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Abstract Phenotypic evolution is often caused by variation in gene expression resulting from altered gene regulatory mechanisms. Genetic variation affecting chromatin remodeling has been identified as a potential source of variable gene expression; however, the roles of specific chromatin remodeling factors remain unclear. Here, we address this knowledge gap by examining the relationship between variation in gene expression, variation in chromatin structure, and variation in binding of the pioneer factor Grainy head between imaginal wing discs of two divergent strains of Drosophila melanogaster and their F1 hybrid. We find that (1) variation in Grainy head binding is mostly due to sequence changes that act in cis but are located outside of the canonical Grainy head binding motif, (2) variation in Grainy head binding correlates with changes in chromatin accessibility, and (3) this variation in chromatin accessibility, coupled with variation in Grainy head binding, correlates with variation in gene expression in some cases but not others. Interactions among these three molecular layers is complex, but these results suggest that genetic variation affecting the binding of pioneer factors contributes to variation in chromatin remodeling and the evolution of gene expression.

Abstract Heritable variation in gene expression is common within species. Much of this variation is due to genetic differences outside of the gene with altered expression and is trans‐acting. This trans‐regulatory variation is often polygenic, with individual variants typically having small effects, making the genetic architecture and evolution of trans‐regulatory variation challenging to study. Consequently, key questions about trans‐regulatory variation remain, including the variability of trans‐regulatory variation within a species, how selection affects trans‐regulatory variation, and how trans‐regulatory variants are distributed throughout the genome and within a species. To address these questions, we isolated and measured trans‐regulatory differences affecting TDH3 promoter activity among 56 strains of Saccharomyces cerevisiae, finding that trans‐regulatory backgrounds varied approximately twofold in their effects on TDH3 promoter activity. Comparing this variation to neutral models of trans‐regulatory evolution based on empirical measures of mutational effects revealed that despite this variability in the effects of trans‐regulatory backgrounds, stabilizing selection has constrained trans‐regulatory differences within this species. Using a powerful quantitative trait locus mapping method, we identified ∼100 trans‐acting expression quantitative trait locus in each of three crosses to a common reference strain, indicating that regulatory variation is more polygenic than previous studies have suggested. Loci altering expression were located throughout the genome, and many loci were strain specific. This distribution and prevalence of alleles is consistent with recent theories about the genetic architecture of complex traits. In all mapping experiments, the nonreference strain alleles increased and decreased TDH3 promoter activity with similar frequencies, suggesting that stabilizing selection maintained many trans‐acting variants with opposing effects. This variation may provide the raw material for compensatory evolution and larger scale regulatory rewiring observed in developmental systems drift among species.

Drosophila pigmentation has been a fruitful model system for understanding the genetic and developmental mechanisms underlying phenotypic evolution. For example, prior work has shown that divergence of the tan gene contributes to pigmentation differences between two members of the virilis group: Drosophila novamexicana, which has a light yellow body color, and D. americana, which has a dark brown body color. Quantitative trait locus (QTL) mapping and expression analysis has suggested that divergence of the ebony gene might also contribute to pigmentation differences between these two species. Here, we directly test this hypothesis by using CRISPR/Cas9 genome editing to generate ebony null mutants in D. americana and D. novamexicana and then using reciprocal hemizygosity testing to compare the effects of each species’ ebony allele on pigmentation. We find that divergence of ebony does indeed contribute to the pigmentation divergence between species, with effects on both the overall body color as well as a difference in pigmentation along the dorsal abdominal midline. Motivated by recent work in D. melanogaster, we also used the ebony null mutants to test for effects of ebony on cuticular hydrocarbon (CHC) profiles. We found that ebony affects CHC abundance in both species, but does not contribute to qualitative differences in the CHC profiles between these two species. Additional transgenic resources for working with D. americana and D. novamexicana, such as white mutants of both species and yellow mutants in D. novamexicana, were generated in the course of this work and are also described. Taken together, this study advances our understanding of loci contributing to phenotypic divergence and illustrates how the latest genome editing tools can be used for functional testing in non-model species.

Pleiotropic genes are genes that affect more than one trait. For example, many genes required for pigmentation in the fruit fly Drosophila melanogaster also affect traits such as circadian rhythms, vision, and mating behavior. Here, we present evidence that two pigmentation genes, ebony and tan, which encode enzymes catalyzing reciprocal reactions in the melanin biosynthesis pathway, also affect cuticular hydrocarbon (CHC) composition in D. melanogaster females. More specifically, we report that ebony loss-of-function mutants have a CHC profile that is biased toward long (>25C) chain CHCs, whereas tan loss-of-function mutants have a CHC profile that is biased toward short (

Genome editing via the CRISPR/Cas9 RNA-guided nuclease system has opened up exciting possibilities for genetic analysis. However, technical challenges associated with homology-directed repair have proven to be roadblocks for producing changes in the absence of unwanted, secondary mutations commonly known as “scars.” To address these issues, we developed a 2-stage, marker-assisted strategy to facilitate precise, “scarless” edits in Drosophila with a minimal requirement for molecular screening. Using this method, we modified 2 base pairs in a gene of interest without altering the final sequence of the CRISPR cut sites. We executed this 2-stage allele swap using a novel transformation marker that drives expression in the pupal wings, which can be screened for in the presence of common eye-expressing reporters. The tools we developed can be used to make a single change or a series of allelic substitutions in a region of interest in any D. melanogaster genetic background as well as in other Drosophila species.

The regulation of gene expression controls development, and changes in this regulation often contribute to phenotypic evolution. Drosophila pigmentation is a model system for studying evolutionary changes in gene regulation, with differences in expression of pigmentation genes such as yellow that correlate with divergent pigment patterns among species shown to be caused by changes in cis- and trans-regulation. Currently, much more is known about the cis-regulatory component of divergent yellow expression than the trans-regulatory component, in part because very few trans-acting regulators of yellow expression have been identified. This study aims to improve our understanding of the trans-acting control of yellow expression by combining yeast-one-hybrid and RNAi screens for transcription factors binding to yellow cis-regulatory sequences and affecting abdominal pigmentation in adults, respectively. Of the 670 transcription factors included in the yeast-one-hybrid screen, 45 showed evidence of binding to one or more sequence fragments tested from the 5′ intergenic and intronic yellow sequences from D. melanogaster, D. pseudoobscura, and D. willistoni, suggesting that they might be direct regulators of yellow expression. Of the 670 transcription factors included in the yeast-one-hybrid screen, plus another TF previously shown to be genetically upstream of yellow, 125 were also tested using RNAi, and 32 showed altered abdominal pigmentation. Nine transcription factors were identified in both screens, including four nuclear receptors related to ecdysone signaling (Hr78, Hr38, Hr46, and Eip78C). This finding suggests that yellow expression might be directly controlled by nuclear receptors influenced by ecdysone during early pupal development when adult pigmentation is forming.

To identify genetic variants underlying changes in phenotypes within and between species, researchers often utilize transgenic animals to compare the function of alleles in different genetic backgrounds. In Drosophila, targeted integration mediated by the ΦC31 integrase allows activity of alternative alleles to be compared at the same genomic location. By using the same insertion site for each transgene, position effects are generally assumed to be controlled for because both alleles are surrounded by the same genomic context. Here, we test this assumption by comparing the activity of tan alleles from two Drosophila species, D. americana and D. novamexicana, at five different genomic locations in D. melanogaster. We found that the relative effects of these alleles varied among insertion sites, with no difference in activity observed between them at two sites. One of these sites simply silenced both transgenes, but the other allowed expression of both alleles that was sufficient to rescue a mutant phenotype yet failed to reveal the functional differences between the two alleles. These results suggest that more than one insertion site should be used when comparing the activity of transgenes because failing to do so could cause functional differences between alleles to go undetected.

Genomic imprinting occurs when expression of an allele differs based on the sex of the parent that transmitted the allele. In D. melanogaster, imprinting can occur, but its impact on allelic expression genome-wide is unclear. Here, we search for imprinted genes in D. melanogaster using RNA-seq to compare allele-specific expression between pools of 7- to 10-day-old adult female progeny from reciprocal crosses. We identified 119 genes with allelic expression consistent with imprinting, and these genes showed significant clustering within the genome. Surprisingly, additional analysis of several of these genes showed that either genomic heterogeneity or high levels of intrinsic noise caused imprinting-like allelic expression. Consequently, our data provide no convincing evidence of imprinting for D. melanogaster genes in their native genomic context. Elucidating sources of false-positive signals for imprinting in allele-specific RNA-seq data, as done here, is critical given the growing popularity of this method for identifying imprinted genes.

Genetic networks are surprisingly robust to perturbations caused by new mutations. This robustness is conferred in part by compensation for loss of a gene's activity by genes with overlapping functions, such as paralogs. Compensation occurs passively when the normal activity of one paralog can compensate for the loss of the other, or actively when a change in one paralog's expression, localization, or activity is required to compensate for loss of the other. The mechanisms of active compensation remain poorly understood in most cases. Here we investigate active compensation for the loss or reduction in expression of the Saccharomyces cerevisiae gene TDH3 by its paralog TDH2. TDH2 is upregulated in a dose-dependent manner in response to reductions in TDH3 by a mechanism requiring the shared transcriptional regulators Gcr1p and Rap1p. TDH1, a second and more distantly related paralog of TDH3, has diverged in its regulation and is upregulated by another mechanism. Other glycolytic genes regulated by Rap1p and Gcr1p show changes in expression similar to TDH2, suggesting that the active compensation by TDH3 paralogs is part of a broader homeostatic response mediated by shared transcriptional regulators.

The fruitless (fru) gene is a member of the Drosophila melanogaster somatic sex determination genetic pathway. Although it has been hypothesized that the primary function of fru is to regulate a genetic hierarchy specifying development of adult male courtship behavior, genes acting downstream of fru have not yet been identified. Here we demonstrate that the yellow (y) gene is genetically downstream of fru in the 3rd-instar larval brain. Yellow protein is present at elevated levels in neuroblasts, which also show expression of male-specific FRU proteins, compared to control neuroblasts without FRU. A location for y downstream of fru in a genetic pathway was experimentally demonstrated by analysis of fru mutants lacking transcription of zinc-finger DNA binding domains, and of animals with temporal, spatial, or sexual mis-expression of male-specific FRU. A subset of fru and y mutants is known to reduce levels of a specific behavioral component of the male courtship ritual, wing extension, and FRU and Yellow were detected in the general region of the brain whose maleness is necessary for development of that behavior. We therefore hypothesized that ectopic expression of Yellow in the 3rd-instar brain, in a y null background, would rescue low levels of wing extension and male competitive mating success, and this was found to be the case. Overall, these data suggest that y is a downstream member of the fru branch of the D. melanogaster sex determination hierarchy, where it plays a currently unknown role in the development of adult male wing extension during courtship. © 2003 Wiley Periodicals, Inc. J Neurobiol 55: 53–72, 2003 [ABSTRACT FROM AUTHOR]

Heritable variation in a gene’s expression arises from mutations impacting cis- and trans-acting components of its regulatory network. Here, we investigate how trans-regulatory mutations are distributed within the genome and within a gene regulatory network by identifying and characterizing 69 mutations with trans-regulatory effects on expression of the same focal gene in Saccharomyces cerevisiae. Relative to 1766 mutations without effects on expression of this focal gene, we found that these trans-regulatory mutations were enriched in coding sequences of transcription factors previously predicted to regulate expression of the focal gene. However, over 90% of the trans-regulatory mutations identified mapped to other types of genes involved in diverse biological processes including chromatin state, metabolism, and signal transduction. These data show how genetic changes in diverse types of genes can impact a gene’s expression in trans, revealing properties of trans-regulatory mutations that provide the raw material for trans-regulatory variation segregating within natural populations.

Drosophila melanogaster males perform a series of courtship behaviors that, when successful, result in copulation with a female. For over a century, mutations in the yellow gene, named for its effects on pigmentation, have been known to reduce male mating success. Prior work has suggested that yellow influences mating behavior through effects on wing extension, song, and/or courtship vigor. Here, we rule out these explanations, as well as effects on the nervous system more generally, and find instead that the effects of yellow on male mating success are mediated by its effects on pigmentation of male-specific leg structures called sex combs. Loss of yellow expression in these modified bristles reduces their melanization, which changes their structure and causes difficulty grasping females prior to copulation. These data illustrate why the mechanical properties of anatomy, not just neural circuitry, must be considered to fully understand the development and evolution of behavior.

Gene expression noise is an evolvable property of biological systems that describes differences in expression among genetically identical cells in the same environment. Prior work has shown that expression noise is heritable and can be shaped by selection, but the impact of variation in expression noise on organismal fitness has proven difficult to measure. Here, we quantify the fitness effects of altering expression noise for the TDH3 gene in Saccharomyces cerevisiae. We show that increases in expression noise can be deleterious or beneficial depending on the difference between the average expression level of a genotype and the expression level maximizing fitness. We also show that a simple model relating single-cell expression levels to population growth produces patterns consistent with our empirical data. We use this model to explore a broad range of average expression levels and expression noise, providing additional insight into the fitness effects of variation in expression noise.

Genetic variation within and between species can be shaped by population-level processes and mutation; however, the relative impact of "survival of the fittest" and "arrival of the fittest" on phenotypic evolution remains unclear. Assessing the influence of mutation on evolution requires understanding the relative rates of different types of mutations and their genetic properties, yet little is known about the functional consequences of new mutations. Here, we examine the spectrum of mutations affecting a focal gene in Saccharomyces cerevisiae by characterizing 231 novel haploid genotypes with altered activity of a fluorescent reporter gene. 7% of these genotypes had a nonsynonymous mutation in the coding sequence for the fluorescent protein and were classified as "coding" mutants; 2% had a change in the S. cerevisiae TDH3 promoter sequence controlling expression of the fluorescent protein and were classified as "cis-regulatory" mutants; 10% contained two copies of the reporter gene and were classified as "copy number" mutants; and the remaining 81% showed altered fluorescence without a change in the reporter gene itself and were classified as "trans-acting" mutants. As a group, coding mutants had the strongest effect on reporter gene activity and always decreased it. By contrast, 50%-95% of the mutants in each of the other three classes increased gene activity, with mutants affecting copy number and cis-regulatory sequences having larger median effects on gene activity than trans-acting mutants. When made heterozygous in diploid cells, coding, cis-regulatory, and copy number mutant genotypes all had significant effects on gene activity, whereas 88% of the trans-acting mutants appeared to be recessive. These differences in the frequency, effects, and dominance among functional classes of mutations might help explain why some types of mutations are found to be segregating within or fixed between species more often than others.

cis-regulatory DNA sequences known as enhancers control gene expression in space and time. They are central to metazoan development and are often responsible for changes in gene regulation that contribute to phenotypic evolution. Here, we examine the sequence, function, and genomic location of enhancers controlling tissue- and cell-type specific expression of the yellow gene in six Drosophila species. yellow is required for the production of dark pigment, and its expression has evolved largely in concert with divergent pigment patterns. Using Drosophila melanogaster as a transgenic host, we examined the expression of reporter genes in which either 5' intergenic or intronic sequences of yellow from each species controlled the expression of Green Fluorescent Protein. Surprisingly, we found that sequences controlling expression in the wing veins, as well as sequences controlling expression in epidermal cells of the abdomen, thorax, and wing, were located in different genomic regions in different species. By contrast, sequences controlling expression in bristle-associated cells were located in the intron of all species. Differences in the precise pattern of spatial expression within the developing epidermis of D. melanogaster transformants usually correlated with adult pigmentation in the species from which the cis-regulatory sequences were derived, which is consistent with cis-regulatory evolution affecting yellow expression playing a central role in Drosophila pigmentation divergence. Sequence comparisons among species favored a model in which sequential nucleotide substitutions were responsible for the observed changes in cis-regulatory architecture. Taken together, these data demonstrate frequent changes in yellow cis-regulatory architecture among Drosophila species. Similar analyses of other genes, combining in vivo functional tests of enhancer activity with in silico comparative genomics, are needed to determine whether the pattern of regulatory evolution we observed for yellow is characteristic of genes with rapidly evolving expression patterns.

This primer discusses a study from Bradley et al. in which changes in transcription factor binding between two Drosophila species are revealed by chromatin immunoprecipitation and high-throughput sequencing.