Multiple protein-binding domains and functional cis-elements in the 5'-flanking region of the human pyruvate dehydrogenase .alpha.-subunit gene

Abstract

We have characterized the 5'-flanking region of the alpha-subunit gene of the human pyruvate dehydrogenase (E1). DNase I footprinting with rat liver nuclear extracts identified 7 major protein-binding domains termed P1 through P7 in a 796 base pair DNA fragment (base pairs -763 to +33). P1 through P4 are clustered in the -221/+33 region. These protein-binding domains contain several known consensus sequences such as a TATA box, CAAT box, Sp1, and CRE, which all have previously been implicated in the constitutive transcription of several genes. Oligonucleotide competition studies indicate that oligonucleotides specific for CTF/NF-1 and Sp1 displaced the nuclear proteins bound to the CAAT box (within P3) and an Sp1 site (within P4), respectively. Several other well-characterized and purified transactivators (c-Fos, c-Jun, C/EBP, AP-2, and Sp1) have been shown to bind to the -221/+33 region. Other elements located upstream of the -221/+33 region, which includes nuclease protection domains P5-P7, are required for enhanced promoter activity of the 796 bp sequence. Promoter activity was measured by transient expression of a chloramphenicol acetyltransferase gene ligated to deletion fragments of the 5'-flanking region. Crucial element(s) for promoter activity and complex DNA-nuclear protein interactions were confined within a region spanning -221/+33. This region also retained more than 75% of the promoter activity of the 796 bp sequence. Additionally, this promoter region shows characteristics of both facultative and housekeeping gene promoters, suggesting complex transcriptional regulation.

Pretranslational regulation of pyruvate dehydrogenase complex subunits in white adipose tissue during the suckling-weaning transition in the rat

“…The Elas gene could belong to the rapidly expanding family of genes that are regulated by specific nutrients, although a role for insulin cannot be ruled out at this stage [36,37]. The recent characterization of its promoter [38] should allow us to investigate what factors and DNA elements are involved in this control.…”

Thioltransferase (Glutaredoxin) Reactivates the DNA-binding Activity of Oxidation-inactivated Nuclear Factor I

“…Here we report that thioltransferase can reactivate oxidized NFI protein in vitro in a GSH-dependent manner. In addition, we demonstrate that treatment with buthionine sulfoximine, a known inhibitor of GSH synthesis (24), potentiates the oxidative inactivation of NFI in cultured HeLa cells and that inclusion of N-acetylcysteine (precursor of intracellular GSH) in the culture medium during the recovery period following diamide treatment increases the extent of restoration of the DNA-binding activity of NFI. These findings suggest an important role for thioltransferase and intracellular GSH in the reduction of oxidation-sensitive cysteine residues in the NFI family of transcription factors and potentially other site-specific DNA-binding proteins.…”

“…Subsequent recovery of the cells was performed for 2 h at 37°C in the presence or absence of the protein synthesis inhibitor cycloheximide (25 g/ml). To deplete the cells of glutathione, cells were incubated with 100 M buthionine sulfoximine (BSO) for 48 h. BSO blocks the synthesis of glutathione by inhibiting the enzyme ␥-glutamylcysteine synthetase (24). More than 90% of the cells were viable under this condition.…”

“…HeLa cells were cultured with two agents known to affect the levels of intracellular glutathione, BSO and N-acetylcysteine. BSO reduces intracellular GSH levels by inhibition of ␥-glutamylcysteine synthetase, whereas N-acetylcysteine can increase GSH levels by providing a high intracellular concentration of cysteine, a precursor of GSH (24). HeLa cells were cultured in the presence and absence of BSO or N-acetylcysteine as described under "Materials and Methods."…”

Regulation of mammalian pyruvate dehydrogenase α subunit gene expression by glucose in HepG2 cells

“…The 5.8 kb (k5.8 kb to j33 bp ; J. Tan and M. S. Patel, unpublished work) or the 796 bp (k763 to j33 bp) [25] human E1α promoter was inserted upstream of the chloramphenicol acetyltransferase (CAT) structure gene in pBluescript (Stratagene, but the β-galactosidase gene in the plasmid was deleted). Deletion constructs were generated by using restriction enzyme digestion at AccI, ApaI and StuI sites of the k763 bp promoter.…”