Assay of Activator Recruitment of Chromatin-Modifying Complexes

Publisher Summary This chapter describes an in vitro assay for analyzing activator recruitment of chromatin-modifying complexes to a nucleosomal promoter. The template used in this assay contains five Gal4 binding sites upstream of the Adenovirus E4 core promoter and five sea urchin 5S nucleosome positioning sequences flanking each at end of the 5XGal4-E4 promoter. These positioning sequences result in twelve nucleosomes, with two over the 5XGal-E4 promoter. The use of a nucleosomal template immobilized on paramagnetic beads is central to this assay. The immobilization of the template allows removal and addition of soluble components in the recruitment reactions by collecting templates on a magnetic concentrator and washing the beads. For example, chromatin-modifying complexes, such as the SAGA histone acetyltransferase and SWI/SNF ATP-dependent chromatin-remodeling complexes can be targeted to a nucleosomal promoter by a transcription activator protein. Afterwards, the activator protein is competed from the template using sequence-specific competitor DNA and the template is washed. The ability of the complexes to remain bound to the promoter in the absence of an activator can then be assessed by western analysis for different components of the reaction, such as acetylated histones, the activator protein, and chromatin-modifying complex components.