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Human Molecular Genetics Advance Access originally published online on August 19, 2003
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Human Molecular Genetics, 2003, Vol. 12, No. 20 2711-2721
DOI: 10.1093/hmg/ddg282
© 2003 Oxford University Press

Genomic microarray analysis reveals distinct locations for the CENP-A binding domains in three human chromosome 13q32 neocentromeres

Alicia Alonso1, Radma Mahmood1, Shulan Li1, Fanny Cheung1, Kinya Yoda2 and Peter E. Warburton1,*

1Department of Human Genetics, Mount Sinai School of Medicine, New York, NY 10029, USA and 2Bioscience Center, Nagoya University, Nagoya 464-8601, Japan

Received July 9, 2003; Accepted August 13, 2003

Human neocentromeres are fully functional centromeres that provide mitotic stability to rearranged chromosomes that have separated from endogenous centromeres. A disproportionate number of neocentromeres has been observed in certain regions such as chromosome 3q (n=6), 15q (n=9) and 13q32 (n=7), suggesting that these regions contain DNA sequences with a high propensity for neocentromere formation. Therefore, we have addressed the role of primary DNA sequence in neocentromere formation by asking whether multiple independent neocentromeres that were cytologically localized to chromosome 13q32 are in fact localized to the same underlying genomic DNA. Analysis of four independent 13q32 neocentromeres using simultaneous FISH with ordered YAC probes and immunofluorescence with antibodies to CENP-C have localized three neocentromeres to a distal ~7 Mb domain in chromosome 13q32, and one to an overlapping proximal domain of ~7 Mb. DNA was obtained from three of these neocentromeres by CENP-A chromatin immunoprecipitation (ChIP) and used to screen ordered BACs using both a slot-blotted BAC pool approach and a genomic microarray that contiguously spans 13q31.3–13q33.1. The CENP-A binding domains from each of these neocentromeres was identified to distinct genomic locations of ~130, 215 and 275 kb within an ~6.5 Mb region. Thus, the lack of coincidence of these neocentromeres to the same underlying DNA sequence refutes the idea of a DNA sequence based neocentromere ‘hotspot’ in 13q32 and further supports the sequence-independent epigenetic formation of human neocentromeres. The screening of genomic microarrays with ChIP DNA provides a powerful method to identify mammalian DNA sequences associated with particular functional chromatin states.

* To whom correspondence should be addressed at: Department of Human Genetics, Box 1498, Mount Sinai School of Medicine, 1425 Madison Ave, East Bldg 14-52A, New York, NY 10029, USA. Tel: +1 2126596728; Fax: +1 2128492508; Email: peter.warburton{at}mssm.edu


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