Figure 7. Loss-of-function hairy mutants abolished suppression on the expression of genes encoding TCA cycle enzymes and reduced hypoxia-tolerance in D. melanogaster. A) Abolished suppression of genes encoding TCA cycle enzymes in hairy loss-of- function mutants. The differences in gene expression were measured in yw control and hairy mutants using real-time PCR. Individual genes encoding TCA cycle enzymes had a significantly higher expression level in all hairy mutants than those in control except gene CG10219. Data were presented as means6SD (p,0.01). Gene description: l(1)G0030: citrate synthase; Idh: isocitrate dehydrogenase; l(1)G0156: isocitrate dehydrogenase; CG6439: isocitrate dehydrogenase; SdhB: succinate dehydrogenase; CG10219: succinate dehydrogenase; CG6666: succinate dehydrogenase; l(1)G0255: fumarase. B) Significant reduction in hypoxia survival in hairy loss-of-function mutants. Embryos from loss-of-function hairy mutants (h1 and h1j3) and controls (Canton-S and yw) were cultured at normoxic or 6% O2 hypoxic condition. The ratio of adult flies to pupae was determined as survival rate of each stock. The loss-of- function hairy mutants exhibited a significantly reduced rate of hypoxia survival. Data were presented as means6SD (** p,0.01). doi:10.1371/journal.pgen.1000221.g007 by Primer 3 software [36] and synthesized at ValueGene (San Diego, CA) (Table S5). First strand cDNA was synthesized using SuperScriptII reverse transcriptase and Oligo-(dT) primer. Real- time PCR amplification was performed using ABI Prism 7900HT Sequence Detection System (Applied Biosystems, Foster City, CA). For each reaction, 10 ml of 26 SYBR green PCR master mix (Applied Biosystems, Foster City, CA) and 0.5 mM of both forward and reverse primers along with 100 ng of each appropriate cDNA samples were mixed (total reaction volume: 20 ml). Melting curves were determined and the final products were isolated with 4% agarose gel to ensure specificity of the reaction. The relative expression level was calculated using 22DDCt method, as described previously [37–39]. Drosophila melanogaster b-actin was used as Adaptation to Long-Term Hypoxia in Drosophila internal control. The final results were presented as fold change of AF over NF or hairy mutants over yw control. All experiments were done in triplicate. Enzyme Activity Assays The enzymatic activity of Triose Phosphate Isomerase (TPI) and Pyruvate Kinase (PK) were determined as previously described with modifications [40]. Briefly, the assay samples were extracted from 3rd instar larvae cultured under either normoxic or hypoxic condition (4% O2). 0.2 ml (100 mg wet weight/ml) of isolation medium (0.25 M sucrose,1 mM EDTA-K, 5 mM HEPES-Tris, pH 7.4, with protease inhibitors) was added and the suspension of larval tissue in isolation medium was transferred into a 2 ml all glass homogenizer. 10% (v/v) Triton X-100 was added to this suspension making the final concentration of Triton X-100 0.5% (v/v). Then the tissue was homogenized with the B (i.e., tight) pestle. Aliquots of this homogenate were employed for enzymatic activity measurements. The enzymatic activities of TPI or PK were determined using 10, 20, or 30 ml of the homogenate with proper substrates. The dynamic of color formation was recorded using Beckman Coulter DU800 Spectrophotometer at selected wave length for each substrate. The kinetic parameters of the reaction were calculated by curve fitting. Identification of Transcription Factor Binding Element in cis-Regulatory Region The common transcription factor binding sites in the defined promoter regions of candidate genes were analyzed using GenomatixSuite (Genomatix Software GmbH, Germany). The genes encoding TCA cycle or b-oxidation enzymes were separated into down-regulated or reference groups. The genome DNA sequences from 2000 bp upstream of the first transcription starting site (TSS) to 500 bp downstream of the last TSS of each gene was downloaded and used as cis-regulatory region of the gene (Drosophila genome R5.2) [41]. These sequences were subjected to GEMS analysis to identify common transcription factor binding elements [42]. The statistical significance of the transcription factor binding element frequency in the AF and NF population was analyzed by CHI-test. ChIP Assay Chromatin immunoprecipitation and PCR detection of hairy binding in the cis-regulatory regions of genes encoding TCA cycle enzymes were performed in cultured Drosophila Kc cells (obtained from Dr. Amy Kiger, UCSD) [43,44]. The Kc cells were treated with 0.5% O2 for 4 hours. About 106 cells were used in each ChIP experiment. Chromatin immunoprecipitation was carried out using ChIP assay kit (Upstate, Temecula, CA) according to the manufacture’s instructions. The immunoprecipitation was per- formed overnight at 4uC with 2 mg of hairy antibody (Abcam, Cambridge, MA). DNA fragments were purified with phenol:- chloroform (Invitrogen, Carlsbad, CA). For PCR, 2 ml of a 25 ml DNA extraction was amplified with specific primers (Table S5). DNA Sequencing and Analysis Genomic DNA was extracted from 15 male AF or NF adult flies. The samples were ground in 400 ml of homogenate buffer (100 mM Tris/HCl, 100 mM EDTA, 100 mM NaCl and 0.5% SDS, pH7.5) and incubated at 65uC for 30 min. Genomic DNA was extracted by adding in 800 ml extraction solution (1 2.5 (v/v) of 5 M KAc and 6 M LiCl). After 15 min of centrifugation at 13,000 rpm, the supernatant was transferred into a new tube and the genomic DNA was precipitated by adding 600 ml of PLoS Genetics | www.plosgenetics.org 9 October 2008 | Volume 4 | Issue 10 | e1000221