THE ANAEROBIC RESPONSIVE ELEMENT CONTAINS 2 GC-RICH SEQUENCES ESSENTIAL FOR BINDING A NUCLEAR-PROTEIN AND HYPOXIC ACTIVATION OF THE MAIZE ADH1 PROMOTER

We have identified a protein (GCBP-1) in nuclear extracts from maize suspension cell cultures that binds to specific sequences within the Anaerobic Responsive Element (ARE) of the maize Adh1 promoter. Competition analyses show that the GCBP-1 binding activity distinguishes ARE sequence motifs from other enhancer elements or pUC19 sequences. The binding activities of several mutant ARE sequences define two regions of the ARE important for GCBP-1 binding in vitro, between nucleotides -135 to -131 and nucleotides -120 to -112 of the maize Adh1 promoter. Both regions are required for efficient GCBP-1 binding to occur in vitro. The minimum consensus binding site for GCBP-1 is 5'-GC(G/C)CC-3'. This sequence is similar to a part of the binding site of the human transcription factor Sp1 (1). We demonstrate that maize GCBP-1 and human Sp1 have similar recognition properties. Using ARE mutants in a transient assay in maize protoplasts we have shown that mutation of the GCBP-1 binding sites prevents significant hypoxic activation of the maize Adh1 promoter. These results suggest a direct role for GCBP-1 in the hypoxic activation of Adhl gene expression. GCBP-1 is present in both uninduced and induced nuclei, indicating that inducible gene expression is not dependent upon synthesis of GCBP-1 and suggesting that post-translational modification of bound GCBP-1 may be important for enhanced transcription to occur.