A naturally-occurring 22-bp coding deletion in Ugt86Dd reduces nicotine resistance in Drosophila melanogaster.

OBJECTIVE: Segregating genetic variants contribute to the response to toxic, xenobiotic compounds, and identifying these causative sites can help describe the mechanisms underlying metabolism of toxic compounds. In previous work we implicated the detoxification gene Ugt86Dd in the genetic control of larval nicotine resistance in Drosophila melanogaster. Furthermore, we suggested that a naturally-occurring 22-bp deletion that leads to a stop codon in exon 2 of the gene markedly reduces resistance. Here we use homology directed CRISPR/Cas9 gene editing to specifically test this hypothesis. RESULTS: We edited chromosome three from an inbred strain named A4 which carries the insertion allele at Ugt86Dd, successfully generated four alleles carrying the 22-bp Ugt86Dd deletion, and substituted edited chromosomes back into the A4 background. The original A4 strain, and an un-edited control strain in the same A4 background, show no significant difference in egg-to-adult or larva-to-adult viability on either control media or nicotine-supplemented media, and only slightly delayed development in nicotine media. However, strains carrying the 22-bp deletion showed reduced viability in nicotine conditions, and significantly longer development. Our data strongly suggest that the naturally-occurring 22-bp insertion/deletion event in Ugt86Dd directly impacts variation in nicotine resistance in D. melanogaster.

Genetics of cocaine and methamphetamine consumption and preference in Drosophila melanogaster.

Illicit use of psychostimulants, such as cocaine and methamphetamine, constitutes a significant public health problem. Whereas neural mechanisms that mediate the effects of these drugs are well-characterized, genetic factors that account for individual variation in susceptibility to substance abuse and addiction remain largely unknown. Drosophila melanogaster can serve as a translational model for studies on substance abuse, since flies have a dopamine transporter that can bind cocaine and methamphetamine, and exposure to these compounds elicits effects similar to those observed in people, suggesting conserved evolutionary mechanisms underlying drug responses. Here, we used the D. melanogaster Genetic Reference Panel to investigate the genetic basis for variation in psychostimulant drug consumption, to determine whether similar or distinct genetic networks underlie variation in consumption of cocaine and methamphetamine, and to assess the extent of sexual dimorphism and effect of genetic context on variation in voluntary drug consumption. Quantification of natural genetic variation in voluntary consumption, preference, and change in consumption and preference over time for cocaine and methamphetamine uncovered significant genetic variation for all traits, including sex-, exposure- and drug-specific genetic variation. Genome wide association analyses identified both shared and drug-specific candidate genes, which could be integrated in genetic interaction networks. We assessed the effects of ubiquitous RNA interference (RNAi) on consumption behaviors for 34 candidate genes: all affected at least one behavior. Finally, we utilized RNAi knockdown in the nervous system to implicate dopaminergic neurons and the mushroom bodies as part of the neural circuitry underlying experience-dependent development of drug preference.

Naturally Segregating Variation at Ugt86Dd Contributes to Nicotine Resistance in Drosophila melanogaster.

Identifying the sequence polymorphisms underlying complex trait variation is a key goal of genetics research, since knowing the precise causative molecular events allows insight into the pathways governing trait variation. Genetic analysis of complex traits in model systems regularly starts by constructing QTL maps, but generally fails to identify causative sequence polymorphisms. Previously we mapped a series of QTL contributing to resistance to nicotine in a Drosophila melanogaster multiparental mapping resource and here use a battery of functional tests to resolve QTL to the molecular level. One large-effect QTL resided over a cluster of UDP-glucuronosyltransferases, and quantitative complementation tests using deficiencies eliminating subsets of these detoxification genes revealed allelic variation impacting resistance. RNAseq showed that Ugt86Dd had significantly higher expression in genotypes that are more resistant to nicotine, and anterior midgut-specific RNA interference (RNAi) of this gene reduced resistance. We discovered a segregating 22-bp frameshift deletion in Ugt86Dd, and accounting for the InDel during mapping largely eliminates the QTL, implying the event explains the bulk of the effect of the mapped locus. CRISPR/Cas9 editing of a relatively resistant genotype to generate lesions in Ugt86Dd that recapitulate the naturally occurring putative loss-of-function allele, leads to a large reduction in resistance. Despite this major effect of the deletion, the allele appears to be very rare in wild-caught populations and likely explains only a small fraction of the natural variation for the trait. Nonetheless, this putatively causative coding InDel can be a launchpad for future mechanistic exploration of xenobiotic detoxification.

Genetic analysis of variation in lifespan using a multiparental advanced intercross Drosophila mapping population.

BACKGROUND: Considerable natural variation for lifespan exists within human and animal populations. Genetically dissecting this variation can elucidate the pathways and genes involved in aging, and help uncover the genetic mechanisms underlying risk for age-related diseases. Studying aging in model systems is attractive due to their relatively short lifespan, and the ability to carry out programmed crosses under environmentally-controlled conditions. Here we investigate the genetic architecture of lifespan using the Drosophila Synthetic Population Resource (DSPR), a multiparental advanced intercross mapping population. RESULTS: We measured lifespan in females from 805 DSPR lines, mapping five QTL (Quantitative Trait Loci) that each contribute 4-5 % to among-line lifespan variation in the DSPR. Each of these QTL co-localizes with the position of at least one QTL mapped in 13 previous studies of lifespan variation in flies. However, given that these studies implicate >90 % of the genome in the control of lifespan, this level of overlap is unsurprising. DSPR QTL intervals harbor 11-155 protein-coding genes, and we used RNAseq on samples of young and old flies to help resolve pathways affecting lifespan, and identify potentially causative loci present within mapped QTL intervals. Broad age-related patterns of expression revealed by these data recapitulate results from previous work. For example, we see an increase in antimicrobial defense gene expression with age, and a decrease in expression of genes involved in the electron transport chain. Several genes within QTL intervals are highlighted by our RNAseq data, such as Relish, a critical immune response gene, that shows increased expression with age, and UQCR-14, a gene involved in mitochondrial electron transport, that has reduced expression in older flies. CONCLUSIONS: The five QTL we isolate collectively explain a considerable fraction of the genetic variation for female lifespan in the DSPR, and implicate modest numbers of genes. In several cases the candidate loci we highlight reside in biological pathways already implicated in the control of lifespan variation. Thus, our results provide further evidence that functional genetics tests targeting these genes will be fruitful, lead to the identification of natural sequence variants contributing to lifespan variation, and help uncover the mechanisms of aging.

Overcoming chemo/radio-resistance of pancreatic cancer by inhibiting STAT3 signaling.

Chemo/radio-therapy resistance to the deadly pancreatic cancer is mainly due to the failure to kill pancreatic cancer stem cells (CSCs). Signal transducer and activator of transcription 3 (STAT3) is activated in pancreatic CSCs and, therefore, may be a valid target for overcoming therapeutic resistance. Here we investigated the potential of STAT3 inhibition in sensitizing pancreatic cancer to chemo/radio-therapy. We found that the levels of nuclear pSTAT3 in pancreatic cancer correlated with advanced tumor grade and poor patient outcome. Liposomal delivery of a STAT3 inhibitor FLLL32 (Lip-FLLL32) inhibited STAT3 phosphorylation and STAT3 target genes in pancreatic cancer cells and tumors. Consequently, Lip-FLLL32 suppressed pancreatic cancer cell growth, and exhibited synergetic effects with gemcitabine and radiation treatment in vitro and in vivo. Furthermore, Lip-FLLL32 reduced ALDH1-positive CSC population and modulated several potential stem cell markers. These results demonstrate that Lip-FLLL32 suppresses pancreatic tumor growth and sensitizes pancreatic cancer cells to radiotherapy through inhibition of CSCs in a STAT3-dependent manner. By targeting pancreatic CSCs, Lip-FLLL32 provides a novel strategy for pancreatic cancer therapy via overcoming radioresistance.

Identifying Loci Contributing to Natural Variation in Xenobiotic Resistance in Drosophila.

Natural populations exhibit a great deal of interindividual genetic variation in the response to toxins, exemplified by the variable clinical efficacy of pharmaceutical drugs in humans, and the evolution of pesticide resistant insects. Such variation can result from several phenomena, including variable metabolic detoxification of the xenobiotic, and differential sensitivity of the molecular target of the toxin. Our goal is to genetically dissect variation in the response to xenobiotics, and characterize naturally-segregating polymorphisms that modulate toxicity. Here, we use the Drosophila Synthetic Population Resource (DSPR), a multiparent advanced intercross panel of recombinant inbred lines, to identify QTL (Quantitative Trait Loci) underlying xenobiotic resistance, and employ caffeine as a model toxic compound. Phenotyping over 1,700 genotypes led to the identification of ten QTL, each explaining 4.5-14.4% of the broad-sense heritability for caffeine resistance. Four QTL harbor members of the cytochrome P450 family of detoxification enzymes, which represent strong a priori candidate genes. The case is especially strong for Cyp12d1, with multiple lines of evidence indicating the gene causally impacts caffeine resistance. Cyp12d1 is implicated by QTL mapped in both panels of DSPR RILs, is significantly upregulated in the presence of caffeine, and RNAi knockdown robustly decreases caffeine tolerance. Furthermore, copy number variation at Cyp12d1 is strongly associated with phenotype in the DSPR, with a trend in the same direction observed in the DGRP (Drosophila Genetic Reference Panel). No additional plausible causative polymorphisms were observed in a full genomewide association study in the DGRP, or in analyses restricted to QTL regions mapped in the DSPR. Just as in human populations, replicating modest-effect, naturally-segregating causative variants in an association study framework in flies will likely require very large sample sizes.