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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