Regulatory mechanisms underlying eukaryotic gene expression, and many other DNA metabolic pathways, are tightly coupled to dynamic changes in chromatin architecture in the nucleus. Activation of gene expression generally requires the recruitment of histone acetyltransferases (HATs) to gene promoters by sequence-specific DNA-binding transcriptional activators. HATs often target specific lysines in the core histone amino-terminal "tail" domains (NTDs), which have the potential ability to alter higher order chromatin structure. In order to better characterize the impact targeted histone acetylation has on chromatin structure and function, we have characterized a novel model system derived from the human T-cell lymphoma virus type 1 promoter. Using this system as an example, here we describe the use of a combination of biochemical and biophysical methods to investigate the effect of activator-dependent acetylation on higher order chromatin structure and transcription by RNA polymerase II.