Characterization of the human P-type 6-phosphofructo-1-kinase gene promoter in neural cell lines.
In humans three isoforms of 6-phosphofructo-1-kinase (PFK) exist. Among them platelet-type PFK (PFKP) is highly abundant in the brain. With its distinct allosteric properties PFKP is regarded to be the key enzyme for the regulation of glycolysis in this organ. We cloned 1.7 kb of the 5' upstream promoter of the human PFKP gene and analyzed the promoter activity by deletion and mutation analysis using a luciferase reporter. The transcription start point was determined at 48 bp upstream of the start codon. In deletion studies the region -65 to +48 turned out to be sufficient for promoter activity while fragment -153 to +48 showed the highest promoter activity. Sequence analysis of the region from -153 to +48 revealed a stretch of eight adjacent putative transcription factor binding sites, seven of which are Sp-family specific sites. Sp1 and Sp3 were shown to bind to most if not all of them. Additionally, an NF-Y binding site was identified. Results of deletion and mutation analysis suggest that all of these transcription factors contribute positively to promoter activity. The methylation status of the promoter region was analyzed in different neural tumor cell lines and compared with that in human leukocytes and muscle.
Hannemann
Anke
A
Institute of Biochemistry, University of Leipzig (Medical Faculty), Liebigstrasse 16, D-04103 Leipzig, Germany.
Jandrig
Burkhard
B
Gaunitz
Frank
F
Eschrich
Klaus
K
Bigl
Marina
M
D001483:Base Sequence; D003001:Cloning, Molecular; D019175:DNA Methylation; D004268:DNA-Binding Proteins / Q000378:metabolism; D017930:Genes, Reporter; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009474:Neurons / Q000378:metabolism*; D010732:Phosphofructokinase-1 / Q000235:genetics*; D011401:Promoter Regions, Genetic; D016329:Sp1 Transcription Factor / Q000378:metabolism; D051705:Sp3 Transcription Factor; D014157:Transcription Factors / Q000378:metabolism; D024363:Transcription Initiation Site; D014162:Transfection; D014407:Tumor Cells, Cultured
D004268:DNA-Binding Proteins; C497121:SP3 protein, human; D016329:Sp1 Transcription Factor; D014157:Transcription Factors; D051705:Sp3 Transcription Factor; D010732:Phosphofructokinase-1; C068652:1-phosphofructokinase