Promoter DNA sequence guides factors that position the +1 nucleosome and facilitate TBP binding [array]

Here we present evidence that precise positioning of the +1 promoter nucleosome in yeast is critical for efficient TBP binding and pre-initiation complex assembly, and is determined, at least in part, by the action of two key factors, the essential chromatin remodeler RSC and one (or more) of a small set of ubiquitous pioneer transcription factors (PTFs). Despite their widespread co-localization, we show that RSC and PTFs often act independently to generate accessible chromatin. Furthermore, we present evidence that RSC binding, as well as the strength and directionality of its action on nucleosomes, depends upon the arrangement of two specific DNA motifs relative to nearby PTF binding sites. Our results provide insights into how promoter DNA sequence instructs trans-acting factors to precisely control nucleosome architecture and stimulate transcription initiation. Two-channel microarrays were used. RNA isolated from a large amount of wt yeast from a single culture was used as a common reference. This common reference was used in one of the channels for each hybridization and used in the statistical analysis to obtain an average expression-profile for each 30 minute depletion timepoint relative to time zero. Two independent cultures were hybridized on two separate microarrays. For the first hybridization, the Cy5 (red) labeled cRNA from the sample is hybridized together with the Cy3 (green) labeled cRNA from the common reference. For the replicate hybridization, the labels are swapped. Each gene is represented twice on the microarray, resulting in four measurements per strain/timepoint.