Role of an adenovirus E 2 promoter binding factor in ElA - mediated coordinate gene control ( transcription control / trans - activation / promoter methylation )

A product of the adenovirus gene EIA is responsible for the stimulation of transcription from six viral promoters as well as at least two cellular promoters. We have detected a HeLa cell factor, termedE2 promoter binding factor (E2F), that appears to mediate the transcriptional stimulation of the viral E2 promoter. Competition experiments revealed that E2F did not recognize and bind to the EIB, E3, E4, or major late promoter sequences. Furthermore, three additional promoters stimulated by ElA, heat shock protein 70, f3-globin, and early simian virus 40, do not bind E2F. In contrast, the factor does recognize sequences in the EIA enhancer, and within the EIA enhancer are duplicated binding sites for E2F. Finally, a single E2F binding site from the EIA enhancer can confer increased transcription to a mouse /3-globin promoter, dependent on the action of the EIA gene product. This stimulation requires binding of E2F since methylation of the binding site, which blocks binding in vitro, reduces transcription stimulation in vivo. We, therefore, conclude that E2F is likely to be responsible for the ElA-mediated stimulation of the EIA gene as well as the E2 gene but is not involved in the activation of the other ElA-inducible promoters. The basis for coordinate control of transcription of a set of genes, in response to any given stimulus, is a crucial aspect of gene control in eukaryotic cells. In some cases, such control may be due to a single factor that recognizes multiple genes while in other cases multiple promoter-specific factors, controlled by a common regulatory stimulus, may be responsible. One system of coordinate gene control that is particularly amenable to study is the set of adenovirus early genes that are regulated by the product of the EIA gene (1-3). We have suggested (4) a mechanism for activation by ElA whereby there is enhanced binding of a cellular factor to critical promoter sequences. Indeed, we have identified a cellular factor, termed E2 promoter binding factor (E2F), that appears to be responsible for the transcriptional stimulation of one of the viral genes (5). In this report, we have investigated the involvement ofE2F in the coordinate control of the set of early genes. MATERIALS AND METHODS Cells and Virus. HeLa cells grown either in suspension in Joklik's modified minimal essential medium containing 5% (vol/vol) calf serum or in monolayer in Dulbecco's modified Eagle's medium (DMEM) containing 10% (vol/vol) fetal calf serum were used. The growth and preparation of wild-type adenovirus type S (AdS) and ofthe ElA-deletion mutant d1312 have been described (3, 6). Nuclear Extracts. The preparation of nuclear extracts by the procedure of Dignam et al. (7) from adenovirus-infected HeLa cells was described (5). Binding Assays. Binding assays were performed using an end-labeled E2 probe, depicted in Fig. 1, by procedures described (5). Hha I Methylation. Methylation with the Hha I methylase was carried out according to the instructions of the supplier (New England Biolabs). Transfection Assays. HeLa cells were infected with 1000 particles per cell of Ad5 or d1312. After 8 hr, cells were transfected with 10 jg of 3globin-specific plasmids and 10,g of pUC19 using calcium phosphate coprecipitation, and 4 hr later cells were glycerol-shocked (8, 9). The cells were maintained throughout in the presence of cytosine arabinonucleoside (25 ,g/ml). Total RNA was prepared 48 hr after transfection by cesium chloride centrifugation (10) and analyzed for ,8-globin RNA using an SP6 RNA probe specific for the first two exons of /3-globin (gift of R. Costa). Details ofthe procedures for hybridization of SP6 probes and analysis of protected fragments have been described (11). Plasmids. The pE2 plasmid has been described (12). The pElb plasmid is an Hpa I/HindIII fragment of adenovirus type 2 (4.4 to 8.0 map units) in pBR322. The pE3 plasmid (gift of D.-W. Huang) is an EcoRI/Sst I fragment of adenovirus type 2 (76.0-76.8 map units) in pUC12. The pE4 plasmid (gift of P. Fischer, Columbia Univ.) contains AdS sequences from 93.5 to 100 map units in pBR322. pML is a Pst I/HindIII fragment of adenovirus type 2 (14.0-17.0 map units) in pGEM1. pp-glo contains mouse globin sequences from position -1221 to position +482 (relative to the cap site at +1) in pUC13. pSV40 is the pSV2-neo vector (13). The pElA plasmid contains Ad5 sequences from the left terminus of the genome (0 map units) to the Xba I site at 3.8 map units, and the pElA(-188) plasmid has been described (14). The ElA-Enh plasmid contains AdS sequences from the Hpa II site at position 188 to the Sac II site at position 353 inserted in pUC13. The Enh-A is an Hpa I/BalI (position 188 to position 270) insert; Enh-B is a BalI/Sac II (position 270 to position 353) insert; Enh-C is a HinPI/HinPI (position 216 to position 335) insert; Enh-D is a HinPI/FnuDII (position 216 to position 279) insert; and Enh-E is a FnuDII/HinPI (position 279 to position 335) insert.