Genetic changes that altered the function of gene regulatory elements have been implicated in the evolution of human traits such as the expansion of the cerebral cortex. However, identifying the particular changes that modified regulatory activity during human evolution remain challenging. Here we used massively parallel enhancer assays in neural stem cells to quantify the functional impact of >32,000 human-specific substitutions in >4,300 human accelerated regions (HARs) and human gain enhancers (HGEs), which include enhancers with novel activities in humans. We found that >30% of active HARs and HGEs exhibited differential activity between human and chimpanzee. We isolated the effects of human-specific substitutions from background genetic variation to identify the effects of genetic changes most relevant to human evolution. We found that substitutions interacted in both additive and nonadditive ways to modify enhancer function. Substitutions within HARs, which are highly constrained compared to HGEs, showed smaller effects on enhancer activity, suggesting that the impact of human-specific substitutions is buffered in enhancers with constrained ancestral functions. Our findings yield insight into how human-specific genetic changes altered enhancer function and provide a rich set of candidates for studies of regulatory evolution in humans.

Developmental gene expression patterns are orchestrated by thousands of distant-acting transcriptional enhancers. However, identifying enhancers essential for the expression of their target genes has proven challenging. Maps of long-range regulatory interactions may provide the means to identify enhancers crucial for developmental gene expression. To investigate this hypothesis, we used circular chromosome conformation capture coupled with interaction maps in the mouse limb to characterize the regulatory topology of , which is essential for hindlimb development. We identified a robust hindlimb-specific interaction between and a putative hindlimb-specific enhancer. To interrogate the role of this interaction in regulation, we used genome editing to delete this enhancer in mouse. Although deletion of the enhancer completely disrupts the interaction, expression in the hindlimb is only mildly affected, without any detectable compensatory interactions between the promoter and potentially redundant enhancers. enhancer null mice did not exhibit any of the characteristic morphological defects of the mutant. Our results suggest that robust, tissue-specific physical interactions at essential developmental genes have limited predictive value for identifying enhancer mutations with strong loss-of-function phenotypes.

The evolution of uniquely human traits likely entailed changes in developmental gene regulation. Human Accelerated Regions (HARs), which include transcriptional enhancers harboring a significant excess of human-specific sequence changes, are leading candidates for driving gene regulatory modifications in human development. However, insight into whether HARs alter the level, distribution, and timing of endogenous gene expression remains limited. We examined the role of the HAR HACNS1 (HAR2) in human evolution by interrogating its molecular functions in a genetically humanized mouse model. We find that HACNS1 maintains its human-specific enhancer activity in the mouse embryo and modifies expression of Gbx2, which encodes a transcription factor, during limb development. Using single-cell RNA-sequencing, we demonstrate that Gbx2 is upregulated in the limb chondrogenic mesenchyme of HACNS1 homozygous embryos, supporting that HACNS1 alters gene expression in cell types involved in skeletal patterning. Our findings illustrate that humanized mouse models provide mechanistic insight into how HARs modified gene expression in human evolution.

Genetic changes that altered the function of gene regulatory elements have been implicated in the evolution of the human brain. However, identifying the particular changes that modified regulatory activity during neurodevelopment remains challenging. Here we used massively parallel enhancer assays in human neural stem cells to measure the impact of 32,776 human-specific substitutions on enhancer activity in 1,363 Human Accelerated Regions (HARs) and 3,027 Human Gain Enhancers (HGEs), which include enhancers with novel activities in humans. We found that 31.9% of active HARs and 36.4% of active HGEs exhibited differential activity between human and chimpanzee. This enabled us to isolate the effects of 401 human-specific substitutions from other types of genetic variation in HARs and HGEs. Substitutions acted in both an additive and non-additive manner to alter enhancer activity. Human-specific substitutions altered predicted binding sites for a specific set of human transcription factors (TFs) that were a subset of TF binding sites associated with enhancer activity in our assay. Substitutions within HARs, which are overall highly constrained compared to HGEs, showed smaller effects on enhancer activity, suggesting that the impact of human-specific substitutions may be buffered in enhancers with constrained ancestral functions. Our findings yield insight into the mechanisms by which human-specific genetic changes impact enhancer function and provide a rich set of candidates for experimental studies of regulatory evolution in humans. Figure 1. Experimental design. (A) We synthesized 137-bp MPRA fragments overlapping 30,708 hSubs in 3,075 HGEs and 1,316 HARs. (B) MPRA fragments (human in blue, chimpanzee in green)were cloned in front of a luc2 reporter gene and a random oligonucleotide barcode tag (in yellow). Sequencing and counting barcodes provides a quantitative measure of enhancer activity. (C) In stage 1 of the experiment, we screened 50,268 orthologous human-chimpanzee fragment pairs. In stage 2, the impact of genetic differences within all fragments exhibiting species-specific changes in activity was dissected by testing all possible combinations of hSubs and ancestral states in both the human and chimpanzee background reference sequences.

Bacterial strain variation exists in natural populations of bacteria and can be generated experimentally through directed or random mutation. The advent of rapid and cost-efficient whole-genome sequencing has facilitated strain-level genotyping. Even with modern tools, however, it often remains a challenge to map specific traits to individual genetic loci, especially for traits that cannot be selected under culture conditions (e.g., colonization level or pathogenicity). Using a combination of classical and modern approaches, we analyzed strain-level variation in Vibrio fischeri and identified the basis by which some strains lack the ability to utilize glycerol as a carbon source. We proceeded to reconstruct the lineage of the commonly used V. fischeri laboratory strains. Compared to the wild-type ES114 strain, we identify in ES114-L a 9.9-kb deletion with endpoints in tadB2 and glpF; restoration of the missing portion of glpF restores the wild-type phenotype. The widely used strains ESR1, JRM100, and JRM200 contain the same deletion, and ES114-L is likely a previously unrecognized intermediate strain in the construction of many ES114 derivatives. ES114-L does not exhibit a defect in competitive squid colonization but ESR1 does, demonstrating that glycerol utilization is not required for early squid colonization. Our genetic mapping approach capitalizes on the recently discovered chitin-based transformation pathway, which is conserved in the Vibrionaceae; therefore, the specific approach used is likely to be useful for mapping genetic traits in other Vibrio species.I dentifying relevant differences in bacterial strains is fundamental to determining the genetic basis of microbial phenotypes. In many cases, the number of polymorphisms between strains is so high that elucidating which locus or loci contribute to specific phenotypes cannot be achieved simply by determining the genome sequence of the isolates. This challenge is especially pronounced in identifying loci that contribute to colonization and/or pathogenicity phenotypes. The study of genomic islands has made it clear that the acquisition of large regions of DNA can profoundly influence a bacterium's ability to engage with a eukaryotic host (1, 2). Recently, it has become increasingly apparent that defined genetic changes in bacteria at individual loci, single genes, or even nucleotide changes have led to dramatic effects in the evolution of colonizing bacteria. As some examples, the acquisition of the nil locus in Xenorhabdus nematophila contributed to the species-specific association with the worm host Steinernema carpocapsae, inactivation of the RscA biofilm regulator was critical in the evolution of Yersinia pestis from Yersinia pseudotuberculosis, and acquisition of the biofilm regulation of RscS facilitated colonization of north Pacific squid by Vibrio fischeri (3-8).In most cases, identification of factors that contribute to host colonization specificity has relied first on identifying the factor as being necessary for host colonization by standard g...