Regulation of transcription of the adenovirus EII promoter by EIa gene products: absence of sequence specificity.

Abstract

During adenovirus infection, the ElI promoter is positively regulated by products of the Ela region. We have studied this regulation by fusing a DNA segment containing the adenovirus EII promoter to a dihydrofolate reductase cDNA segment. Expression of this hybrid gene is stimulated in trans when ceUl lines containing an integrated copy are either transfected with plasmids carrying the EIa region or infected with adenovirus. This suggests that Ela activity regulates transcription of the EII promoter in the absence of other viral proteins and that this stimulation can occur when the EII promoter is organized in cellular chromatin. Transcription from the ElI promoter is initiated at two sites in cell lines lacking EIa activity. Introduction of the Ela region preferentially stimulated transcription from one of these two sites. A sensitive, stable cotransfection assay was used to test for specific EU sequences required for stimulation. Ela activity stimulates all mutant promoters; the most extensive deletion retained only 18 base pairs of sequences upstream of the initiation site. We suggest that regulation of a promoter by the Ela region does not depend on the presence of a set of specific sequences, but instead reflects a characteristic of promoters that have been exogenously introduced into ceUs. Insertion of the 72-base-pair repeat of simian-virus 40 in cis enhances transcription from the ElI promoter. The stimulatory effects of ETa activity and of the simian virus 40 sequence are additive and appear to differ mechanistically.The Ela region of adenovirus encodes two interesting activities. First, this region positively regulates transcription from viral and cellular promoters (2,8,14,23,24,33). Second, the region encodes proteins that can immortalize primary cells (14). The latter activity appears to be a critical component in the transformation of a normal cell to a tumorforming cell and is an activity shared by a class of oncogenes that includes myc, myb, and the large tumor antigen of polyomavirus (18,27,30). It is not clear at this time whether the ability of the Ela products to regulate transcription is related to their ability to alter regulation of cellular growth. Determination of the mechanism by which the Ela region regulates transcription will help elucidate the relationship between these two activities.The Ela region encodes three mRNAs of sizes 9S, 12S, and 13S (9, 26). The 289-amino-acid product of the 13S message stimulates expression of other adenovirus early transcription units by increasing the rate of transcription initiation (23, 28). Recently, it has been recognized that several non-adenoviral promoters are also stimulated by the Ela region (6a, 8, 33). In most positively regulated eucaryotic promoters, specific regulatory sequences have been identified which mediate this stimulation. For promoters stimulated by the Ela products, however, it has not been possible to separate sequences required for regulation from promoter sequences required for general transcription (3,4,8 The CHO DHFR-(DUK...

Cis and trans activation of adenovirus IVa2gene transcription

“…In order for the IVa2 gene to be transcribed in a transient assay, the IVa2 promoter requires an enhancer in cis as do many other promoters (16,31,33,34,41 For the IVa2 and MLPs, the effects of EIA gene product in trans and enhancer in cis are additive. In studies by others, when the ElA gene product is provided in trans and an enhancer is present in cis, either an additive or inhibitory effect, or no effect has been observed, depending on the promoter (13,16,43,44). The mechanisms of activation by enhancer in cis and ElA gene in trans are not known, but they are thought to be different (13,16,43).…”

“…In studies by others, when the ElA gene product is provided in trans and an enhancer is present in cis, either an additive or inhibitory effect, or no effect has been observed, depending on the promoter (13,16,43,44). The mechanisms of activation by enhancer in cis and ElA gene in trans are not known, but they are thought to be different (13,16,43). It has been proposed that stimulation of transcription by the ElA gene product is through its inactivation of transcription repressors, even though its direct interaction with promoter sequence has not been ruled out (45,46).…”

“…After it is transfected into suitable cells, the amount of enzyme activity produced is assumed to provide a measure of the RNA synthesized from the promoter (12,13,14). This approach has been successfully used in identifying the cell and tissue specific nature of enhancers (35,36,37) and also in defining the control regions of promoters (12,13,14). For the IVa2 promoter, we have found that the results obtained by measuring enzyme levels did not agree with the results obtained by measuring the RNA synthesized from various deletion mutants.…”

“…In the identification of transcriptional control elements in vivo, one effective approach is to fuse the promoter to be studied to the coding region of a second gene such as thymidine kinase, dihydrofolate reductase, or chloramphenicol acetyl transferase (CATase) genes (12,13,14) whose function can be easily assayed. In this report, we have analyzed the adenovirus IVa2 promoter control sequences by fusing this promoter to the chloramphenicol acetyltransferase gene coding region (15).…”

An adenovirus type 5 E1A protein with a single amino acid substitution blocks wild-type E1A transactivation.

“…(ii) A specific set of sequence homologies is not observed within the DNA region 5' proximal to the start site of transcription of ElA-inducible promoters (1). It is important to note that extensive deletion analysis of the Ad5 E2 and E3 promoter regions has failed to reveal an ElA-responsive element that can be separated from promoter elements required for transcription per se (10,27,31,35,40). (iii) The immediate early (IE) gene of pseudorabies virus and the X gene of human T-cell leukemia virus type II are both able to transactivate adenovirus early promoters (7,11); it seems unlikely that gene products of such divergent viruses would recognize a common DNA sequence.…”