Rd. Shelby et al., ASSEMBLY OF CENP-A INTO CENTROMERIC CHROMATIN REQUIRES A COOPERATIVE ARRAY OF NUCLEOSOMAL DNA CONTACT SITES, The Journal of cell biology, 136(3), 1997, pp. 501-513
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.