Propagation of centromeric chromatin requires exit from mitosis

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Abstract

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-A–containing 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.

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RADIOAUTOGRAPHIC STUDIES OF CHOLINE INCORPORATION INTO PERIPHERAL NERVE MYELIN

Walter J. Hendelman, Richard Bunge (1969)

This radioautographic study was designed to localize the cytological sites involved in the incorporation of a lipid precursor into the myelin and the myelin-related cell of the peripheral nervous system. Both myelinating and fully myelinated cultures of rat dorsal root ganglia were exposed to a 30-min pulse of tritiated choline and either fixed immediately or allowed 6 or 48 hr of chase incubation before fixation. After Epon embedding, light and electron microscopic radioautograms were prepared with Ilford L-4 emulsion. Analysis of the pattern of choline incorporation into myelinating cultures indicated that radioactivity appeared all along the length of the internode, without there being a preferential site of initial incorporation. Light microscopic radioautograms of cultures at varying states of maturity were compared in order to determine the relative degree of myelin labeling. This analysis indicated that the myelin-Schwann cell unit in the fully myelinated cultures incorporated choline as actively as did this unit in the myelinating cultures. Because of technical difficulties, it was not possible to determine the precise localization of the incorporated radioactivity within the compact myelin. These data are related to recent biochemical studies indicating that the mature myelin of the central nervous system does incorporate a significant amount of lipid precursor under the appropriate experimental conditions. These observations support the concept that a significant amount of myelin-related metabolic activity occurs in mature tissue; this activity is considered part of an essential and continuous process of myelin maintenance and repair.

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A general method for the covalent labeling of fusion proteins with small molecules in vivo.

Antje Keppler, Susanne Gendreizig, Thomas Gronemeyer … (2003)

Characterizing the movement, interactions, and chemical microenvironment of a protein inside the living cell is crucial to a detailed understanding of its function. Most strategies aimed at realizing this objective are based on genetically fusing the protein of interest to a reporter protein that monitors changes in the environment of the coupled protein. Examples include fusions with fluorescent proteins, the yeast two-hybrid system, and split ubiquitin. However, these techniques have various limitations, and considerable effort is being devoted to specific labeling of proteins in vivo with small synthetic molecules capable of probing and modulating their function. These approaches are currently based on the noncovalent binding of a small molecule to a protein, the formation of stable complexes between biarsenical compounds and peptides containing cysteines, or the use of biotin acceptor domains. Here we describe a general method for the covalent labeling of fusion proteins in vivo that complements existing methods for noncovalent labeling of proteins and that may open up new ways of studying proteins in living cells.

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The human CENP-A centromeric nucleosome-associated complex.

Daniel Foltz, Lars E T Jansen, Ben Black … (2006)

The basic element for chromosome inheritance, the centromere, is epigenetically determined in mammals. The prime candidate for specifying centromere identity is the array of nucleosomes assembled with CENP-A, the centromere-specific histone H3 variant. Here, we show that CENP-A nucleosomes directly recruit a proximal CENP-A nucleosome associated complex (NAC) comprised of three new human centromere proteins (CENP-M, CENP-N and CENP-T), along with CENP-U(50), CENP-C and CENP-H. Assembly of the CENP-A NAC at centromeres is dependent on CENP-M, CENP-N and CENP-T. Facilitates chromatin transcription (FACT) and nucleophosmin-1 (previously implicated in transcriptional chromatin remodelling and as a multifunctional nuclear chaperone, respectively) are absent from histone H3-containing nucleosomes, but are stably recruited to CENP-A nucleosomes independent of CENP-A NAC. Seven new CENP-A-nucleosome distal (CAD) centromere components (CENP-K, CENP-L, CENP-O, CENP-P, CENP-Q, CENP-R and CENP-S) are identified as assembling on the CENP-A NAC. The CENP-A NAC is essential, as disruption of the complex causes errors of chromosome alignment and segregation that preclude cell survival despite continued centromere-derived mitotic checkpoint signalling.

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Author and article information

Journal

Journal ID (nlm-ta): J Cell Biol

Journal ID (publisher-id): jcb

Title: The Journal of Cell Biology

Publisher: The Rockefeller University Press

ISSN (Print): 0021-9525

ISSN (Electronic): 1540-8140

Publication date (Print): 12 March 2007

Volume: 176

Issue: 6

Pages: 795-805

Affiliations

[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

Author notes

Correspondence to Don W. Cleveland: dcleveland@ 123456ucsd.edu

Article

Publisher ID: 200701066

DOI: 10.1083/jcb.200701066

PMC ID: 2064054

PubMed ID: 17339380

SO-VID: 69c8015f-040a-491d-80e1-50759ecb1a5c

History

Date received : 11 January 2007

Date accepted : 7 February 2007

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