Published online
doi:10.1083/jcb.200701066
The Journal of Cell Biology, Vol. 176, No. 6, 795-805
The Rockefeller University Press, 0021-9525 $30.00
© Jansen et al.
Propagation of centromeric chromatin requires exit from mitosis
Lars E.T. Jansen1,
Ben E. Black1,2,
Daniel R. Foltz1, and
Don W. Cleveland1
1 Ludwig Institute for Cancer Research and Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, CA 92093
2 Department of Biochemistry and Biophysics, University of Pennsylvania School of Medicine, Philadelphia, PA 19104
Correspondence to Don W. Cleveland: dcleveland{at}ucsd.edu
Centromeres direct chromosomal inheritance by nucleating assembly of the kinetochore, a large multiprotein complex required for microtubule attachment during mitosis. Centromere identity in humans is epigenetically determined, with no DNA sequence either necessary or sufficient. A prime candidate for the epigenetic mark is assembly into centromeric chromatin of centromere protein A (CENP-A), a histone H3 variant found only at functional centromeres. A new covalent fluorescent 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 domains of replicated centromere DNA is shown to require passage through mitosis but not microtubule attachment. Very surprisingly, assembly and stabilization of new CENP-Acontaining nucleosomes is restricted exclusively to the subsequent G1 phase, demonstrating direct coupling between progression through mitosis and assembly/maturation of the next generation of centromeres.
Abbreviations used in this paper: BG, benzylguanine; CENP-A, centromere protein A; PEG, polyethylene glycol; TMR, tetramethylrhodamine.

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