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.