2 DOMAINS OF ISGF3-GAMMA THAT MEDIATE PROTEIN-DNA AND PROTEIN-PROTEIN INTERACTIONS DURING TRANSCRIPTION FACTOR ASSEMBLY CONTRIBUTE TO DNA-BINDING SPECIFICITY

Alpha interferon (IFN-alpha) induces the transcription of a large set of genes through activation of multimeric transcription factor ISGF3. This factor can be dissociated into two protein components, termed ISGF3gamma and ISGF3alpha. ISGF3gamma is a 48-kDa protein related at the amino terminus to members of the IFN-regulatory factor (IRF) and Myb families of DNA-binding proteins; ISGF3alpha consists of three polypeptides of 84, 91, and 113 kDa that self-assemble to form an activated component in response to IFN-alpha. DNA-binding studies indicated that ISGF3gamma binds DNA alone, recognizing the IFN-stimulated response element, while the ISGF3alpha polypeptides alone display no specific interactions with DNA. A complex between ISGF3gamma and activated ISGF3alpha binds the IFN-stimulated response element with much greater affinity than does the 48-kDa ISGF3gamma protein alone. The DNA-binding domain of ISGF3gamma and regions responsible for protein-protein interaction with ISGF3alpha were identified by using deleted forms of ISGF3gamma expressed in vitro. The amino-terminal region of ISGF3gamma homologous to the IRF and Myb proteins was sufficient for interaction with DNA and displayed the binding specificity of the intact protein; phosphorylation of this region was necessary for activity. A second region of 160 amino acids separated from the DNA-binding domain by over 100 amino acids contained a domain capable of associating with ISGF3alpha and was sufficient to confer specific ISGF3alpha interaction to a heterologous protein. Interaction of the ISGF3alpha component with the protein interaction domain of ISGF3gamma altered the DNA-binding specificity of the resulting complex, suggesting that one or more of the ISGF3alpha polypeptides make base-specific contacts with DNA. This interaction defines a mechanism through which IRF-like proteins complexed with regulatory components can display novel DNA-binding specificities.