The IE2 regulatory protein of human cytomegalovirus induces expression of the human transforming growth factor beta1 gene through an Egr-1 binding site.
Increases in transforming growth factor beta1 (TGF-beta1) mRNA and biological activity in the early phase of human cytomegalovirus (CMV) infection in fibroblasts are paralleled by increased TGF-beta1-chloramphenicol acetyltransferase (CAT) reporter gene activity. To determine how CMV infection transactivates the TGF-beta1 promoter, we examined the effects of the cotransfected IE2 regulatory protein of human CMV on 5'-deleted TGF-beta1 promoter-CAT reporter genes in transient DNA transfection assays. Two upstream TGF-beta1 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-l 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-l-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 beads. 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-beta1 gene as well as other potential cellular targets by virtue of its ability to interact with the Egr-1 DNA-binding protein.
Yoo
Y D
YD
Laboratory of Chemoprevention, National Cancer Institute, Bethesda, Maryland 20892, USA.
Chiou
C J
CJ
Choi
K S
KS
Yi
Y
Y
Michelson
S
S
Kim
S
S
Hayward
G S
GS
Kim
S J
SJ
D001254:Astrocytoma / Q000378:metabolism; D003587:Cytomegalovirus / Q000235:genetics* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D051766:Early Growth Response Protein 1; D015967:Gene Expression Regulation, Viral; D006801:Humans; D017874:Immediate-Early Proteins; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism; D015533:Transcriptional Activation; D016212:Transforming Growth Factor beta / Q000096:biosynthesis / Q000235:genetics*; D014407:Tumor Cells, Cultured; D014764:Viral Proteins / Q000235:genetics* / Q000378:metabolism
D004268:DNA-Binding Proteins; C497325:EGR1 protein, human; D051766:Early Growth Response Protein 1; D017874:Immediate-Early Proteins; D014157:Transcription Factors; D016212:Transforming Growth Factor beta; D014764:Viral Proteins