Human-specific gain of function in the HACNS1 enhancer

Figure 1. Genomic location of HACNS1 and its level of conservation across terrestrial vertebrate genomes. Top: HACNS1 is located in an intron of AGAP1 and 300 kb downstream of GBX2 on chromosome 2. Bottom: Sequence alignment of HACNS1 with orthologs from other vertebrate genomes. Positions identical to human are shown in black. A plot of sequence conservation is shown in blue above the alignment. The location of each human-specific substitution is indicated by a vertical red line. The depth of nonhuman conservation at human-substituted positions is shown by a vertical yellow line that indicates whether each sequence is identical to chimpanzee and rhesus at that position. The location of a cluster of 13 substitutions in 81 bp is underlined.

HACNS1 is the most rapidly evolving conserved non-coding sequence that we have identified. Although this sequence is highly conserved among vertebrates, it has experienced 16 human-specific nucleotide changes in the ~6 million years since humans and chimpanzees shared a common ancestor, 13 of which are clustered in a span of 81 base pairs (Fig. 1). Using mouse transgenic reporter assays, we have demonstrated that HACNS1 is an enhancer that drives strong tissue-specific expression in the ear, branchial arches, and primordial limb (Fig. 2). Chimpanzee and macaque orthologs of HACNS1 also function as enhancers, but are much weaker overall and fail to drive reproducible expression in the developing limb. The human-specific robust limb expression domain is consistent across two developmental time points and includes the presumptive anterior wrist and proximal thumb. Using synthetic enhancers, we have shown that the 13 clustered human-specific sequence changes are sufficient to confer the human-specific gain of function in HACNS1 (Fig. 3).

Figure 2. Human-specific gain of function in HACNS1. A. HACNS1 acts as an enhancer in transgenic mouse embryos, driving robust expression of a lacZ reporter gene in the developing anterior limb and other structures at embryonic day 11.5 (E11.5) and E13.5. However, the chimpanzee and rhesus orthologs fail to drive consistent expression in the limb at either time point. B. In the E13.5 limb, the human-specific expression domain extends into the handplate (and footplate, not shown) and includes digit 1, homologous to the human thumb and great toe.

These findings suggest that the gain of function in HACNS1 may have contributed to uniquely human aspects of digit and limb patterning by altering the expression of nearby genes during limb development. We are therefore taking a number of approaches to study the functional impact of HACNS1 on human development and characterize its evolutionary history. We are fully characterizing the enhancer activity of HACNS1 and its chimpanzee ortholog by establishing stable transgenic lines of mice expressing the lacZ reporter under control of HACNS1 and the orthologous sequence from chimpanzee. We are also using novel combinations of proven biochemical techniques to identify the trans-factors that interact with HACNS1 and its orthologs.

Figure 3. Molecular basis of the human-specific gain of function in HACNS1. A. Alignment of HACNS1 with orthologous sequences from other genomes, focused on an 81-bp region in the element containing 13 human specific substitutions. Each human-specific nucleotide is highlighted in red. These 13 substitutions are sufficient to confer the gain of function. B. Expression pattern of a synthetic enhancer in which the 13 human-specific substitutions (red box) are introduced into the orthologous chimpanzee sequence background (black bar). C. Expression pattern of a synthetic enhancer obtained by reversion of these substitutions (black box) in the human sequence (red bar) to the nucleotides in chimpanzee and rhesus.

We are also modeling the phenotypic impact of the gain of function in HACNS1 on human development by selectively “humanizing” the mouse genome. Using homologous recombination, we are replacing the native enhancer in the mouse genome with HACNS1 and, independently, the chimpanzee ortholog. These mice will be phenotyped at different stages of development for changes in the expression of genes near HACNS1, as well as for morphological changes in the limb and other structures. We will then extend this approach to analyze other regulatory sequences with human-specific functions. The phenotypes obtained in the mouse will be evaluated to the extent that they alter mouse biology to recapitulate aspects of human-specific biology and development. In this manner the course of molecular evolution that produced specific human traits can be reconstructed in vivo.