In this work we have used transgenic flies to demonstrate and characterize in vivo the operation of a hypoxia inducible transcription response in Drosophila that is homologous to mammalian HIF. The work confirms the candidacy of Sima and Tgo as the Drosophila homologues of mammalian HIF-α and HIF-β, respectively, defines a conserved multistep mode of regulation for Sima and provides new insights into the mechanisms regulating HIF proteins, as well as into the spatial and temporal operation of the hypoxia-responsive system during Drosophila development.
By tracking reporter gene activation in developing flies, we analyzed the oxygen concentration dependence, developmental regulation, and spatial distribution of the transcriptional response. Serial studies of the hypoxia response during development indicated that induction by hypoxia is modest in early embryogenesis and mid-embryogenesis and then rises sharply to peak levels at the end of embryogenesis, thereafter remaining relatively high throughout the larval stages. This developmentally restricted capacity fits well with the adaptive requirements of Drosophila larvae. After eclosion larvae usually dig into the substrate, while feeding actively, and are probably subjected to major variations in environmental oxygen tension so that enhanced activity of the HIF system is likely to be of critical importance at this stage.
Interestingly and somewhat unexpectedly, analysis of reporter expression patterns in developing flies showed enhanced hypoxia-inducible activity in the cells of the tracheal system. Although experiments using severe hypoxia and genetic inactivation of Sima proteolysis demonstrated a widespread potential for transcriptional activation by this system, exposure to more moderate hypoxia clearly demonstrated enhanced activity in tracheal cells. This was reflected both in higher expression levels of Sima and in higher activity of different HRE-linked reporter genes and, moreover, was shown to be a cell autonomous function that was preserved in cells of tracheal fate even in the face of mutations that disrupt tracheal architecture.
The existence of enhanced responses to hypoxia in cells composing the organ of oxygen delivery is clearly of interest and raises questions as to its function, particularly since current models indicate that the regulation of tracheal development by oxygen is guided by signals arising in the metabolizing tissues outside the tracheae (24
). Interestingly, some of the branches of the tracheal system run alongside the Drosophila
nervous system (14
), and one possibility is that the tracheae function as sensory organs for hypoxia, as the carotid body does in mammals. A hypoxia pathway affecting behavioral responses has been described in flies (54
), and it will be interesting to determine whether hypoxia-induced behavioral responses share a regulatory mechanism with the HIF system.
In the current work we also utilized the HRE transgenic reporter system to define upstream control mechanisms operating on the Drosophila
HIF system. Our studies identify Sima as the regulatory Drosophila
HIF subunit and demonstrate a major mode of regulation through oxygen-dependent proteolysis that involves a central ODDD. Interestingly, both of the sites of prolyl hydroxylation that operate in mammalian HIF-α subunit ODDD (34
) appear to be conserved in Sima. Furthermore, genetic ablation of the Drosophila
HIF prolyl hydroxylase homologue CG1114 results in striking upregulation of both Sima and reporter gene activity in vivo. This strongly supports a conserved mode of proteolytic regulation of Sima following prolyl hydroxylation at one or both of these sites.
In contrast with the mammalian system, where HIF prolyl hydroxylase activity is represented by the three PHD isoforms (4
), survey of the Drosophila
genome revealed only one homologue (48
), raising questions about the potential of this activity to regulate precisely tuned physiological responses. Interestingly, however, we found that the CG1114 gene is itself a Sima target, demonstrating the operation of a conserved feedback control with the potential to contribute to the complex demands of physiological oxygen homeostasis.
Studies of Sima regulation also demonstrated an additional regulatory step. Transgenic overexpression of Sima in normoxic embryos resulted in cytoplasmic accumulation of the protein and little transcriptional activity. In contrast, similar levels of overexpression in hypoxia resulted in nuclear accumulation and a strong transcriptional response, demonstrating the presence of a second oxygen-regulated mechanism controlling Sima subcellular localization. An oxygen-regulated nuclear localization step has previously been demonstrated for mammalian HIF-α (2
), although not in every study. Our demonstration of conservation of this mode of regulation in Drosophila
Sima, however, provides strong support for the physiological relevance of this process. Our findings suggest that Sima subcellular localization is controlled by an active mechanism that maintains the protein in the cytoplasm in normoxia as opposed to an hypoxia-dependent machinery that mediates nuclear import. Although the strong transcriptional activity of mammalian HIF-α that is observed after deletion of the ODDD (12
), mutation of the VHL binding sites (34
), or inactivation of VHL (36
) is consistent with a role for this domain in cytoplasmic localization in normoxia, this has not been tested in studies of mammalian HIF-α that have examined subcellular localization directly. Moreover, although induction of nuclear localization by iron chelators and cobaltous ions (26
) suggests a similar mode of regulation to proteolytic regulation, neither the source of the oxygen-sensitive signal nor the mechanism of transduction have been defined. Our in vivo studies in flies show induction of nuclear Sima after inactivation of CG1114 either by RNAi or by mutation, thus clearly implicating this gene product in the process of cytoplasmic localization in normoxia. Moreover, Sima nuclear localization was also observed in flies bearing the SimaΔ692-863 transgene, indicating that this sequence is absolutely required for cytoplasmic localization.
Very recently nonproteolytic regulation of mammalian HIF-α subunits involving the C-terminal transactivation domains has been shown to be regulated by hydroxylation of a specific asparaginyl residue by an enzymatic activity that, like the prolyl hydroxylases involved in HIF proteolysis, demonstrates the properties of an α-ketoglutarate-dependent dioxygenase (30
). Thus, regulatory hydroxylation of HIF-α residues by this class of enzyme appears to extend to both specific asparaginyl and prolyl residues. Currently, the precise substrate requirements of the CG1114 gene product are not defined, and it is not clear whether effects on nuclear localization are mediated through the conserved prolyl residues, possibly reflecting additional functions of the VHL ubiquitylation complex, or whether other sequences within the Sima ODDD mediate this process. Further biochemical and genetic studies should clarify these new insights into the HIF system.
Overall, the high degree of conservation in the Drosophila system indicates that genetic studies in this organism should be highly informative in analyses of both the upstream pathways regulating the HIF system, and the downstream physiological effects in an intact organism.