title: Sp/Krüppel-like transcription factors are essential for the expression of mitochondrial glycerol phosphate dehydrogenase promoter B.
abstract: The human mitochondrial glycerol phosphate dehydrogenase (hmGPD) is abundant in the normal pancreatic insulin cell and its level is lowered to 50% by high glucose and diabetes. Using Drosophila and INS-1 cells, we have analysed the hmGPD gene promoter B to characterize cis-elements and trans-acting factors that affect its regulation. We identified ten efficient Sp/Krüppel-like transcription-factor-binding sites in the promoter sequence. These sites include four GC-boxes (CCCGCCC at -227, -68, -46 and GGGCGAG at -382), three GT-boxes (CCCCACCC at -350, CCCACACCC at -257, and CACCCGCCC at -48), and three CT/GA-boxes (TCCCTCCC at -262, GGGAGGGAG at -129, and GGGAGGAGGA at -107). Transfection of Drosophila SL2 cells, which lack Sp/Krüppel-like factors, with constructs encoding either Sp1, Sp3, Sp4 or erythroid Krüppel-like factor (EKLF) specifically activates the hmGPD promoter B up to 50-fold. Promoter activation requires the Sp1 activation and the DNA binding domains. Co-transfected EKLF was synergistic with either Sp1 or Sp3. On the other hand, the basic Krüppel-like factor (BKLF) inhibited Sp1-and EKLF-mediated promoter activation. Similarly, constructs encoding either GA-binding protein (GABP) or PU.1 inhibited Sp1-mediated promoter activation. Oligonucleotide 'decoys' with potential transcription factor binding sites decreased promoter activity in both INS-1 and Drosophila cells. Significant loss of Sp- and EKLF-mediated activation was observed with some internal as well as sequential 5' deletions of the promoter DNA. The level of Sp1 protein was reduced by 50% in INS-1 cells chronically exposed to a high concentration of glucose. The results demonstrate that Sp/Krüppel-like factors are essential for mGPD gene expression.
authors.affiliation: The Children's Diabetes Center, The University of Wisconsin, Room 3550 Medical Science Center, 1300 University Avenue, Madison 53706, USA. nmhasan@facstaff.wisc.edu
authors.forename: Noaman M
authors.initials: NM
authors.lastname: Hasan
authors.forename: Michael J
authors.initials: MJ
authors.lastname: MacDonald
mesh_terms: D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002460:Cell Line; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D051268:GA-Binding Protein Transcription Factor; D015971:Gene Expression Regulation, Enzymologic; D005993:Glycerolphosphate Dehydrogenase / Q000235:genetics*; D006367:HeLa Cells; D006801:Humans; D051741:Kruppel-Like Transcription Factors; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008928:Mitochondria / Q000201:enzymology*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016254:Mutagenesis, Insertional; D009154:Mutation; D009841:Oligonucleotides / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011485:Protein Binding; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins; D017384:Sequence Deletion; D016329:Sp1 Transcription Factor / Q000378:metabolism; D051705:Sp3 Transcription Factor; D051706:Sp4 Transcription Factor; D015534:Trans-Activators / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism*; D014162:Transfection; D014407:Tumor Cells, Cultured; D000099232:Proto-Oncogene Protein Spi-1
chemical_list: D004247:DNA; D004268:DNA-Binding Proteins; D051268:GA-Binding Protein Transcription Factor; D005993:Glycerolphosphate Dehydrogenase; D051741:Kruppel-Like Transcription Factors; D008156:Luciferases; D009841:Oligonucleotides; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D016329:Sp1 Transcription Factor; D051705:Sp3 Transcription Factor; D051706:Sp4 Transcription Factor; D015534:Trans-Activators; D014157:Transcription Factors; D000099232:Proto-Oncogene Protein Spi-1; C501168:KLF3 protein, human; C497121:SP3 protein, human; C497124:SP4 protein, human; C497123:Sp3 protein, rat; C080774:erythroid Kruppel-like factor