Patricia J. Wittkopp

Evolutionary changes in cis and trans gene regulation

Differences in gene expression are central to evolution. Such differences can arise from cis-regulatory changes that affect transcription initiation, transcription rate and/or transcript stability in an allele-specific manner, or from trans-regulatory changes that modify the activity or expression of factors that interact with cis-regulatory sequences. Both cis- and trans-regulatory changes contribute to divergent gene expression, but their respective contributions remain largely unknown. Here we examine the distribution of cis- and trans-regulatory changes underlying expression differences between closely related Drosophila species, D. melanogaster and D. simulans, and show functional cis-regulatory differences by comparing the relative abundance of species-specific transcripts in F1 hybrids. Differences in trans-regulatory activity were inferred by comparing the ratio of allelic expression in hybrids with the ratio of gene expression between species. Of 29 genes with interspecific expression differences, 28 had differences in cis-regulation, and these changes were sufficient to explain expression divergence for about half of the genes. Trans-regulatory differences affected 55% (16 of 29) of genes, and were always accompanied by cis-regulatory changes. These data indicate that interspecific expression differences are not caused by select trans-regulatory changes with widespread effects, but rather by many cis-acting changes spread throughout the genome.

Chance caught on the wing: cis-regulatory evolution and the origin of pigment patterns in Drosophila.

The gain, loss or modification of morphological traits is generally associated with changes in gene regulation during development. However, the molecular bases underlying these evolutionary changes have remained elusive. Here we identify one of the molecular mechanisms that contributes to the evolutionary gain of a male-specific wing pigmentation spot in Drosophila biarmipes, a species closely related to Drosophila melanogaster. We show that the evolution of this spot involved modifications of an ancestral cis-regulatory element of the yellow pigmentation gene. This element has gained multiple binding sites for transcription factors that are deeply conserved components of the regulatory landscape controlling wing development, including the selector protein Engrailed. The evolutionary stability of components of regulatory landscapes, which can be co-opted by chance mutations in cis-regulatory elements, might explain the repeated evolution of similar morphological patterns, such as wing pigmentation patterns in flies.

Cis-regulatory elements: molecular mechanisms and evolutionary processes underlying divergence

Cis-regulatory sequences, such as enhancers and promoters, control development and physiology by regulating gene expression. Mutations that affect the function of these sequences contribute to phenotypic diversity within and between species. With many case studies implicating divergent cis-regulatory activity in phenotypic evolution, researchers have recently begun to elucidate the genetic and molecular mechanisms that are responsible for cis-regulatory divergence. Approaches include detailed functional analysis of individual cis-regulatory elements and comparing mechanisms of gene regulation among species using the latest genomic tools. Despite the limited number of mechanistic studies published to date, this work shows how cis-regulatory activity can diverge and how studies of cis-regulatory divergence can address long-standing questions about the genetic mechanisms of phenotypic evolution.

Regulatory changes underlying expression differences within and between Drosophila species

Differences in gene expression are an important source of phenotypic variation, and can be caused by changes in cis and/or trans regulation. cis-regulatory variants alter allele-specific expression, whereas trans-regulatory variants influence expression of both alleles in a diploid cell. Because of this difference, we hypothesize that natural selection may favor one type of change over the other. Here, we investigate contributions of cis- and trans-regulatory changes to variable intra- and interspecific gene expression using four strains of Drosophila melanogaster, three strains of D. simulans and a total of 78 genes. We show that cis-regulatory changes account for a greater proportion of the expression differences observed between rather than within species. These data are inconsistent with a neutral model assuming equal probabilities of fixation for cis- and trans-regulatory polymorphisms, suggesting that natural selection influences the molecular mechanisms underlying divergent gene expression. Specifically, cis-regulatory changes seem to accumulate preferentially over time.

Regulatory divergence in Drosophila revealed by mRNA-seq

The regulation of gene expression is critical for organismal function and is an important source of phenotypic diversity between species. Understanding the genetic and molecular mechanisms responsible for regulatory divergence is therefore expected to provide insight into evolutionary change. Using deep sequencing, we quantified total and allele-specific mRNA expression levels genome-wide in two closely related Drosophila species (D. melanogaster and D. sechellia) and their F(1) hybrids. We show that 78% of expressed genes have divergent expression between species, and that cis- and trans-regulatory divergence affects 51% and 66% of expressed genes, respectively, with 35% of genes showing evidence of both. This is a relatively larger contribution of trans-regulatory divergence than was expected based on prior studies, and may result from the unique demographic history of D. sechellia. Genes with antagonistic cis- and trans-regulatory changes were more likely to be misexpressed in hybrids, consistent with the idea that such regulatory changes contribute to hybrid incompatibilities. In addition, cis-regulatory differences contributed more to divergent expression of genes that showed additive rather than nonadditive inheritance. A correlation between sequence similarity and the conservation of cis-regulatory activity was also observed that appears to be a general feature of regulatory evolution. Finally, we examined regulatory divergence that may have contributed to the evolution of a specific trait--divergent feeding behavior in D. sechellia. Overall, this study illustrates the power of mRNA sequencing for investigating regulatory evolution, provides novel insight into the evolution of gene expression in Drosophila, and reveals general trends that are likely to extend to other species.

Evolution in black and white: genetic control of pigment patterns in Drosophila

Coloration is one of the most variable characters among animals and is a rich source of models of phenotypic evolution. The great diversity of pigment patterns in Drosophila, coupled with the availability of genetic approaches in both model and more exotic species, has recently spawned efforts to elucidate the genetic architecture and molecular basis of pigment pattern evolution. Pigmentation differences are often polygenic and correlate with regulatory changes in both transcription factor genes and structural genes. Understanding the developmental genetic basis of color differences in Drosophila could provide inroads to classic evolutionary problems such as industrial melanism, mimicry and phenotypic convergence.

Development and evolution of insect pigmentation : Genetic mechanisms and the potential consequences of pleiotropy

Insect pigmentation is a premier model system in evolutionary and developmental biology. It has been at the heart of classical studies as well as recent breakthroughs. In insects, pigments are produced by epidermal cells through a developmental process that includes pigment patterning and synthesis. Many aspects of this process also impact other phenotypes, including behavior and immunity. This review discusses recent work on the development and evolution of insect pigmentation, with a focus on pleiotropy and its effects on color pattern diversification.

Compensatory cis-trans Evolution and the Dysregulation of Gene Expression in Interspecific Hybrids of Drosophila

Hybrids between species are often characterized by novel gene-expression patterns. A recent study on allele-specific gene expression in hybrids between species of Drosophila revealed cases in which cis- and trans-regulatory elements within species had coevolved in such a way that changes in cis-regulatory elements are compensated by changes in trans-regulatory elements. We hypothesized that such coevolution should often lead to gene misexpression in the hybrid. To test this hypothesis, we estimated allele-specific expression and overall expression levels for 31 genes in D. melanogaster, D. simulans, and their F1 hybrid. We found that 13 genes with cis-trans compensatory evolution are in fact misexpressed in the hybrid. These represent candidate genes whose dysregulation might be the consequence of coevolution of cis- and trans-regulatory elements within species. Using a mathematical model for the regulation of gene expression, we explored the conditions under which cis-trans compensatory evolution can lead to misexpression in interspecific hybrids.

Evaluating the role of natural selection in the evolution of gene regulation

Surveys of gene expression reveal extensive variability both within and between a wide range of species. Compelling cases have been made for adaptive changes in gene regulation, but the proportion of expression divergence attributable to natural selection remains unclear. Distinguishing adaptive changes driven by positive selection from neutral divergence resulting from mutation and genetic drift is critical for understanding the evolution of gene expression. Here, we review the various methods that have been used to test for signs of selection in genomic expression data. We also discuss properties of regulatory systems relevant to neutral models of gene expression. Despite some potential caveats, published studies provide considerable evidence for adaptive changes in gene expression. Future challenges for studies of regulatory evolution will be to quantify the frequency of adaptive changes, identify the genetic basis of expression divergence and associate changes in gene expression with specific organismal phenotypes.

Intraspecific Polymorphism to Interspecific Divergence: Genetics of Pigmentation in Drosophila

Drosophila Body Color Fly body color is controlled by a variety of genes and alleles. Now Wittkopp et al. (p. 540) describe how two genes at the ebony and tan genetic loci control body color among two closely related species, Drosophila americana and D. novamexicana. Variations at the tan locus and linked to the ebony locus also contribute to intraspecific pigmentation changes with geography in D. americana. The sequencing of multiple isolates suggests that some strains of D. americana carry alleles of tan and ebony that are more closely related to the D. novamexicana alleles than they are to other D. americana alleles. Thus, the genetic determinants of both inter- and intraspecies color variation is due to shared alleles. Color-determining genes in flies show an ancestral polymorphism that contributes to variation, both within and between species. Genetic changes contributing to phenotypic differences within or between species have been identified for a handful of traits, but the relationship between alleles underlying intraspecific polymorphism and interspecific divergence is largely unknown. We found that noncoding changes in the tan gene, as well as changes linked to the ebony gene, contribute to pigmentation divergence between closely related Drosophila species. Moreover, we found that alleles linked to tan and ebony fixed in one Drosophila species also contribute to variation within another species, and that multiple genotypes underlie similar phenotypes even within the same population. These alleles appear to predate speciation, which suggests that standing genetic variation present in the common ancestor gave rise to both intraspecific polymorphism and interspecific divergence.