Combining chromatin immunoprecipitation and DNA footprinting: a novel method to analyze protein-DNA interactions in vivo.
A variety of methods are available to analyze protein-DNA interactions in vivo. Two of the most prominent of these methods are chromatin immunoprecipitation (ChIP) and in vivo footprinting. Both of these procedures have specific limitations. For example, the ChIP assay fails to document where exactly a protein binds in vivo. The precipitation of a specific segment of DNA with antibodies directed against DNA-binding proteins does not necessarily indicate that the protein directly interacts with a sequence in the precipitate but could rather reflect protein-protein interactions. Furthermore, the results of in vivo footprinting studies are inconclusive if a DNA sequence is analyzed that is bound by a specific protein in only a certain fraction of cells. Finally, in vivo footprinting does not indicate which protein is bound at a specific site. We have developed a new procedure that combines the ChIP assay and DMS footprinting techniques. Using this method we show here that antibodies specific for USF1 and NF-E2 precipitate the murine beta-globin promoter in MEL cells. DMS footprinting analysis of the DNA precipitated with NF-E2 antibodies revealed a protection over a partial NF-E2-binding site in the beta-globin downstream promoter region. We believe that this novel method will generally benefit investigators interested in analyzing protein-DNA interactions in vivo.
Kang
Sung-Hae Lee
SH
Department of Biochemistry and Molecular Biology, Powell Gene Therapy Center, Center for Mammalian Genetics, College of Medicine, University of Florida, 1600 SW Archer Road, Gainesville, FL 32610, USA.
Vieira
Karen
K
Bungert
Jörg
J
D000818:Animals; D001483:Base Sequence; D002843:Chromatin / Q000235:genetics / Q000378:metabolism*; D004247:DNA / Q000235:genetics / Q000378:metabolism; D018983:DNA Footprinting / Q000379:methods*; D004268:DNA-Binding Proteins / Q000378:metabolism; D050981:Erythroid-Specific DNA-Binding Factors; D005914:Globins / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D050983:NF-E2 Transcription Factor; D050984:NF-E2 Transcription Factor, p45 Subunit; D011233:Precipitin Tests / Q000379:methods*; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding; D014157:Transcription Factors / Q000378:metabolism; D014407:Tumor Cells, Cultured; D051720:Upstream Stimulatory Factors
D002843:Chromatin; D004268:DNA-Binding Proteins; D050981:Erythroid-Specific DNA-Binding Factors; D050983:NF-E2 Transcription Factor; D050984:NF-E2 Transcription Factor, p45 Subunit; C494692:NFE2 protein, human; D014157:Transcription Factors; C497181:USF1 protein, human; D051720:Upstream Stimulatory Factors; D005914:Globins; D004247:DNA