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Nucleic Acids Research 2005 33(20):e176; doi:10.1093/nar/gni180
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Published online 27 November 2005

© The Author 2005. Published by Oxford University Press. All rights reserved
The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact journals.permissions{at}oxfordjournals.org

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Footprinting of mammalian promoters: use of a CpG DNA methyltransferase revealing nucleosome positions at a single molecule level

Mehrnaz Fatemi, Martha M. Pao, Shinwu Jeong, Einav Nili Gal-Yam, Gerda Egger, Daniel J. Weisenberger and Peter A. Jones*

Department of Urology, Biochemistry and Molecular Biology, USC/Norris Comprehensive Cancer Center, Keck School of Medicine, University of Southern California Los Angeles, CA 90089-9181, USA

*To whom correspondence should be addressed. Tel: +1 323 865 0816; Fax: +1 323 865 0102; Email: jones_p{at}ccnt.hsc.usc.edu

Received June 7, 2005. Revised September 9, 2005. Accepted October 31, 2005.

Promoters are molecular ‘modules’, which are controlled as individual entities yet are often analyzed by nuclease digestion methodologies which, a priori, destroy this modularity. About 40% of mammalian genes contain CpG islands in their promoters and exonic regions, which are normally unmethylated. We developed a footprinting strategy to map the chromatin structure at unmethylated CpG islands by treatment of isolated nuclei with the CpG-specific DNA methyltransferase SssI (M.SssI), followed by genomic bisulfite sequencing of individual progeny DNA molecules. This gave single molecule resolution over the promoter region and allowed for the physical linkage between binding sites on individual promoter molecules to be maintained. Comparison of the p16 promoters in two human cell lines, J82 and LD419, expressing the p16 gene at 25-fold different levels showed that the two cell lines contain remarkably different, heterogeneously positioned nucleosomes over the promoter region, which were not distinguishable by standard methods using nucleases. Our high resolution approach gives a ‘digitized’ visualization of each promoter providing information regarding nucleosome occupancy and may be utilized to define transcription factor binding and chromatin remodeling.


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