The adenovirus EII early promoter has multiple EIA-sensitive elements, two of which function cooperatively in basal and virus-induced transcription

Abstract

The mechanism by which the adenovirus-encoded nuclear oncogene EIA activates transcription of several viral and host promoters is an important issue in the regulation of eucaryotic gene expression and virus-host cell interactions. Identffication of cis-acting elements of the promoters and the cognate host transcription factors that are targets for EIA action is crucial for our understanding of the EIA-mediated control of coordinately regulated genes. The adenovirus EII early promoter has a complex architecture and contains two overlapping promoters with start sites at +1 (major promoter) and-26 (minor promoter). The major promoter responds strongly to virus-encoded trans activators EIA and EIV and contains four elements: a TAGA motif analogous to the TATA box, two EIIF sites present in an inverted orientation, and an ATF/CREB site. To determine precisely the roles played by these cis-acting elements in both basal and virus-induced transcription when the promoter is situated in its natural context, we investigated the phenotype of a series of linker scan promoter substitution mutants inserted into the viral chromosome. Promoter constructs harboring linker scan mutations in each element were rebuilt into a novel EIA-adenovirus vector, and transcriptional activity was monitored in virus-infected cells. In the absence of virus-encoded trans activators, basal activity in vivo was dependent on all four cis-acting elements. Surprisingly, a promoter mutant with only one of the two ETTF sites intact could not promote transcription in vivo, suggesting that the two ETTF sites function cooperatively even in basal transcription. Promoters harboring mutations in either of these two EIIF sites also failed to bind to an infection-specific form of EIIF in gel shift assays and competed only very weakly for EIIF binding with the wild-type promoter fragment. The dramatic cooperativity shown by the two inverted EIIF sites of the ETT promoter both in vivo and in vitro could reflect simultaneous contact of both sites by the transcription factor EIIF. Furthermore, promoter mutants with mutations in the TAGA motif, the two ETTF sites, and the single ATF site all failed to respond to virus-encoded trans activators. Whereas recent results demonstrate that EIIF activity can be modulated independently by EIV, leading to transactivation of this promoter, our results and those published previously strongly indicate that the three different transcription factors that bind to TAGA, EIIF, and ATF motifs of the ETT early promoter are all targets for EIA regulation in vivo. Thus, strong transactivation of the EII early promoter through these multiple EIA-sensitive elements and independently by the recently discovered EIV pathway suggests that the EII early promoter is stringently regulated in virus-infected cells. Such a stringent regulation of this promoter is consistent with the vital roles played by the three gene products of this transcription unit in the viral lytic cycle.

Co‐amplification and concomitant high levels of expression of a DEAD box gene with MYCN in human neuroblastoma

“…According to the Kozak hypothesis (Kozak, 1981), the upstream AUG codon in clone 9-14[3] appears to be in a slightly better context for initiation of translation than is the downstream AUG codon reported by Godbout and colleagues (Godbout et al, 1994). Primer extension analysis of RNA was performed as described before (Manohar et al, 1990) to map the 5' end of DDXZ mRNA in two different NB cell lines; two potential major and one relatively minor RNA start sites were identified (data not shown). Genomic sequence analysis will help determine if these are indeed the DDXZ mRNA start sites.…”

Identification of a Cellular Repressor of Transcription of the Adenoviral Late IVa2 Gene That Is Unaltered in Activity in Infected Cells

“…The CR1 and CR2 sequences present in both E1A proteins also regulate transcription. For example, they mediate binding of E1A proteins to the cellular retinoblastoma protein (Rb) to induce the release of members of the E2F family of transcriptional activators from association with Rb and thus transcription of E2Fdependent genes, including several that are crucial for progression from the G1 to the S phase of the cell cycle (see Bartek et al, 1996;Flint and Shenk, 1997;Nevins, 1992;Weinberg, 1995), and the adenoviral E2 early promoter (Kovesdi et al, 1986a,b;Manohar et al, 1990;Zajchowski et al, 1985). Once E2 replication proteins have accumulated to sufficient concentrations, viral DNA synthesis begins within the infected cell nucleus.…”

The C-terminal 70 amino acids of the adenovirus E4-ORF6/7 protein are essential and sufficient for E2F complex formation

“…An ATF binding site is located between -68 and -77 (10)(11)(12), and two E2F binding sites, inverted with respect to each other, are located between -35 and -68 (13,14). Each of these elements contributes to basal E2aE expression and each has been implicated in the ability of ElA to trans-activate the E2aE promoter (8,9,(15)(16)(17)(18)(19).…”