Sp/Krüppel-like transcription factors are essential for the expression of mitochondrial glycerol phosphate dehydrogenase promoter B.
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.
Hasan
Noaman M
NM
The Children's Diabetes Center, The University of Wisconsin, Room 3550 Medical Science Center, 1300 University Avenue, Madison 53706, USA. nmhasan@facstaff.wisc.edu
MacDonald
Michael J
MJ
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
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