Accelerated Evolution of Conserved Noncoding Sequences in Humans

Figure 1. Identification of “human accelerated” conserved noncoding sequences (HACNSs). A. HACNSs show a statistically significant excess of human-specific sequence changes (circled) at positions highly conserved in other species. B. Distribution of observed human-acceleration P values (red) for 110,549 CNSs versus the P value distribution expected by chance (dashed blue line). The 992 CNSs with P values < 0.005 (highlighted in yellow) are considered HACNSs. HACNSs are numbered based on their P value rank (e.g., HACNS1,HACNS2, etc.) C. Genomic distribution of HACNSs. The location of each HACNS is indicated in red.

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.

Figure 2. A representative HACNS, HACNS4 . Top: Genomic location of HACNS4. Bottom: Alignment of HACNS4 to orthologous sequences from nonhuman genomes. The location of each human-specific substitution is indicated by a red box above the alignment.

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.

Figure 3. HACNS enhancer discovery by mouse transgenesis. Selected HACNSs are tested for enhancer activity using standard mouse transgenic strategies. Each candidate enhancer sequence is PCR amplified from genomic DNA, cloned into an expression vector upstream of a minimal promoter driving expression of a reporter gene (e.g., lacZ) and the resulting construct injected into single cell mouse embryos. These embryos are transplanted into foster mothers, collected at a subsequent developmental time point (e.g. embryonic day (E) 11.5) and stained for reporter gene activity.

We are using in vivo mouse transgenic assays to examine particular HACNSs from our current dataset for human-specific gene regulatory functions by comparison to chimpanzee and rhesus orthologs (Fig. 3). We have shown that several of the most rapidly evolving HACNSs function as developmental enhancers in transgenic mice. These experiments have also provided evidence of functional differences between human and chimpanzee orthologs. We are dissecting a number of elements in detail using a synthetic approach, in which subsets of the human-specific sequence changes in each element are transferred into the chimpanzee enhancer sequence. This will allow us to precisely identify the sequence changes conferring the novel human function, and will provide insight into how adaptive processes can alter cis-regulatory codes. Our long-term goal is to model the phenotypic effect of human-specific sequence change in selected elements by generating “humanized” mice, using homologous recombination to replace endogenous mouse elements with their human orthologs. We anticipate that these studies will contribute to our understanding of how uniquely human traits arose and how they are encoded in the genome.