Rapid in vivo footprinting technique identifies proteins bound to the TTR gene in the mouse liver

Abstract

In vivo examination of the occupancy of DNA elements that can regulate transcription is critical to reveal which proteins actually take part in establishing and maintaining gene expression. We describe a new genomic sequencing method involving the rapid purification of relevant DNA segments from the bulk of the genomic DNA using a biotinylated riboprobe. The purified sequences are revealed by a single primer extension using Taq DNA polymerase. We used this technique to study the promoter and the enhancer of mouse transthyretin (TTR), a gene highly expressed in the liver. Footprints showed high liver-specific occupancy of some, but not all, of the DNA sites that had been identified as important for expression by transfection studies in hepatoma cells. In addition, several previously undetected sites were observed that bound proteins specifically in liver. These results suggest that not all demonstrable binding sites are involved in ongoing transcription and that in vivo studies may reveal additional and probably more relevant sites.