Although the genomic pattern of nucleosome positioning is broadly conserved, 
quantitative aspects vary over evolutionary timescales. We identify the cis and 
trans determinants of nucleosome positioning using a functional evolutionary 
approach involving S. cerevisiae strains containing large genomic regions from 
other yeast species. In a foreign species, nucleosome depletion at promoters is 
maintained over poly(dA:dT) tracts, whereas internucleosome spacing and all 
other aspects of nucleosome positioning tested are not. Interestingly, the 
locations of the +1 nucleosome and RNA start sites shift in concert. Strikingly, 
in a foreign species, nucleosome-depleted regions occur fortuitously in coding 
regions, and they often act as promoters that are associated with a positioned 
nucleosome array linked to the length of the transcription unit. We suggest a 
three-step model in which nucleosome remodelers, general transcription factors, 
and the transcriptional elongation machinery are primarily involved in 
generating the nucleosome positioning pattern in vivo.