Critical role of p53 in histone deacetylase inhibitor-induced Epstein-Barr virus Zta expression - PubMed
p53 is a determinant of the HDACi-mediated susceptibility of Zp. (A) Requirement of p53 for HDACi-induced expression of BZLF1 transcripts. NA cells were pretreated with p53 siRNA (sip53) or an irrelevant siRNA (siE7) and then manipulated with TSA for 24 h or SB for 48 h. RT-PCR analysis was performed to measure the BZLF1 transcripts. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression was detected as an internal control. RT(+) and RT(−) denote experiments with and without reverse transcriptase in the RT-PCR mix, respectively. The numeric data below the panels are the relative intensities of BZLF1 transcripts, which were determined by ImageQuant quantification and normalized with their corresponding glyceraldehyde-3-phosphate dehydrogenase signals. (B) Confirmation of the requirement of p53 for Zta protein expression from a plasmid containing the BamH1 Z fragment (BamH1 Zf). After transfection with the sip53 and siE7 siRNAs, TW01 cells were transiently transfected with BamH1 Zf, followed by TSA treatment for 24 h. The control represents no drug treatment. Zta protein expression was detected by immunoblotting assay. β-Actin was used as the internal control. The intensities of Zta and p53 relative to that of β-actin are shown. (C) p53 is an essential factor for TSA-induced Zp activation. A reporter construct driven by Zp (positions −554 to +12) and a control vector (pGL2-basic) were used in this luciferase reporter assay. TW01 cells treated with siRNA or TSA are indicated (+). A t test analysis was used to evaluate the statistical significance of the difference in Zp activities in TSA-treated (bar 6) and mock-treated (bar 5) TW01 cells expressing siE7 (P = 0.015). A significant difference in Zp activities between TW01 cells transfected with siE7 (bar 6) and those transfected with sip53 (bar 8) was seen (P = 0.011). (D) TSA promotes Zp histone acetylation status in a time-dependent manner. NA cells treated with TSA for the indicated times were subjected to ChIP assay as described previously (21). Briefly, formaldehyde cross-linked cells were washed with ice-cold phosphate-buffered saline and harvested after centrifugation at 10,000 × g. Pellets were then resuspended in nuclear lysis buffer (50 mM Tris, pH 8.1, 10 mM EDTA, 1% SDS) with Complete protease inhibitor cocktail (Roche). Lysates were sonicated to yield 500- to 1,000-bp DNA fragments. Subsequent lysates were diluted with 10 volumes of ChIP dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl, pH 8.1, 167 mM NaCl, 1 mM dithiothreitol, and 50 μg salmon sperm DNA) and incubated overnight at 4°C with anti-acetylated histone H3 (anti-AcH3; Upstate), anti-AcH4 (Upstate), or nonspecific immunoglobulin G (Dako). The immunocomplexes were precipitated by incubation with salmon sperm DNA and bovine serum albumin preblocked 50% protein A-Sepharose (GE Healthcare), followed by washing sequentially with buffer I (20 mM Tris, pH 8.0, 2 mM EDTA, 150 mM NaCl, 1% Triton, 0.1% SDS), buffer II (20 mM Tris, pH 8.0, 2 mM EDTA, 500 mM NaCl, 1% Triton, 0.1% SDS), buffer III (10 mM Tris, pH 8.1, 0.25 M LiCl, 1 mM EDTA, 1% NP-40, 1% Igepal), and Tris-EDTA buffer. The immunocomplexes were then eluted using buffer containing 1% SDS, 0.25 M NaCl, and 0.1 M NaHCO3. Cross-linked DNAs were then reversed by being heated to 65°C overnight. After being treated with proteinase K, DNAs were extracted with phenol-chloroform, dissolved in Tris buffer (pH 8.0), and analyzed by PCR, using specific primers encompassing positions −221 to +12 of Zp. The ImageQuant-determined intensities of anti-AcH3- and anti-AcH4-captured Zp signals relative to those of their corresponding input controls are shown. (E to G) p53 did not affect TSA-mediated acetylation of H3 and H4 on Zp. p53-depleted NA cells (E and F) and p53-transfected H1299A cells (G) were subjected to histone H3 and H4 acetylation status analysis using ChIP assay. The regions of positions −221 to +12 (E and G) and −554 to −221 (F) of Zp were amplified by PCR. The relative intensities of anti-AcH3- and anti-AcH4-precipitated Zp DNA amounts were provided after being normalized with those of their input controls and standardized with that of TSA-treated siE7 (E and F, lane 2) or SB-treated vector control (G, lane 2).