Adaptation to Long-Term Hypoxia in Drosophila Figure 6. Effect of the transcriptional suppressor, hairy, on TCA cycle gene expression and hypoxia tolerance. A) Genomic localization of hairy binding elements in the cis-regulatory region of genes encoding TCA cycle enzymes. Arrowheads indicate consensus hairy binding sites and arrows indicate transcriptional start sites. B) Significant up-regulation of hairy expression in hypoxia-selected AF populations (p,0.01). C) Chromatin immunoprecipitation assay of gene l(1)G0030, SdhB and CG12344 under normoxia or hypoxia. doi:10.1371/journal.pgen.1000221.g006 The hairy loss-of-function mutants (h1 and h1j3) were obtained from Drosophila Stock Center (Bloomington, IN). The survival rate of these stocks in hypoxia was determined by culturing them in a 6% O2 environment. After 3 weeks in culture, the number of live adult flies and pupae were counted. The ratio between live adult flies to the total number of pupae was calculated and presented as survival rate. The statistical significance of survival between hairy mutants and controls was calculated by CHI-test. cDNA Microarray Analysis cDNA microarrays containing 13,061 known or predicted genes of the D. melanogaster genome were processed according to previous descriptions [16,35]. Nine larval samples from AF or NF, 6 adult samples from AF or NF were included in this analysis. Each larval sample contained a pool of 25 3rd instar larvae, and each adult sample contained 25 male and 25 female adult flies from each individual population. Total RNA was extracted from the samples using TRIzol (Invitrogen, Carlsbad, CA) followed by a clean-up with RNeasy kit (Qiagen, Valencia, CA). Three mg of total RNA from each sample was amplified with an in vitro transcription-based strategy [13]. A common reference design was applied for the hybridizations, and the reference RNA sample was created using a balanced pool of 3rd instar larvae (for the larval samples) or adult flies (for the adult samples) from each parental line. This reference was chosen so that relative abundance of each transcript could be calculated individually, and the relative levels of each transcript among biological replicates could be compared. A total of 30 arrays were included in this analysis, and the hybridizations were done in different days using arrays printed from different batches. Microarray images were acquired by GenePix 4000 microarray scanner using GenePix Pro 3 microarray analysis software (Axon Instruments, Sunnyvale, CA, USA). The statistical significance (q- value, i.e., false discover rate (FDR)) and the ratio of the changes in expression was calculated using Significance Analysis of Micro- array (SAM) software [19] following LOWESS normalization. The fold changes were presented as ratios, if up-regulated, or 21/ ratio, if down-regulated. The gene ontology (GO) based analyses were performed using GenMAPP software [20]. The microarray data can be retrieved using access number GSE8803 in the GEO database at http://www.ncbi.nlm.nih.gov/geo. Semi-Quantitative Real-Time RT-PCR Semi-quantitative real-time RT-PCR was used to evaluate the result of microarrays and to determine the differences in expression levels of genes encoding TCA cycle enzymes between the hairy mutants and control. All specific primers were designed PLoS Genetics | www.plosgenetics.org 8 October 2008 | Volume 4 | Issue 10 | e1000221