Frequent Gain and Loss of Functional Transcription Factor Binding Sites

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Abstract
Cis-regulatory sequences are not always conserved across species. Divergence within cis-regulatory sequences may result from the evolution of species-specific patterns of gene expression or the flexible nature of the cis-regulatory code. The identification of functional divergence in cis-regulatory sequences is therefore important for both understanding the role of gene regulation in evolution and annotating regulatory elements. We have developed an evolutionary model to detect the loss of constraint on individual transcription factor binding sites (TFBSs). We find that a significant fraction of functionally constrained binding sites have been lost in a lineage-specific manner among three closely related yeast species. Binding site loss has previously been explained by turnover, where the concurrent gain and loss of a binding site maintains gene regulation. We estimate that nearly half of all loss events cannot be explained by binding site turnover. Recreating the mutations that led to binding site loss confirms that these sequence changes affect gene expression in some cases. We also estimate that there is a high rate of binding site gain, as more than half of experimentally identified S. cerevisiae binding sites are not conserved across species. The frequent gain and loss of TFBSs implies that cis-regulatory sequences are labile and, in the absence of turnover, may contribute to species-specific patterns of gene expression. Research in the field of molecular evolution is focused on understanding the genetic basis of functional differences between species. Protein coding sequences have traditionally been the focus of these studies, as the genetic code enables a detailed study of the strength of selection acting on amino acid sequences. However, from the earliest cross-species sequence comparisons, it was clear that protein sequences among closely related species are too similar to explain the observed phenotypic diversity. This led to the hypothesis that the evolution of gene regulation has played a key role in generating diversity between species. The availability of numerous complete genome sequences has made it possible to begin testing this hypothesis. In this work, the authors use an evolutionary model to identify functional divergence within transcription factor binding sites, the core functional elements involved in gene regulation. Applying this model to the baker's yeast, Saccharomyces cerevisiae, and its three closest relatives, the authors find that a substantial fraction of the ancestral binding sites have been lost in a species-specific manner. In some cases the loss of the binding site creates gene expression differences that may be indicative of species-specific changes in gene regulation. This work provides a useful computational framework that will allow further study of the conservation of cis-regulatory sequences and their role in molecular evolution.