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Transcriptional enhancers orchestrate cell type- and time point-specific gene expression programs. Genetic variation within enhancer sequences is an important contributor to phenotypic variation including evolutionary adaptations and human disease. Certain genes and pathways may be more prone to regulatory evolution than others, with different patterns across diverse organisms, but whether such patterns exist has not been investigated at a sufficient scale. To address this question, we identified signatures of accelerated sequence evolution in conserved enhancer elements throughout the mammalian phylogeny at an unprecedented scale. While different genes and pathways were enriched for regulatory evolution in different parts of the tree, we found a striking overall pattern of pleiotropic genes involved in gene regulatory and developmental processes being enriched for accelerated enhancer evolution. These genes were connected to more enhancers than other genes, which was the basis for having an increased amount of sequence acceleration over all their enhancers combined. We provide evidence that sequence acceleration is associated with turnover of regulatory function. Detailed study of one acceleration event in an enhancer of HES1 revealed that sequence evolution led to a new activity domain in the developing limb that emerged concurrently with the evolution of digit reduction in hoofed mammals. Our results provide evidence that enhancer evolution has been a frequent contributor to regulatory innovation at conserved developmental signaling genes in mammals. [ABSTRACT FROM AUTHOR]

Transcriptional enhancers orchestrate cell type- and time point-specific gene expression programs. Evolution of enhancer sequences can alter target gene expression without causing detrimental misexpression in other contexts. It has long been thought that this modularity allows evolutionary changes in enhancers to escape pleiotropic constraints, which is especially important for evolutionary constrained developmental patterning genes. However, there is still little data supporting this hypothesis. Here we identified signatures of accelerated evolution in conserved enhancer elements across the mammalian phylogeny. We found that pleiotropic genes involved in gene regulatory and developmental processes were enriched for accelerated sequence evolution within their enhancer elements. These genes were associated with an excess number of enhancers compared to other genes, and due to this they exhibit a substantial degree of sequence acceleration over all their enhancers combined. We provide evidence that sequence acceleration is associated with turnover of regulatory function. We studied one acceleration event in depth and found that its sequence evolution led to the emergence of a new enhancer activity domain that may be involved in the evolution of digit reduction in hoofed mammals. Our results provide tangible evidence that enhancer evolution has been a frequent contributor to modifications involving constrained developmental signaling genes in mammals.

Genetic changes that modify the function of transcriptional enhancers have been linked to the evolution of biological diversity across species. Multiple studies have focused on the role of nucleotide substitutions, transposition, and insertions and deletions in altering enhancer function. Here we show that turnover of CpG islands (CGIs), which contribute to enhancer activation, is broadly associated with changes in enhancer activity across mammals, including humans. We integrated maps of CGIs and enhancer activity-associated histone modifications obtained from multiple tissues in nine mammalian species and found that CGI content in enhancers was strongly associated with increased histone modification levels. CGIs showed widespread turnover across species and species-specific CGIs were strongly enriched for enhancers exhibiting species-specific activity across all tissues and species we examined. Genes associated with enhancers with species-specific CGIs showed concordant biases in their expression, supporting that CGI turnover contributes to gene regulatory innovation. Our results also implicate CGI turnover in the evolution of Human Gain Enhancers (HGEs), which show increased activity in human embryonic development and may have contributed to the evolution of uniquely human traits. Using a humanized mouse model, we show that a highly conserved HGE with a large CGI absent from the mouse ortholog shows increased activity at the human CGI in the humanized mouse diencephalon. Collectively, our results point to CGI turnover as a mechanism driving gene regulatory changes potentially underlying trait evolution in mammals.