Guanine-adenine ligation-mediated PCR in vivo footprinting

The identification of relevant DNA regulatory sequences involved in transcriptional control is critical to establishing which proteins mediate cell-specific gene expression. As an approach to investigating the mechanisms of gene regulation, in vivo footprinting studies reveal protein-DNA interactions as they actually occur in situ. We have used in vivo footprinting to complement in vitro studies of the human globin locus control regions (LCRs) in erythroid cells. To further enhance the detection of protein contacts with this technique, we have modified the dimethyl sulfate-based ligation-mediated PCR (LMPCR) in vivo footprinting procedure to permit the assessment of protein binding at guanine and adenine residues, rather than exclusively at guanines. This modification, termed GA-LMPCR in vivo footprinting, was essential for the analysis of GATA-1 motifs in the alpha-LCR and HS-3 of the beta-LCR. Moreover, GA-LMPCR in vivo footprinting provided high-resolution analysis of AP-1/NF-E2 elements and revealed protein contacts at sequences that are not coincident with previously described regulatory motifs. A comprehensive discussion of the GA-LMPCR in vivo footprinting methodology and representative analyses from our studies, including GATA-1 and AP-1/NF-E2 motifs, are presented to illustrate the modified technique.