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.

Genetic studies in non-human primates serve as a potential strategy for identifying genomic intervals where polymorphisms impact upon human disease-related phenotypes. It remains unclear, however, whether independently arising polymorphisms in orthologous regions of non-human primates leads to similar variation in a quantitative trait found in both species. To explore this paradigm, we studied a baboon apolipoprotein gene cluster (APOA1/C3/A4/A5) for which the human gene orthologs have well established roles in influencing plasma HDL-cholesterol and triglyceride concentrations. Our extensive polymorphism analysis of this 68 kb gene cluster in 96 pedigreed baboons identified several haplotype blocks each with limited diversity, consistent with haplotype findings in humans. To determine whether baboons, like humans, also have particular haplotypes associated with lipid phenotypes, we genotyped 634 well characterized baboons using 16 haplotype tagging SNPs. Genetic analysis of single SNPs, as well as haplotypes, revealed an association of APOA5 and APOC3 variants with HDL cholesterol and triglyceride concentrations, respectively. Thus, independent variation in orthologous genomic intervals does associate with similar quantitative lipid traits in both species, supporting the possibility of uncovering human QTL genes in a highly controlled non-human primate model.

Sequence changes in regulatory regions have often been invoked to explain phenotypic divergence among species, but molecular examples of this have been difficult to obtain. In this study, we identified an anthropoid primate specific sequence element that contributed to the regulatory evolution of the LDL receptor. Using a combination of close and distant species genomic sequence comparisons coupled with in vivo and in vitro studies, we show that a functional cholesterol-sensing sequence motif arose and was fixed within a pre-existing enhancer in the common ancestor of anthropoid primates. Our study demonstrates one molecular mechanism by which ancestral mammalian regulatory elements can evolve to perform new functions in the primate lineage leading to human.