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Co-localization of CENP-C and CENP-H to discontinuous domains of CENP-A chromatin at human neocentromeres

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Mammalian centromere formation is dependent on chromatin that contains centromere protein (CENP)-A, which is the centromere-specific histone H3 variant. Human neocentromeres have acquired CENP-A chromatin epigenetically in ectopic chromosomal locations on low-copy complex DNA. Neocentromeres permit detailed investigation of centromeric chromatin organization that is not possible in the highly repetitive alpha satellite DNA present at endogenous centromeres. Results We have examined the distribution of CENP-A, as well as two additional centromeric chromatin-associated proteins (CENP-C and CENP-H), across neocentromeric DNA using chromatin immunoprecipitation (ChIP) on CHIP assays on custom genomic microarrays at three different resolutions. Analysis of two neocentromeres using a contiguous bacterial artificial chromosome (BAC) microarray spanning bands 13q31.3 to 13q33.1 shows that both CENP-C and CENP-H co-localize to the CENP-A chromatin domain. Using a higher resolution polymerase chain reaction (PCR)-amplicon microarray spanning the neocentromere, we find that the CENP-A chromatin is discontinuous, consisting of a major domain of about 87.8 kilobases (kb) and a minor domain of about 13.2 kb, separated by an approximately 158 kb region devoid of CENPs. Both CENP-A domains exhibit co-localization of CENP-C and CENP-H, defining a distinct inner kinetochore chromatin structure that is consistent with higher order chromatin looping models at centromeres. The PCR microarray data suggested varying density of CENP-A nucleosomes across the major domain, which was confirmed using a higher resolution oligo-based microarray. Conclusion Centromeric chromatin consists of several CENP-A subdomains with highly discontinuous CENP-A chromatin at both the level of individual nucleosomes and at higher order chromatin levels, raising questions regarding the overall structure of centromeric chromatin.

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Published 01 January 2007
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Language English
2eAVt0looal0lnu7.smoe8,Issue7,ArticleR148Open Access Research Co-localization of CENP-C and CENP-H to discontinuous domains of CENP-A chromatin at human neocentromeres * †‡* * Alicia Alonso, Björn Fritz, Dan Hasson, György Abrusan, * §Fanny Cheung, Kinya Yoda, Bernhard Radlwimmer, † * Andreas G Ladurnerand Peter E Warburton
* Addresses: Departmentof Genetics and Genomic Sciences, Mount Sinai School of Medicine, 1425 Madison Avenue, New York, New York † ‡ 10029, USA.Gene Expression Unit, Meyerhofstrasse, European Molecular Biology Laboratory (EMBL), 69117 Heidelberg, Germany.Abbott § Germany, Max-Planck-Ring, 65205 Wiesbaden, Germany.Bioscience and Biotechnology Center, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan.Deutsches Krebsforschungszentrum (DKFZ), Im Neuenheimer Feld, 69120 Heidelberg, Germany.
Correspondence: Peter E Warburton. Email: peter.warburton@mssm.edu
Published: 25 July 2007 GenomeBiology2007,8:R148 (doi:10.1186/gb-2007-8-7-r148) The electronic version of this article is the complete one and can be found online at http://genomebiology.com/2007/8/7/R148
Received: 10 April 2007 Revised: 28 June 2007 Accepted: 25 July 2007
© 2007 Alonsoet al.; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. o<Hfpus>emTvaehnreanldeiCsotcEreiNbnPtur-tioAo-mnceoseeriatncrtfntrsomrutgcnuiesihttahtswohsNADiceromtrenoctssonsiincomatchrunibtaosms-ad-ncsaciim/op.r<hruhamnenarcsosp>roteinsociated
Abstract Background:Mammalian centromere formation is dependent on chromatin that contains centromere protein (CENP)-A, which is the centromere-specific histone H3 variant. Human neocentromeres have acquired CENP-A chromatin epigenetically in ectopic chromosomal locations on low-copy complex DNA. Neocentromeres permit detailed investigation of centromeric chromatin organization that is not possible in the highly repetitive alpha satellite DNA present at endogenous centromeres.
Results:We have examined the distribution of CENP-A, as well as two additional centromeric chromatin-associated proteins (CENP-C and CENP-H), across neocentromeric DNA using chromatin immunoprecipitation (ChIP) on CHIP assays on custom genomic microarrays at three different resolutions. Analysis of two neocentromeres using a contiguous bacterial artificial chromosome (BAC) microarray spanning bands 13q31.3 to 13q33.1 shows that both CENP-C and CENP-H co-localize to the CENP-A chromatin domain. Using a higher resolution polymerase chain reaction (PCR)-amplicon microarray spanning the neocentromere, we find that the CENP-A chromatin is discontinuous, consisting of a major domain of about 87.8 kilobases (kb) and a minor domain of about 13.2 kb, separated by an approximately 158 kb region devoid of CENPs. Both CENP-A domains exhibit co-localization of CENP-C and CENP-H, defining a distinct inner kinetochore chromatin structure that is consistent with higher order chromatin looping models at centromeres. The PCR microarray data suggested varying density of CENP-A nucleosomes across the major domain, which was confirmed using a higher resolution oligo-based microarray.
Conclusion:Centromeric chromatin consists of several CENP-A subdomains with highly discontinuous CENP-A chromatin at both the level of individual nucleosomes and at higher order chromatin levels, raising questions regarding the overall structure of centromeric chromatin.
GenomeBiology2007,8:R148