The Importance of Being Cis: Evolution of Orthologous Fish and Mammalian Enhancer Activity

Conserved noncoding elements (CNEs) in vertebrate genomes often act as developmental enhancers, but a critical issue is how well orthologous CNE sequences retain the same activity in their respective species, a characteristic important for generalization of model organism studies. To quantify how well CNE enhancer activity has been preserved, we compared the anatomy-specific activities of 41 zebra fish CNEs in zebra fish embryos with the activities of orthologous human CNEs in mouse embryos. We found that 13/41 (∼30%) of the orthologous CNE pairs exhibit conserved positive activity in zebra fish and mouse. Conserved positive activity is only weakly associated with either sequence conservation or the absence of bases undergoing accelerated evolution. A stronger effect is that disparate activity is associated with transcription factor binding site divergence. To distinguish the contributions of cis- versus trans-regulatory changes, we analyzed 13 CNEs in a three-way experimental comparison: human CNE tested in zebra fish, human CNE tested in mouse, and orthologous zebra fish CNE tested in zebra fish. Both cis- and trans-changes affect a significant fraction of CNEs, although human and zebra fish sequences exhibit disparate activity in zebra fish (indicating cis regulatory changes) twice as often as human sequences show disparate activity when tested in mouse and zebra fish (indicating trans regulatory changes). In all four cases where the zebra fish and human CNE display a similar expression pattern in zebra fish, the human CNE also displays a similar expression pattern in mouse. This suggests that the endogenous enhancer activity of ∼30% of human CNEs can be determined from experiments in zebra fish alone, and to identify these CNEs, both the zebra fish and the human sequences should be tested.