Functional characterization of the 5'-flanking region of the gene encoding human 2-oxoglutarate dehydrogenase.
The 5'-flanking region of the gene (OGDH) encoding human 2-oxoglutarate dehydrogenase (OGDH) does not contain TATA or CAAT boxes, but contains an inverted GC box. To identify a functional OGDH promoter, systematic transient expression analysis of the 5'-flanking region (wild type, -3276/+212) of the OGDH was performed using serially nested deletions and linker-scanning mutations. The OGDH (wild type)-luciferase (LUC) reporter vector (pGV-E) construct and its deletion or linker-mutant constructs were transfected into BHK-21 cells and LUC activity was assessed. The OGDH-LUC construct expressed reproducibly >12-fold more LUC activity than did a control pGV-E vector. The promoter activity was up-regulated by treatment with 2-oxoglutarate and 2-oxoglutarate/glutamate. Deletions of sequences between nt -563 and -62 (M6 and M7) resulted in a 2-fold increase in LUC activity. Further deletion of the sequence between nt -61 and +212 (M8-M10) abolished LUC activity. High resolution mutagenesis within the -113 to -14 region indicated that the -53 to -44 and -33 to -24 sequences were required for positive regulation and the -93 to -84 sequence for negative regulation. We have identified a nuclear factor that binds to nt -63 to -24 including two cis-acting sites.
Koike
K
K
Department of Pathological Biochemistry, Atomic Disease Institute, Nagasaki University School of Medicine, Sakamoto, Japan.
Matsuo
S
S
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D002478:Cells, Cultured; D002874:Chromosome Mapping; D006224:Cricetinae; D017930:Genes, Reporter; D006367:HeLa Cells / Q000378:metabolism; D006801:Humans; D007655:Ketoglutarate Dehydrogenase Complex / Q000235:genetics*; D007668:Kidney / Q000166:cytology / Q000378:metabolism; D008156:Luciferases / Q000096:biosynthesis / Q000235:genetics; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid; D017384:Sequence Deletion; D014162:Transfection
D009687:Nuclear Proteins; D011994:Recombinant Proteins; D008156:Luciferases; D007655:Ketoglutarate Dehydrogenase Complex