Human-specific gain of function in a developmental enhancer

Abstract

Changes in gene regulation are thought to have contributed to the evolution of human development. However, in vivo evidence for uniquely human developmental regulatory function has remained elusive. In transgenic mice, a conserved noncoding sequence (HACNS1) that evolved extremely rapidly in humans acted as an enhancer of gene expression that has gained a strong limb expression domain relative to the orthologous elements from chimpanzee and rhesus macaque. This gain of function was consistent across two developmental stages in the mouse and included the presumptive anterior wrist and proximal thumb. In vivo analyses with synthetic enhancers, in which human-specific substitutions were introduced into the chimpanzee enhancer sequence or reverted in the human enhancer to the ancestral state, indicated that 13 substitutions clustered in an 81-base pair module otherwise highly constrained among terrestrial vertebrates were sufficient to confer the human-specific limb expression domain.

Figure 1. Human-specific gain of function in HACNS1

A. Top: Location of HACNS1 in NCBI build 36.1 of the human genome assembly. Bottom: Sequence alignment of HACNS1 with orthologs from other vertebrate genomes; positions identical to human are shown in black. A quantitative plot of sequence conservation is shown in blue above the alignment (26-28). The location of each human-specific substitution is indicated by a vertical red line, and the depth of nonhuman evolutionary 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 cluster of 13 human–specific substitutions in 81 basepairs is also indicated. B. Expression patterns obtained from the HACNS1 enhancer and orthologous sequences from chimpanzee and rhesus driving expression of a lacZ reporter gene in E11.5 mouse embryos. Arrows indicate positions in the anterior limb bud where reproducible reporter gene expression is present or absent. A representative HACNS1 embryo is shown at top to illustrate the relevant anatomical structures. Three embryos resulting from independent transgene integration events are shown for each orthologous sequence tested. C. Number of embryos transgenic for each sequence displaying the limb expression patterns described in the text.

Figure 2. Gain of function in HACNS1 persists at E13.5

A. Expression patterns obtained from HACNS1 and its chimpanzee ortholog in E13.5 mouse embryos. Three embryos resulting from independent transgene integration events are shown for each construct. Close-up views of forelimb and hindlimb expression in a representative embryo for each construct are shown at left, and arrows indicate positions where limb expression is present or absent. B. Dorsal view of reporter gene expression in the distal anterior forelimb of a HACNS1 E13.5 transgenic embryo. Arrows indicate the most anterior digit. C. Number of embryos transgenic for each construct that display the limb expression patterns described in the text.

Figure 3. Identification of human-specific substitutions contributing to the gain of function in HACNS1

A. Alignment of HACNS1 with orthologous sequences from other vertebrate genomes, focused on an 81-bp region in the element that contains 13 human-specific substitutions. The position of each substitution is indicated by a red box above the alignment and each human-specific nucleotide is highlighted in red. Positions in the nonhuman genomes that are identical to the human sequence are displayed as dots. B. Expression pattern of a synthetic enhancer in which the 13 human-specific substitutions (red box) are introduced into the orthologous 1.2 kb 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 nucleotide states in chimpanzee and rhesus. D. Number of embryos transgenic for each synthetic enhancer that show full, partial or no expression in the limb at E11.5.

Figure 4. Human-specific substitution rate and proportion of AT to GC substitutions in HACNS1 and flanking genomic region

The rate of human-specific substitutions (solid red line) and the fraction of human substitutions that are AT to GC events (solid blue line) were estimated in sliding windows across a 9 kb interval around HACNS1. The average values of each metric for 1 Mb of genomic sequence centered on HACNS1 are shown for reference (dashed lines). The 546 bp interval corresponding to HACNS1 is highlighted in yellow.