We present evidence that transcriptional activation of the human interferon-beta 
(IFN beta) gene requires the assembly of a higher order transcription enhancer 
complex (enhanceosome). This multicomponent complex includes at least three 
distinct transcription factors and the high mobility group protein HMG I(Y). 
Both the in vitro assembly and in vivo transcriptional activity of this complex 
require a precise helical relationship between individual transcription 
factor-binding sites. In addition, HMG I(Y), which binds specifically to three 
sites within the enhancer, promotes cooperative binding of transcriptional 
factors in vitro and is required for transcriptional synergy between these 
factors in vivo. Thus, HMG I(Y) plays an essential role in the assembly and 
function of the IFN beta gene enhanceosome.