DNA-binding-defective mutants of the Epstein-Barr virus lytic switch activator Zta transactivate with altered specificities.

The Epstein-Barr virus BRLF1 and BZLF1 genes are the first viral genes transcribed upon induction of the viral lytic cycle. The protein products of both genes (referred to here as Rta and Zta, respectively) activate expression of other viral genes, thereby initiating the lytic cascade. Among the viral antigens expressed upon induction of the lytic cycle, however, Zta is unique in its ability to disrupt viral latency; expression of the BZLF1 gene is both necessary and sufficient for triggering the viral lytic cascade. We have previously shown that Zta can activate its own promoter (Zp), through binding to two Zta recognition sequences (ZIIIA and ZIIIB). Here we describe mutant Zta proteins that do not bind DNA (referred to as Zta DNA-binding mutants [Zdbm]) but retain the ability to transactivate Zp. Consistent with the inability of these mutants to bind DNA, transactivation of Zp by Zdbm is not dependent on the Zta recognition sequences. Instead, transactivation by Zdbm is dependent upon promoter elements that bind cellular factors. An examination of other viral and cellular promoters identified promoters that are weakly responsive or unresponsive to Zdbm. An analysis of a panel of artificial promoters containing one copy of various promoter elements demonstrated a specificity for Zdbm activation that is distinct from that of Zta. These results suggest that non-DNA-binding forms of some transactivators retain the ability to transactivate specific target promoters without direct binding to DNA.     

Identification of novel TGF-beta /Smad gene targets in dermal fibroblasts using a combined cDNA microarray/promoter transactivation approach

Despite major advances in the understanding of the intimate mechanisms of transforming growth factor-beta (TGF-beta) signaling through the Smad pathway, little progress has been made in the identification of direct target genes. In this report, using cDNA microarrays, we have focussed our attention on the characterization of extracellular matrix-related genes rapidly induced by TGF-beta in human dermal fibroblasts and attempted to identify the ones whose up-regulation by TGF-beta is Smad-mediated. For a gene to qualify as a direct Smad target, we postulated that it had to meet the following criteria: (1) rapid (30 min) and significant (at least 2-fold) elevation of steady-state mRNA levels upon TGF-beta stimulation, (2) activation of the promoter by both exogenous TGF-beta and co-transfected Smad3 expression vector, (3) up-regulation of promoter activity by TGF-beta blocked by both dominant-negative Smad3 and inhibitory Smad7 expression vectors, and (4) promoter transactivation by TGF-beta not possible in Smad3(-/-) mouse embryo fibroblasts. Using this stringent approach, we have identified COL1A2, COL3A1, COL6A1, COL6A3, and tissue inhibitor of metalloproteases-1 as definite TGF-beta/Smad3 targets. Extrapolation of this approach to other extracellular matrix-related gene promoters also identified COL1A1 and COL5A2, but not COL6A2, as novel Smad targets. Together, these results represent a significant step toward the identification of novel, early-induced Smad-dependent TGF-beta target genes in fibroblasts.     

Smad3-ATF3 signaling mediates TGF-beta suppression of genes encoding Phase II detoxifying proteins

This study provides evidence that in mammary epithelial cells the pluripotent cytokine TGF-beta1 repressed expression of multiple genes involved in Phase II detoxification. GCLC, the gene that encodes the catalytic subunit of the enzyme glutamate cysteine ligase, the rate-limiting enzyme in the biosynthesis of glutathione, was used as a molecular surrogate for investigating the mechanisms by which TGF-beta suppressed Phase II gene expression. TGF-beta was found to suppress luciferase reporter activity mediated by the human GCLC proximal promoter, as well as reporter activity mediated by the GCLC antioxidant response element, ARE4. TGF-beta downregulated expression of endogenous GCLC mRNA and GCLC protein. TGF-beta suppression of the Phase II genes correlated with a decrease in cellular glutathione and an increase in cellular reactive oxygen species. Ectopic expression of constitutively active Smad3E was sufficient to inhibit both reporters in the absence of TGF-beta, whereas dominant negative Smad3A blocked TGF-beta suppression. Smad3E suppressed Nrf2-mediated activation of the GCLC reporter. We demonstrate that TGF-beta increased ATF3 protein levels, as did transient overexpression of Smad3E. Ectopic expression of ATF3 was sufficient to suppress the GCLC reporter activity, as well as endogenous GCLC expression. These results demonstrate that Smad3-ATF3 signaling mediates TGF-beta repression of ARE-dependent Phase II gene expression and potentially provide critical insight into mechanisms underlying TGF-beta1 function in carcinogenesis, tissue repair, and fibrosis.     

Epstein-Barr Virus Zta Upregulates Matrix Metalloproteinases 3 and 9 That Synergistically Promote Cell Invasion In Vitro

Zta is a lytic transactivator of Epstein-Barr virus (EBV) and has been shown to promote migration and invasion of epithelial cells. Although previous studies indicate that Zta induces expression of matrix metalloproteinase (MMP) 9 and MMP1, direct evidence linking the MMPs to Zta-induced cell migration and invasion is still lacking. Here we performed a series of in vitro studies to re-examine the expression profile and biologic functions of Zta-induced MMPs in epithelial cells derived from nasopharyngeal carcinoma. We found that, in addition to MMP9, MMP3 was a new target gene upregulated by Zta. Ectopic Zta expression in EBV-negative cells increased both mRNA and protein production of MMP3. Endogenous Zta also contributed to induction of MMP3 expression, migration and invasion of EBV-infected cells. Zta activated the MMP3 promoter through three AP-1 elements, and its DNA-binding domain was required for the promoter binding and MMP3 induction. We further tested the effects of MMP3 and MMP9 on cell motility and invasiveness in vitro. Zta-promoted cell migration required MMP3 but not MMP9. On the other hand, both MMP3 and MMP9 were essential for Zta-induced cell invasion, and co-expression of the two MMPs synergistically increased cell invasiveness. Therefore, this study provides integrated evidence demonstrating that, at least in the in vitro cell models, Zta drives cell migration and invasion through MMPs.     

TGF-beta 1 inhibition of transin/stromelysin gene expression is mediated through a Fos binding sequence

Transforming growth factor beta 1 (TGF-beta 1) inhibits the growth factor and oncogene induction of transin/stromelysin, a secreted matrix-degrading metalloprotease. We demonstrate that a 10 bp element in the transin promoter is required for the TGF-beta 1 inhibitory effects and that this sequence is conserved in the promoter regions of several other TGF-beta 1-inhibited genes. The TGF-beta 1 inhibitory element (TIE) specifically binds a nuclear protein complex from TGF-beta 1-stimulated rat fibroblasts. Interestingly, this complex contained the c-fos proto-oncogene product, Fos, and induction of Fos expression was required for the inhibitory effect of TGF-beta 1 on transin gene expression. These results suggest that TGF-beta 1 inhibition of gene expression is mediated by the binding of a Fos-containing protein complex to the TIE promoter sequences.