Flexibility of transcription factor target site position in conserved cis-regulatory modules

Eight Strongylocentrotus purpuratus cis-regulatory modules, in each of which up to three different transcription factor target sites had been previously authenticated in gene transfer and mutagenesis studies, were compared to the orthologous modules in the genome of Lytechinus variegatus. These species diverged about 50 million years ago. The orthologous modules were identified in sequenced Lytechinus BACs, as conserved sequence patches in similar regions of the respective genes. The similar functionality of several of these control modules in the two species was confirmed by cross-species gene transfer experiments. In each case the repertoire of transcription factor target sites was the same in the orthologous modules, but the positions of the individual sites with respect to one another was evolutionarily flexible, even though the intervening sequence was often strongly conserved. The most invariably conserved features, as seen also in other systems, were pairs of target sites that are immediately adjacent to one another. Their conservation is probably due to the necessity for interaction of proximally bound transcription factors, while a facilitated form of sequence conversion might be the mechanism of site position change.