Two Sp Sites Are ImportantcisElements Regulating the Upstream Promoter Region of the Gene for Rat Type I Hexokinase

Multiple transcriptional start sites have been identified for the gene encoding the rat Type I isozyme of hexokinase (White, J. A., Liu, W., and Wilson, J. E.,Arch. Biochem. Biophys.335, 161–172, 1996); these are clustered at positions approximately −460, −300, and −100 relative to the translational start codon (ATG, with A being +1). PC12 cells and H9c2 cells were transfected with luciferase reporter constructs containing genomic sequence between positions −3366 and −171. Marked (85%) decrease in promoter activity was associated with deletion of sequence between −742 and −516. In DNase I footprinting experiments, two regions, called P1 (−552 to −529) and P2 (−480 to −458) boxes, were protected by proteins present in nuclear extracts from PC12 cells. Mutation or deletion of the P2 box had no effect on promoter activity; protection in this region, which includes the most upstream cluster of transcriptional start sites, is attributed to binding of RNA polymerase II or associated factors. In contrast, mutations or deletions in the P1 box had markedly detrimental effects on promoter activity and on binding of proteins in PC12 cell nuclear extracts. Maintenance of a consensus Sp1 binding site centrally located in the P1 box was critical for both promoter activity and binding. A second Sp1 site (−570), just upstream from the P1 box, was also shown to be functionally important but no protection of this region was detected in footprinting experiments, presumably reflecting lower affinity at this site under the conditions used. Supershift experiments demonstrated the involvement of Sp1, Sp3, and Sp4 in formation of complexes with the P1 box region and implicate these transcription factors in regulating promoter activity associated with this region. Another series of reporter constructs, including sequence between −171 and −1, permitted detection of an additional promoter activity downstream from −364. While not yet extensively characterized, it is already evident that theciselements influencing the downstream promoter activity are distinct from the Sp factors determined to be important in expression from the upstream promoter region.