The polyomavirus enhancer is composed of multiple DNA sequence elements serving as binding sites for proteins present in mouse nuclear extracts that activate transcription and DNA replication. We have identified three such proteins and their binding sites and correlate them with enhancer function. Mutation of nucleotide (nt) 5140 in the enhancer alters the binding site (TGACTAA, nt 5139-5145) for polyomavirus enhancer A binding protein 1 (PEA1), a murine homolog of the human transcription factor activator protein 1 (AP1). This mutation simultaneously reduces polyomavirus transcription and DNA replication. Reversion of this mutation simultaneously restores binding of PEA1 and both DNA replication and transcription. Binding of a second protein, PEA2, adjacent to the PEA1 site at nt 5147-5155 is enhanced by PEA1 binding, suggesting that these proteins interact. A third protein, PEA3, binds to the sequence AGGAAG (nt 5133-5138) adjacent to the PEA1 binding site; integrity of this late-proximal PEA3 binding site or an additional earlyproximal site (nt 5228-5233) is important for enhancer function. We correlate binding of PEA1 and PEA2 with the induction of a DNase 1-hypersensitive site in polyomavirus minichromosomes isolated from mouse fibroblasts.The polyomavirus enhancer activates the viral early promoter in vivo and is required for viral DNA replication (1)(2)(3)(4)(5)(6)(7)(8)(9)(10). It is composed of multiple functionally redundant DNA elements whose activities vary with cell type and growth state (2, 5-7, 9, 11, 12). These elements serve as binding sites for cellular proteins (13-20) that most likely help form initiation complexes at cis-linked origins and promoters (5,14,21).Two cellular proteins (15) bind to the polyomavirus enhancer at nucleotides (nt) 5139-5155 of the A3 strain (22) or nt 5114-5130 of the A2 strain (23). Polyomavirus enhancer A binding protein 1 (PEA1) binds to nt 5139-5145 (A2 strain nt 5114-5120), which make up the consensus sequence for the HeLa cell transcription factor activator protein 1 (AP1) (TGACTAA). AP1 activates the human metallothionein and simian virus 40 enhancers (24); it is most likely encoded by the human protooncogene c-jun (25), and it shares substantial sequence homology with the DNA-binding domains of yeast . PEA1 is probably the murine homolog of AP1 (21, 30).PEA2 binds to nt 5147-5154 (A2 strain nt 5122-5129), adjacent to the PEA1 binding site. An additional factor, polyomavirus enhancer B binding protein 1 (PEB1), binds to other enhancer sequences between nt 5180 and 5220 (A2 strain nt 5155-5195) (13,14,21), and several proteins- Because of the functional redundancy of the polyomavirus enhancer, we chose to inactivate multiple important elements by introducing numerous random point mutations. Using this approach, we identified several polyomaviruses whose DNA replication and transcription were greatly reduced because of point mutations in the enhancer (5). In this report we provide evidence for the involvement of PEA1 and PEA2, with a third factor, PEA3, ...

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.

We have studied the assembly of GA-binding protein (GABP) in solution and established the role of DNA in the assembly of the transcriptionally active GABP␣ 2 ␤ 2 heterotetrameric complex. GABP binds DNA containing a single PEA3/Ets-binding site (PEA3/EBS) exclusively as the ␣␤ heterodimer complex, but readily binds as the GABP␣ 2 ␤ 2 heterotetramer complex on DNA containing two PEA3/EBSs. Positioning of the PEA3/EBSs on the same face of the DNA helix stabilizes heterotetramer complex binding. These observations suggest that GABP␣␤ heterodimers are the predominant molecular species in solution and that DNA containing two PEA3/ EBSs promotes formation of the GABP␣ 2 ␤ 2 heterotetrameric complex. We analyzed the assembly of GABP␣ 2 ␤ 2 heteromeric complexes in solution by analytical ultracentrifugation. GABP␣ exists as a monomer in solution while GABP␤ exists in a monomer-dimer equilibrium (K d ‫؍‬ 1.8 ؎ 0.27 M). In equimolar mixtures of the two subunits, GABP␣ and GABP␤ formed a stable heterodimer, with no heterotetramer complex detected. Thus, GABP exists in solution as the heterodimer previously shown to be a weak transcriptional activator. Assembly of the transcriptionally active GABP␣ 2 ␤ 2 heterotetramer complex requires the presence of specific DNA containing at least two PEA3/EBSs.

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.