Adaptation to Long-Term Hypoxia in Drosophila Figure 3. Summary of alterations in signal transduction pathways. The expression levels of genes encoding five signal transduction pathways were significantly altered in hypoxia-selected Drosophila melanogaster (p,0.05). The majority of the changes was an up-regulation. Significantly altered genes encoding the components A) of EGF receptor pathway, B) of the Insulin receptor pathway, C) of the JNK cascade, D) of the Notch pathway, and E) of the Toll/Imd pathway. doi:10.1371/journal.pgen.1000221.g003 mammalian tissues/cells [22–28]. For example, we and others have shown that the Notch receptor and its targets were altered in mouse heart [29] and cultured cells [30,31] following chronic hypoxia. Such similarities in hypoxia responses demonstrated that some of the mechanisms underlying hypoxia-tolerance are conserved across species. Fourth, previous studies have demon- strated an enhancement of glycolytic activity as a major metabolic response in cells/tissues following acute (in minutes or hours) hypoxia, in contrast, our present data showed that many genes encoding glycolytic enzymes were down-regulated in the AF. Moreover, no significant lactate accumulation was found in the AF flies (unpublished observations from our laboratory). Finally, although hypoxia-tolerance is a complex trait, a single gene alteration, such as that of hairy, can remarkably make a significant difference in terms of survival. For more than three decades, physiologists have debated the importance of down-regulation of metabolism in hypoxia tolerance in some tolerant animals such as the turtle. For example, Hochachka and others have argued that anoxia-tolerant organ- isms depress their metabolism in order to minimize the mismatch between supply and demand [32–34]. While this idea, based on metabolic data, is intuitively appealing, there is no information about how various metabolic enzymes could be coordinated in order to survive severe long lasting hypoxia. Our current work provides the first evidence showing that such coordination can be achieved at a transcriptional level by a seemingly metabolic switch, i.e., hairy, which is a transcriptional suppressor. This notion is supported by a number of observations from current study: a) expression of hairy was up-regulated in AF, b) hairy-binding region was presented in the cis-regulatory regions of the down-regulated genes but not others, c) the functional ChIP analysis led us to believe that the down-regulation of these metabolic genes is based on the changes of hairy, especially that its binding was not increased for other, non-metabolic target genes, e.g., CG12344, and d) hairy loss-of-function mutations abolished the suppression on the expression of TCA cycle genes and significantly reduced hypoxia survival in Drosophila. This decreased survival in flies carrying the hairy mutation is particularly important since these experiments strongly demonstrate that hairy contributes to hypoxia tolerance through the regulation of TCA cycle enzymes. While it is possible that the decreased survival is not related to the role that hairy plays in metabolic regulation but due to an inherent weakness of the hairy mutants, this is much less likely because a) we have studied in the past different mutation alleles in flies and they do not PLoS Genetics | www.plosgenetics.org 5 October 2008 | Volume 4 | Issue 10 | e1000221