Functional antagonism between the retinoic acid receptor and the viral transactivator BZLF1 is mediated by protein-protein interactions.

Abstract

The Epstein-Barr virus-encoded protein BZLF1 is a member of the basic leucine zipper (bZip) Epstein-Barr virus is a human herpesvirus that infects B lymphocytes and epithelial cells of the nasopharynx (1). Infection of B cells typically results in a latent state, whereas infection of epithelial cells leads to viral replication and cytolysis (2). The switch from latency to the productive (replicative) cycle is induced by various agents such as calcium ionophores, sodium butyrate, and tumor promoters like phorbol 12-myristate 13-acetate, all of which activate expression of the Epstein-Barr virus immediate-early gene product BZLF1 (2). Overexpression of BZLF1 is sufficient to trigger the switch to the lytic cycle (2). Like the c-Jun/c-Fos proteins BZLF1 is a member of the basic leucine zipper (bZIP) transcription factor family (3, 4). BZLF1 binds as a homodimer to DNA (4) and transactivates several early viral promoters required in the cytolytic cycle of the virus. The BZLF1 response elements (ZREs) in these promoters are similar and sometimes identical to AP-1 binding sites (4).Retinoids play an important role in development and differentiation and are well-known inhibitors of cell growth (for review, see ref. 5). Retinoic acid (RA) exerts biological effects by acting through at least two distinct classes of intracellular proteins including the RA receptors (RARs) and the retinoid X receptors (RXRs), both of which are members of the nuclear receptor superfamily (5). While both the RARs and RXRs are effective activators of some genes, RA is also known to repress expression of AP-1-dependent genes (6). RAR has beenThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.shown to inhibit transcriptional activity of the AP-1 complex by a mechanism that is not dependent on the receptor's ability to bind DNA and most likely reflects protein-protein interactions (6). This interaction of distinct regulatory pathways, termed "cross-talk," suggested a means by which nuclear hormone receptors could modulate the action of growthinducing factors. Recent data suggested that RARa is able to repress the BZLF1-mediated transcriptional activation of the BMFR1 promoter via direct protein-protein interactions between RARa and BZLF1 (7).To further extend our understanding of the cross-talk between RAR and members of the bZIP transcription factor family, we were interested in examining the mutual repression effects of RARa and BZLF1 in more detail. In this report, we show that RARa and BZLF1 repress each other's transcriptional activity. Transfection analysis of RARa and BZLF1 mutants reveals that the transactivation domain and the dimerization domain of the BZLF1 and the DNA binding domain (DBD) of RARa are required for repression. Glutathione S-transferase (GST)-pulldown assays and coimmunoprecipitation experiments demonstrate direct protein-protein interactions between BZ...

Transcriptional regulation and induction of apoptosis: implications for the use of monomeric p53 variants in gene therapy

“…Nuclear extracts of transiently transfected SKBR3 cells were prepared as described previously. 25 Plasmids All p53 expression vectors used in this study were constructed from the pCMX-pL1 65 vector containing the CMV immediate-early promoter/enhancer and human p53 cDNA. Truncated p53 variants, p53⌬326 and p53⌬300 were derived from wild-type cDNA using PCR methods.…”

bZIP proteins of human gammaherpesviruses

“…Evidence in favour of the functional relevance of this interaction comes from the observation that retinoic acid is able to inhibit the reactivation of EBV from latency (Sista et al, 1993). The bZIP region and the transactivation domain of Zta appear to be involved in this interaction (Pfitzner et al, 1995;Sista et al, 1995).…”

“…Zta is able to interact directly with the retinoic acid receptors RAR a and RXR a (Sista et al, 1993(Sista et al, , 1995Pfitzner et al, 1995). Each transcription factor is able to inhibit the other when expressed at high levels.…”

Molecular cloning of the Matrix Gla Protein gene from Xenopus laevis. Functional analysis of the promoter identifies a calcium sensitive region required for basal activity

“…Cells were seeded at 60% confluency in 12-well plates and transient transfections were carried out using the standard calcium phosphate coprecipitation technique [22]. To evaluate dose-dependent effects of extracellular calcium on MGP transcription cells were grown in medium supplemented with either calcium chloride (Sigma) or water 24 h after transfection.…”