Evolution of transcription factor binding through sequence variations and turnover of binding sites

Variations in noncoding regulatory sequences play a central role in evolution. Interpreting such variations, however, remains difficult even in the context of defined attributes such as transcription factor (TF) binding sites. Here, we systematically link variations in cis-regulatory sequences to TF binding by profiling the allele-specific binding of 27 TFs expressed in a yeast hybrid, in which two related genomes are present within the same nucleus. TFs localize preferentially to sites containing their known consensus motifs but occupy only a small fraction of the motif-containing sites available within the genomes. Differential binding of TFs to the orthologous alleles was well explained by variations that alter motif sequence, whereas differences in chromatin accessibility between alleles were of little apparent effect. Motif variations that abolished binding when present in only one allele were still bound when present in both alleles, suggesting evolutionary compensation, with a potential role for sequence conservation at the motif's vicinity. At the level of the full promoter, we identify cases of binding-site turnover, in which binding sites are reciprocally gained and lost, yet most interspecific differences remained uncompensated. Our results show the flexibility of TFs to bind imprecise motifs and the fast evolution of TF binding sites between related species.