Genome-wide gene expression responses to experimental manipulation of Saccharomyces cerevisiae repressor activator protein 1 (Rap1) expression level.

Precise regulation of transcription in gene expression is critical for all aspects of normal organism form, fitness, and function and even minor alterations in the level, location, and timing of gene expression can result in phenotypic variation within and between species including evolutionary innovations and human disease states. Eukaryotic transcription is regulated by a complex interplay of multiple factors working both at a physical and molecular levels influencing this process. In Saccharomyces cerevisiae, the TF with the greatest number of putative regulatory targets is the essential gene Repressor Activator Protein 1 (RAP1). While much is known about the roles of Rap1 in gene regulation and numerous cellular processes, the response of Rap1 target genes to systematic titration of RAP1 expression level remains unknown. To fill this knowledge gap, we used a strain with a tetracycline-titratable promoter replacing wild-type regulatory sequences of RAP1 to systematically reduce the expression level of RAP1 and followed this with RNA sequencing (RNA-seq) to measure genome-wide gene expression responses. Previous research indicated that Rap1 plays a significant regulatory role in particular groups of genes including telomere-proximal genes, homothallic mating (HM) loci, glycolytic genes, DNA repair genes, and ribosomal protein genes; therefore, we focused our analyses on these groups and downstream targets to determine how they respond to reductions in RAP1 expression level. Overall, despite being known as both an activator and as a repressor of its target genes, we found that Rap1 acts as an activator for more target genes than as a repressor. Additionally, we found that Rap1 functions as an activator of ribosomal protein genes and a repressor for HM loci genes consistent with predictions from the literature. Unexpectedly, we found that Rap1 functions as a repressor of glycolytic enzyme genes contrary to prior reports of it having the opposite effect. We also compared the expression of RAP1 to five different genes related to DNA repair pathway and found that decreasing RAP1 downregulated four of those five genes. Finally, we found no effect of RAP1 depletion on telomere-proximal genes despite its functioning to silence telomeric repeat-containing RNAs. Together our results enrich our understanding of this important transcriptional regulator.

Derived esterase activity in Drosophila sechellia contributes to evolved octanoic acid resistance.

The dietary specialist fruit fly Drosophila sechellia has evolved resistance to the secondary defence compounds produced by the fruit of its host plant, Morinda citrifolia. The primary chemicals that contribute to lethality of M. citrifolia are the medium-chain fatty acids octanoic acid (OA) and hexanoic acid. At least five genomic regions contribute to this adaptation in D. sechellia and whereas the fine-mapped major effect locus for OA resistance on chromosome 3R has been thoroughly analysed, the remaining four genomic regions that contribute to toxin resistance remain uncharacterized. To begin to identify the genetic basis of toxin resistance in this species, we removed the function of well-known detoxification gene families to determine whether they contribute to toxin resistance. Previous work found that evolution of cytochrome P450 enzymatic activity or expression is not responsible for the OA resistance in D. sechellia. Here, we tested the role of the two other major detoxification gene families in resistance to Morinda fruit toxins - glutathione-S-transferases and esterases - through the use of the pesticide synergists diethyl maleate and tribufos that inhibit the function of these gene families. This work suggests that one or more esterase(s) contribute to evolved OA resistance in D. sechellia.

Analysis of cytochrome P450 contribution to evolved plant toxin resistance in Drosophila sechellia.

Drosophila sechellia is a dietary specialist species of fruit fly that has evolved resistance to the toxic secondary defence compounds produced by the fruit of its preferred host plant Morinda citrifolia. The genetic basis of adult toxin resistance is the result of evolution at five loci across the genome. Genetic mapping between D. sechellia and Drosophila simulans and subsequent functional studies in Drosophila melanogaster have identified candidate genes potentially underlying one locus involved in toxin resistance but the remainder of the genes involved are unknown. Genes in the mixed function oxidase or cytochrome P450 gene family are frequently utilized in evolved toxin resistance in insects, yet whether they play a role in D. sechellia's resistance to the toxins found in its host plant is unknown. Here we test the role of cytochrome P450 enzymatic activity in evolved resistance to the two primary toxins found in M. citrifolia fruit: octanoic acid and hexanoic acid. We found that although cytochrome P450 enzymatic activity is involved in basal resistance it is not involved in derived toxin resistance in D. sechellia.

Genetic basis of octanoic acid resistance in Drosophila sechellia: functional analysis of a fine-mapped region.

Drosophila sechellia is a species of fruit fly endemic to the Seychelles islands. Unlike its generalist sister species, D. sechellia has evolved to be a specialist on the host plant Morinda citrifolia. This specialization is interesting because the plant's fruit contains secondary defence compounds, primarily octanoic acid (OA), that are lethal to most other Drosophilids. Although ecological and behavioural adaptations to this toxic fruit are known, the genetic basis for evolutionary changes in OA resistance is not. Prior work showed that a genomic region on chromosome 3R containing 18 genes has the greatest contribution to differences in OA resistance between D. sechellia and D. simulans. To determine which gene(s) in this region might be involved in the evolutionary change in OA resistance, we knocked down expression of each gene in this region in D. melanogaster with RNA interference (RNAi) (i) ubiquitously throughout development, (ii) during only the adult stage and (iii) within specific tissues. We identified three neighbouring genes in the Osiris family, Osiris 6 (Osi6), Osi7 and Osi8, that led to decreased OA resistance when ubiquitously knocked down. Tissue-specific RNAi, however, showed that decreasing expression of Osi6 and Osi7 specifically in the fat body and/or salivary glands increased OA resistance. Gene expression analyses of Osi6 and Osi7 revealed that while standing levels of expression are higher in D. sechellia, Osi6 expression is significantly downregulated in salivary glands in response to OA exposure, suggesting that evolved tissue-specific environmental plasticity of Osi6 expression may be responsible for OA resistance in D. sechellia.