The IE2 regulatory protein of human cytomegalovirus induces expression of the human transforming growth factor β1 gene through an Egr-1 binding site

Young Do Yoo, Chuang Jiun Chiou, Kyeong Sook Choi, Youngsuk Yi, Susan Michelson, Sunyoung Kim, Gary S. Hayward, Seong Jin Kim

Research output: Contribution to journalArticle

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Abstract

Increases in transforming growth factor β1 (TGF-β1) mRNA and biological activity in the early phase of human cytomegalovirus (CMV) infection in fibroblasts are paralleled by increased TGF-β1-chloramphenicol acetyltransferase (CAT) reporter gene activity. To determine how CMV infection transactivates the TGF-β1 promoter, we examined the effects of the cotransfected IE2 regulatory protein of human CMV on 5'-deleted TGF-β1 promoter-CAT reporter genes in transient DNA transfection assays. Two upstream TGF-β1 promoter regions each containing an Egr-1 consensus site were shown to be important for IE2-induced transactivation in a cell type that displayed greatly reduced nonspecific activity. Furthermore, transfer of an Egr-1 site from between positions -125 and -98, but not point mutant versions of this site, to a heterologous promoter also conveyed IE2 responsiveness. Addition of an IE2 expression vector or use of the U373 A45 astrocytoma cell line expressing IE2 also produced synergistic stimulation of GAL4-Egr-1-mediated activation of a target promoter containing GAL4 binding sites. The 80-kDa IE2 protein present in A45 cells proved to selectively bind to glutathione S-transferase (GST)-Egr-1 heads. The results of in vitro protein binding assays also revealed that an intact in vitro-translated IE2 protein bound directly to the GST-Egr-1 fusion protein through the zinc finger domain of the Egr-1 protein and that this binding activity was abolished by deletion of parts of the zinc finger DNA-binding domain. Similarly, the Egr-1 protein was found to associate preferentially with a small region within the C-terminal half of the IE2 protein adjacent to the DNA-binding and dimerization domains that are important for both transactivation and downregulation. We conclude from these observations that IE2 may regulate transcription of the TGF-β1 gene as well as other potential cellular targets by virtue of its ability to interact with the Egr-1 DNA- binding protein.

Original languageEnglish
Pages (from-to)7062-7070
Number of pages9
JournalJournal of Virology
Volume70
Issue number10
Publication statusPublished - 1996 Oct 1
Externally publishedYes

Fingerprint

Transforming Growth Factors
Cytomegalovirus
Binding Sites
Genes
Proteins
Chloramphenicol O-Acetyltransferase
Zinc Fingers
Cytomegalovirus Infections
Glutathione Transferase
Reporter Genes
Protein Binding
Transcriptional Activation
DNA
Astrocytoma
DNA-Binding Proteins
Dimerization
Genetic Promoter Regions
Transfection
Down-Regulation
Fibroblasts

ASJC Scopus subject areas

  • Immunology

Cite this

The IE2 regulatory protein of human cytomegalovirus induces expression of the human transforming growth factor β1 gene through an Egr-1 binding site. / Yoo, Young Do; Chiou, Chuang Jiun; Choi, Kyeong Sook; Yi, Youngsuk; Michelson, Susan; Kim, Sunyoung; Hayward, Gary S.; Kim, Seong Jin.

In: Journal of Virology, Vol. 70, No. 10, 01.10.1996, p. 7062-7070.

Research output: Contribution to journalArticle

Yoo, YD, Chiou, CJ, Choi, KS, Yi, Y, Michelson, S, Kim, S, Hayward, GS & Kim, SJ 1996, 'The IE2 regulatory protein of human cytomegalovirus induces expression of the human transforming growth factor β1 gene through an Egr-1 binding site', Journal of Virology, vol. 70, no. 10, pp. 7062-7070.
Yoo, Young Do ; Chiou, Chuang Jiun ; Choi, Kyeong Sook ; Yi, Youngsuk ; Michelson, Susan ; Kim, Sunyoung ; Hayward, Gary S. ; Kim, Seong Jin. / The IE2 regulatory protein of human cytomegalovirus induces expression of the human transforming growth factor β1 gene through an Egr-1 binding site. In: Journal of Virology. 1996 ; Vol. 70, No. 10. pp. 7062-7070.
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abstract = "Increases in transforming growth factor β1 (TGF-β1) mRNA and biological activity in the early phase of human cytomegalovirus (CMV) infection in fibroblasts are paralleled by increased TGF-β1-chloramphenicol acetyltransferase (CAT) reporter gene activity. To determine how CMV infection transactivates the TGF-β1 promoter, we examined the effects of the cotransfected IE2 regulatory protein of human CMV on 5'-deleted TGF-β1 promoter-CAT reporter genes in transient DNA transfection assays. Two upstream TGF-β1 promoter regions each containing an Egr-1 consensus site were shown to be important for IE2-induced transactivation in a cell type that displayed greatly reduced nonspecific activity. Furthermore, transfer of an Egr-1 site from between positions -125 and -98, but not point mutant versions of this site, to a heterologous promoter also conveyed IE2 responsiveness. Addition of an IE2 expression vector or use of the U373 A45 astrocytoma cell line expressing IE2 also produced synergistic stimulation of GAL4-Egr-1-mediated activation of a target promoter containing GAL4 binding sites. The 80-kDa IE2 protein present in A45 cells proved to selectively bind to glutathione S-transferase (GST)-Egr-1 heads. The results of in vitro protein binding assays also revealed that an intact in vitro-translated IE2 protein bound directly to the GST-Egr-1 fusion protein through the zinc finger domain of the Egr-1 protein and that this binding activity was abolished by deletion of parts of the zinc finger DNA-binding domain. Similarly, the Egr-1 protein was found to associate preferentially with a small region within the C-terminal half of the IE2 protein adjacent to the DNA-binding and dimerization domains that are important for both transactivation and downregulation. We conclude from these observations that IE2 may regulate transcription of the TGF-β1 gene as well as other potential cellular targets by virtue of its ability to interact with the Egr-1 DNA- binding protein.",
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