ROLE OF THE STIMULATORY PROTEINS SP1 AND SP3 IN THE REGULATION OF TRANSCRIPTION OF THE RAT PYRUVATE-KINASE-M GENE

Site-directed mutagenesis of cis-regulatory elements in the 5' flankin g region of the rat pyruvate kinase M gene revealed that two out of th e three GC boxes (-133/-124 and -48/-39) are involved in the stimulati on of a core promoter (-35/+46). These two regions were also protected in DNaseI footprinting assays. Spl and Sp3 were identified as binding proteins to all three GC boxes by supershift experiments. Cotransfect ions in Drosophila SL2 cells revealed a strong stimulatory function of Sp1 and a synergistic effect of Sp3 to Sp1 in the activation of the p yruvate kinase M promoter. No inhibitory effect of Sp3 was detected. T hese data indicate that binding of Sp1 at two GC boxes is required for full promoter activity of the pyruvate kinase M gene and thus contrib utes to the observed cell-cycle-dependent expression of this enzyme in proliferating rat thymocytes.