Binding of Zta, MCAF1 and Rta to ZRE. (A) Probe Rp-SZ contains the sequence from −64 to −28 in the BRLF1 promoter. It also includes an Sp1 site (oval) and ZRE (rectangle). Probes Rp-mS, Rp-mZ and Rp-mSZ have the same sequence as Rp-SZ, except that the Sp1 site and ZRE in Rp-SZ were mutated as indicated by a crossed rectangle and a crossed oval. The number represents the nucleotide position relative to the transcriptional start site, ‘+1’ (arrow). ‘TATA’ represents TATA box. (C) A probe, MRP-RZ, contained the region between −174 and −36 in the BMRF1 promoter. Probes MRP-mR, MRP-mZ and MRP-mRZ contained a mutated RRE, ZREs and both RRE and ZREs as indicated by crossed rectangles. (E) Probe HLp-Z contained a ZRE from the BHLF1 promoter. In HLp-Z, the ZRE was mutated. (B, D, E) The probes were added to a lysate from P3HR1 cells that had been treated with TPA and sodium butyrate. Proteins that were bound to the probes were captured using streptavidin magnetic beads and analyzed by immunoblotting with anti-MCAF1, anti-Rta, anti-Sp1 and anti-Zta antibodies.

Quantitation of the binding of Zta, MCAF1, and Rta to the ZREs in the promoters of BHLF1 (A), BHRF1 (B), BMRF1 (C) and BMLF1 (D). P3HR1 cells were treated with TPA and sodium butyrate. The binding of Zta, MCAF1 and Rta to the ZREs was investigated by CHIP analysis using anti-Zta, anti-MCAF1 and anti-Rta antibodies and by qPCR. The reaction was performed with and without the addition of anti-IgG antibody as negative controls. The error bar represents standard error.

Mapping the interaction domains in MCAF1 and Zta. (A) Various regions in Zta were fused with GFP. Numbers represent the positions of amino acid in Zta. TA, transactivation domain; DBD, DNA-binding domain; DIM, dimerization domain. (B) Plasmids that expressed GFP-ZN (lanes 1 and 4), GFP-ZM (lanes 2 and 5) and GFP-ZC (lanes 3 and 6) were transfected into 293T cells. Proteins in the lysates were analyzed by immunoblotting (IB) with anti-GFP antibody (lanes 1–3). Proteins in the lysate were immunoprecipitated with anti-MCAF1 antibody and examined by IB with anti-GFP antibody (lanes 4–6). (C) Plasmids that expressed a segment of MCAF1 that was fused with GFP were used to identify the region in MCAF1 that interacted with Zta. Numbers represent positions of amino acids in MCAF1. Domains 1 and 2 were delineated by Fujita et al. (23). (D) The plasmids were transfected into P3HR1 cells that were treated with TPA and sodium butyrate. Proteins in the cell lysate that contained GFP-MCAF1-N (lane 1), GFP-MCAF1-DM1 (lane 2), GFP-MCAF1-M (lane 3) and GFP-MCAF1-DM2 (lane 4) were detected by IB with anti-GFP antibody (lanes 1–4). Proteins in the lysate were immunoprecipitated with anti-Zta antibody and analyzed by immunoblotting with anti-GFP antibody (lanes 5–8). (E) The lysate from E. coli BL21(DE3)(pET-Zta) was mixed with GST-Rta-, GST-MCAF1-C1-, GST-MCAF1-C2- or GST-glutathione-Sepharose-beads. Proteins that were bound to beads were detected by immunoblotting using anti-Zta antibody (lanes 1–5). E. coli lysates that contained His-Rta, His-MCAF1-C1 and His were mixed with GST-Zta-glutathione-Sepharose beads. Proteins that were pulled down by the beads were examined by IB with anti-Rta antibody (lanes 6–8). Lanes 1 and 6 were loaded with 0.5% of the lysate. Lane 9 shows the immunoblot of His-MCAF1-C1 that was purified by Ni-NTA agarose beads.

MCAF1 and synCergistic activation of transcription by Rta and Zta. (A) 293T cells were cotransfected with a reporter plasmid, pBHLF1 or pEAD, and an empty vector, pCMV-3 (CMV), plasmids that expressed Rta (R), Zta (Z) and MCAF1 (M). (B) Cells were also cotransfected with pCMV-R, pCMV-Z and 0–0.5 μg of pcDNA-MCAF1. (C) A similar study was conducted in BJAB cells. The luciferase activity of the cells was determined 48 h following transfection. Each transfection experiment was performed three times, and each sample in the experiment was prepared in duplicate.

Interaction among Zta, MCAF1 and Rta in vivo. (A) P3HR1 cells were treated with TPA and sodium butyrate. Proteins in the lysate (lanes 1 and 5) were immunoprecipitated (IP) with anti-IgG (lanes 2 and 6), anti-MCAF1 (lanes 4 and 7) and anti-Zta (lanes 3 and 8) antibodies. Immunoprecipitated proteins were analyzed by immunoblotting (IB) with anti-MCAF1 (lanes 1–4) and anti-Zta (lanes 5–8) antibodies. (B) Proteins in the lysate (lanes 1 and 5) were immunoprecipitated with anti-IgG (lanes 2 and 6), anti-Rta (lanes 3 and 7) and anti-Zta (lanes 4 and 8) antibodies. Immunoprecipitated proteins were detected by immunoblotting with anti-Rta (lanes 1–4) and anti-Zta (lanes 5–8) antibodies. Lane 1 in (A) and (B) was loaded with 1% of the cell lysate. Lane 5 in (A) and (B) was loaded with 3% of the cell lysate.

Subcellular localization of MCAF1, Rta and Zta. P3HR1 cells were transfected with pEGFP-Rta (A–D) or an empty vector, pEGFP (E–H), and treated with TPA and sodium butyrate (SB). Cells were also treated (I–L) or untreated (M–P) with TPA and sodium butyrate without transfection. Cells were incubated with monoclonal anti-Zta antibody (C, G, K and O) and rabbit anti-MCAF1 polyclonal antibody (J and N). DAPI staining revealed the nucleus (A, E, I, M). Cells were observed under a confocal laser-scanning microscope. D, H, L and P are merged images.

ZRE and synergistic activation by Rta and Zta. (A) A reporter plasmid, pBMRF1, contains a luciferase gene that is transcribed from the −172 to +20 region in BMRF1. In this region, the BMRF1 promoter contains an RRE and three ZREs. In pBMRF1-mRRE and pBMRF1-mZRE, the RRE and ZREs, respectively, are mutated. RRE and ZRE are represented as empty boxes. Mutant sequences are shown as crossed rectangles. (B) 293T cells were cotransfected with the reporter plasmids, pCMV-R and pCMV-Z. Cells that were transfected with pCMV-3 were used as a control. (C) 293T cells were also cotransfected with pBMRF1 and pCMV-R(K213A), pTag2-Zta or pCMV-3. (D) Plasmid pRRE contained an RRE from the BMLF1 promoter and pZRE contained a ZRE from the BRLF1 promoter. 293T cells were cotransfected with the reporter plasmids, pCMV-R, pCMV-Z and pCMV-3. The luciferase activity of the cells was determined at 24 h after transfection. Each transfection experiment was performed three times, and each sample in the experiment was prepared in duplicate. (E) 293T cells were transfected with pCMV-3, pCMV-R, pCMV-Z or cotransfected with pCMV-R and pCMV-Z. Rta, Zta and α-tubulin expressed by the cells were examined by immunoblotting.

Knockdown of MCAF1 and synergistic activation. (A) 293T cells were transfected with MCAF1 siRNA (lane 2) or control siRNA (lane 1). The expression of MCAF1 was determined by immunoblotting using anti-MCAF1 antibody at 48 h following transfection. The amount of α-tubulin in the cell was used as a control. (B) 293T cells were cotransfected with pBHLF1, pEAD, or pBMLF1 with pCMV-R, pCMV-Z and MCAF1 siRNA or control siRNA. The luciferase activity of the cells was determined 48 h following transfection. Each transfection experiment was performed three times, and each sample in the experiment was prepared in duplicate.