Transcriptional activation of the IFN-β gene in response to virus infection requires the assembly of an enhanceosome, which instructs a recruitment program of chromatin modifiers/remodelers and general transcription factors to the promoter. This program culminates with sliding of a nucleosome blocking the core promoter to a downstream position, a prerequisite for transcriptional activation. We show that delivery of this nucleosome to the same downstream position to create an accessible IFN-β core promoter prior to enhanceosome assembly results in major changes in the gene expression program with regard to the temporal pattern and the signal specificity of the transcriptional response. Thus, the identity of a gene expression program is achieved and maintained by the dynamic interplay between specific enhanceosomes and specific local chromatin structure.