Nucleic Acids Research In vivo photocrosslinking reveals that transcription factor binding to the mammalian ATF recognition sequence is required for ElA-induced transactivation in injected Xenopus laevis oocytes

Abstract

The adenovirus El A 13S mRNA product transactivates genes injected into Xenopus laevis oocytes that are normally E1Ainducible in mammalian cells. However, ElA-stimulated transcription, but not basal (uninduced) transcription, was inhibited if oocytes were incubated in the presence of protein synthesis inhibitors. This suggests that a cellular protein(s) is required for ElA-induced transactivation, but that it is dispensable for basal transcription. In order to identify such a protein from Xenopus oocytes that interacts with the adenovirus E3 promoter, gel shift assays, a new in vivo photocrosslinking assay, and immunoselection of biotinylated oligonucleotides were employed. A protein of molecular size 75 kd, which bound to the mammalian ATF recognition sequence in vivo, was found to be essential for ElA-induced transactivation. Although cycloheximide treatment of oocytes inhibited factor binding, E1A exerted no effect on factor binding. These data suggest that E1A modulates the activity of an oocyte transcription factor, either directly or indirectly, but not its ability to bind DNA. INTRODUCTION Defined protein products of viruses that transactivate viral genes have proven to be exceptionally useful paradigms for the study of differential gene transcription in eukaryotic cells. The 289 amino acid product of the adenovirus El A 13S mRNA, for example, induces the transcription of all the other early viral genes, whose products in turn are required for a productive infection by the virus (1). El A also activates the transcription of some cellular genes such as HSP70 (2,3) and B-tubulin (4). Antithetically, El A can repress the transcription of some genes by interfering with enhancer activity (5,6,7). DNA sequence and mutational analysis of several gene promoters that are induced by El A have not revealed conserved elements that could indicate a specific site of El A interaction (8,9, reviewed in ref. 1). These observations, coupled with experiments that indicate that El A is not a sequence-specific DNA binding protein (10), suggest that diverse cellular factors may mediate the regulatory activity of E1A. One approach for the identification of such factors has been to use gel mobility shift assays and enzymatic or chemical 'footprinting' techniques to examine the cellular proteins that bind to El A inducible promoters, and to assess whether their binding activities are enhanced by El A. At least two HeLa cell factors bind the adenovirus E2A promoter (11), and the binding activity of one, termed EIIF, is enhanced when protein extracts are prepared from adenovirus infected cells (11,12). The adenovirus E3 promoter is bound by four cellular factors (13,14), although the binding of none of these appears to be regulated by E1A (14). Three factors interact with the adenovirus E4 promoter (15). One of these, E4F1, is required for ElA-induced transcription in vitro (i.e., in extracts prepared from 293 cells, which constitutively express the El A gene), but its binding activity is unaffected

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