Guanine-adenine ligation-mediated polymerase chain reaction in vivo footprinting.
The analysis of functional DNA regulatory sequences involved in transcriptional control is critical to establishing which proteins mediate cell-specific gene expression. The organization of erythroid LCRs is complex, consisting of multiple, interdigested cis elements. As in situ binding to these sites is determined by the accessibility of these regulatory regions in native chromatin and the availability of relevent cell-specific and ubiquitous factors, in vivo footprinting was used to define protein DNA interactions in human globin LCRs. To further enhance the detection of protein contacts with this technique, we have modified the dimethyl sulfate-based ligation-mediated PCR in vivo footprinting procedure to permit the assessment of protein binding at guanine and adenine resides, 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 this modification and sample illustrations from our studies have been presented to demonstrate the enhanced detection and resolution obtained with this procedure.
Strauss
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Department of Ophthalmology, Massachusetts Eye and Ear Infirmary, Boston 02114, USA.
Orkin
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D000225:Adenine; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002478:Cells, Cultured; D004247:DNA / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D018983:DNA Footprinting / Q000379:methods*; D019175:DNA Methylation; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000378:metabolism*; D050981:Erythroid-Specific DNA-Binding Factors; D050982:GATA1 Transcription Factor; D006147:Guanine; D006801:Humans; D006822:Hybrid Cells; D007202:Indicators and Reagents; D051379:Mice; D016133:Polymerase Chain Reaction / Q000379:methods*; D014157:Transcription Factors / Q000378:metabolism; D014407:Tumor Cells, Cultured
D017931:DNA Primers; D004268:DNA-Binding Proteins; D050981:Erythroid-Specific DNA-Binding Factors; D050982:GATA1 Transcription Factor; C494688:GATA1 protein, human; C494689:Gata1 protein, mouse; D007202:Indicators and Reagents; D014157:Transcription Factors; D006147:Guanine; D004247:DNA; D000225:Adenine