+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
Full Article
+
+
+ 