The packaging of eukaryotic genomes into nuclesomes plays critical roles in 
chromatin organization and gene regulation. Studies in Saccharomyces cerevisiae 
indicate that nucleosome occupancy is partially encoded by intrinsic 
antinucleosomal DNA sequences, such as poly(A) sequences, as well as by binding 
sites for trans-acting factors that can evict nucleosomes, such as Reb1 and the 
Rsc3/30 complex. Here, we use genome-wide nucleosome occupancy maps in 13 
Ascomycota fungi to discover large-scale evolutionary reprogramming of both 
intrinsic and trans determinants of chromatin structure. We find that poly(G)s 
act as intrinsic antinucleosomal sequences, comparable to the known function of 
poly(A)s, but that the abundance of poly(G)s has diverged greatly between 
species, obscuring their antinucleosomal effect in low-poly(G) species such as 
S. cerevisiae. We also develop a computational method that uses nucleosome 
occupancy maps for discovering trans-acting general regulatory factor (GRF) 
binding sites. Our approach reveals that the specific sequences bound by GRFs 
have diverged substantially across evolution, corresponding to a number of major 
evolutionary transitions in the repertoire of GRFs. We experimentally validate a 
proposed evolutionary transition from Cbf1 as a major GRF in pre-whole-genome 
duplication (WGD) yeasts to Reb1 in post-WGD yeasts. We further show that the 
mating type switch-activating protein Sap1 is a GRF in S. pombe, demonstrating 
the general applicability of our approach. Our results reveal that the 
underlying mechanisms that determine in vivo chromatin organization have 
diverged and that comparative genomics can help discover new determinants of 
chromatin organization.