Repeated shifts in sociality are associated with fine-tuning of highly conserved and lineage-specific enhancers in a socially flexible bee.

Comparative genomic studies of social insects suggest that changes in gene regulation are associated with evolutionary transitions in social behavior, but the activity of predicted regulatory regions has not been tested empirically. We used STARR-seq, a high-throughput enhancer discovery tool, to identify and measure the activity of enhancers in the socially variable sweat bee, Lasioglossum albipes. We identified over 36,000 enhancers in the L. albipes genome from three social and three solitary populations. Many enhancers were identified in only a subset of L. albipes populations, revealing rapid divergence in regulatory regions within this species. Population-specific enhancers were often proximal to the same genes across populations, suggesting compensatory gains and losses of regulatory regions may preserve gene activity. We also identified 1182 enhancers with significant differences in activity between social and solitary populations, some of which are conserved regulatory regions across species of bees. These results indicate that social trait variation in L. albipes is associated with the fine-tuning of ancient enhancers as well as lineage-specific regulatory changes. Combining enhancer activity with population genetic data revealed variants associated with differences in enhancer activity and identified a subset of differential enhancers with signatures of selection associated with social behavior. Together, these results provide the first empirical map of enhancers in a socially flexible bee and highlight links between cis-regulatory variation and the evolution of social behavior.

Evolutionary Innovations in Conserved Regulatory Elements Associate With Developmental Genes in Mammals.

Transcriptional enhancers orchestrate cell type- and time point-specific gene expression programs. Genetic variation within enhancer sequences is an important contributor to phenotypic variation including evolutionary adaptations and human disease. Certain genes and pathways may be more prone to regulatory evolution than others, with different patterns across diverse organisms, but whether such patterns exist has not been investigated at a sufficient scale. To address this question, we identified signatures of accelerated sequence evolution in conserved enhancer elements throughout the mammalian phylogeny at an unprecedented scale. While different genes and pathways were enriched for regulatory evolution in different parts of the tree, we found a striking overall pattern of pleiotropic genes involved in gene regulatory and developmental processes being enriched for accelerated enhancer evolution. These genes were connected to more enhancers than other genes, which was the basis for having an increased amount of sequence acceleration over all their enhancers combined. We provide evidence that sequence acceleration is associated with turnover of regulatory function. Detailed study of one acceleration event in an enhancer of HES1 revealed that sequence evolution led to a new activity domain in the developing limb that emerged concurrently with the evolution of digit reduction in hoofed mammals. Our results provide evidence that enhancer evolution has been a frequent contributor to regulatory innovation at conserved developmental signaling genes in mammals.

Massively parallel discovery of human-specific substitutions that alter enhancer activity.

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.

Perspectives on Gene Regulatory Network Evolution.

Animal development proceeds through the activity of genes and their cis-regulatory modules (CRMs) working together in sets of gene regulatory networks (GRNs). The emergence of species-specific traits and novel structures results from evolutionary changes in GRNs. Recent work in a wide variety of animal models, and particularly in insects, has started to reveal the modes and mechanisms of GRN evolution. I discuss here various aspects of GRN evolution and argue that developmental system drift (DSD), in which conserved phenotype is nevertheless a result of changed genetic interactions, should regularly be viewed from the perspective of GRN evolution. Advances in methods to discover related CRMs in diverse insect species, a critical requirement for detailed GRN characterization, are also described.

Favorable genomic environments for cis-regulatory evolution: A novel theoretical framework.

Cis-regulatory changes are arguably the primary evolutionary source of animal morphological diversity. With the recent explosion of genome-wide comparisons of the cis-regulatory content in different animal species is now possible to infer general principles underlying enhancer evolution. However, these studies have also revealed numerous discrepancies and paradoxes, suggesting that the mechanistic causes and modes of cis-regulatory evolution are still not well understood and are probably much more complex than generally appreciated. Here, we argue that the mutational mechanisms and genomic regions generating new regulatory activities must comply with the constraints imposed by the molecular properties of cis-regulatory elements (CREs) and the organizational features of long-range chromatin interactions. Accordingly, we propose a new integrative evolutionary framework for cis-regulatory evolution based on two major premises for the origin of novel enhancer activity: (i) an accessible chromatin environment and (ii) compatibility with the 3D structure and interactions of pre-existing CREs. Mechanisms and DNA sequences not fulfilling these premises, will be less likely to have a measurable impact on gene expression and as such, will have a minor contribution to the evolution of gene regulation. Finally, we discuss current comparative cis-regulatory data under the light of this new evolutionary model, and propose that the two most prominent mechanisms for the evolution of cis-regulatory changes are the overprinting of ancestral CREs and the exaptation of transposable elements.

Accelerated Evolution of Enhancer Hotspots in the Mammal Ancestor.

Mammals have evolved remarkably different sensory, reproductive, metabolic, and skeletal systems. To explore the genetic basis for these differences, we developed a comparative genomics approach to scan whole-genome multiple sequence alignments to identify regions that evolved rapidly in an ancestral lineage but are conserved within extant species. This pattern suggests that ancestral changes in function were maintained in descendants. After applying this test to therian mammals, we identified 4,797 accelerated regions, many of which are noncoding and located near developmental transcription factors. We then used mouse transgenic reporter assays to test if noncoding accelerated regions are enhancers and to determine how therian-specific substitutions affect their activity in vivo. We discovered enhancers with expression specific to the therian version in brain regions involved in the hormonal control of milk ejection, uterine contractions, blood pressure, temperature, and visual processing. This work underscores the idea that changes in developmental gene expression are important for mammalian evolution, and it pinpoints candidate genes for unique aspects of mammalian biology.

CpG island turnover events predict evolutionary changes in enhancer activity.

BACKGROUND: Genetic changes that modify the function of transcriptional enhancers have been linked to the evolution of biological diversity across species. Multiple studies have focused on the role of nucleotide substitutions, transposition, and insertions and deletions in altering enhancer function. CpG islands (CGIs) have recently been shown to influence enhancer activity, and here we test how their turnover across species contributes to enhancer evolution. RESULTS: We integrate maps of CGIs and enhancer activity-associated histone modifications obtained from multiple tissues in nine mammalian species and find that CGI content in enhancers is strongly associated with increased histone modification levels. CGIs show widespread turnover across species and species-specific CGIs are strongly enriched for enhancers exhibiting species-specific activity across all tissues and species. Genes associated with enhancers with species-specific CGIs show concordant biases in their expression, supporting that CGI turnover contributes to gene regulatory innovation. Our results also implicate CGI turnover in the evolution of Human Gain Enhancers (HGEs), which show increased activity in human embryonic development and may have contributed to the evolution of uniquely human traits. Using a humanized mouse model, we show that a highly conserved HGE with a large CGI absent from the mouse ortholog shows increased activity at the human CGI in the humanized mouse diencephalon. CONCLUSIONS: Collectively, our results point to CGI turnover as a mechanism driving gene regulatory changes potentially underlying trait evolution in mammals.

Extensive Loss and Gain of Conserved Noncoding Elements During Early Teleost Evolution.

Conserved noncoding elements in vertebrates are enriched around transcription factor loci associated with development. However, loss and rapid divergence of conserved noncoding elements has been reported in teleost fish, albeit taking only few genomes into consideration. Taking advantage of the recent increase in high-quality teleost genomes, we focus on studying the evolution of teleost conserved noncoding elements, carrying out targeted genomic alignments and comparisons within the teleost phylogeny to detect conserved noncoding elements and reconstruct the ancestral teleost conserved noncoding elements repertoire. This teleost-centric approach confirms previous observations of extensive vertebrate conserved noncoding elements loss early in teleost evolution, but also reveals massive conserved noncoding elements gain in the teleost stem-group over 300 million years ago. Using synteny-based association to link conserved noncoding elements to their putatively regulated target genes, we show the most teleost gained conserved noncoding elements are found in the vicinity of orthologous loci involved in transcriptional regulation and embryonic development that are also associated with conserved noncoding elements in other vertebrates. Moreover, teleost and vertebrate conserved noncoding elements share a highly similar motif and transcription factor binding site vocabulary. We suggest that early teleost conserved noncoding element gains reflect a restructuring of the ancestral conserved noncoding element repertoire through both extreme divergence and de novo emergence. Finally, we support newly identified pan-teleost conserved noncoding elements have potential for accurate resolution of teleost phylogenetic placements in par with coding sequences, unlike ancestral only elements shared with spotted gar. This work provides new insight into conserved noncoding element evolution with great value for follow-up work on phylogenomics, comparative genomics, and the study of gene regulation evolution in teleosts.

A common cis-regulatory variant impacts normal-range and disease-associated human facial shape through regulation of PKDCC during chondrogenesis.

Genome-wide association studies (GWAS) identified thousands of genetic variants linked to phenotypic traits and disease risk. However, mechanistic understanding of how GWAS variants influence complex morphological traits and can, in certain cases, simultaneously confer normal-range phenotypic variation and disease predisposition, is still largely lacking. Here, we focus on rs6740960, a single nucleotide polymorphism (SNP) at the 2p21 locus, which in GWAS studies has been associated both with normal-range variation in jaw shape and with an increased risk of non-syndromic orofacial clefting. Using in vitro derived embryonic cell types relevant for human facial morphogenesis, we show that this SNP resides in an enhancer that regulates chondrocytic expression of PKDCC - a gene encoding a tyrosine kinase involved in chondrogenesis and skeletal development. In agreement, we demonstrate that the rs6740960 SNP is sufficient to confer chondrocyte-specific differences in PKDCC expression. By deploying dense landmark morphometric analysis of skull elements in mice, we show that changes in Pkdcc dosage are associated with quantitative changes in the maxilla, mandible, and palatine bone shape that are concordant with the facial phenotypes and disease predisposition seen in humans. We further demonstrate that the frequency of the rs6740960 variant strongly deviated among different human populations, and that the activity of its cognate enhancer diverged in hominids. Our study provides a mechanistic explanation of how a common SNP can mediate normal-range and disease-associated morphological variation, with implications for the evolution of human facial features.

Evolved Repression Overcomes Enhancer Robustness.

Biological systems display extraordinary robustness. Robustness of transcriptional enhancers results mainly from clusters of binding sites for the same transcription factor, and it is not clear how robust enhancers can evolve loss of expression through point mutations. Here, we report the high-resolution functional dissection of a robust enhancer of the shavenbaby gene that has contributed to morphological evolution. We found that robustness is encoded by many binding sites for the transcriptional activator Arrowhead and that, during evolution, some of these activator sites were lost, weakening enhancer activity. Complete silencing of enhancer function, however, required evolution of a binding site for the spatially restricted potent repressor Abrupt. These findings illustrate that recruitment of repressor binding sites can overcome enhancer robustness and may minimize pleiotropic consequences of enhancer evolution. Recruitment of repression may be a general mode of evolution to break robust regulatory linkages.

Evolution of lineage-specific functions in ancient cis-regulatory modules.

Morphological evolution is driven both by coding sequence variation and by changes in regulatory sequences. However, how cis-regulatory modules (CRMs) evolve to generate entirely novel expression domains is largely unknown. Here, we reconstruct the evolutionary history of a lens enhancer located within a CRM that not only predates the lens, a vertebrate innovation, but bilaterian animals in general. Alignments of orthologous sequences from different deuterostomes sub-divide the CRM into a deeply conserved core and a more divergent flanking region. We demonstrate that all deuterostome flanking regions, including invertebrate sequences, activate gene expression in the zebrafish lens through the same ancient cluster of activator sites. However, levels of gene expression vary between species due to the presence of repressor motifs in flanking region and core. These repressor motifs are responsible for the relatively weak enhancer activity of tetrapod flanking regions. Ray-finned fish, however, have gained two additional lineage-specific activator motifs which in combination with the ancient cluster of activators and the core constitute a potent lens enhancer. The exploitation and modification of existing regulatory potential in flanking regions but not in the highly conserved core might represent a more general model for the emergence of novel regulatory functions in complex CRMs.

Measuring transcription factor-binding site turnover: a maximum likelihood approach using phylogenies.

A major mode of gene expression evolution is based on changes in cis-regulatory elements (CREs) whose function critically depends on the presence of transcription factor-binding sites (TFBS). Because CREs experience extensive TFBS turnover even with conserved function, alignment-based studies of CRE sequence evolution are limited to very closely related species. Here, we propose an alternative approach based on a stochastic model of TFBS turnover. We implemented a maximum likelihood model that permits variable turnover rates in different parts of the species tree. This model can be used to detect changes in turnover rate as a proxy for differences in the selective pressures acting on TFBS in different clades. We applied this method to five TFBS in the fungi methionine biosynthesis pathway and three TFBS in the HoxA clusters of vertebrates. We find that the estimated turnover rate is generally high, with half-life ranging between approximately 5 and 150 My and a mode around tens of millions of years. This rate is consistent with the finding that even functionally conserved enhancers can show very low sequence similarity. We also detect statistically significant differences in the equilibrium densities of estrogen- and progesterone-response elements in the HoxA clusters between mammal and nonmammal vertebrates. Even more extreme clade-specific differences were found in the fungal data. We conclude that stochastic models of TFBS turnover enable the detection of shifts in the selective pressures acting on CREs in different organisms. The analysis tool, called CRETO (Cis-Regulatory Element Turn-Over) can be downloaded from http://www.bioinf.uni-leipzig.de/Software/creto/.