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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 transfection of epitope-tagged CENP-A derivatives into HeLa cells. Centromeric targeting is driven solely by the conserved histone fold domain of CENP-A. Using the...

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Detalles Bibliográficos
Autores principales: Shelby, Richard D., Vafa, Omid, Sullivan, Kevin F.
Formato: Texto
Lenguaje:English
Publicado: The Rockefeller University Press 1997
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2134286/
https://www.ncbi.nlm.nih.gov/pubmed/9024683
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author Shelby, Richard D.
Vafa, Omid
Sullivan, Kevin F.
author_facet Shelby, Richard D.
Vafa, Omid
Sullivan, Kevin F.
author_sort Shelby, Richard D.
collection PubMed
description We investigated the requirements for targeting the centromeric histone H3 homologue CENP-A for assembly at centromeres in human cells by transfection of epitope-tagged CENP-A derivatives into HeLa cells. Centromeric targeting is driven solely by the conserved histone fold domain of CENP-A. Using the crystal structure of histone H3 as a guide, a series of CENPA/histone H3 chimeras was constructed to test the role of discrete structural elements of the histone fold domain. Three elements were identified that are necessary for efficient targeting to centromeres. Two correspond to contact sites between histone H3 and nucleosomal DNA. The third maps to a homotypic H3–H3 interaction site important for assembly of the (H3/H4)(2) heterotetramer. Immunoprecipitation confirms that CENP-A self-associates in vivo. In addition, targeting requires that CENP-A expression is uncoupled from histone H3 synthesis during S phase. CENP-A mRNA accumulates later in the cell cycle than histone H3, peaking in G2. Isolation of the gene for human CENP-A revealed a regulatory motif in the promoter region that directs the late S/G2 expression of other cell cycle–dependent transcripts such as cdc2, cdc25C, and cyclin A. Our data suggest a mechanism for molecular recognition of centromeric DNA at the nucleosomal level mediated by a cooperative 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.
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spelling pubmed-21342862008-05-01 Assembly of CENP-A into Centromeric Chromatin Requires a Cooperative Array of Nucleosomal DNA Contact Sites Shelby, Richard D. Vafa, Omid Sullivan, Kevin F. J Cell Biol Article We investigated the requirements for targeting the centromeric histone H3 homologue CENP-A for assembly at centromeres in human cells by transfection of epitope-tagged CENP-A derivatives into HeLa cells. Centromeric targeting is driven solely by the conserved histone fold domain of CENP-A. Using the crystal structure of histone H3 as a guide, a series of CENPA/histone H3 chimeras was constructed to test the role of discrete structural elements of the histone fold domain. Three elements were identified that are necessary for efficient targeting to centromeres. Two correspond to contact sites between histone H3 and nucleosomal DNA. The third maps to a homotypic H3–H3 interaction site important for assembly of the (H3/H4)(2) heterotetramer. Immunoprecipitation confirms that CENP-A self-associates in vivo. In addition, targeting requires that CENP-A expression is uncoupled from histone H3 synthesis during S phase. CENP-A mRNA accumulates later in the cell cycle than histone H3, peaking in G2. Isolation of the gene for human CENP-A revealed a regulatory motif in the promoter region that directs the late S/G2 expression of other cell cycle–dependent transcripts such as cdc2, cdc25C, and cyclin A. Our data suggest a mechanism for molecular recognition of centromeric DNA at the nucleosomal level mediated by a cooperative 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. The Rockefeller University Press 1997-02-10 /pmc/articles/PMC2134286/ /pubmed/9024683 Text en This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/4.0/).
spellingShingle Article
Shelby, Richard D.
Vafa, Omid
Sullivan, Kevin F.
Assembly of CENP-A into Centromeric Chromatin Requires a Cooperative Array of Nucleosomal DNA Contact Sites
title Assembly of CENP-A into Centromeric Chromatin Requires a Cooperative Array of Nucleosomal DNA Contact Sites
title_full Assembly of CENP-A into Centromeric Chromatin Requires a Cooperative Array of Nucleosomal DNA Contact Sites
title_fullStr Assembly of CENP-A into Centromeric Chromatin Requires a Cooperative Array of Nucleosomal DNA Contact Sites
title_full_unstemmed Assembly of CENP-A into Centromeric Chromatin Requires a Cooperative Array of Nucleosomal DNA Contact Sites
title_short Assembly of CENP-A into Centromeric Chromatin Requires a Cooperative Array of Nucleosomal DNA Contact Sites
title_sort assembly of cenp-a into centromeric chromatin requires a cooperative array of nucleosomal dna contact sites
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2134286/
https://www.ncbi.nlm.nih.gov/pubmed/9024683
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