Centromeres of higher eukaryotes are epigenetically marked by the 
centromere-specific CENP-A nucleosome. New CENP-A recruitment requires the 
CENP-A histone chaperone HJURP. In this paper, we show that a LacI (Lac 
repressor) fusion of HJURP drove the stable recruitment of CENP-A to a LacO (Lac 
operon) array at a noncentromeric 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, the microtubule-binding protein NDC80, 
and the formation of stable kinetochore-microtubule attachments. An 
amino-terminal fragment of HJURP was able to assemble CENP-A nucleosomes in 
vitro, demonstrating that HJURP is a chromatin assembly factor. Furthermore, 
HJURP recruitment to endogenous centromeres required the Mis18 complex. 
Together, these data suggest that the role of the Mis18 complex in CENP-A 
deposition is to recruit HJURP and that the CENP-A nucleosome assembly activity 
of HJURP is responsible for centromeric chromatin assembly to maintain the 
epigenetic mark.