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

Masayo Kornuc, Steven Kliewer, Joseph Garcia, David Harrich, Ching Li, Richard Gaynor

Research output: Contribution to journalArticle

10 Citations (Scopus)

Abstract

Transcription of the adenovirus early region 3 promoter is strongly induced by the adenovirus El A 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), API (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 rf/434, an E1A/E1B deletion mutant, or wild-type adenovirus. Mutations of either the ATF- or API-binding sites but not the TATA- and NFl-binding sites resulted in severe decreases in both basal and ElA/ElB-induced transcriptional levels. These constructs were also assayed in in vitro transcription assays with cellular extracts prepared from rf/434-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 API-binding site decreased both basal and E1A/EIB-induced transcriptional levels. Gel retardation analysis using these extracts indicated that the binding to ATF, API, 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 tf434-infected cells indicated that the levels of these RNAs were not altered by the E1A/E1B proteins. Immunoprecipitation of API and CREB from both U434- and wild-type-adenovirus-infected cells indicated that the amounts of these proteins were not significantly altered. These results suggest that ElA/ElB-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.

Original languageEnglish (US)
Pages (from-to)2004-2013
Number of pages10
JournalJournal of Virology
Volume64
Issue number5
StatePublished - 1990

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Activating Transcription Factors
gene activation
Adenoviridae
Genetic Promoter Regions
Transcriptional Activation
transcription factors
promoter regions
DNA
binding sites
Binding Sites
Proteins
proteins
transcription (genetics)
extracts
RNA
oligonucleotides
HeLa Cells
cells
deoxyribonuclease I
mutation

ASJC Scopus subject areas

  • Immunology

Cite this

Adenovirus early region 3 promoter regulation by E1A/E1B is independent of alterations in DNA binding and gene activation of CREB/ATF and AP1. / Kornuc, Masayo; Kliewer, Steven; Garcia, Joseph; Harrich, David; Li, Ching; Gaynor, Richard.

In: Journal of Virology, Vol. 64, No. 5, 1990, p. 2004-2013.

Research output: Contribution to journalArticle

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abstract = "Transcription of the adenovirus early region 3 promoter is strongly induced by the adenovirus El A 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), API (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 rf/434, an E1A/E1B deletion mutant, or wild-type adenovirus. Mutations of either the ATF- or API-binding sites but not the TATA- and NFl-binding sites resulted in severe decreases in both basal and ElA/ElB-induced transcriptional levels. These constructs were also assayed in in vitro transcription assays with cellular extracts prepared from rf/434-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 API-binding site decreased both basal and E1A/EIB-induced transcriptional levels. Gel retardation analysis using these extracts indicated that the binding to ATF, API, 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 tf434-infected cells indicated that the levels of these RNAs were not altered by the E1A/E1B proteins. Immunoprecipitation of API and CREB from both U434- and wild-type-adenovirus-infected cells indicated that the amounts of these proteins were not significantly altered. These results suggest that ElA/ElB-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.",
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T1 - Adenovirus early region 3 promoter regulation by E1A/E1B is independent of alterations in DNA binding and gene activation of CREB/ATF and AP1

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AU - Kliewer, Steven

AU - Garcia, Joseph

AU - Harrich, David

AU - Li, Ching

AU - Gaynor, Richard

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N2 - Transcription of the adenovirus early region 3 promoter is strongly induced by the adenovirus El A 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), API (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 rf/434, an E1A/E1B deletion mutant, or wild-type adenovirus. Mutations of either the ATF- or API-binding sites but not the TATA- and NFl-binding sites resulted in severe decreases in both basal and ElA/ElB-induced transcriptional levels. These constructs were also assayed in in vitro transcription assays with cellular extracts prepared from rf/434-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 API-binding site decreased both basal and E1A/EIB-induced transcriptional levels. Gel retardation analysis using these extracts indicated that the binding to ATF, API, 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 tf434-infected cells indicated that the levels of these RNAs were not altered by the E1A/E1B proteins. Immunoprecipitation of API and CREB from both U434- and wild-type-adenovirus-infected cells indicated that the amounts of these proteins were not significantly altered. These results suggest that ElA/ElB-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.

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