Regulation of Drosophila Hypoxia-inducible Factor (HIF) Activity in SL2 Cells

Abstract

Although hypoxia-inducible factor-␣ (HIF␣) subunitspecific hydroxylation and proteolytic breakdown explain the binary switch between the presence (hypoxia) and absence (normoxia) of HIFs, little is known of the mechanisms that fine-tune HIF activity under constant, rather than changing, oxygen tensions. Here, we report that the Drosophila HIF␣ homolog, the basic helix-loophelix/PAS protein Sima (Similar), in hypoxic cultures of SL2 cells is expressed in full-length (fl) and splice variant (sv) isoforms. The following evidence supports the role of flSima as functional HIF␣ and the role of SL2 HIF as a transcriptional activator or suppressor. The pO 2 dependence of Sima abundance matched that of HIF activity. HIF-dependent changes in candidate target gene expression were detected through variously effective stimuli: hypoxia (strong) > iron chelation, e.g. desferrioxamine (moderate) > > transition metals, e.g. cobalt Ӎ normoxia (ineffective). Sima overexpression augmented hypoxic induction or suppression of different targets. In addition to the full-length exon 1-12 transcript yielding the 1510-amino acid HIF␣ homolog, the sima gene also expressed, specifically under hypoxia, an exon 1-7/12 splice variant, which translated into a 426-amino acid Sima truncation termed svSima. svSima contains basic helix-loop-helix and PAS sequences identical to those of flSima, but, because of deletion of exons 8 -11, lacks the oxygen-dependent degradation domain and nuclear localization signals. Overexpressed svSima failed to transactivate reporter genes. However, it attenuated HIF (Sima⅐Tango)-stimulated reporter expression in a dose-dependent manner. Thus, svSima has the potential to regulate Drosophila HIF function under steady and hypoxic pO 2 by creating a cytosolic sink for the Sima partner protein Tango.

Hypoxia-induced Synthesis of Hemoglobin in the Crustacean Daphnia magna Is Hypoxia-inducible Factor-dependent

“…In each transfection, the pUC18 plasmid was added to achieve a total of 20 g of DNA. Following 16-h transfections, post-transfection processing of Hep3B and SL2 cells was identical and is described in detail in the accompanying article (69).…”

Daphnia and Drosophila: two invertebrate models for O2 responsive and HIF-mediated regulation of genes and genomes

“…One important discovery with relevance for a possible HIF contribution to confer hypoxic tolerance, is that HIF, once bound to DNA, can act either as transactivator ( Y gene induction) or -inactivator ( Y gene silencing). This functional duality of HIF was found to apply to the hypoxic expression of daphnid versus drosophilid globin genes [1,4]. Despite Daphnia's tolerance toward acute anoxic stresses and strong anaerobic capacity (see above and [6]), a gradual decline in pO 2 over days rather than minutes-hours, will be met through the HIF-mediated induced transcription of several of its globin genes (notably hb2, see Fig.…”

“…For example, D. magna (Crustacea, Cladocera) or D. melanogaster (Insecta, Diptera) are particularly suited to further our understanding of oxy-genes and -genomes. They both express HIF, the key regulator protein in the transcriptional response to hypoxia (Daphnia: [1]; Drosophila: flies [2], cell culture [3,4]) and therefore make use of a phylogenetically conserved signaling pathway (i.e., oxygen sensing) to relay environmental cues such as declining oxygen tensions through the hypoxia-specific stabilization of the HIFa subunit protein, followed by heterodimerization with its partner protein ARNT, into the nucleus. In contrast, under normoxic conditions, the HIFa subunit is oxidatively modified and subsequently degraded by proteolysis, thus abrogating HIF function.…”

Induction of the Heat Shock Pathway during Hypoxia Requires Regulation of Heat Shock Factor by Hypoxia-inducible Factor-1

“…We transfected Kc 167 cells with an epitope-tagged HIF-1␣ expression vector (26). DNA bound to HIF-1 protein during hypoxia was immunoprecipitated, and the Hsf intron genomic region as well as control genomic regions were PCR amplified to test for enrichment compared with DNA immunoprecipitated from a mock transfection.…”