The CCAN recruits CENP-A to the centromere and forms the structural core for kinetochore assembly

• DOI: 10.1083/jcb.201210106
• Publication Date: 2013-01-01T02:46:42Z
• Authors: Tetsuya Hori; Wei-Hao Shang; Kozo Takeuchi; Tatsuo Fukagawa
• Keywords: 0301 basic medicine; Chromosomal Proteins, Non-Histone; Autoantigens; Cell Line; Protein Structure, Tertiary; DNA-Binding Proteins; 03 medical and health sciences; Multiprotein Complexes; Animals; Kinetochores; Chickens; Research Articles; Centromere Protein A

CENP-A acts as an important epigenetic marker for kinetochore specification. However, the mechanisms by which CENP-A is incorporated into centromeres and the structural basis for kinetochore formation downstream of CENP-A remain unclear. Here, we used a unique chromosome-engineering system in which kinetochore proteins are targeted to a noncentromeric site after the endogenous centromere is conditionally removed. Using this system, we created two distinct types of engineered kinetochores, both of which were stably maintained in chicken DT40 cells. Ectopic targeting of full-length HJURP, CENP-C, CENP-I, or the CENP-C C terminus generated engineered kinetochores containing major kinetochore components, including CENP-A. In contrast, ectopic targeting of the CENP-T or CENP-C N terminus generated functional kinetochores that recruit the microtubule-binding Ndc80 complex and chromosome passenger complex (CPC), but lack CENP-A and most constitutive centromere-associated network (CCAN) proteins. Based on the analysis of these different engineered kinetochores, we conclude that the CCAN has two distinct roles: recruiting CENP-A to establish the kinetochore and serving as a structural core to directly recruit kinetochore proteins.