The systematic identification of conserved developmental regulatory elements has made it possible to study the impact of human-specific changes in regulatory DNAs on a genome-wide scale. To this end, we have focused on identifying noncoding sequences highly conserved in nonhuman vertebrate species that display high rates of nucleotide substitution on the human lineage, which may indicate positive selection for new regulatory functions. There are two motivations for this approach. First, human-specific developmental features, as elaborations on the common theme of mammalian development, are likely to arise in part from changes in the conserved genetic architecture of the developmental regulatory machinery. The second, more practical consideration is that evolutionary conservation provides a means to interpret the statistical and ultimately biological importance of human-specific sequence changes.
To identify putative regulatory sequences evolving rapidly on the human lineage, we developed a test statistic (in collaboration with Shyam Prabhakar of the Genome Institute of Singapore) for assessing the significance of human-specific sequence acceleration in conserved noncoding sequences (CNSs). In our approach, sites within CNSs were binned based on their degree of sequence conservation in six nonhuman species: chimpanzee, rhesus macaque, mouse, rat, dog and chicken, and human-specific substitutions were identified by parsimony (Fig. 1A). Human-specific substitutions at sites conserved from chimpanzee to chicken are expected to be uncommon events, and were consequently given more weight than substitutions at less conserved sites. This partitioning strategy facilitated a formal test of the likelihood of observing the exact configuration of human-specific substitutions in a CNS, compared with what would be expected in a typical CNS showing a comparable level of conservation and located in region of the genome of comparable background neutral rate.
Using this approach, we identified 992 conserved noncoding sequences that are evolving rapidly on the human lineage (Fig. 1B and 1C). We termed these elements human-accelerated conserved noncoding sequences (HACNSs). HACNSs are disproportionately associated with genes involved in neuronal migration, adhesion, axon guidance and synapse formation, suggesting that cis-regulatory changes in human evolution may have contributed to changes in brain development and function. We have also identified several loci with a significant excess of rapidly evolving noncoding sequences. These loci may have been hotspots of cis-regulatory change throughout human evolution.