Deep conservation of the enhancer regulatory code in animals

Enhancer function, from sponges to humans Identifying the function of enhancers, DNA regions that help to regulate gene expression and evolve rapidly, has been difficult. This area of research has been hampered by the difficultly in identifying functional conservation. Wong et al. now show that despite low sequence conservation, enhancer function is strongly conserved through the animal kingdom (see the Perspective by Harmston). Transgenic expression of sponge enhancers in zebrafish and mice demonstrates that these sequences can drive cell type–specific gene expression across species. These results suggest an unexpectedly deep level of conservation of gene regulation across the animal kingdom maintained over the course of metazoan evolution. Science, this issue p. eaax8137; see also p. 657 Across the animal kingdom, enhancer function is conserved throughout 700 million years of evolution. INTRODUCTION In animals, gene regulatory networks specify cell identity in space and time. Transcription of genes in these networks is modulated by a class of cis-regulatory elements called enhancers that contain short (~10 base pairs) DNA sequence motifs recognized by transcription factors (TFs). In contrast to TFs, whose histories have been largely traced to the origin of the animal kingdom or earlier, the origin and evolution of enhancers have been relatively difficult to discern. Although not a single enhancer has been shown to be conserved across the animal kingdom, enhancers may be as ancient and conserved as the TFs with which they interact. This inability to identify conserved enhancers is apparently because they evolve faster than both the TFs they interact with and the genes they regulate. RATIONALE Putative enhancers in the sponge Amphimedon queenslandica had previously been identified on the basis of combinatorial patterns of histone modifications. Here, we sought to determine whether sponges share functionally conserved enhancers with bilaterians. We primarily focused on deeply conserved metazoan microsyntenic gene pairs. These pairs are thought to be conserved because the cis-regulatory elements that regulate the developmental expression of one gene (the target gene) are located in the other gene (the bystander gene). This proposed regulatory linkage may underlie the maintenance of these microsyntenic gene pairs across 700 million years of independent evolution. RESULTS We found that enhancers present in Amphimedon microsyntenic regions drive consistent patterns of cell type–specific gene expression in zebrafish and mouse embryos. Although these sponge enhancers do not share significant sequence identity with vertebrates, they are in microsyntenic regions that are orthologous with microsyntenic regions in other metazoans and have strong histone H3 Lys4 methylation (H3K4me1) enhancer signals. Focusing on an Islet enhancer in the Islet-Scaper microsyntenic region, we found that the sponge 709–base pair enhancer, independent of its orientation, drives green fluorescent protein (GFP) expression in zebrafish cells in the hindbrain neuroepithelial region, the roof plate around the midline, the pectoral fin, and the otic vesicle; the activity overlaps with endogenous Isl2a expression. Systematic removal of sequences from the Amphimedon Islet enhancer revealed that both the 5′ and 3′ regions of this enhancer are required for consistent cell type–specific activity in zebrafish. We then used the number and frequency of TF binding motifs in the Amphimedon Islet enhancer to identify putative enhancers in human, mouse, and fly Islet-Scaper regions. The candidate orthologous enhancers from humans and mice drove gene expression patterns similar to those in sponges and endogenous Islet enhancers in zebrafish. We also demonstrated that a number of putative Amphimedon enhancers, which are outside conserved microsyntenic regions, can also drive unique expression patterns: Enhancers of sponge housekeeping genes drive broader expression patterns in zebrafish. CONCLUSION These results suggest the existence of an ancient and conserved, yet flexible, genomic regulatory syntax that (i) can be interpreted by the available TFs present in cells constituting disparate developmental systems and cell types, and (ii) has been repeatedly co-opted into cell type–specific networks across the animal kingdom. This common regulatory code maintains a repertoire of conserved TF binding motifs that stabilize and preserve enhancer functionality over evolution. Once established, these enhancers may be maintained as part of conserved gene regulatory network modules over evolution. Although robust, these enhancers can evolve through the expansion and integration of new TF binding motifs and the loss of others. We posit that the expansion of TFs and enhancers may underlie the evolution of complex body plans. Islet enhancer activity is conserved across animal evolution. Enhancers located within conserved microsyntenic units in the sponge Amphimedon queenslandica are tested in a zebrafish transgenic reporter system. In zebrafish, the sponge Islet enhancer drives a GFP reporter expression pattern similar to that of human, mouse, and zebrafish enhancers identified within the Islet-Scaper microsyntenic region. This suggests the conservation of regulatory syntax specified by flexible organizations of motifs. Interactions of transcription factors (TFs) with DNA regulatory sequences, known as enhancers, specify cell identity during animal development. Unlike TFs, the origin and evolution of enhancers has been difficult to trace. We drove zebrafish and mouse developmental transcription using enhancers from an evolutionarily distant marine sponge. Some of these sponge enhancers are located in highly conserved microsyntenic regions, including an Islet enhancer in the Islet-Scaper region. We found that Islet enhancers in humans and mice share a suite of TF binding motifs with sponges, and that they drive gene expression patterns similar to those of sponge and endogenous Islet enhancers in zebrafish. Our results suggest the existence of an ancient and conserved, yet flexible, genomic regulatory syntax that has been repeatedly co-opted into cell type–specific gene regulatory networks across the animal kingdom.