Figure 1.
NAP1 and TAF-I contribute to EBV lytic replication.
(A) AGS-EBV cells were transfected with siRNAs against NAP1, TAF-I, or negative control (NC) siRNA and, 48 hours later, treated with TSA for 24 hours to induce the EBV lytic cycle (lanes 4-6) or left untreated (lanes 1-3). Equal amounts of extracts from these cells were Western blotted using antibodies against NAP1 [52] [46] [45] [45] [45], TAF-I, actin, BZLF1 and BMRF1. (B) qPCR analysis of mRNA levels of BZLF1 and BMRF1 in TSA-treated AGS-EBV cells transfected with the indicated siRNAs as in A. Total RNA was isolated and BZLF1 and BMRF1 transcripts were amplified and normalized to GAPDH transcripts. Relative mRNA levels are shown where NC siRNA samples were set to 100. (C) EBV episome copy number was determined from samples in B by qPCR of the DS region, which was normalized to cellular GAPDH. The fold increase in EBV genomes after TSA treatment (compared to no TSA treatment) is shown for each siRNA treatment. The data from B and C are from three independent experiments with PCR performed in duplicate for each. (D) AGS-EBV cells grown on coverslips were transfected with TAF-I siRNA or NC-siRNA, treated with TSA for 6 or 16 hours, then stained for BZLF1. The percentage of BZLF1-positive cells was counted by immunofluorescence microscopy for 3 independent experiments. A representative image is shown of BZLF1 staining after treatment with TAF-I siRNA or NC-siRNA and 16 hr TSA treatment (all cells were confirmed to be depleted in TAF-I after siTAF-I treatment in an independent experiment). (E) HONE1-Akata cells were transfected with siRNA against TAF-I, treated with TSA for 16 hours, then stained for BZLF1. The percentage of BZLF1-positive cells was counted by immunofluorescence microscopy for 3 independent experiments and average values are shown in the bar graph. A Western blot confirming TAF-Iβ silencing and a representative image of the BZLF1 staining are also shown. For all the bar graphs ** = P<0.01, * = 0.01<P<0.05.
Figure 2.
Overexpression of TAF-Iβ or NAP1 induces BZLF1 expression.
(A) AGS-EBV cells grown on coverslips were transfected with pCMVmyc-TAFIβ, treated with TSA for 16 hours or left untreated then stained with anti-myc and anti-BZLF1 antibodies. The percentage of BZLF1-positive cells was counted separately for myc-positive (myc-TAF-I) and myc-negative (mock) cells on the same slides and average values are plotted from 3 independent experiments. ** = P<0.01. * = 0.01<P<0.05. (B) AGS-EBV cells were transfected with pCMVmyc-TAF-Iα, pCMVmyc-TAF-Iβ, pCMVmyc-NAP1 or negative control pCMVmyc and 24 hours later stained with anti-myc and anti-BZLF1 antibodies. The percentage of myc-positive cells that expressed BZLF1 was determined in two independent experiments. (C) Representative microscopy images from B. (D) A Western blot of the cells in B after transfection with TAF-Iα, TAF-Iβ and NAP1 expression plasmids as compared to mock transfected cells (C).
Figure 3.
TAF-I binds to the BZLF1 promoter upon EBV lytic reactivation and affects histone modifications.
(A) ChIP assays were performed on AGS-EBV cells before (grey bars) or after (black bars) TSA treatment using antibodies against TAF-I or nonspecific rabbit IgG. Recovered DNA fragments were quantified by qPCR, using primer sets specific to the DS or the BZLF1 promoter regions as indicated and normalized to signals from total EBV DNA. The signals from the TAF-I samples are shown relative to those from the nonspecific IgG samples, which were set to 1. (B) AGS-EBV cells were transfected with siRNAs against TAF-I (black bars) or negative control siRNA against GFP (grey bars), and then treated with TSA for 24 hours or left untreated as indicated. ChIP assays were performed as in A using antibodies against H3K4me2 (left panels), H4K8ac (right panels), or total H4. The amplified signals from the promoter region or DS element were normalized to those from total histone H4. Data is shown from 3 independent experiments with PCR performed in duplicate for each. ** = P<0.01. * = 0.01<P<0.05. (C) Equal amounts of total cell lysates from AGS-EBV cells treated with siRNA against TAF-I, NAP1 or GFP with and without TSA treatment were analyzed by Western blotting with the indicated antibodies.
Figure 4.
TAF-I recruits MLL1 to the BZLF1 promoter.
(A) ChIP assays were performed on AGS-EBV cells with (black bars) or without (grey bars) TSA treatment using antibody against MLL1 or nonspecific IgG and primers to amplify the DS or BZLF1 promoter region as indicated. The amplified signals from the ChIP samples were normalized to those from total EBV DNA and shown relative to the nonspecific IgG negative control. (B) ChIP assays were performed with MLL1 antibody on TSA-treated AGS-EBV cells transfected with siRNA against TAF-I (black bars) or GFP (grey bars). A Western blot is also shown confirming TAF-I depletion. All ChIP data is from 3 independent experiments with PCR performed in duplicate. ** = P<0.01. (C) AGS-EBV cells were transfected with siRNA against TAF-I, MLL1 or negative control siRNA (NC), then treated with TSA (+TSA) or left untreated (-TSA). Western blots were then performed on equal amounts of cell lysates using the indicated antibodies. The image for the BZLF1/BMRF1 blot with –TSA samples was developed using a longer exposure time than that of the +TSA samples in order to detect the low level of spontaneous BZLF1 expression.
Figure 5.
TAF-I binding to the BZLF1 promoter is influenced by EBNA1 upon lytic reactivation.
(A) ChIP assays for the BZLF1 promoter region were performed on AGS-EBV cells treated with siRNA against EBNA1 (black bars) or GFP (grey bars) before (left graph) or after TSA (right graph) treatment using TAF-I antibody as in Fig. 3A. ** = P<0.01 (B) A Western blot showing EBNA1 depletion and TAF-Iβ levels.