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.