THE JOURNAL OF CELL BIOLOGYJCB: ARTICLE© The Rockefeller University Press $15.00The Journal of Cell Biologyhttp://www.jcb.org/cgi/doi/10.1083/jcb.200701066Cite by DOI: 10.1083/jcb.200701066 JCB 1 of 11IntroductionKinetochores mediate the interaction between chromosomes and spindle microtubules, thereby enabling mitotic chromo-some movement, and produce a mitotic checkpoint signal that ensures bipolar attachment of all chromosomes before ana-phase onset (Cleveland et al., 2003; Chan et al., 2005). Assembly of the kinetochore during mitosis takes place at the centromere, a megabase-sized specialized chromatin region typically formed on arrays of α satellite DNA (Cleveland et al., 2003; Amor et al., 2004b; Carroll and Straight, 2006).Despite the prevalence of centromeres at adenine- thymine–rich repetitive α satellite DNA, the DNA sequences themselves appear to play a nonessential role in centromere specifi cation. This is most clearly exemplifi ed by the characterization of hu-man neocentromeres. In these rare but naturally occurring patient cases, a specifi c centromere has relocated to another site on the chromosome without any apparent DNA rearrangements, con-comitant with vacating the original α satellite–containing locus (Amor and Choo, 2002; Amor et al., 2004a; Ventura et al., 2004). This shows that DNA sequences normally associated with centromeres are neither necessary nor suffi cient to promote centromere propagation and that maintenance of centromeres is determined predominantly in an epigenetic manner.Centromere protein A (CENP-A) is a conserved histone H3 variant that replaces canonical H3 specifi cally at centro-meres in all known eukaryotes (Palmer et al., 1987; Meluh et al., 1998; Henikoff et al., 2000; Oegema et al., 2001) and has been shown to be required for the localization of nearly all other centromere and kinetochore components (Howman et al., 2000; Oegema et al., 2001; Goshima et al., 2003; Amor et al., 2004a; Regnier et al., 2005; Foltz et al., 2006; Liu et al., 2006). We have recently shown that the loop1 and α2 helix of the CENP-A histone fold domain is responsible for forming a rigid/inaccessible interface with histone H4 and that this re-gion, when transplanted into canonical histone H3, confers cen-tromere targeting (Black et al., 2004, 2007a) and provides an essential function of CENP-A (Black et al., 2007b). CENP-A chromatin directly recruits a six-component CENP-A nu-cleosome-associated complex (CENP-ANAC) that forms the foundation for the assembly of other centromere components and the kinetochore during mitosis (Foltz et al., 2006). The existence of a CENP-A–directed centromere-specifi c chroma-tin structure makes CENP-A a prime candidate for the epigen-etic propagation of centromere identity. This directly implies that CENP-A propagation at the centromere is a partially or completely self-directed process. It is, however, unknown how CENP-A is discriminated from canonical histone H3 and how its specific incorporation at centromeric nucleosomes is achieved.Propagation of centromeric chromatin requires exit from mitosisLars E.T. Jansen,1 Ben E. Black,1,2 Daniel R. Foltz,1 and Don W. Cleveland11Ludwig Institute for Cancer Research and Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, CA 920932Department of Biochemistry and Biophysics, University of Pennsylvania School of Medicine, Philadelphia, PA 19104 Centromeres direct chromosomal inheritance by nucleating assembly of the kinetochore, a large multiprotein complex required for microtubule at-tachment during mitosis. Centromere identity in humans is epigenetically determined, with no DNA sequence either necessary or suffi cient. A prime candidate for the epigene-tic mark is assembly into centromeric chromatin of centro-mere protein A (CENP-A), a histone H3 variant found only at functional centromeres. A new covalent fl uorescent pulse-chase labeling approach using SNAP tagging has now been developed and is used to demonstrate that CENP-A bound to a mature centromere is quantitatively and equally partitioned to sister centromeres generated during S phase, thereby remaining stably associated through multiple cell divisions. Loading of nascent CENP-A on the megabase do-mains of replicated centromere DNA is shown to require pas-sage through mitosis but not microtubule attachment. Very surprisingly, assembly and stabilization of new CENP-A–containing nucleosomes is restricted exclusively to the sub-sequent G1 phase, demonstrating direct coupling between progression through mitosis and assembly/ maturation of the next generation of centromeres.Correspondence to Don W. Cleveland: [email protected] used in this paper: BG, benzylguanine; CENP-A, centromere pro-tein A; PEG, polyethylene glycol; TMR, tetramethylrhodamine.The online version of this article contains supplemental material. on March 6, 2007 www.jcb.orgDownloaded from http://www.jcb.org/cgi/content/full/jcb.200701066/DC1Supplemental Material can be found at:JCB 2 of 11Earlier models have suggested that differences in timing of replication of centromeric DNA versus the genome overall may provide a temporal window permissive for CENP-A load-ing (O’Keefe et al., 1992; Csink and Henikoff, 1998). However, this appears not to be the case, as replication of centromeric DNA is not restricted to a specifi c time during S phase (Shelby et al., 2000; Sullivan and Karpen, 2001). Alternatively, CENP-A loading could be separate from assembly of canonical histones altogether by allowing CENP-A loading outside S phase. In-deed, DNA replication is not required for CENP-A assembly and CENP-A mRNA, and protein levels peak only after S phase during late G2 phase, consistent with a disconnect between the timing of CENP-A and H3 assembly (Shelby et al., 1997, 2000). Whether propagation of centromeric chromatin and general chromatin is indeed temporally distinct and how and when CENP-A nucleosomes turn overis not known. This we now test by developing and exploiting a novel, covalent fl uorescent pulse-labeling strategy with SNAP tagging.ResultsTiming of assembly and turnover of CENP-A at centromeres using the SNAP tagThe SNAP tag, a modifi ed variant of the suicide enzyme O6- alkylguanine-DNA alkyltransferase, whose normal function is in DNA repair, has been extensively engineered to covalently and irreversibly modify (and inactivate) itself through acceptance of the cell-permeable