ASSEMBLY OF CENP-A INTO CENTROMERIC CHROMATIN REQUIRES A COOPERATIVE ARRAY OF NUCLEOSOMAL DNA CONTACT SITES

We investigated the requirements for targeting the centromeric histone H3 homologue CENP-A for assembly at centromeres in human cells by tra nsfection of epitope-tagged CENP-A derivatives into HeLa cells, Centro meric targeting is driven solely by the conserved histone fold domain of CENP-A. Using the crystal structure of histone H3 as a guide, a ser ies of CENP-A/histone H3 chimeras was constructed to test the role of discrete structural elements of the histone fold domain. Three element s were identified that are necessary for efficient targeting to centro meres. Two correspond to contact sites between histone H3 and nucleoso mal DNA. The third maps to a homotypic H3-H3 interaction site importan t for assembly of the (H3/H4)(2) heterotetramer. Immunoprecipitation c onfirms that CENP-A self-associates in vivo In addition, targeting req uires that CENP-A expression is uncoupled from histone H3 synthesis du ring S phase, CENP-A mRNA accumulates later in the cell cycle than his tone H3, peaking in G2. Isolation of the gene for human CENP-A reveale d a regulatory motif in the promoter region that directs the late S/G2 expression of other cell cycle-dependent transcripts such as cdc2, cd c25C, and cyclin A, Our data suggest a mechanism for molecular recogni tion of centromeric DNA at the nucleosomal level mediated by a coopera tive 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.