2A, lane 8), and coimmunoprecipitated by anti-Rta antibody (Fig. that this Rta-MCAF1-ATF2 complex binds to a typical AP-1 binding sequence around the promoter of BMRF2, a key viral gene for EBV contamination. Mutation of this sequence decreased Rta-mediated promoter activity significantly. Taken together, these results indicate a critical role for MCAF1 in AP-1-dependent Rta activation of BZLF1 transcription. Introduction Epstein-Barr computer virus (EBV) is usually a human herpesvirus that infects lymphoid and epithelial cells. During the switch from latency to the lytic cycle, EBV expresses two transcription factors, Rta Enfuvirtide Acetate(T-20) and Zta, which activate the transcription of EBV lytic genes [1]C[4]. These two factors are crucial to lytic progression, as infectious EBV particles cannot be produced in the absence of either factor [5]. It is Enfuvirtide Acetate(T-20) known that Rta binds to the Rta-response element (RRE) to activate viral promoters [2], [6], [7]. Rta also activates Sp1-dependent transcription via its conversation with an Sp1-binding protein, MCAF1 [8]. Zta, a member of the b-ZIP family, is encoded by the viral gene BZLF1, and regulates EBV lytic replication via binding to and recruitment of viral replication factors [9], [10]. Emerging evidence suggests that Zta binds to canonical Zta-response elements (ZRE) Enfuvirtide Acetate(T-20) in viral promoters, with a preference for CpG-methylated ZREs (meZRE), such as those found in the promoters of BBLF4, BMRF1 and BALF5 [11]. During the switch to lytic phase, Zta induces chromatin remodeling at target promoters, and can bind to meZRE in viral promoters without requiring active DNA demethylation [12], suggesting that EBV relies on Zta to reverse epigenetic silencing for induction of the lytic cycle [13]. Additionally, Zta acts in concert with Rta via MCAF1 to activate lytic promoters that contain ZREs [14]. Since Zta is crucial to the switch from latency to lytic progression in EBV, the regulation of the BZLF1 promoter (Zp) has been examined extensively [15]. The and to and to sequence in the ATF2 site was altered to for a mutant probe, mZII. A cell lysate prepared from P3HR1 cells treated with sodium butyrate and TPA was mixed with 0.2 g of biotinylated probe, in a binding buffer containing 60 mM KCl, 12 mM HEPES (pH 7.9), 4 mM Tris-HCl, 5% glycerol, 0.5 mM EDTA, 1 mM dithiothreitol, and 10 g/ml each of Enfuvirtide Acetate(T-20) leupeptin, aprotinin, and 4-(2-aminoethyl)-benzenesulfonyl fluoride. After incubating on ice for 45 min, the DNA-protein complex was incubated Enfuvirtide Acetate(T-20) with 30 l of Streptavidin MagneSphere Paramagnetic particles (Promega), which were pre-equilibrated in the binding buffer Rabbit Polyclonal to KNG1 (H chain, Cleaved-Lys380) for 1 h at 4C. The DNA-protein complex was then washed three times with the binding buffer, after which 2X electrophoresis sample buffer was added to the precipitated DNA-protein complex and the solution boiled for 5 min to dissociate the proteins. Finally, the proteins were separated by SDS-polyacrylamide gel electrophoresis, and detected using immunoblotting with specific antibodies. Immunoprecipitation 293T cells (1107) were transfected with plasmids using Turbofect (Thermo). Cell lysates were prepared 24 h after transfection using HEPES buffer (12 mM HEPES and 0.5% Nonidet P-40). Proteins in the lysate were immunoprecipitated with anti-Rta (Argene), anti-MCAF1 (Invitrogen), anti-ATF2 (Abcam), or anti-Flag (Sigma) antibodies. Protein-A/G agarose beads (Oncogene) were then added to the lysate. After shaking for 1 hr at 4C, the beads were collected by centrifugation and washed three times. Proteins bound to the beads were eluted by adding 2X electrophoresis sample buffer, and analyzed by immunoblotting. A cell lysate was also prepared from P3HR1 cells that had been treated with TPA and sodium butyrate for 24 h, to examine the conversation between Rta, MCAF1 and ATF2. Immunoprecipitated proteins were detected by immunoblotting using appropriate antibodies. GST pull-down assay GST and GST-ATF2 were purified from BL21(DE3) using glutathione-Sepharose 4B beads (Amersham Biosciences), according to a method described previously [27]. Immunoblot analysis Proteins in SDS-polyacrylamide gels were.
