vertebrates. During cell division, chromosomes are segregated to nascent daughter cells by attaching to the microtubules of the mitotic spindle through the kinetochore. Kinetochores are assembled on a specialized chromatin domain called the centromere, which is characterized by the replacement of nucleosomal histone H3 with the histone H3 variant centromere protein A (CENP-A). CENP-A is essential for centromere and kinetochore formation in all eukaryotes but it is unknown how CENP-A chromatin directs centromere and kinetochore assembly1. Here we generate synthetic CENP-A chromatin that recapitulates essential steps of centromere and kinetochore assembly in vitro. We show that reconstituted CENP-A chromatin when added to cell-free extracts is sufficient for the assembly of centromere and kinetochore proteins, microtubule binding and stabilization, and mitotic checkpoint function. Using chromatin assembled from histone H3/CENP-A chimaeras, we demonstrate that the conserved carboxy terminus of CENP-A is necessary and sufficient for centromere and kinetochore protein recruitment and function but that the CENP-A targeting domain—required for new CENP-A histone assembly2—is not. These data show that two of the primary requirements for accurate chromosome segregation, the assembly of the kinetochore and the propagation of CENP-A chromatin, are specified by different elements in the CENP-A histone. Our unique cell-free system enables complete control and manipulation of the chromatin substrate and thus presents a powerful tool to study centromere and kinetochore assembly.