Abstract

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.