-Summary:
-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 thatmight 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 mM sodium acetate), or lead-treated food (made with 250 mM 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 12major 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 leadinduced
-expression.
-Materials and Methods:
-The 75 Drosophila roo lines were obtained from Trudy Mackay.
-To avoid batch effects (Zakharkin et al., 2005), the growth of the
-flies, the RNA extraction and the order of running the arrays, and
-the fluidics well used for each array was completely randomized
-for the 75 lines in two treatments. Control food consisted of
-standard cornmeal, agar, sugar, yeast, and 250 mM NaAc (Ashburner,
-1989). Lead-contaminated food consisted of standard food
-plus 250 mM PbAc (lead exposure at this concentration has been
-shown to affect locomotion in adults; Hirsch et al., 2003). Flies
-from each of the 75 roo lines (20 males and 20 females) were
-placed in a vial with 10 ml of food (control or PbAc) for 3 days at
-25 8C and allowed to lay eggs; the adults were subsequently
-discarded. Newly enclosed adult males were placed on the same
-medium (control or PbAc) as had been present during pre-adult
-development for 5–10 days before being used as subjects. Male
-progeny were pooled from each vial (65 males per vial) and frozen
-at 80 8C. RNA samples were extracted in groups of 24 and arrays
-hybridization run in groups of 4 with 3 groups run per day. Effects
-of RNA extraction and array hybridizations day were examined by
-ANOVA and Support Vector approaches and no obvious day effects
-were observed.
-Data Source Acknowledgements:
-
-
This work was supported by the Environmental Health Sciences
-Center in Molecular and Cellular Toxicology with Human
-Applications Grant P30 ES06639 at Wayne State University, NIH
-R01 grants ES012933 and CA105349 to D.M.R., DK071073 to X.L.,
-and UAB-CNGI grant to M.D.G. We thank H. Ghiradella for critical
-comments on the manuscript. The microarray data is freely
-available to the public, in the MIAME format in 150 CEL files, in the
-GEO database under GSE 11695.
Please cite this article in press as: Ruden DM, et al. Genetical toxicogenomics in Drosophila identifies master-modulatory loci that are
-regulated by developmental exposure to lead, Neurotoxicology (2009), doi:10.1016/j.neuro.2009.08.011
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