Characterizing transcription factor binding sites using formaldehyde crosslinking and immunoprecipitation.
In their article, A.S. Weinmann and P.J. Farnham (2002, Methods 26) described new techniques for isolating in vivo binding sites for any DNA-binding protein. In this article, we describe complementary methods for detailed in vivo characterizations of such identified protein-DNA interactions. First, we describe how formaldehyde crosslinking and chromatin immunoprecipitation (ChIP), in conjunction with transient transfections or the use of cell lines containing stably integrated constructs or episomes, can be employed to identify which specific nucleotides of a region of DNA are required for recruitment of a particular transcription factor. In contrast to in vivo footprinting, this method not only specifies which nucleotides are bound, but also identifies the protein(s) involved in binding. Next, we discuss the use of the ChIP assay to study how binding of a transcription factor is altered by passage through the cell cycle, by overexpression or deletion of another factor, or during tumorigenesis. Finally, a look toward the future suggests that the ChIP assay may be combined with Western blot analysis or mass spectrometry to identify additional proteins that interact with a transcription factor of interest.
Wells
Julie
J
McArdle Laboratory for Cancer Research, University of Wisconsin Medical School, 1400 University Avenue, Madison 53706, USA.
Farnham
Peggy J
PJ
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D003432:Cross-Linking Reagents / Q000378:metabolism; D004247:DNA / Q000378:metabolism*; D005557:Formaldehyde / Q000378:metabolism*; D016259:Genes, myc; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D010957:Plasmids; D011233:Precipitin Tests / Q000379:methods*; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D014157:Transcription Factors / Q000378:metabolism*; D014162:Transfection; D016662:Virus Integration
D003432:Cross-Linking Reagents; D014157:Transcription Factors; D005557:Formaldehyde; D004247:DNA