HYPOXIA REGULATES BETA-ENOLASE AND PYRUVATE KINASE-M PROMOTERS BY MODULATING SP1 SP3 BINDING TO A CONSERVED GC ELEMENT/

The transcription rates of glycolytic enzyme genes are coordinately in duced when cells are exposed to low oxygen tension. This effect has be en described in many cell types and is not restricted to species or ph yla. In mammalian cells, there are 11 distinct glycolytic enzymes, at least 9 of which are induced by hypoxia. Recent reports described a ro le for the hypoxia-inducible factor-1 (HIF-1) in the transcriptional a ctivation of lactate dehydrogenase A, aldolase-A, phosphoglycerate kin ase, and enolase-l genes. It is not known whether the HIF-1 factor act s exclusively to regulate these genes during hypoxia, or how the other genes of the pathway are regulated. In this paper, we describe analys es of the muscle-specific pyruvate kinase-M and beta-enolase promoters that implicate additional mechanisms for the regulation of glycolytic enzyme gene transcription by hypoxia, Transient transcription of a re porter gene directed by either promoter was activated when transfected muscle cells were exposed to hypoxia. Neither of these promoters cont ain HIF-1 binding sites. Instead, the hypoxia response was localized t o a conserved GC-rich element positioned immediately upstream of a GAT AA site in the proximal promoter regions of both genes. The GC element was essential for both basal and hypoxia-induced expression and bound the transcription factors Spl and Sp3. Hypoxia caused the progressive depletion of Sp3 determined by DNA binding studies and Western analys es, whereas Spl protein levels remained unchanged, Overexpression of S p3 repressed expression of p-enolase promoters. It is concluded that h ypoxia activates these glycolytic enzyme gene promoters by down-regula ting Sp3, thereby removing the associated transcriptional repression.