The liver-type and muscle-type isozymes of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase are encoded by one gene that uses two alternative promoters. We have identified cis-acting sequences and protein-binding sites on the liver-type promoter. Transfection assays with deleted promoters showed that maximal promoter activity is contained within 360 bp upstream of the cap site. DNase I footprinting experiments with liver and spleen nuclear extracts and with purified proteins revealed several protein-binding sites in this region. These included four binding sites for nuclear factor I, one site that contains an octamer consensus but showed a liver-specific footprint pattern, two liver-specific protein-binding sites, and one poly(dG)-containing binding site. Transfection of cells of hepatic origin suggested that all these sites except one are involved in transcriptional regulation. The region between -360 and -2663 contained an element that functioned as a silencer in a nonhepatic cell line. We conclude that in liver transcription from the liver-type promoter of the 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase gene is controlled by ubiquitous and tissue-specific factors and involves activating and derepressing mechanisms.Fructose-2,6-bisphosphate is the most potent stimulator of 6-phosphofructo-1-kinase, a key enzyme of glycolysis. The synthesis and degradation of fructose-2,6-bisphosphate are catalyzed, respectively, by 6-phosphofructo-2-kinase (PFK-2) and fructose-2,6-bisphosphatase (FBPase-2). In liver these two activities are borne by separate domains of a homodimeric protein which is therefore one ofthe rare bifunctional enzymes. Several PFK-2/FBPase-2 isozymes whose expression differs depending on the tissue have been described (for a review, see reference 12). The molecular structure of the isozymes is unknown, except for the liver (L) and muscle (M) isozymes. We have indeed cloned a 55-kb rat gene, containing 15 exons, that encodes the L and M isozymes by alternative use of two different promoters, referred to as L and M promoters. The L and M mRNAs share the same 13 exons. They have an additional exon which is specific for either the M isozyme (first exon of the gene) or the L isozyme (second exon of the gene). Thus, the L promoter region lies between these two exons (7). The concentration of L mRNA is highest in liver, but this mRNA is detectable in other tissues (5). Its concentration in liver is regulated by hormones and is increased by insulin in diabetic rats (3) and by triiodothyronine (T3) in hypothyroid rats (31). Transcription of the liver mRNA is also increased by glucocorticoids in adrenalectomized rats (16). The PFK-2/ FBPase-2 L promoter is therefore an interesting model for studying the tissue-specific and hormonal regulation of transcription. We have delineated here the 5'-flanking sequences medium supplemented with 5% fetal calf serum (FCS), 1 ,uM dexamethasone, 1 ,uM T3, and 10 nM insulin. Twenty-four hours before transfection, 1.5 x 106 ATIII-SV40 cells per 60-mm dish were pla...

DNase I footprinting experiments showed that binding activities of Spl and of GHF-1 to its distal site on the human growth hormone gene promoter are mutually exclusive. The kinetics of GHF-1 binding were indicative of positive cooperativity. The Spl site did not affect promoter activity in cell-free transcription. Still, Spl could compensate partially for the decreased stimulation of transcription seen at low GHF-1 concentrations.