Characterizing transcription factor binding sites using formaldehyde crosslinking and immunoprecipitation

Julie Wells, Peggy J. Farnham

Research output: Contribution to journalArticle

200 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)48-56
Number of pages9
JournalMethods
Volume26
Issue number1
DOIs
StatePublished - 2002
Externally publishedYes

Keywords

  • Cell cycle regulation
  • Crosslinking
  • Immunoprecipitation
  • Protein-protein interactions
  • Synchronization
  • Tissues
  • Transcription factor
  • Tumorigenesis

ASJC Scopus subject areas

  • Molecular Biology

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