ACTIVATION OF HYPOXIA-INDUCIBLE FACTOR-I - DEFINITION OF REGULATORY DOMAINS WITHIN THE ALPHA-SUBUNIT

Hypoxia-inducible factor-1 (HIF-1), a heterodimeric DNA binding comple x composed of two basic-helix-loop-helix Per-AHR-ARNT-Sim proteins (HI F-1 alpha and -1 beta), is a key component of a widely operative trans criptional response activated by hypoxia, cobaltous ions, and iron che lation, To identify regions of HIF-1 subunits responsible for oxygen-r egulated activity, we constructed chimeric genes in which portions of coding sequence from HIF-1 genes were either linked to a heterologous DNA binding domain or encoded between such a DNA binding domain and a constitutive activation domain, Sequences from HIF-1 alpha but not HIF -1 beta conferred oxygen-regulated activity, Two minimal domains withi n HIF-1 alpha (amino acids 549-582 and amino acids 775-826) were defin ed by deletional analysis, each of which could act independently to co nvey inducible responses. Both these regions confer transcriptional ac tivation, and in both cases adjacent sequences appeared functionally r epressive in transactivation assays. The inducible operation of the fi rst domain, but not the second, involved major changes in the level of the activator fusion protein in transfected cells, inclusion of this sequence being associated with a marked reduction of expressed protein level in normoxic cells, which was relieved by stimulation with hypox ia, cobaltous ions, or iron chelation, These results lead us to propos e a dual mechanism of activation in which the operation of an inducibl e activation domain is amplified by regulation of transcription factor abundance, most likely occurring through changes in protein stability .