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

Abstract

Although hypoxia-inducible factor-? (HIF?) subunit- specific hydroxylation and proteolytic breakdown ex- plain 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-loop- helix/PAS protein Sima (Similar), in hypoxic cultures of SL2 cells is expressed in full-length (fl) and splice vari- ant (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 pO2 dependence of Sima abundance matched that of HIF activity. HIF-dependent changes in candidate target gene expression were detected through variously ef- fective stimuli: hypoxia (strong) > iron chelation, e.g. desferrioxamine (moderate) >> transition metals, e.g. cobalt ? normoxia (ineffective). Sima overexpression augmented hypoxic induction or suppression of differ- ent targets. In addition to the full-length exon 1–12 tran- script 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 con- tains 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 atten- uated HIF (Sima?Tango)-stimulated reporter expression in a dose-dependent manner. Thus, svSima has the po- tential to regulate Drosophila HIF function under steady and hypoxic pO2 by creating a cytosolic sink for the Sima partner protein Tango.