Adaptation to Long-Term Hypoxia in Drosophila Figure 1. Phenotypic changes in hypoxia-selected Drosophila melanogaster. A) Survival rate of parental Drosophila melanogaster population in hypoxic conditions. The survival rates of the parental Drosophila melanogaster population were determined in hypoxic conditions, i.e., 8%, 6% and 4% oxygen. Data were presented as means6SD (* p,0.05, **p,0.01; student’s t-test). The parental population survived well at 8% O2 but their survival rate was reduced at 6%; and 4% O2 was lethal. B) Lifespan of NF and AF. No significant alteration in lifespan was found in AF flies (p.0.05, student’s t-test) at 25uC in normoxia. Survivorship curves were analyzed with GraphPad prism software (San Diego, CA) to calculate the mean lifespan. C) and D) Body size and weight in AF. The body size and weight of AF were significantly reduced, as compared to NF. Data were presented as means6SD (* p,0.05, **p,0.01; student’s t-test). doi:10.1371/journal.pgen.1000221.g001 genes were down-regulated, and only 3 genes were up-regulated. Interestingly, the suppression of these metabolic genes occurred only in the larvae and not in the adult fly (Figure 5C). Coordinated Suppression of TCA Cycle Enzymes Since many genes encoding metabolic enzymes were signifi- cantly down-regulated, we asked whether such down-regulation was coordinated at a transcriptional level. Therefore, the GenomatixSuite (GEMS) software was used to identify transcrip- tion factor binding elements in defined cis-regulatory regions of the TCA cycle, glycolysis and lipid b-oxidation genes. The TCA cycle related genes were separated into two groups, one containing the significantly down-regulated genes (down-regulated group, 16 genes) and the other containing those that were not significantly altered or up-regulated genes (reference group, 8 genes) (Table S6). The binding elements of the Drosophila transcriptional suppressor hairy were present in the regulatory region of the down-regulated genes (15 of 16 cis-regulatory regions, 0.88/kb) but not in the reference group (1 out of 7 cis-regulatory regions, 0.15/ kb) (p,0.0001, CHI-Test) (Figure 6A). Of particular interest, the expression level of hairy was significantly up-regulated in AF (Figure 6B, Table S1). This result suggested that hairy, a key transcriptional suppressor, reduced the expression of the TCA cycle genes. No such specific transcription factor binding elements were found in genes encoding glycolysis or b-oxidation enzymes. To confirm that hairy directly binds to the cis-regulatory regions of these candidate TCA cycle genes, chromatin immunoprecip- itation (ChIP) assay was performed using a specific antibody to hairy in Drosophila Kc cells. The candidate hairy binding targets were tested using specific primers in both hypoxia and normoxia (Table S5). For a negative control, we included a cis-regulatory region of another TCA cycle gene (i.e., CG6629) that was up- regulated in AF and had no hairy binding elements detected. We found that hairy did bind to the cis-regulatory region of gene l(1)G0030 in hypoxia but not in normoxia. Similarly, hairy was also found to bind to the cis-regulatory region of gene SdhB under both hypoxic and normoxic conditions, and its binding activity was significantly higher in hypoxia than in normoxia. Such hypoxia- induced increase in hairy binding did not occur in CG12344, a hairy target gene, which encodes for a non-metabolic gene (i.e., an isoform of GABA-A receptor). This result demonstrates that the down-regulated TCA cycle genes are direct targets of hairy, and hairy specifically suppresses their expression under hypoxia. Furthermore, such hypoxia-induced suppression of TCA cycle genes was abolished in the hairy loss-of-function mutants, h1 or h1j3 (Figure 7A). To further evaluate the role of hairy in hypoxia PLoS Genetics | www.plosgenetics.org 3 October 2008 | Volume 4 | Issue 10 | e1000221