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

Alicia Alonso¹ , Björn Fritz²^,3 , Dan Hasson¹ , György Abrusan¹ , Fanny Cheung¹ , Kinya Yoda⁴ , Bernhard Radlwimmer⁵ , Andreas G Ladurner² and Peter E Warburton¹

¹Department of Genetics and Genomic Sciences, Mount Sinai School of Medicine, 1425 Madison Avenue, New York, New York 10029, USA

²Gene Expression Unit, Meyerhofstrasse, European Molecular Biology Laboratory (EMBL), 69117 Heidelberg, Germany

³Abbott Germany, Max-Planck-Ring, 65205 Wiesbaden, Germany

⁴Bioscience and Biotechnology Center, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan

⁵Deutsches Krebsforschungszentrum (DKFZ), Im Neuenheimer Feld, 69120 Heidelberg, Germany

author email corresponding author email

Genome Biology 2007, 8:R148doi:10.1186/gb-2007-8-7-r148


Published:	25 July 2007

Subject areas: Molecular biology, Cell biology, Genome studies

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.