Picking Up the Trail of Phylogenetic Footprints

Non-coding sequence in eukaryotes encodes functionally important signals for the regulation of gene expression. Since these elements are evolutionary conserved among related taxa due to stabilizing selection they accumulate less mutations than adjacent non-functional DNA. These conserved non-coding sequences with potential regulatory activity, are known as phylogenetic footprints. They can be detected by comparison of the sequences surrounding orthologous genes in (distantly) related species. Loss or acquisition of phylogenetic footprints in some of these lineages provides evidence for the evolutionary modification of cis-regulatory elements. In order to observe the distribution of phylogenetic footprints among whole gene clusters and various species we developed the software tool tracker. It is designed for large scale analysis and identifies corresponding footprints in long sequences from multiple species more efficiently than other available algorithms. We apply our novel method to the published sequences of HoxA clusters to study the footprint evolution after the most recent cluster duplication in Danio rerio (zebrafish) and Takifugu rubripes (pufferfish). We introduce a statistical model that allows us to estimate the loss of non-coding sequence conservation that can be attributed to gene loss and other structural reasons. According to this model we observe an unexpectedly high loss of sequence conservation suggesting that binding site turnover and/or adaptive modification also contribute to the massive loss of sequence conservation. The statistical analysis of phylogenetic footprints in the two known Hox clusters of Heterodontus francisci (horn shark) and the four mammalian Hox clusters A,B,C and D shows that the shark HoxN cluster is HoxD-like. From this finding we conclude that the most recent common ancestor of gnathostomes (jawed vertebrates) had at least four Hox clusters, including those which are orthologous to the four mammalian Hox clusters. Within the intergenic region from hoxA13 to hoxA11 we discovered a set of footprints specifically conserved among the available representatives of the tetrapods. Since exclusive expression domains of hoxA13 and hoxA11 are known to determine limb development we propose that these footprints are crucial for the fin limb transition. Consequently we predict the presence of homologous footprints in amphibians and reptiles.