Gene regulatory divergence between populations of Drosphila melanogaster

The pervasiveness of gene expression variation at both the population and species level is well-documented. Underlying this expression variation are gene regulatory changes. It has been hypothesized that regulatory changes, especially cis-regulatory changes, are especially important in phenotypic evolution since they are more easily fine-tuned both temporally and tissue-specifically than protein-coding changes. This dissertation aims to examine the genetic basis of adaptive regulatory changes, including the effects of adaptive regulatory polymorphisms on both gene expression and organismal phenotype. This thesis centers around the analysis of adaptive cis-regulatory changes associated with two Drosophila melanogaster genes: CG9509, a gene of unknown function, and Metallothionein A (MtnA), a gene involved in metal homeostasis and the general stress response. Chapters 1 and 2 provide an in-depth analysis of a case of previously identified adaptive regulatory divergence in a cis-regulatory element of CG9509. Adult CG9509 expression is 2–3-fold higher in a European population in comparison to an ancestral, sub- Saharan African population and transgenic reporter gene assays have previously shown that this expression divergence is driven by a 1.2-kb cis-regulatory enhancer element upstream of the CG9509 coding region, which shows signs of recent positive selection in the European population. In Chapter 1, I show that the observed CG9509 expression divergence extends to adults in other cosmopolitan and sub-Saharan African populations, and in chapter 2 I show that it extends to larvae as well. This suggests that the previously identified positive selection on the cosmopolitan variant of the CG9509 enhancer element occurred during or shortly after D. melanogaster’s expansion out of Africa, before the separation of European and Asian populations. In chapter 2, I use site directed-mutagenesis and transgenic reporter gene constructs to identify the three single nucleotide polymorphisms (SNPs) within the CG9509 enhancer responsible for the observed expression divergence. Interestingly, two of these SNPs have a relatively small effect on expression and appear to have been the targets of a selective sweep, while the third SNP has a much larger effect on expression and appears to have been a recent target of balancing selection. In chapter 2, I further use a series of functional and tolerance assays to show that CG9509 expression affects D. melanogaster growth and propose that the organismal phenotype under selection is reduced wing loading, which likely improves flight ability at cooler temperatures. Chapter 3 identifies a new case of adaptive cis-regulatory divergence in the 3’ untranslated region (UTR) of MtnA. MtnA expression in the brain is 5-fold higher in a European in comparison to an African population and within the MtnA 3’UTR is a 49- basepair insertion/deletion (indel) polymorphism. I performed transgenic reporter gene assays to show that the deletion in the 3’UTR, which is the derived variant and is at high frequency in the European population, is associated with increased MtnA expression. In conjunction with population genetic evidence, this suggests that the deletion in the MtnA 3’UTR has been the target of selection for an increase in MtnA expression in the European population. Using hydrogen peroxide tolerance assays, I further show that MtnA expression is involved in oxidative stress tolerance and that the 3’UTR indel polymorphism is associated with oxidative stress tolerance variation in natural populations, suggesting that improved oxidative stress tolerance is the organismal phenotype under selection

Gene regulatory divergence between populations of Drosphila melanogaster

The pervasiveness of gene expression variation at both the population and species level is well-documented. Underlying this expression variation are gene regulatory changes. It has been hypothesized that regulatory changes, especially cis-regulatory changes, are especially important in phenotypic evolution since they are more easily fine-tuned both temporally and tissue-specifically than protein-coding changes. This dissertation aims to examine the genetic basis of adaptive regulatory changes, including the effects of adaptive regulatory polymorphisms on both gene expression and organismal phenotype. This thesis centers around the analysis of adaptive cis-regulatory changes associated with two Drosophila melanogaster genes: CG9509, a gene of unknown function, and Metallothionein A (MtnA), a gene involved in metal homeostasis and the general stress response. Chapters 1 and 2 provide an in-depth analysis of a case of previously identified adaptive regulatory divergence in a cis-regulatory element of CG9509. Adult CG9509 expression is 2–3-fold higher in a European population in comparison to an ancestral, sub- Saharan African population and transgenic reporter gene assays have previously shown that this expression divergence is driven by a 1.2-kb cis-regulatory enhancer element upstream of the CG9509 coding region, which shows signs of recent positive selection in the European population. In Chapter 1, I show that the observed CG9509 expression divergence extends to adults in other cosmopolitan and sub-Saharan African populations, and in chapter 2 I show that it extends to larvae as well. This suggests that the previously identified positive selection on the cosmopolitan variant of the CG9509 enhancer element occurred during or shortly after D. melanogaster’s expansion out of Africa, before the separation of European and Asian populations. In chapter 2, I use site directed-mutagenesis and transgenic reporter gene constructs to identify the three single nucleotide polymorphisms (SNPs) within the CG9509 enhancer responsible for the observed expression divergence. Interestingly, two of these SNPs have a relatively small effect on expression and appear to have been the targets of a selective sweep, while the third SNP has a much larger effect on expression and appears to have been a recent target of balancing selection. In chapter 2, I further use a series of functional and tolerance assays to show that CG9509 expression affects D. melanogaster growth and propose that the organismal phenotype under selection is reduced wing loading, which likely improves flight ability at cooler temperatures. Chapter 3 identifies a new case of adaptive cis-regulatory divergence in the 3’ untranslated region (UTR) of MtnA. MtnA expression in the brain is 5-fold higher in a European in comparison to an African population and within the MtnA 3’UTR is a 49- basepair insertion/deletion (indel) polymorphism. I performed transgenic reporter gene assays to show that the deletion in the 3’UTR, which is the derived variant and is at high frequency in the European population, is associated with increased MtnA expression. In conjunction with population genetic evidence, this suggests that the deletion in the MtnA 3’UTR has been the target of selection for an increase in MtnA expression in the European population. Using hydrogen peroxide tolerance assays, I further show that MtnA expression is involved in oxidative stress tolerance and that the 3’UTR indel polymorphism is associated with oxidative stress tolerance variation in natural populations, suggesting that improved oxidative stress tolerance is the organismal phenotype under selection

Functional characterization of adaptive variation within a cis-regulatory element influencing Drosophila melanogaster growth

Gene expression variation is a major contributor to phenotypic diversity within species and is thought to play an important role in adaptation. However, examples of adaptive regulatory polymorphism are rare, especially those that have been characterized at both the molecular genetic level and the organismal level. In this study, we perform a functional analysis of the Drosophila melanogaster CG9509 enhancer, a cis-regulatory element that shows evidence of adaptive evolution in populations outside the species' ancestral range in sub-Saharan Africa. Using site-directed mutagenesis and transgenic reporter gene assays, we determined that 3 single nucleotide polymorphisms are responsible for the difference in CG9509 expression that is observed between sub-Saharan African and cosmopolitan populations. Interestingly, while 2 of these variants appear to have been the targets of a selective sweep outside of sub-Saharan Africa, the variant with the largest effect on expression remains polymorphic in cosmopolitan populations, suggesting it may be subject to a different mode of selection. To elucidate the function of CG9509, we performed a series of functional and tolerance assays on flies in which CG9509 expression was disrupted. We found that CG9509 plays a role in larval growth and influences adult body and wing size, as well as wing loading. Furthermore, variation in several of these traits was associated with variation within the CG9509 enhancer. The effect on growth appears to result from a modulation of active ecdysone levels and expression of growth factors. Taken together, our findings suggest that selection acted on 3 sites within the CG9509 enhancer to increase CG9509 expression and, as a result, reduce wing loading as D. melanogaster expanded out of sub-Saharan Africa

The discovery, distribution and diversity of DNA viruses associated with Drosophila melanogaster in Europe

International audienceDrosophila melanogaster is an important model for antiviral immunity in arthropods, but very few DNA viruses have been described from the family Drosophilidae. This deficiency limits our opportunity to use natural host-pathogen combinations in experimental studies, and may bias our understanding of the Drosophila virome. Here we report fourteen DNA viruses detected in a metagenomic analysis of approximately 6500 pool-sequenced Drosophila, sampled from 47 European locations between 2014 and 2016. These include three new nudiviruses, a new and divergent entomopoxvirus, a virus related to Leptopilina boulardi filamentous virus, and a virus related to Musca domestica salivary gland hypertrophy virus. We also find an endogenous genomic copy of galbut virus, a dsRNA partitivirus, segregating at very low frequency. Remarkably, we find that Drosophila Vesanto virus, a small DNA virus previously described as a bidnavirus, may be composed of up to 12 segments and thus represent a new lineage of segmented DNA viruses. Two of the DNA viruses, Drosophila Kallithea nudivirus and Drosophila Vesanto virus are relatively common, found in 2% or more of wild flies. The others are rare, with many likely to be represented by a single infected fly. We find that virus prevalence in Europe reflects the prevalence seen in publicly-available datasets, with Drosophila Kallithea nudivirus and Drosophila Vesanto virus the only ones commonly detectable in public data from wild-caught flies and large population cages, and the other viruses being rare or absent. These analyses suggest that DNA viruses are at lower prevalence than RNA viruses in D. melanogaster, and may be less likely to persist in laboratory cultures. Our findings go some way to redressing an earlier bias toward RNA virus studies in Drosophila, and lay the foundation needed to harness the power of Drosophila as a model system for the study of DNA viruses

Drosophila evolution over space and time (DEST):A new population genomics resource

Drosophila melanogaster is a leading model in population genetics and genomics, and a growing number of whole-genome datasets from natural populations of this species have been published over the last years. A major challenge is the integration of disparate datasets, often generated using different sequencing technologies and bioinformatic pipelines, which hampers our ability to address questions about the evolution of this species. Here we address these issues by developing a bioinformatics pipeline that maps pooled sequencing (Pool-Seq) reads from D. melanogaster to a hologenome consisting of fly and symbiont genomes and estimates allele frequencies using either a heuristic (PoolSNP) or a probabilistic variant caller (SNAPE-pooled). We use this pipeline to generate the largest data repository of genomic data available for D. melanogaster to date, encompassing 271 previously published and unpublished population samples from over 100 locations in > 20 countries on four continents. Several of these locations have been sampled at different seasons across multiple years. This dataset, which we call Drosophila Evolution over Space and Time (DEST), is coupled with sampling and environmental meta-data. A web-based genome browser and web portal provide easy access to the SNP dataset. We further provide guidelines on how to use Pool-Seq data for model-based demographic inference. Our aim is to provide this scalable platform as a community resource which can be easily extended via future efforts for an even more extensive cosmopolitan dataset. Our resource will enable population geneticists to analyze spatio-temporal genetic patterns and evolutionary dynamics of D. melanogaster populations in unprecedented detail.DrosEU is funded by a Special Topic Networks (STN) grant from the European Society for Evolutionary Biology (ESEB). MK (M. Kapun) was supported by the Austrian Science Foundation (grant no. FWF P32275); JG by the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (H2020-ERC-2014-CoG-647900) and by the Spanish Ministry of Science and Innovation (BFU-2011-24397); TF by the Swiss National Science Foundation (SNSF grants PP00P3_133641, PP00P3_165836, and 31003A_182262) and a Mercator Fellowship from the German Research Foundation (DFG), held as a EvoPAD Visiting Professor at the Institute for Evolution and Biodiversity, University of Münster; AOB by the National Institutes of Health (R35 GM119686); MK (M. Kankare) by Academy of Finland grant 322980; VL by Danish Natural Science Research Council (FNU) grant 4002-00113B; FS Deutsche Forschungsgemeinschaft (DFG) grant STA1154/4-1, Project 408908608; JP by the Deutsche Forschungsgemeinschaft Projects 274388701 and 347368302; AU by FPI fellowship (BES-2012-052999); ET Israel Science Foundation (ISF) grant 1737/17; MSV, MSR and MJ by a grant from the Ministry of Education, Science and Technological Development of the Republic of Serbia (451-03-68/2020-14/200178); AP, KE and MT by a grant from the Ministry of Education, Science and Technological Development of the Republic of Serbia (451-03-68/2020-14/200007); and TM NSERC grant RGPIN-2018-05551.Peer reviewe

Corrigendum to: Drosophila Evolution over Space and Time (DEST): a New Population Genomics Resource

Drosophila melanogaster is a leading model in population genetics and genomics, and a growing number of whole-genome datasets from natural populations of this species have been published over the last years. A major challenge is the integration of disparate datasets, often generated using different sequencing technologies and bioinformatic pipelines, which hampers our ability to address questions about the evolution of this species. Here we address these issues by developing a bioinformatics pipeline that maps pooled sequencing (Pool-Seq) reads from D. melanogaster to a hologenome consisting of fly and symbiont genomes and estimates allele frequencies using either a heuristic (PoolSNP) or a probabilistic variant caller (SNAPE-pooled). We use this pipeline to generate the largest data repository of genomic data available for D. melanogaster to date, encompassing 271 previously published and unpublished population samples from over 100 locations in > 20 countries on four continents. Several of these locations have been sampled at different seasons across multiple years. This dataset, which we call Drosophila Evolution over Space and Time (DEST), is coupled with sampling and environmental meta-data. A web-based genome browser and web portal provide easy access to the SNP dataset. We further provide guidelines on how to use Pool-Seq data for model-based demographic inference. Our aim is to provide this scalable platform as a community resource which can be easily extended via future efforts for an even more extensive cosmopolitan dataset. Our resource will enable population geneticists to analyze spatio-temporal genetic patterns and evolutionary dynamics of D. melanogaster populations in unprecedented detail.DrosEU is funded by a Special Topic Networks (STN) grant from the European Society for Evolutionary Biology (ESEB). MK (M. Kapun) was supported by the Austrian Science Foundation (grant no. FWF P32275); JG by the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (H2020-ERC-2014-CoG-647900) and by the Spanish Ministry of Science and Innovation (BFU-2011-24397); TF by the Swiss National Science Foundation (SNSF grants PP00P3_133641, PP00P3_165836, and 31003A_182262) and a Mercator Fellowship from the German Research Foundation (DFG), held as a EvoPAD Visiting Professor at the Institute for Evolution and Biodiversity, University of Münster; AOB by the National Institutes of Health (R35 GM119686); MK (M. Kankare) by Academy of Finland grant 322980; VL by Danish Natural Science Research Council (FNU) grant 4002-00113B; FS Deutsche Forschungsgemeinschaft (DFG) grant STA1154/4-1, Project 408908608; JP by the Deutsche Forschungsgemeinschaft Projects 274388701 and 347368302; AU by FPI fellowship (BES-2012-052999); ET Israel Science Foundation (ISF) grant 1737/17; MSV, MSR and MJ by a grant from the Ministry of Education, Science and Technological Development of the Republic of Serbia (451-03-68/2020-14/200178); AP, KE and MT by a grant from the Ministry of Education, Science and Technological Development of the Republic of Serbia (451-03-68/2020-14/200007); and TM NSERC grant RGPIN-2018-05551.Peer reviewe

Gene expression dynamics in Zambian and Dutch populations.

Gene expression dynamics in Zambian and Dutch populations.</p

Genes included in our dataset but detected as not expressed in a subset of stages and/or populations.

Shown are the number of genes detected as expressed only in a given subset of stages or populations (pop) but not expressed in the others. For a gene to be considered as expressed in a particular population and stage, we required a minimum of an average of 15 reads for that population/stage combination. (XLSX)</p