Genomic comparisons between human and distant, non-primate mammals are commonly used to identify cis-regulatory elements based on constrained sequence evolution. However, these methods fail to detect cryptic functional elements, which are too weakly conserved among mammals to distinguish from nonfunctional DNA. To address this problem, we explored the potential of deep intra-primate sequence comparisons. We sequenced the orthologs of 558 kb of human genomic sequence, covering multiple loci involved in cholesterol homeostasis, in 6 nonhuman primates. Our analysis identified 6 noncoding DNA elements displaying significant conservation among primates, but undetectable in more distant comparisons. In vitro and in vivo tests revealed that at least three of these 6 elements have regulatory function. Notably, the mouse orthologs of these three functional human sequences had regulatory activity despite their lack of significant sequence conservation, indicating that they are cryptic ancestral cis-regulatory elements. These regulatory elements could still be detected in a smaller set of three primate species including human, rhesus and marmoset. Since the human and rhesus genome sequences are already available, and the marmoset genome is actively being sequenced, the primate-specific conservation analysis described here can be applied in the near future on a whole-genome scale, to complement the annotation provided by more distant species comparisons.
