Genetical toxicogenomics in Drosophila identifies master-modulatory loci that are regulated by developmental exposure to lead.

The genetics of gene expression in recombinant inbred lines (RILs) can be mapped as expression quantitative trait loci (eQTLs). So-called "genetical genomics" studies have identified locally acting eQTLs (cis-eQTLs) for genes that show differences in steady-state RNA levels. These studies have also identified distantly acting master-modulatory trans-eQTLs that regulate tens or hundreds of transcripts (hotspots or transbands). We expand on these studies by performing genetical genomics experiments in two environments in order to identify trans-eQTL that might be regulated by developmental exposure to the neurotoxin lead. Flies from each of 75 RIL were raised from eggs to adults on either control food (made with 250 microM sodium acetate), or lead-treated food (made with 250 microM lead acetate, PbAc). RNA expression analyses of whole adult male flies (5-10 days old) were performed with Affymetrix DrosII whole genome arrays (18,952 probesets). Among the 1389 genes with cis-eQTL, there were 405 genes unique to control flies and 544 genes unique to lead-treated ones (440 genes had the same cis-eQTLs in both samples). There are 2396 genes with trans-eQTL which mapped to 12 major transbands with greater than 95 genes. Permutation analyses of the strain labels but not the expression data suggests that the total number of eQTL and the number of transbands are more important criteria for validation than the size of the transband. Two transbands, one located on the 2nd chromosome and one on the 3rd chromosome, co-regulate 33 lead-induced genes, many of which are involved in neurodevelopmental processes. For these 33 genes, rather than allelic variation at one locus exerting differential effects in two environments, we found that variation at two different loci are required for optimal effects on lead-induced expression.

Massively parallel resequencing of the isogenic Drosophila melanogaster strain w(1118); iso-2; iso-3 identifies hotspots for mutations in sensory perception genes.

We used the Illumina reversible-short sequencing technology to obtain 17-fold average depth (s.d. approximately 8) of approximately 94% of the euchromatic genome and approximately 1-5% of the heterochromatin sequence of the Drosophila melanogaster isogenic strain w(1118); iso-2; iso-3. We show that this strain has a approximately 9 kb deletion that uncovers the first exon of the white (w) gene, approximately 4 kb of downstream promoter sequences, and most of the first intron, thus demonstrating that whole-genome sequencing can be used for mutation characterization. We chose this strain because there are thousands of transposon insertion lines and hundreds of isogenic deficiency lines available with this genetic background, such as the Exelixis, Inc., and the DrosDEL collections. We compared our sequence to Release 5 of the finished reference genome sequence which was made from the isogenic strain y(1); cn(1) bw(1) sp(1) and identified 356,614 candidate SNPs in the approximately 117 Mb unique sequence genome, which represents a substitution rate of approximately 1/305 nucleotides ( approximately 0.30%). The distribution of SNPs is not uniform, but rather there is a approximately 2-fold increase in SNPs on the autosome arms compared with the X chromosome and a approximately 7-fold increase when compared to the small 4(th) chromosome. This is consistent with previous analyses that demonstrated a correlation between recombination frequency and SNP frequency. An unexpected finding was a SNP hotpot in a approximately 20 Mb central region of the 4(th) chromosome, which might indicate higher than expected recombination frequency in this region of this chromosome. Interestingly, genes involved in sensory perception are enriched in SNP hotspots and genes encoding developmental genes are enriched in SNP coldspots, which suggests that recombination frequencies might be proportional to the evolutionary selection coefficient. There are currently 12 Drosophila species sequenced, and this represents one of many isogenic Drosophila melanogaster genome sequences that are in progress. Because of the dramatic increase in power in using isogenic lines rather than outbred individuals, the SNP information should be valuable as a test bed for understanding genotype-by-environment interactions in human population studies.

The EDGE hypothesis: epigenetically directed genetic errors in repeat-containing proteins (RCPs) involved in evolution, neuroendocrine signaling, and cancer.

Trans-generational epigenetic phenomena, such as contamination with endocrine-disrupting chemicals (EDCs) that decrease fertility and the global methylation status of DNA in the offspring, are of great concern because they may affect health, particularly the health of children. However, of even greater concern is the possibility that trans-generational changes in the methylation status of the DNA might lead to permanent changes in the DNA sequence itself. By contaminating the environment with EDCs, mankind might be permanently affecting the health of future generations. In this section, we present evidence from our laboratory and others that trans-generational epigenetic changes in DNA might lead to mutations directed to genes encoding amino acid repeat-containing proteins (RCPs) that are important for adaptive evolution or cancer progression. Such epigenetic changes can be induced "naturally" by hormones or "unnaturally" by EDCs or environmental stress. To illustrate the phenomenon, we present new bioinformatic evidence that the only RCP ontological categories conserved from Drosophila to humans are "regulation of splicing," "regulation of transcription," and "regulation of synaptogenesis," which are classes of genes likely to be important for evolutionary processes. Based on that and other evidence, we propose a model for evolution that we call the EDGE (Epigenetically Directed Genetic Errors) hypothesis for the mechanism by which mutations are targeted at epigenetically modified "contingency genes" encoding RCPs. In the model, "epigenetic assimilation" of metastable epialleles of RCPs over many generations can lead to mutations directed to those genes, thereby permanently stabilizing the adaptive phenotype.

Potential long-term consequences of fad diets on health, cancer, and longevity: lessons learned from model organism studies.

While much of the third world starves, many in the first world are undergoing an obesity epidemic, and the related epidemics of type II diabetes, heart disease, and other diseases associated with obesity. The amount of economic wealth being directly related to a decline in health by obesity is ironic because rich countries contribute billions of dollars to improve the health of their citizens. Nevertheless, nutritional experiments in model organisms such as yeast, C. elegans, Drosophila, and mice confirm that "caloric restriction" (CR), which is defined generally as a 30-40% decrease in caloric intake, a famine-like condition for humans seen only in the poorest of countries, promotes good health and increases longevity in model organisms. Because caloric restriction, and dieting in general, requires a great deal of will power to deal with the feelings of deprivation, many fad diets, such as the Atkins, South Beach, and Protein Power, have been developed which allow people to lose weight purportedly without the severe feelings of deprivation. However, the long-term effects of such fad diets are not known and few experiments have been performed in the laboratory to investigate possible side affects and adverse consequences. In this paper, we review studies with fad-like dietary conditions in humans and model organisms, and we propose a "Dietary Ames Test" to rapidly screen fad diets, dietary supplements, and drugs for potential long-term health consequences in model organisms.

Possible effects of early treatments of hsp90 inhibitors on preventing the evolution of drug resistance to other anti-cancer drugs.

Hsp90 is a chaperone that is critically important for both cancer progression and tumor survival. Hsp90 is an exciting target for anti-cancer drugs because most of the proteins that interact with Hsp90 are known to be in the cell cycle, signaling and chromatin-remodeling pathways. Recent work in fungi has shown that reduction of Hsp90 activity dramatically increases the efficacy of many fungicides. Furthermore, in studies on the evolution of drug resistance in fungi, it has been shown that high levels of Hsp90 increase the rate of the development of fungicide resistance, whereby low levels of Hsp90 decrease the rate of fungicide resistance. Similarly, in humans and mammalian models, Hsp90 inhibitors have been shown to act additively or synergistically with many other cancer therapies for killing both solid tumors and leukemias. Also, several recent studies have shown that Hsp90 inhibitors potentiate the activity of drugs in cancer cells lines that are otherwise resistant to the drug. However, during the evolution of drug resistance in cancer cells, it has not yet been determined whether early exposure to Hsp90 inhibitors slows the rate of developing resistance to other anti-cancer drugs, as would be expected from the fungal studies. In this review, we summarize the effects of the Hsp90 inhibitors geldanamycin and its derivatives with other anti-cancer drugs on killing cancer cells. We also discuss other basic science and clinical studies that need to be done to determine the optimum exposure regimens for Hsp90 inhibitor treatments to maximize its cancer-killing activities, and to minimize the evolution of resistance to other anti-cancer drugs.