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PMID: 2139139 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, Non-P.H.S. Research Support, U.S. Gov't, P.H.S.

Adenovirus early region 3 promoter regulation by E1A/E1B is independent of alterations in DNA binding and gene activation of CREB/ATF and AP1.

Journal of virology ·Vol. 64 ·No. 5 ·1990-05-00 ·Pages 2004-13

Kornuc M, Kliewer S, Garcia J, Harrich D, Li C, Gaynor R

Abstract

Transcription of the adenovirus early region 3 promoter is strongly induced by the adenovirus E1A protein. Previous DNase I footprinting has indicated that four regions in this promoter serve as binding sites for HeLa nuclear proteins. These include binding sites for NF-1 (site IV), AP1 (site III), CREB/activating transcription factor (ATF) (site II), and TATA (site I). To determine the relative importance of these sites in both the in vivo and in vitro transcriptional regulation of the E3 promoter, oligonucleotide-directed mutagenesis of these sites was performed. Each of these constructs was assayed by transfection onto HeLa cells in the presence of either dl434, an E1A/E1B deletion mutant, or wild-type adenovirus. Mutations of either the ATF- or AP1-binding sites but not the TATA- and NF1-binding sites resulted in severe decreases in both basal and E1A/E1B-induced transcriptional levels. These constructs were also assayed in in vitro transcription assays with cellular extracts prepared from dl434-infected or wild-type-adenovirus-infected HeLa cells. The wild-type E3 promoter was transcribed approximately 30 times more efficiently in extracts containing the E1A/E1B proteins compared with extracts lacking these proteins. Mutations of either the TATA element, the ATF site, or the AP1-binding site decreased both basal and E1A/E1B-induced transcriptional levels. Gel retardation analysis using these extracts indicated that the binding to ATF, AP1, or NF1 oligonucleotides was not altered in the presence of the E1A/E1B proteins compared with extracts lacking these proteins. Northern (RNA) blot analysis of c-jun and CREB RNA prepared from wild-type adenovirus and dl434-infected cells indicated that the levels of these RNAs were not altered by the E1A/E1B proteins. Immunoprecipitation of AP1 and CREB from both dl434- and wild-type-adenovirus-infected cells indicated that the amounts of these proteins were not significantly altered. These results suggest that E1A/E1B-induced activation of the E3 promoter does not involve activation of transcription factor genes nor a change in the DNA binding activity of important promoter-binding components. Our results are consistent with a model in which the E1A/E1B proteins either directly or indirectly alter the interactions of factors that bind to the basal E3 promoter transcription complex, thereby inducing transcription.

MeSH Terms
Adenovirus Early Proteins Adenoviruses, Human/genetics Base Sequence Blotting, Northern Cell Nucleus/metabolism Chloramphenicol O-Acetyltransferase/genetics Cyclic AMP Response Element-Binding Protein DNA-Binding Proteins/genetics,metabolism Gene Expression Regulation, Viral HeLa Cells/metabolism Humans Molecular Sequence Data Mutation Nuclear Proteins/metabolism Oligonucleotide Probes Oncogene Proteins, Viral/genetics,metabolism Plasmids Promoter Regions, Genetic Proto-Oncogene Proteins c-jun Transcription Factors/metabolism Transcription, Genetic Transcriptional Activation Transfection
Chemicals
Adenovirus Early Proteins Cyclic AMP Response Element-Binding Protein DNA-Binding Proteins Nuclear Proteins Oligonucleotide Probes Oncogene Proteins, Viral Proto-Oncogene Proteins c-jun Transcription Factors Chloramphenicol O-Acetyltransferase
Authors & Affiliations
6 authors, click to expand affiliations / ORCID
Kornuc M
Department of Medicine, University of California, Los Angeles School of Medicine 90024.
Kliewer S
Garcia J
Harrich D
Li C
Gaynor R
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Article Info
Journal
Journal of virology
Abbr.
J Virol
ISSN
0022-538X
DOI
10.1128/JVI.64.5.2004-2013.1990
Published
1990-05-00
Pages
2004-13
Language
English
Region
United States
NLM ID
0113724
PMCID
PMC249355
Subset
IM
Grants
NCI NIH HHS · CA30981 · United States
External Links
PubMed source DOI: 10.1128/JVI.64.5.2004-2013.1990 PMC Full Text View journal details
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