AR1 Is an Integral Part of the Adenovirus Type 2 E1A-CR3 Transactivation Domain

Abstract

We have previously shown that the nonconserved carboxy-terminal exon of the adenovirus type 2 E1A-289R protein contains two interchangeable sequence elements, auxiliary region (AR) 1 and AR2, that are required for efficient CR3-mediated transcriptional activation of the viral E4 promoter (M. Bondesson, C. Svensson, S. Linder, and G. Akusjärvi, EMBO J. 11:3347–3354, 1992). Here we show that CR3-mediated transactivation of all adenovirus early promoters and the HSP70 promoter requires the AR1 element. We further show that AR2 can substitute for AR1 only when artificially juxtaposed to CR3. AR1 consists of six tandem glutamic acid-proline (EP) repeats and is positioned immediately downstream of CR3. Genetic dissection of AR1 showed that the number of EP repeats in AR1 is critical for CR3 function. Thus, reducing or increasing the number of EP repeats reduces the CR3 transactivation capacity. Furthermore, the introduction of amino acid substitutions into AR1 suggested that the net negative charge in AR1 is of critical importance for its function as an enhancer of CR3-mediated transcriptional activation. Using an in vitro binding approach, we showed that the AR1 element is not part of the CR3 promoter localization signal mediating contact with the Sp1, ATF-2, or c-Jun upstream-binding transcription factors. Previous studies have suggested that the 49-amino-acid sequence constituting CR3 represents the minimal domain required for E1A-induced activation of viral early promoters. Since AR1 was required for efficient CR3-mediated transcriptional activation of all tested promoters, we suggest that the carboxy-terminal boundary for the CR3 transactivation domain should be extended to include the AR1 element.

Adenovirus E1A Activation Domain Regulates H3 Acetylation Affecting Varied Steps in Transcription at Different Viral Promoters

“…Taken together, these experiments indicate that the acidic region C terminal to CR3, the (EP) 6 repeat, named AR1, for auxiliary region 1, by Ström et al ( 17 ), contributes to E1A activation of early promoters E2, E3, and E4 during viral infection of human respiratory epithelial cells through an interaction with p300 and most likely the closely related host protein CBP. Further, E1A(aa 183–188) is essential for E1A activation of the early viral promoters E2, E3, and E4 and additionally the low MLP activity early in infection, probably because they are required for E1A to interact with multisubunit mediator complexes.…”

“…E1A(aa 140–178), including a C 4 -type zinc finger, is essential for activation ( 14 , 15 , 18 ). In addition, peptide regions that closely overlap those that we propose to contribute to p300 and multisubunit mediator binding (aa 190 to 205 and aa 179 to 189, respectively) are required for high levels of activation ( 16 – 18 ). To determine if activation by the LacI-mCherry-truncated E1A fragments analyzed here is consistent with previously demonstrated activation by E1A fragments in transient-transfection assays, we assayed activation by these LacI fusions using a luciferase reporter with eight lacO sites upstream of the E1B promoter ( Fig.…”

“…Further studies using Gal4-DBD fusions to progressive N- and C-terminal deletions of E1A(aa 121–223) suggested that the minimal region required for activation is aa 141 to 178, closely overlapping CR3 ( 15 ). Six tandem glutamic acid-proline repeats (EP 6 ) just C terminal to CR3 (aa 189 to 200; designated AR1) were later shown to be essential for activation of early viral promoters by native E1A (not fused to the Gal4-DBD) in transient-transfection assays ( 16 , 17 ).…”

Requirements for E1A dependent transcription in the yeast Saccharomyces cerevisiae

“…The 289R E1A protein is thought to be primarily responsible for activation of gene expression, as mutations within CR3 generally abolish E1A transactivation [ 7 - 11 ]. An adjacent acidic region spanning residues 189–200, termed Auxiliary Region 1 (AR1), is also essential for efficient transactivation of early viral promoters by E1A [ 12 ].…”

Two Domains within the Adenovirus Type 12 E1A UniqueSpacerHave Disparate Effects on the Interaction of E1A with P105-Rb and the Transformation of Primary Mouse Cells

“…Thus in contrast to the E1A proteins of Ad2, a p300independent transcriptional trans-activation domain is located within the 29 amino acids of the amino-terminus (Lipinski et al, 1997). Moreover, Ad12 E1A lacks two transcriptional activation domains (AR1 and AR2) that were found in Ad5 E1A to be involved in CR3-mediated transcriptional activation (Bondesson et al, 1992;Strom et al, 1998). Another important difference is the presence of a unique spacer region in Ad12 E1A that is located between CR2 and CR3.…”