Formation of Regulatory Modules by Local Sequence Duplication

Turnover of regulatory sequence and function is an important part of molecular evolution. But what are the modes of sequence evolution leading to rapid formation and loss of regulatory sites? Here we show that a large fraction of neighboring transcription factor binding sites in the fly genome have formed from a common sequence origin by local duplications. This mode of evolution is found to produce regulatory information: duplications can seed new sites in the neighborhood of existing sites. Duplicate seeds evolve subsequently by point mutations, often towards binding a different factor than their ancestral neighbor sites. These results are based on a statistical analysis of 346 cis-regulatory modules in the Drosophila melanogaster genome, and a comparison set of intergenic regulatory sequence in Saccharomyces cerevisiae. In fly regulatory modules, pairs of binding sites show significantly enhanced sequence similarity up to distances of about 50 bp. We analyze these data in terms of an evolutionary model with two distinct modes of site formation: (i) evolution from independent sequence origin and (ii) divergent evolution following duplication of a common ancestor sequence. Our results suggest that pervasive formation of binding sites by local sequence duplications distinguishes the complex regulatory architecture of higher eukaryotes from the simpler architecture of unicellular organisms. Author Summary: Since Jacob and Monod stressed the importance of gene regulation in evolution, our understanding of the mechanisms of regulation has substantially advanced. In higher eukaryotes, genes often have complex regulatory input, which is encoded in cis-regulatory sequence with multiple transcription factor binding sites. However, the modes of genome evolution generating regulatory complexity are much less understood. This study reports a surprising finding: in fly regulatory modules, the majority of transcription factor binding sites show evidence of a local sequence duplication in their evolutionary history, which relates their sequence information to that of neighboring binding sites. Our analysis suggests that local sequence duplications are a pervasive production mode of regulatory information. This mode appears to be specific to higher eukaryotes; we have not found evidence of frequent local duplications in the yeast genome. Our results affect genomic sequence analysis, in particular, computational identification of cis-regulatory elements and alignment of regulatory DNA. At the same time, they address fundamental questions on the evolution of regulation: How much of the regulatory “grammar” observed in higher eukaryotes is due to optimization of function, and how much reflects the underlying sequence evolution modes? What is the result and what is the substrate of natural selection?