Identification of phorbol ester response elements in the promoter of Epstein-Barr virus putative lytic switch gene BZLF1

The product of the Epstein-Barr virus BZLF1 gene encodes a protein which is related to c-fos, it has been shown to bind specifically to a consensus AP-1 site, and its expression in latently Epstein-Barr virus-infected lymphocytes is sufficient to trigger the viral lytic cycle. We identified several elements within the BZLF1 promoter (Zp) which are responsive to the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), an inducer of the viral lytic cycle. These elements fall into two classes based on the factors which bind to these sequences and their resulting functional behavior. Four of the elements are homologous (ZI elements) and share homology to a protein-binding domain in the promoter region of the coordinately expressed BRLF1 gene. When cloned upstream of heterologous promoters, the ZI elements function as silencers which exhibit TPA-inducible enhancer activity. A distinct TPA-responsive element (ZII) is located near the TATA box and shares homology with the AP-1-binding site in the c-jun promoter. A synthetic oligonucleotide with a sequence corresponding to the ZII element effectively competes for binding of nuclear factors to the c-jun AP-1 site. Furthermore, we found that a complex of c-jun and c-fos bound to the ZII domain.     

佛波酯诱导内皮素和FOS／JUN基因在血管内皮细胞中的表达及AP－1结合活性

佛波酯诱导内皮素和ＦＯＳ／ＪＵＮ基因在血管内皮细胞中的表达及ＡＰ－１结合活性温进坤,魏素珍（河北医学院生化教研室,石家庄０５００１７）张晨晖,姚阿卿,周爱儒,汤健（北京医科大学心血管基础研究所,北京,１０００８３）关键词内皮素基因表达;ＡＰ－１转录因...     

FOS／JUN介导佛波酯对内皮素基因表达的诱导作用

利用凝胶电泳迁移率改变实验、RNA印迹和蛋白质印迹分析分别检查了c-jun抗体对内皮素-1(ET-1)基因AP-1位点与核蛋白结合的影响及肿瘤促进剂佛波酯(TPA)对c-fos/c-jun基因表达的作用.结果发现,c-jun抗体可使AP-1位点-核蛋白复合物的电泳迁移率发生改变,TPA显著促进c-fos/c-jun基因表达和血管内皮细胞的AP-1结合活性.实验表明,TPA对ET-1基因表达的诱导作用是通过促进AP-1转录因子c-fos/c-jun合成来介导的.     

Suppression of albumin enhancer activity by H-ras and AP-1 in hepatocyte cell lines.

We demonstrated, using a transient transfection assay, that the albumin enhancer increased the expression of the albumin promoter in a highly differentiated, simian virus 40 (SV40)-immortalized hepatocyte cell line, CWSV1, but was not functional in two ras-transformed cell lines (NR3 and NR4) derived from CWSV1 by stable transfection with the T24ras oncogene. A transient cotransfection assay showed that T24ras and normal c-Ha-ras were each able to inhibit the activity of the albumin enhancer in an immortal hepatocyte cell line. DNase I footprinting and gel mobility shift assays demonstrated that the DNA binding activities specific to the albumin enhancer were not decreased in the ras-transformed cells. ras also did not diminish the expression of HNF1 alpha, C/EBP alpha, HNF3 alpha, HNF3 beta, or HNF3 gamma but did significantly increase AP-1 binding activity. Three AP-1 binding sites were identified within the albumin enhancer, and DNA binding activities specific to these AP-1 sites were induced in the ras-transformed hepatocytes. Subsequent functional assays showed that overexpression of c-jun and c-fos inhibited the activity of the albumin enhancer. Site-directed mutagenesis of the AP-1 binding sites in the albumin enhancer partially abrogated the suppressing effect of ras and c-jun/c-fos on the enhancer. These functional studies therefore supported the results of the structural studies with AP-1. We conclude that the activity of the albumin enhancer is subject to regulation by ras signaling pathways and that the effect of ras on the albumin enhancer activity may be mediated by AP-1.     

Identification of the 12-O-tetradecanoylphorbol-13-acetate-responsive enhancer of the MS gene of the Epstein-Barr virus.

We previously located two 12-O-tetradecanoylphorbol-13-acetate (TPA)-responsive enhancers, MSTRE-I and MSTRE-II, in the upstream sequence of the MS gene of Epstein-Barr virus (Liu, Q., and Summers, W.C. (1989) J. Virol. 63, 5062-5068). The core sequence of the MSTRE-I enhancer is now determined to be between -718 and -708 of the upstream sequence of the MS gene. The activity of the enhancer is also sensitive to its immediate surrounding sequence on either side. A single copy of a 30-base pair (bp) fragment containing the MSTRE-I sequence was able to confer TPA responsiveness upon the MS promoter even in the absence of an AP-1 binding site. Multiple tandem copies of this 30-bp fragment, regardless of their relative orientations to each other, could function synergistically to enhance the MS promoter activity. At least two copies of the 30-bp fragment were required to bestow TPA induction upon the thymidine kinase gene promoter of herpes simplex virus type 1. The MSTRE-I sequence could also be bound by a Fos-GCN4 chimeric protein but with an affinity much lower than that between the chimeric protein and the AP-1 binding site. This MSTRE-I region has strong homology to one of the TPA-responsive elements (the ZII domain) in the upstream sequence of the EBV BZLF1 gene. In addition, a putative negative regulatory region or silencer was found immediately downstream of the MSTRE-I enhancer. This potential silencer region contains a 14-bp sequence that is homologous to the silencer consensus sequence of the BZLF1 gene. Therefore, the regulation of the MS gene may share the same pathway with the immediate early gene BZLF1.     

Rapid and preferential activation of the c-jun gene during the mammalian UV response.

Exposure of mammalian cells to DNA-damaging agents leads to activation of a genetic response known as the UV response. Because several previously identified UV-inducible genes contain AP-1 binding sites within their promoters, we investigated the induction of AP-1 activity by DNA-damaging agents. We found that expression of both c-jun and c-fos, which encode proteins that participate in formation of the AP-1 complex, is rapidly induced by two different DNA-damaging agents: UV and H2O2. Interestingly, the c-jun gene is far more responsive to UV than any other immediate-early gene that was examined, including c-fos. Other jun and fos genes were only marginally affected by UV or H2O2. Furthermore, UV is a much more efficient inducer of c-jun than phorbol esters, the standard inducers of c-jun expression. This preferential response of the c-jun gene is mediated by its 5' control region and requires the TPA response element, suggesting that this element also serves as an early target for the signal transduction pathway elicited by DNA damage. Both UV and H2O2 lead to a long-lasting increase in AP-1 binding activity, suggesting that AP-1 may mediate the induction of other damage-inducible genes such as human collagenase.     

jun-B inhibits and c-fos stimulates the transforming and trans-activating activities of c-jun

We have cloned the human jun-B gene and determined its sequence and transforming and trans-activating activities. jun-B is less potent that c-jun in transforming and immortalizing primary rat embryo cells in cooperation with activated ras (effects enhanced by c-fos and TPA); unlike c-jun, jun-B does not transform Rat-1A cells alone. However, cotransfection of c-jun and jun-B into primary rat embryo cells with c-Ha-ras results in a significant decrease in transformation compared with c-jun alone, an event reversed by TPA. Cotransfection of c-jun and jun-B with or without c-fos into F9 teratocarcinoma cells results in decreased trans-activation of AP-1 compared with either gene alone. Introduction of jun-B into primary rat c-jun/ras transformants or c-jun into jun-B/ras transformants also results in a decrease in trans-activation. These findings demonstrate that, whereas jun-B and c-jun each participate in AP-1 trans-activation and malignant transformation, interactions between them involve negative regulation.