Genomic DNA affinity chromatography: a technique to isolate interferon-inducible DNA binding factors

Cytokines elicit responses in target cells by inducing changes in gene expression. For interferons (IFNs), this involves receptor-mediated activation of specific transcription factors, which then translocate into the nucleus to bind to cognate gene elements in the promoters of IFN-inducible genes. The prototypic IFN-inducible transcription factors are the signal transducer and activator of transcription (STAT) proteins. IFN-receptor interactions invoke Janus kinase activation via phosphorylation events, which in turn leads to the recruitment and phosphorylation of STAT proteins on tyrosine residues. Activated STATs then dimerize to form STAT complexes. IFNs-alpha/beta will activate STAT-1, STAT-2, STAT-3 ,and STAT-5, whereas IFN-gamma will predominantly activate STAT-1. In this chapter, we describe a procedure to identify IFN-inducible deoxyribonucleic acid (DNA) binding factors independently of any knowledge of their target DNA sequences. This procedure permits the identification of IFN-inducible STAT complexes as well as any other IFN-inducible DNA binding factors. This biochemical technique uses genomic DNA affinity chromatography to isolate DNA binding factors from IFN-inducible cytoplasmic or nuclear extracts.