TWO CRM1-DEPENDENT NUCLEAR EXPORT SIGNALS ARE INVOLVED IN THE REGULATION OF HIF1/SIMA.

NURIA ROMERO, MAXIMILIANO IRISARRI, ANA CAHUERFF AND PABLO WAPPNER

Rosario, Santa Fé

Congreso; SAIB 2006; 2006

Sociedad Argentina de Investigación en Buímica y biología molecular

The heterodimeric transcription factor HIF (Hypoxia Inducible Factor) has a central conserved role in oxygen homeostasis and is composed of two basic-helix-loop-helix (bHLH)-PAS protein subunits, HIFa and HIFb. HIFa is regulated by oxygen levels through several different mechanisms that include protein stability, transcriptional co-activator recruitment and subcellular localization. We have previously reported that the Drosophila HIF-a protein, Sima, is mainly nuclear in hypoxia and accumulates in the cytoplasm in normoxia, but so far the molecular basis of this regulation is unclear. We show here that Sima shuttles continuously between the nucleus and the cytoplasm. Removal of the bHLH domain led to Sima nuclear accumulation, consistent with the occurrence of two nuclear export signals (NES) in this region that promote CRM1-dependent Sima nuclear export, both in cell culture and in vivo. Site directed mutagenesis of either NES provoked Sima nuclear retention and increased transcriptional activity of a LacZ reporter and of endogenous target genes, suggesting that nuclear export contributes to Sima regulation. These NES are conserved and functional in the bHLH domain of several other proteins of the bHLH-PAS family and therefore, we propose that these NES are important for rapid nuclear clearance of bHLH-PAS proteins upon cessation of the external stimulus.