The chromatin architecture of eukaryotic gene promoters is generally 
characterized by a nucleosome-free region (NFR) flanked by at least one H2A.Z 
variant nucleosome. Computational predictions of nucleosome positions based on 
thermodynamic properties of DNA-histone interactions have met with limited 
success. Here we show that the action of the essential RSC remodeling complex in 
S. cerevisiae helps explain the discrepancy between theory and experiment. In 
RSC-depleted cells, NFRs shrink such that the average positions of flanking 
nucleosomes move toward predicted sites. Nucleosome positioning at distinct 
subsets of promoters additionally requires the essential Myb family proteins 
Abf1 and Reb1, whose binding sites are enriched in NFRs. In contrast, H2A.Z 
deposition is dispensable for nucleosome positioning. By regulating H2A.Z 
deposition using a steroid-inducible protein splicing strategy, we show that NFR 
establishment is necessary for H2A.Z deposition. These studies suggest an 
ordered pathway for the assembly of promoter chromatin architecture.