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© The Rockefeller University Press, 0021-9525/1997//501 $5.00
The Journal of Cell Biology, Volume 136, Number 3, , 1997 501-513

Article

Assembly of CENP-A into Centromeric Chromatin Requires a Cooperative Array of Nucleosomal DNA Contact Sites



Richard D. Shelby, Omid Vafa, and Kevin F. Sullivan

Department of Cell Biology, The Scripps Research Institute, La Jolla, California 92037

We investigated the requirements for targeting the centromeric histone H3 homologue CENP-A for assembly at centromeres in human cells by transfection of epitope-tagged CENP-A derivatives into HeLa cells. Centromeric targeting is driven solely by the conserved histone fold domain of CENP-A. Using the crystal structure of histone H3 as a guide, a series of CENPA/histone H3 chimeras was constructed to test the role of discrete structural elements of the histone fold domain. Three elements were identified that are necessary for efficient targeting to centromeres. Two correspond to contact sites between histone H3 and nucleosomal DNA. The third maps to a homotypic H3–H3 interaction site important for assembly of the (H3/H4)2 heterotetramer. Immunoprecipitation confirms that CENP-A self-associates in vivo. In addition, targeting requires that CENP-A expression is uncoupled from histone H3 synthesis during S phase. CENP-A mRNA accumulates later in the cell cycle than histone H3, peaking in G2. Isolation of the gene for human CENP-A revealed a regulatory motif in the promoter region that directs the late S/G2 expression of other cell cycle–dependent transcripts such as cdc2, cdc25C, and cyclin A. Our data suggest a mechanism for molecular recognition of centromeric DNA at the nucleosomal level mediated by a cooperative series of differentiated CENP-A–DNA contact sites arrayed across the surface of a CENP-A nucleosome and a distinctive assembly pathway occurring late in the cell cycle.

1. Abbreviation used in this paper: HA, hemagglutinin.

Address all correspondence to Kevin F. Sullivan, Department of Cell Biology, The Scripps Research Institute, 10550 N. Torrey Pines Rd., La Jolla, CA 92037. Tel.: (619) 784-2350. Fax: (619) 784-2345. e-mail: ksulli van{at}scripps.edu


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