Centromeres are unique chromatin domains that direct the site of kinetochore formation during mitosis and mediate the movement of chromosomes during cell division. Centromeres contain a unique nucleosome in which histone H3 is replaced by centromere protein A (CENP-A). Because of their unique position in chromatin, the CENP-A nucleosome was hypothesized to determine the site of centromere and kinetochore assembly. In order to test the long held assumption that CENP-A dictates the location of the centromere; we developed a novel de novo centromere formation assay, which provides a new and powerful constructive approach to studying centromeres. This system is based on a LacO array that is stably integrated into the long arm of chromosome 1, far away from the existing centromere. Targeting the CENP-A chaperone HJURP or the Mis18 complex to the LacO array, by fusing them to the LacI repressor, drove the stable recruitment of CENP-A nucleosomes to the LacO array at the non-centromeric locus. Ectopically-targeted CENP-A chromatin at the LacO array was sufficient to direct the assembly of a functional centromere as indicated by the recruitment of the constitutive centromere-associated network proteins (CCAN) and formed an active kinetochore during mitosis as indicated by the recruitment of the microtubule-binding protein NDC80, inner centromere protein Aurora A and the formation of stable kinetochore-microtubule attachments.
Our work using the de novo centromere assay demonstrates that:
- The assembly of CENP-A nucleosomes is sufficient to recruit the centromere and kinetochore, proving the primary role of CENP-A in building the centromere
- The specific recruitment of the CENP-A chaperone HJURP via the Mis18 complex is a key step in maintaining centromere inheritance
We continue to capitalize on this novel assay to determine the critical roles of proteins on assembly and function of the centromere.