Abstract
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 vivofootprinting studies reveal protein–DNA interactions as they actually occurin situ.We have usedin vivofootprinting to complementin vitrostudies 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 vivofootprinting procedure to permit the assessment of protein binding at guanine and adenine residues, rather than exclusively at guanines. This modification, termed GA-LMPCRin vivofootprinting, was essential for the analysis of GATA-1 motifs in the α-LCR and HS-3 of the β-LCR. Moreover, GA-LMPCRin vivofootprinting 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-LMPCRin vivofootprinting methodology and representative analyses from our studies, including GATA-1 and AP-1/NF-E2 motifs, are presented to illustrate the modified technique.
Published Version
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