We have investigated the reduction/oxidation (redox) regulation of the heteromeric transcription factor GAbinding protein (GABP). GABP, also known as nuclear respiratory factor 2, regulates the expression of nuclear encoded mitochondrial proteins involved in oxidative phosphorylation, including cytochrome c oxidase subunits IV and Vb, as well as the expression of mitochondrial transcription factor 1. GABP is composed of two subunits, the Ets-related GABP-␣, which mediates specific DNA binding, and GABP-, which forms heterodimers and heterotetramers on DNA sequences containing the PEA3/Ets motif ((C/A)GGA(A/T)(G/A)). We demonstrate here that GABP DNA binding activity and GABP-dependent gene expression in 3T3 cells are inhibited by pro-oxidant conditions. DNA binding of recombinant GABP-␣ was activated by chemical reduction (dithiothreitol) and by thioredoxin; however, GSSG inhibited GABP DNA binding activity. Treatment of GABP-␣, but not GABP- 1 , with sulfhydryl-alkylating agents also inhibited GABP DNA binding activity. Our results suggest that GABP DNA binding activity is redox-regulated in vivo, possibly by thioredoxin-mediated reduction and by GSSG-mediated oxidation of the GABP-␣ subunit. The regulation of GABP (nuclear respiratory factor 2) DNA binding activity by cellular redox changes provides an important link between mitochondrial and nuclear gene expression and the redox state of the cell.
The transcription factor GA-binding protein (GABP) is composed of two subunits, GABP␣ and GABP. The DNA-binding subunit, GABP␣, is a member of the Ets family of transcription factors, characterized by the conserved Ets-domain that mediates DNA binding and associates with GABP, which lacks a discernible DNA binding domain, through ankyrin repeats in the NH 2 terminus of GABP. We previously demonstrated that GABP is subject to redox regulation in vitro and in vivo through four COOH-terminal cysteines in GABP␣. To determine the roles of individual cysteines in GABP redox regulation, we generated a series of serine substitution mutants by site-directed mutagenesis and identified three redox-sensitive cysteine residues in GABP␣ (
Many eukaryotic RNA polymerase II promoters contain initiator elements which direct accurate transcription in a TATA-independent manner. The PEA3/Ets-binding site (PEA3/EBS) is a common enhancer element in eukaryotic genes and is also found near the transcriptional start sites of many TATA-less promoters. We demonstrate that two PEA3/EBSs driving expression of the luciferase reporter gene, function as a minimal transcriptional initiator element. Maximal levels of transcription was achieved when two PEA3/EBSs, in either orientation, were located on the same face of the DNA helix, and the sites could be separated by up to three helical turns. In vitro transcription start sites directed by PEA3/EBS elements were clustered on either side of the upstream PEA3/EBS and were abolished by immunodepletion of GA-binding protein (GABP) from FM3A cell nuclear extracts. In vivo, co-transfection of GABPalpha and GABPbeta expression vectors enhanced reporter gene expression driven from PEA3/EBS initiator elements. Like other initiator elements, the PEA3/EBS elements were activated synergistically by upstream Sp1-binding sites. Thus, our results establish GABP as both a transcriptional activator factor and as an initiator factor.
Proteins binding to the PEA3 enhancer motif (AGGAAG) activate the polyomavirus early promoter and help comprise the viral late mRNA initiator element (W. Yoo, M. E. Martin, and W. R. Folk, J. Virol. 65:5391-5400, 1991). Because many developmentally regulated cellular genes have PEA3 motifs near their promoter sequences, and because Ets family gene products activate the PEA3 motif, we have studied the expression of PEA3-binding proteins and Ets-related proteins during differentiation of F9 embryonal carcinoma cells. An -91-kDa protein (PEA3-91) was identified in F9 cell nuclear extracts by UV cross-linking to a radiolabeled PEA3 oligonucleotide probe, and expression of PEA3-91 was down-regulated after differentiation of F9 cells to parietal endoderm. The c-ets-I gene product binds to a sequence in the murine sarcoma virus long terminal repeat that is similar to the PEA3 motif (cGGAAG), but PEA3-91 was not cross-linked to this Ets-l-binding motif, nor did antiserum which recognizes murine c-ets-1 and c-ets-2 proteins have any effect on PEA3-binding activity in mobility shift assays. Furthermore, c-ets-1 mRNA was not detected in undifferentiated or differentiated F9 cells, and c-ets-2 mRNA levels remained high after differentiation. Antiserum against the Drosophila Ets-related E74A protein, however, recognized an -92-kDa protein in F9 cells whose expression during differentiation varied in a manner identical to that of PEA3-91. These data suggest that PEA3-91 is not the product of the ets-l or ets-2 genes but is likely to be the product of a murine homolog of the Drosophila E74 gene.
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