Analysis of transcription factor interactions in osteoblasts using competitive chromatin immunoprecipitation.
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.
Roca
Hernan
H
Department of Periodontics and Oral Medicine, University of Michigan School of Dentistry, Ann Arbor, MI, USA.
Franceschi
Renny T
RT
D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D002460:Cell Line; D002843:Chromatin / Q000378:metabolism; D047369:Chromatin Immunoprecipitation / Q000379:methods*; D050659:Core Binding Factor Alpha 1 Subunit / Q000378:metabolism; D058574:Integrin-Binding Sialoprotein; D051379:Mice; D009841:Oligonucleotides / Q000737:chemistry; D010006:Osteoblasts / Q000378:metabolism*; D015675:Osteocalcin / Q000235:genetics; D011401:Promoter Regions, Genetic; D012795:Sialoglycoproteins / Q000235:genetics; D014157:Transcription Factors / Q000378:metabolism*
D002843:Chromatin; D050659:Core Binding Factor Alpha 1 Subunit; C546872:Ibsp protein, mouse; D058574:Integrin-Binding Sialoprotein; D009841:Oligonucleotides; D012795:Sialoglycoproteins; D014157:Transcription Factors; D015675:Osteocalcin