Tissue-specific and cis-regulatory changes underlie parallel, adaptive gene expression evolution in house mice.

Changes in gene regulation have long been appreciated as a driving force of adaptive evolution, however the relative contributions of cis- and trans-acting changes to gene regulation over short evolutionary timescales remain unclear. Instances of recent, parallel phenotypic evolution provide an opportunity to assess whether parallel patterns are seen at the level of gene expression, and to assess the relative contribution of cis- and trans- changes to gene regulation in the early stages of divergence. Here, we studied gene expression in liver and brown adipose tissue in two wild-derived strains of house mice that independently adapted to cold, northern environments, and we compared them to a strain of house mice from a warm, tropical environment. To investigate gene regulatory evolution, we studied expression in parents and allele-specific expression in F1 hybrids of crosses between warm-adapted and cold-adapted strains. First, we found that the different cold-adapted mice showed both unique and shared changes in expression, but that the proportion of shared changes (i.e. parallelism) was greater than expected by chance. Second, we discovered that expression evolution occurred largely at tissue-specific and cis-regulated genes, and that these genes were over-represented in parallel cases of evolution. Finally, we integrated the expression data with scans for selection in natural populations and found substantial parallelism in the two northern populations for genes under selection. Furthermore, selection outliers were associated with cis-regulated genes more than expected by chance; cis-regulated genes under selection influenced phenotypes such as body size, immune functioning, and activity level. These results demonstrate that parallel patterns of gene expression in mice that have independently adapted to cold environments are driven largely by tissue-specific and cis-regulatory changes, providing insight into the mechanisms of adaptive gene regulatory evolution at the earliest stages of divergence.

Environmentally robust cis-regulatory changes underlie rapid climatic adaptation.

Changes in gene expression are thought to play a major role in adaptive evolution. While it is known that gene expression is highly sensitive to the environment, very few studies have determined the influence of genetic and environmental effects on adaptive gene expression differences in natural populations. Here, we utilize allele-specific expression to characterize cis and trans gene regulatory divergence in temperate and tropical house mice in two metabolic tissues under two thermal conditions. First, we show that gene expression divergence is pervasive between populations and across thermal conditions, with roughly 5 to 10% of genes exhibiting genotype-by-environment interactions. Second, we found that most expression divergence was due to cis-regulatory changes that were stable across temperatures. In contrast, patterns of expression plasticity were largely attributable to trans-effects, which showed greater sensitivity to temperature. Nonetheless, we found a small subset of temperature-dependent cis-regulatory changes, thereby identifying loci underlying expression plasticity. Finally, we performed scans for selection in wild house mice to identify genomic signatures of rapid adaptation. Genomic outliers were enriched in genes with evidence for cis-regulatory divergence. Notably, these genes were associated with phenotypes that affected body weight and metabolism, suggesting that cis-regulatory changes are a possible mechanism for adaptive body size evolution between populations. Our results show that gene expression plasticity, largely controlled in trans, may facilitate the colonization of new environments, but that evolved changes in gene expression are largely controlled in cis, illustrating the genetic and nongenetic mechanisms underlying the establishment of populations in new environments.

The evolution of sex-biased gene expression in the Drosophila brain.

Genes with sex-biased expression in Drosophila are thought to underlie sexually dimorphic phenotypes and have been shown to possess unique evolutionary properties. However, the forces and constraints governing the evolution of sex-biased genes in the somatic tissues of Drosophila are largely unknown. By using population-scale RNA sequencing data, we show that sex-biased genes in the Drosophila brain are highly enriched on the X Chromosome and that most are biased in a species-specific manner. We show that X-linked male-biased genes, and to a lesser extent female-biased genes, are enriched for signatures of directional selection at the gene expression level. By examining the evolutionary properties of gene-flanking regions on the X Chromosome, we find evidence that adaptive cis-regulatory changes are more likely to drive the expression evolution of X-linked male-biased genes than other X-linked genes. Finally, we examine whether constraint owing to broad expression across multiple tissues and genetic constraint owing to the largely shared male and female genomes could be responsible for the observed patterns of gene expression evolution. We find that expression breadth does not constrain the directional evolution of gene expression in the brain. Additionally, we find that the shared genome between males and females imposes a substantial constraint on the expression evolution of sex-biased genes. Overall, these results significantly advance our understanding of the patterns and forces shaping the evolution of sexual dimorphism in the Drosophila brain.

Behavioral and Genomic Sensory Adaptations Underlying the Pest Activity of Drosophila suzukii.

Studying how novel phenotypes originate and evolve is fundamental to the field of evolutionary biology as it allows us to understand how organismal diversity is generated and maintained. However, determining the basis of novel phenotypes is challenging as it involves orchestrated changes at multiple biological levels. Here, we aim to overcome this challenge by using a comparative species framework combining behavioral, gene expression, and genomic analyses to understand the evolutionary novel egg-laying substrate-choice behavior of the invasive pest species Drosophila suzukii. First, we used egg-laying behavioral assays to understand the evolution of ripe fruit oviposition preference in D. suzukii compared with closely related species D. subpulchrella and D. biarmipes as well as D. melanogaster. We show that D. subpulchrella and D. biarmipes lay eggs on both ripe and rotten fruits, suggesting that the transition to ripe fruit preference was gradual. Second, using two-choice oviposition assays, we studied how D. suzukii, D. subpulchrella, D. biarmipes, and D. melanogaster differentially process key sensory cues distinguishing ripe from rotten fruit during egg-laying. We found that D. suzukii's preference for ripe fruit is in part mediated through a species-specific preference for stiff substrates. Last, we sequenced and annotated a high-quality genome for D. subpulchrella. Using comparative genomic approaches, we identified candidate genes involved in D. suzukii's ability to seek out and target ripe fruits. Our results provide detail to the stepwise evolution of pest activity in D. suzukii, indicating important cues used by this species when finding a host, and the molecular mechanisms potentially underlying their adaptation to a new ecological niche.

The evolution and mutational robustness of chromatin accessibility in Drosophila.

The evolution of regulatory regions in the genome plays a critical role in shaping the diversity of life. While this process is primarily sequence-dependent, the enormous complexity of biological systems has made it difficult to understand the factors underlying regulation and its evolution. Here, we apply deep neural networks as a tool to investigate the sequence determinants underlying chromatin accessibility in different tissues of Drosophila. We train hybrid convolution-attention neural networks to accurately predict ATAC-seq peaks using only local DNA sequences as input. We show that a model trained in one species has nearly identical performance when tested in another species, implying that the sequence determinants of accessibility are highly conserved. Indeed, model performance remains excellent even in distantly-related species. By using our model to examine species-specific gains in chromatin accessibility, we find that their orthologous inaccessible regions in other species have surprisingly similar model outputs, suggesting that these regions may be ancestrally poised for evolution. We then use in silico saturation mutagenesis to reveal evidence of selective constraint acting specifically on inaccessible chromatin regions. We further show that chromatin accessibility can be accurately predicted from short subsequences in each example. However, in silico knock-out of these sequences does not qualitatively impair classification, implying that chromatin accessibility is mutationally robust. Subsequently, we demonstrate that chromatin accessibility is predicted to be robust to large-scale random mutation even in the absence of selection. We also perform in silico evolution experiments under the regime of strong selection and weak mutation (SSWM) and show that chromatin accessibility can be extremely malleable despite its mutational robustness. However, selection acting in different directions in a tissue-specific manner can substantially slow adaptation. Finally, we identify motifs predictive of chromatin accessibility and recover motifs corresponding to known chromatin accessibility activators and repressors. These results demonstrate the conservation of the sequence determinants of accessibility and the general robustness of chromatin accessibility, as well as the power of deep neural networks as tools to answer fundamental questions in regulatory genomics and evolution.

The evolution and mutational robustness of chromatin accessibility in Drosophila.

BACKGROUND: The evolution of genomic regulatory regions plays a critical role in shaping the diversity of life. While this process is primarily sequence-dependent, the enormous complexity of biological systems complicates the understanding of the factors underlying regulation and its evolution. Here, we apply deep neural networks as a tool to investigate the sequence determinants underlying chromatin accessibility in different species and tissues of Drosophila. RESULTS: We train hybrid convolution-attention neural networks to accurately predict ATAC-seq peaks using only local DNA sequences as input. We show that our models generalize well across substantially evolutionarily diverged species of insects, implying that the sequence determinants of accessibility are highly conserved. Using our model to examine species-specific gains in accessibility, we find evidence suggesting that these regions may be ancestrally poised for evolution. Using in silico mutagenesis, we show that accessibility can be accurately predicted from short subsequences in each example. However, in silico knock-out of these sequences does not qualitatively impair classification, implying that accessibility is mutationally robust. Subsequently, we show that accessibility is predicted to be robust to large-scale random mutation even in the absence of selection. Conversely, simulations under strong selection demonstrate that accessibility can be extremely malleable despite its robustness. Finally, we identify motifs predictive of accessibility, recovering both novel and previously known motifs. CONCLUSIONS: These results demonstrate the conservation of the sequence determinants of accessibility and the general robustness of chromatin accessibility, as well as the power of deep neural networks to explore fundamental questions in regulatory genomics and evolution.