Control of adenovirus late promoter expression in two human cell lines.

Abstract

We investigated the nucleotide sequence requirements of the adenovirus 2 late promoter when activated by either a trans-acting regulatory protein or a cis-acting enhancer element. Using deletion mutants in transient expression assays, we determined that the 5' limit of the region required for activation by a trans-acting regulatory protein, the adenovirus early region la gene product, and the simian virus 40 enhancer is the same in both 293 and HeLa cells. Surprisingly, the 3' limit of required sequences varied, depending on the mechanism of activation. Activation mediated by the early region la protein endogenous in 293 cells or produced after cotransfection of HeLa cells requires the region around the transcriptional start site, whereas activation brought about by an enhancer element in HeLa cells has no requirement for these sequences. Under no conditions tested did the simian virus 40 enhancer activate the late promoter in 293 cells, even when sequences sufficient for enhancer-mediated activation in HeLa cells, but not for early region la activation, were present. These results suggest the existence of at least two different mechanisms for positive regulation of promoter activity.A major factor in gene regulation is differential promoter activation. Gene expression can be controlled at the level of transcription initiation by a number of mechanisms, all of which require either cis-or trans-acting factors, or both. Transcriptional enhancers are an example of cis-acting regulatory squences. (see references 18 and 35, for reviews.) The prototype for this class of controlling element, the simian virus 40 (SV40) 72-base-pair (bp) repeated sequence, was first identified as a far upstream component of the early promoter of this virus (4, 23). However, the so-called 72-bp repeats were soon shown to have unprecedented properties. When DNA fragments containing these sequences were excised from viral DNA and joined to recombinant molecules containing various heterologous genes, it was found that expression of the linked gene was dramatically enhanced, in a manner essentially independent of the position and orientation of the viral DNA fragment (3, 49).Enhancers have since been identified in a number of viral genomes, including polyomavirus (13), retroviral long terminal repeats (20,37), adenovirus (26, 31) and bovine papillomavirus (41). More recently, controlling elements that behave like enhancers have been found in or near certain cellular genes, including immunoglobulins (2, 17, 51) and insulin and chymotrypsin (62). The enhancers described to date show various degrees of specificity, ranging from a slight species specificity in the case of the papovavirus and retroviral enhancers (37) to an almost absolute cell-type specificity in the case of the immunoglobulin enhancers, which are functional only in lymphoid cells.Enhancers in turn interact with factors in the cell nucleus (55). Such trans-acting factors constitute a second category of transcriptional regulators. One of the best studied is the SV40 T antigen, whi...

Downstream sequences affect transcription initiation from the adenovirus major late promoter.

“…To determine whether the transcriptional control sequences downstream from the major late promoter are active in uninfected cells or whether their activity depends upon a virus-coded protein or virusinduced host factor, we examined transcription from plasmid-borne copies of the late promoter in uninfected and infected cells. Previous work suggested that transcription from the adenovirus late promoter carried on the pAd-SV plasmids would be undetectable in HeLa cells (21,29). We Ad-SVI confirmed this result and also found that transcription from the plasmid-borne late promoter is not detectable 24 h after adenovirus infection of transfected HeLa cells (data not shown).…”

“…We used a transient expression assay to compare the effects of alterations in 3' DNA sequences on transcription initiation from the late promoter in infected and uninfected cells. In agreement with previous results (21,29), no expression of SV40 T antigen under the control of the major late promoter was detected from any of the pAd-SV plasmids after their transfection into HeLa cells. However, by adding the minimal SV40 origin of replication to the pAd-SV plasmids and transfecting them into COS7 cells which allow plasmid replication (17), expression from the plasmid-borne promoters was readily detected.…”

“…Recently, Lewis and Manley (29) have demonstrated a requirement for DNA sequences downstream from the adenovirus late promoter from the cap site to at least position +33 for transcriptional activity in 293 cells or in HeLa cells cotransfected with an Ela-expressing plasmid. These transient expression systems cannot be compared directly with ours because of differences in transfection efficiency, plasmid structure, and plasmid replicaton.…”

The downstream core promoter element, DPE, is conserved fromDrosophila to humans and is recognized by TAFII60 of Drosophila

“…Such downstream promoter sequences have been found in TATA-containing promoters (see, e.g., Lewis and Manley 1985;Nakatani et al 1990; Lee et al 1992;Emanuel and Gilmour 1993;Purnell and Gilmour 1993), as well as in TATA-less promoters (see, e.g., Biggin and Tjian 1988;Perkins et al 1988;Soeller et al 1988;Smale and Baltimore 1989;Jarrell and Meselson 1991;Contursi et al 1995;Minchiotti et al 1997). It appears that many of these downstream promoter sequences are involved in basal transcription, but it is also important to consider that some downstream promoter sequences might be binding sites for sequence-specific transcriptional activators.…”

Transcriptional Regulation of the Rat NHE3 Gene

“…in the adenoviral major late (AdML) (34) and murine terminal transferase (TdT) (35) genes. Although some studies indicated that TFIID, TFII-I, cap-binding protein (CAP), or USF proteins may bind to these regions, downstream elements remain poorly understood.…”