Regulation of Drosophila hypoxia-inducible factor (HIF) activity in SL2 cells: identification of a hypoxia-induced variant isoform of the HIFalpha homolog gene similar.
Although hypoxia-inducible factor-alpha (HIFalpha) subunit-specific 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 HIFalpha homolog, the basic helix-loop-helix/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 HIFalpha 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 approximately 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 HIFalpha 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 (Sima.Tango)-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.
Gorr
Thomas A
TA
Division of Hematology, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA.
Tomita
Takeshi
T
Wappner
Pablo
P
Bunn
H Franklin
HF
D000595:Amino Acid Sequence; D000818:Animals; D051784:Aryl Hydrocarbon Receptor Nuclear Translocator; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D015687:Cell Hypoxia; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004330:Drosophila / Q000378:metabolism*; D029721:Drosophila Proteins / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D008969:Molecular Sequence Data; D017385:Sequence Homology; D015398:Signal Transduction; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism
D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D029721:Drosophila Proteins; C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; C100429:Sima protein, Drosophila; D014157:Transcription Factors; C109766:tgo protein, Drosophila; D051784:Aryl Hydrocarbon Receptor Nuclear Translocator