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.