Nucleic Acids Research Advance Access originally published online on February 7, 2008
Nucleic Acids Research 2008 36(5):1723-1730; doi:10.1093/nar/gkn022
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Nucleic Acids Research, 2008, Vol. 36, No. 5 1723-1730
© 2008 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Molecular Biology |
Analysis of transcription factor interactions in osteoblasts using competitive chromatin immunoprecipitation
1Department of Periodontics and Oral Medicine, University of Michigan School of Dentistry and 2Department of Biological Chemistry, University of Michigan School of Medicine, Ann Arbor, MI, USA
*To whom correspondence should be addressed. Tel: +1 734 763 7381; Fax: +1 734 763 5503; Email: rennyf{at}umich.edu
Received November 11, 2007. Revised January 13, 2008. Accepted January 14, 2008.
Chromatin immunoprecipitation (ChIP) is a widely used technique for quantifying protein–DNA interactions in living cells. This method commonly uses fixed (crosslinked) chromatin that is fragmented by sonication (X-ChIP). We developed a simple new ChIP procedure for the immunoprecipitation of sonicated chromatin isolated from osteoblasts in the absence of crosslinking (N-ChIP). The use of noncrosslinked chromatin allowed development of a new modification of the ChIP assay: the combination of N-ChIP and competition with double-stranded oligonucleotides containing specific binding sites for individual transcription factors (Competitive N-ChIP). Using this approach, we were able to discriminate between individual binding sites for the Runx2 transcription factor in the osteocalcin and bone sialoprotein genes that cannot be resolved by traditional X-ChIP. N-ChIP assays were also able to detect several other types of chromatin interactions including those with Dlx homeodomain factors and nuclear proteins such as Sin3a that lack an intrinsic DNA-binding motif and, therefore, bind to chromatin via interactions with other proteins.