ElA trans-activation, and adenovirus infection results in a stimulation of the DNA- binding activity of OTF-1/NFIII factor

Adenovirus ElA-dependent trans-activation of viral transcription involves the utilization and alteration of multiple sequence-specific transcription factors. Cellular genes are also activated by ElA, one example being the immunoglobulin heavy-chain locus when assayed by transfection into fibroblast cells. We have explored the basis for the E1Adependent activation of this cellular transcription unit. We demonstrate that the ATTTGCAT ("octamer") element found in the heavy-chain enhancer and promoter is a target for ElA trans-activation since this sequence can confer inducibility to the normally unresponsive simian virus 40 early promoter. In addition, adenovirus infection stimulates the DNA-binding activity of the ubiquitous octamer-specific factor, OTF-1, and we presume that this is the basis for the stimulation of transcription. Although there are no octamer elements in the adenovirus genome that are known to be important for transcription, there are octamer elements in the viral terminal repeat sequences. These elements bind the NFIH factor and are important for the initiation ofDNA replication. Since the NFIII factor has been shown to be identical to OTF-1, we suggest that the stimulation of OTF-1/NFUI activity during an adenovirus infection may be important for efficient initiation ofadenovirns DNA synthesis. The use of animal viruses, and especially the DNA tumor viruses, has been invaluable in developing an understanding of eukaryotic gene regulation. Perhaps because of the simplicity of their genomes but also due to the genetics that viruses bring to a mammalian system, these viral systems have provided a unique path to exploring the basis for control of transcription factor activity. In each case, genetic analysis has defined a gene or genes that act in trans to stimulate transcription ofa group ofadditional viral genes (1). One such viral trans-activating gene that has been studied extensively is the ElA gene of adenovirus. The expression of other early genes of adenovirus is dependent on the expression of the ElA gene (2-4), and various studies have shown that this trans-activation is not limited to the early adenovirus genes but extends to a variety of genetically unrelated viral and cellular genes (5, 6). The finding that ElA does not bind to DNA in a sequence-specific manner (7) and that there is no single target sequence shared by all ElA-inducible promoters suggested that ElA does not induce transcription by direct binding to the promoters. Indeed, it is now firmly established that trans-activation by ElA is mediated through cellular transcription factors (1, 8). Moreover, it appears that multiple promoter-specific factors must be targeted as intermediates in the trans-activation event. The E2F factor was initially identified on the basis of an increased DNA-binding activity upon adenovirus infection and the fact that binding required functionally important promoter sequences (9, 10). The same strategy identified an E4-binding protein termed E4F (11). Other studies have identified the TFIIIC transcription factor (12, 13) and a TATA-specific factor (14, 15) as likely to be involved in EMA control. Finally, recent experiments indicate that the AP1 factor is induced by adenovirus infection (16) and, in view of the previous mutagenesis experiments (17), may be important for trans-activation of the E3 promoter. There are other cases, however, where ElA-dependent trans-activation cannot readily be accounted for in terms of the factors already described. One particularly intriguing case is the control of the immunoglobulin heavy-chain locus since, depending on the cell type, ElA can either activate (18) or repress (19) transcription. We have pursued the basis for this control by analyzing factors in extracts of virus-infected cells that interact with regulatory elements of the immunoglobulin enhancer. Although we find no evidence for an alteration that might explain the negative control by ElA, we do find that the ubiquitous OTF-1 factor, which binds specifically to the 8-base-pair sequence 5'-ATTTGCAT-3' ("octamer"), is increased in adenovirus-infected fibroblasts. This correlates with the finding that the octamer element can confer EMA control on a heterologous promoter. MATERIALS AND METHODS Cells and Virus. CV-1 monkey kidney cells were grown as monolayers in Dulbecco's modified Eagle's medium (DMEM) containing 5% bovine calf serum. F9 mouse embryonal carcinoma cells were maintained in the same medium containing 10%o fetal bovine serum and were induced to differentiate by the addition of retinoic acid (0.1 ,uM) and dibutryl cAMP (1 mM) as described (20). The growth and purification of wild-type adenovirus 5 (AdS) and the E1Adeficient mutant d1312 have been described (21). Infection and Whole Cell Extract Preparation. Whole cell extracts were prepared from CV-1 cells infected for 18 hr with 4000 particles of wild-type Ad5 per cell, as described (11). Extracts were made from undifferentiated F9 cells, F9 cells differentiated for 5 days, or F9 cells differentiated for 4 days and then infected with Ad5 for 18 hr by the same protocol. Gel Shift Assay. The Dde I-Hinfl fragment of the immunoglobulin heavy-chain enhancer was 3'-end-labeled by DNA polymerase Klenow fragment and used as a probe. A similar synthetic oligoncleotide fragment with the octamer sequence mutated as shown in Fig. 1 was used for competitions. Gel shift assays were performed essentially as described (22), using 10 gg of whole cell extract and 3 ,jg of poly(dI-dC)poly(dI-dC) as competitor. Abbreviations: AdS, adenovirus 5; CAT, chloramphenicol acetyltransferase; SV40, simian virus 40. *To whom reprint requests should be addressed. 5878 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. Proc. Natl. Acad. Sci. USA 87 (1990) 5879