The Mechanism of Transcriptional Synergy of an In Vitro Assembled Interferon-β Enhanceosome

Abstract

A functional interferon-β gene enhanceosome was assembled in vitro using the purified recombinant transcriptional activator proteins ATF2/c-JUN, IRF1, and p50/p65 of NF-κB. Maximal levels of transcriptional synergy between these activators required the specific interactions with the architectural protein HMG I(Y) and the correct helical phasing of the binding sites of these proteins on the DNA helix. Analyses of the in vitro assembled enhanceosome revealed that the transcriptional synergy is due, at least in part, to the cooperative assembly and stability of the complex. Reconstitution experiments showed that the formation of a stable enhanceosome-dependent preinitiation complex requires cooperative interactions between the enhanceosome; the general transcription factors TFIID, TFIIA, and TFIIB; and the cofactor USA. These studies provide a direct biochemical demonstration of the importance of the structure and function of natural multicomponent transcriptional enhancer complexes in gene regulation.