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 language | English (US) |
|---|---|
| Pages (from-to) | 7062-7070 |
| Number of pages | 9 |
| Journal | Journal of Virology |
| Volume | 70 |
| Issue number | 10 |
| State | Published - Oct 1996 |
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ASJC Scopus subject areas
- Immunology
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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 Selwyn; Kim, Seong Jin.
In: Journal of Virology, Vol. 70, No. 10, 10.1996, p. 7062-7070.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - The IE2 regulatory protein of human cytomegalovirus induces expression of the human transforming growth factor β1 gene through an Egr-1 binding site
AU - Yoo, Young Do
AU - Chiou, Chuang Jiun
AU - Choi, Kyeong Sook
AU - Yi, Youngsuk
AU - Michelson, Susan
AU - Kim, Sunyoung
AU - Hayward, Gary Selwyn
AU - Kim, Seong Jin
PY - 1996/10
Y1 - 1996/10
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=0000465838&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0000465838&partnerID=8YFLogxK
M3 - Article
C2 - 8794351
AN - SCOPUS:0000465838
VL - 70
SP - 7062
EP - 7070
JO - Journal of Virology
JF - Journal of Virology
SN - 0022-538X
IS - 10
ER -