Regulation at Drosophila's Malic Enzyme highlights the complexity of transvection and its sensitivity to genetic background.

Transvection, a type of trans-regulation of gene expression in which regulatory elements on one chromosome influence elements on a paired homologous chromosome, is itself a complex biological phenotype subject to modification by genetic background effects. However, relatively few studies have explored how transvection is affected by distal genetic variation, perhaps because it is strongly influenced by local regulatory elements and chromosomal architecture. With the emergence of the "hub" model of transvection and a series of studies showing variation in transvection effects, it is becoming clear that genetic background plays an important role in how transvection influences gene transcription. We explored the effects of genetic background on transvection by performing two independent genome wide association studies (GWASs) using the Drosophila genetic reference panel (DGRP) and a suite of Malic enzyme (Men) excision alleles. We found substantial variation in the amount of transvection in the 149 DGRP lines used, with broad-sense heritability of 0.89 and 0.84, depending on the excision allele used. The specific genetic variation identified was dependent on the excision allele used, highlighting the complex genetic interactions influencing transvection. We focussed primarily on genes identified as significant using a relaxed P-value cutoff in both GWASs. The most strongly associated genetic variant mapped to an intergenic single nucleotide polymorphism (SNP), located upstream of Tiggrin (Tig), a gene that codes for an extracellular matrix protein. Variants in other genes, such transcription factors (CG7368 and Sima), RNA binding proteins (CG10418, Rbp6, and Rig), enzymes (AdamTS-A, CG9743, and Pgant8), proteins influencing cell cycle progression (Dally and Eip63E) and signaling proteins (Atg-1, Axo, Egfr, and Path) also associated with transvection in Men. Although not intuitively obvious how many of these genes may influence transvection, some have been previously identified as promoting or antagonizing somatic homolog pairing. These results identify several candidate genes to further explore in the understanding of transvection in Men and in other genes regulated by transvection. Overall, these findings highlight the complexity of the interactions involved in gene regulation, even in phenotypes, such as transvection, that were traditionally considered to be primarily influenced by local genetic variation.

Metabolomic analysis of oxidative stress: Superoxide dismutase mutation and paraquat induced stress in Drosophila melanogaster.

Oxidative stress results in substantial biochemical and physiological perturbations in essentially all organisms. To determine the broad metabolic effects of oxidative stress, we have quantified the response in Drosophila melanogaster to both genetically and environmentally derived oxidative stress. Flies were challenged with loss of Superoxide dismutase activity or chronic or acute exposure to the oxidizing chemical paraquat. Metabolic changes were then quantified using a recently developed chemical isotope labeling (CIL) liquid chromatography - mass spectrometry (LC-MS) platform that targets the carboxylic acid and amine/phenol submetabolomes with high metabolic coverage. We discovered wide spread changes in both submetabolomes in response to all three types of stresses including: changes to the urea cycle, tryptophan metabolism, porphyrin metabolism, as well as a series of metabolic pathways involved in glutathione synthesis. Strikingly, while there are commonalities across the conditions, all three resulted in different metabolomic responses, with the greatest difference between the genetic and environmental responses. Genetic oxidative stress resulted in substantially more widespread effects, both in terms of the percent of the metabolome altered, and the magnitude of changes in individual metabolites. Chronic and acute environmental stress resulted in more similar responses although both were distinct from genetic stress. Overall, these results indicate that the metabolomic response to oxidative stress is complex, reaching across multiple metabolic pathways, with some shared features but with more features unique to different, specific stressors.

Transvection-based gene regulation in Drosophila is a complex and plastic trait.

Transvection, a chromosome pairing-dependent form of trans-based gene regulation, is potentially widespread in the Drosophila melanogaster genome and varies across cell types and within tissues in D. melanogaster, characteristics of a complex trait. Here, we demonstrate that the trans-interactions at the Malic enzyme (Men) locus are, in fact, transvection as classically defined and are plastic with respect to both genetic background and environment. Using chromosomal inversions, we show that trans-interactions at the Men locus are eliminated by changes in chromosomal architecture that presumably disrupt somatic pairing. We further show that the magnitude of transvection at the Men locus is modified by both genetic background and environment (temperature), demonstrating that transvection is a plastic phenotype. Our results suggest that transvection effects in D. melanogaster are shaped by a dynamic interplay between environment and genetic background. Interestingly, we find that cis-based regulation of the Men gene is more robust to genetic background and environment than trans-based. Finally, we begin to uncover the nonlocal factors that may contribute to variation in transvection overall, implicating Abd-B in the regulation of Men in cis and in trans in an allele-specific and tissue-specific manner, driven by differences in expression of the two genes across genetic backgrounds and environmental conditions.

A novel ion pairing LC/MS metabolomics protocol for study of a variety of biologically relevant polar metabolites.

We report a method of ion-pairing liquid chromatography coupled to mass spectrometry (IP-LC-MS) that we have developed for the sensitive detection and quantification of a variety of biologically relevant polar molecules. We use the ion-pairing agent diamyl ammonium to improve chromatographic resolution of polar compounds, such as nucleotide cofactors, sugar phosphates, and organic acids, that are generally poorly retained by conventional reverse phase chromatographic methods. This method showed good linearity (average R value of 0.996) and reproducibility (generally RSD values <10%). We demonstrate the utility of this method by investigating the metabolomic signature of three distinct biological systems: the metabolic response to lack of superoxide dismutase activity and to paraquat induced oxidative stress, and the metabolic profiles of four different Drosophila species.

A combination of structural and cis-regulatory factors drives biochemical differences in Drosophila melanogaster malic enzyme.

The evolutionary significance of molecular variation is still contentious, with much current interest focusing on the relative contribution of structural changes in proteins versus regulatory variation in gene expression. We present a population genetic and biochemical study of molecular variation at the malic enzyme locus (Men) in Drosophila melanogaster. Two amino acid polymorphisms appear to affect substrate-binding kinetics, while only one appears to affect thermal stability. Interestingly, we find that enzyme activity differences previously assigned to one of the polymorphisms may, instead, be a function of linked regulatory differences. These results suggest that both regulatory and structural changes contribute to differences in protein function. Our examination of the Men coding sequences reveals no evidence for selection acting on the polymorphisms, but earlier work on this enzyme indicates that the biochemical variation observed has physiological repercussions and therefore could potentially be under natural selection.

Interactions of NADP-reducing enzymes across varying environmental conditions: a model of biological complexity.

Interactions across biological networks are often quantified under a single set of conditions; however, cellular behaviors are dynamic and interactions can be expected to change in response to molecular context and environment. To determine the consistency of network interactions, we examined the enzyme network responsible for the reduction of nicotinamide adenine dinucleotide phosphate (NADP) to NADPH across three different conditions: oxidative stress, starvation, and desiccation. Synthetic, activity-variant alleles were used in Drosophila melanogaster for glucose-6-phosphate dehydrogenase (G6pd), cytosolic isocitrate dehydrogenase (Idh), and cytosolic malic enzyme (Men) along with seven different genetic backgrounds to lend biological relevance to the data. The responses of the NADP-reducing enzymes and two downstream phenotypes (lipid and glycogen concentration) were compared between the control and stress conditions. In general, responses in NADP-reducing enzymes were greater under conditions of oxidative stress, likely due to an increased demand for NADPH. Interactions between the enzymes were altered by environmental stress in directions and magnitudes that are consistent with differential contributions of the different enzymes to the NADPH pool: the contributions of G6PD and IDH seem to be accentuated by oxidative stress, and MEN by starvation. Overall, we find that biological network interactions are strongly influenced by environmental conditions, underscoring the importance of examining networks as dynamic entities.