Identification of the promoter region of human placental 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase gene.
The placenta-type isozyme of human 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (HP2K) is expressed in several tissues such as placenta, brain, testis, liver, kidney, skeletal muscle, and primary blood mononuclear cells. To better understand the regulation of HP2K gene expression, we isolated and characterized its genomic DNA, which includes the promoter region. The results of oligo-capping analysis indicate that the transcription start point (tsp) is an adenine residue 329 bp upstream of the translational start codon. DNA sequence analysis of this gene shows that the promoter region that contains the TATA box sequence and the 5'-UTR is different from the other known PFK-2/F2, 6BPase genes. In addition, its 5'-flanking and 5'-UTR both have G + C-rich sequences containing Sp1 binding sites. To identify the promoter/enhancer region of HP2K gene, we performed transfection analyses of human choriocarcinoma BeWo cells with HP2K promoter-luciferase constructs. These experiments identified a promoter region 164 bp upstream from the tsp and an enhancer region between -1265 and -1329 on the 5'-flanking sequences. We also showed that Sp1 sites were not essential for HP2K transcription. Following transfection, stimulation experiments with serum, progesterone and phorbol 12-myristate 13-acetate showed that only the construct with the enhancer containing putative early growth response-1 binding motif was responsive to serum. We propose that the transcription of HP2K is strictly controlled by tissue-specific factors even though its genomic DNA contains several transcriptional elements.
Fukasawa
M
M
Department of Microbiology, Department of Parasitology, National Defense Medical College, Namiki 3-2, Tokorozawa, Sitama, 359-8513, Japan.
Takayama
E
E
Shinomiya
N
N
Okumura
A
A
Rokutanda
M
M
Yamamoto
N
N
Sakakibara
R
R
D020121:5' Untranslated Regions / Q000235:genetics; D000595:Amino Acid Sequence; D001483:Base Sequence; D002460:Cell Line; D005260:Female; D006597:Fructose-Bisphosphatase / Q000235:genetics*; D017930:Genes, Reporter; D015698:Genomic Library; D006801:Humans; D008156:Luciferases / Q000235:genetics; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000235:genetics*; D010732:Phosphofructokinase-1 / Q000235:genetics*; D025481:Phosphofructokinase-2; D010920:Placenta / Q000201:enzymology*; D011247:Pregnancy; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D014158:Transcription, Genetic; D014162:Transfection
D020121:5' Untranslated Regions; D009097:Multienzyme Complexes; D011993:Recombinant Fusion Proteins; D008156:Luciferases; D025481:Phosphofructokinase-2; D010732:Phosphofructokinase-1; D006597:Fructose-Bisphosphatase