Serine residue 186 is required for Z activation of the methylated Nap <i>in vivo</i>.

<p>HeLa cells were transfected with methylated luciferase vectors driven by the Na or R promoters, in the presence or absence of a co-transfected wild-type Z (WTZ), or the Z(S186A) mutant altered in the DNA binding domain at residue 186. The amount of luciferase activity produced by each condition (obtained from duplicate transfections) is indicated.</p

Serine residue 186 is required for Z binding to the ZREs in the Nap.

<p>(A) EMSA was performed using either methylated or mock-methylated oligonucleotide probes containing the Nap ZRE1, Nap ZRE2, Rp ZRE2, Rp ZRE3 and BMRF1p AP1 sites and <i>in vitro</i> translated wild-type Z (WTZ), ZS186A or ZC189S. The relative efficiency of WTZ, versus mutant Z, binding to each probe is indicated; the amount of WTZ binding to each probe is arbitrarily set as 100%. ND: none detected. (B) The amount of Z protein used in the EMSAs in (a) was quantitated by immunoblot for WT and mutant proteins.</p

Z binds to the Na promoter, and binding is enhanced by Nap methylation.

<p>(A) Latently infected EBV+ 293 Z-KO cells were transfected with a BZLF1 expression vector (+) or an empty vector control (−). Chromatin immunoprecipitation assay was performed 20 hours after transfection using anti-Z and control goat IgG antibodies as indicated to examine Z binding to the Na promoter (Nap), ZREs within the EBV oriLyt (positive control) and EBV sequences in the 3′ end of the EBV BALF5 gene (negative control). (B) The ability of <i>in vitro</i> translated Z to bind to ³²P end-labeled probes (in either the methylated, or unmethylated forms) containing the BRRF1 promoter (Nap) sequences from −280 to −470, −108 to −301, and +77 to −128 (relative to the <i>BRRF1</i> mRNA start site) was examined by EMSA. A probe containing the BRLF1 promoter (Rp) sequences from −1 to −270 (in the methylated form) served as a positive control.</p

Wild-type Z, but not Z(S186A), activates Na and R transcription.

<p>Wild-type Z (WTZ), Z(S186A), Z(C189S) or empty vector (vector) were transfected in to 293 Z-KO cells in the presence or absence of an R expression vector and assayed by western blot for expression of Z, R, Na, EA-D or β-actin.</p

Comparison of ZRE sites.

<p>Sequences of the consensus AP1 site, the three Rp ZRE sites and the two Nap ZREs are shown. Methylated cytosines are indicated with an asterisk.</p

Z preferentially binds to the methylated forms of ZRE sites in the Nap.

<p>(A) The ability of <i>in vitro</i> translated Z to bind to labeled oligonucleotide probes containing potential ZRE sites in the Nap was examined by EMSA using methylated or mock-methylated probes as indicated. Nap sequences (with probable ZRE sites underlined) were (Nap ZRE1, −58 to −41): 5′-CAT TC<u>T CGC CCG</u> TGG GCC-3′ and (Nap ZRE2, −83 to −70): 5′-GT<u>T GAG CGT</u> GGC CA-3′. Binding to the unmethylated and methylated forms of two CpG-containing ZREs in the BRLF1 promoter (Rp) was also examined. The relative efficiency of Z binding to each probe is indicated; the amount of Z binding to the methylated Rp ZRE3 probe is arbitrarily set as 100%. ND: none detected. (B) The ability of <i>in vitro</i> translated Z to bind to the labeled oligonucleotide Nap ZRE1 probe (−58 to −41) containing no methylated cytosine, one methylated cytosine, or two methylated cytosines was compared. The methylated cytosine(s) present in each probe are indicated by a bold C in the sequence.</p

(A) LCL cells were transfected with the indicated amounts of empty pcDNA3 (Vector) or expression vector pcDNA3-Flag-DOK1.

<p>After 24(<b>B</b>) LCL cells were monitored for cell cycle analysis 48 hours after being transfected with the indicated amounts of pcDNA3 empty (Vector) or expression vector pcDNA3-Flag-DOK1. Cells in different cycle phases (SubG0, G0/G1, S, or G2/M) are represented as percentage of total cells. (<b>C</b>) The same cells from (B) were monitored for apoptosis using Annexin V staining. Non transfected cells were used as control (NT). Error bars indicate the SD from two independent experiments. Data were analyzed using Student's t test (*, P<0.05).</p

LMP1 represses <i>DOK1</i> promoter activity through the recruitment of E2F1/pRB/DNMT1 inhibitory complex.

<p>(<b>A</b>) RPMI cells were transfected with the indicated firefly luciferase reporter pGL3-<i>DOK1</i> promoter constructs along with increasing amounts of pcDNA3 LMP1. Renilla luciferase was used as an internal control for the reporter assay. After 48 hours, cells were collected and processed for luciferase activity measurement. The data are average of three independent experiments. (<b>B</b>) RPMI cells, RPMI cells infected with GFP-EBV recombinant virus, or GFP-EBVΔLMP1, RPMI cells transduced with empty pLXSN (V) or expression vector pLXSN-LMP1, and LCLs and their original primary B-cells were subjected to quantitative ChIP assay using anti-E2F1 (KH 95) antibody or IgG. The <i>DOK1</i> promoter was amplified by real-time PCR using specific primers flanking the E2F-response element located at (−498/−486). Data were calculated as percentages of enrichment of input. Error bars indicate the standard deviation from three independent experiments performed in triplicate. (<b>C</b>) The same cells from (B) were subjected to ChIP assay using the anti-H3K27 trimethylation antibody, anti-H3K4 trimethylation antibody or IgG. The <i>DOK1</i> promoter and GAPDH promoter were amplified by real-time PCR. (<b>D</b>) In vitro DNA pull-down assay. The <i>DOK1</i> promoter region containing the original E2F-response element located at (−498/−486) or a mutated one (obtained by replacing the core GGCG of the consensus sequence with AAAA), was amplified by PCR using specific 5′biotinylated primers. The PCR products (agarose gel, bottom panel) were incubated with total lysate from RPMI cells transduced with empty pLXSN (Vector) or expression vector pLXSN-LMP1, and then pulled down using streptavidin-agarose beads. Immunoblotting was used to check the recruitment of E2F1, pRB, DNMT1, EZH2 and p65 to the different PCR fragments. β-Actin was used as a negative control of binding to DNA. (<b>E</b>) Transduced RPMI cells with empty pLXSN (Vector) or expression vectorpLXSN-LMP1 were used for quantitative ChIP Re-ChIP assay. To assess the individual recruitment of E2F1, pRB, DNMT1, and EZH2 to the <i>DOK1</i> promoter, chromatin was immuno-precipitated (IP 1) with the indicated antibodies and IgG was used as a negative control (<b>top</b>). To determine the E2F1 association with the indicated factors, E2F1 chromatin complex (IP 1) was subjected to Re-ChIP (IP 2) using the indicated antibodies (<b>bottom</b>). The <i>DOK1</i> promoter was amplified using real time PCR and data from IP 1 and IP 2 were calculated as percentage of total input.</p

LMP1-mediated NF-κB activation is required for EBV-related <i>DOK1</i> down-regulation.

<p>RPMI cells transduced with empty retroviral pLXSN (Vector), expression vector pLXSN-LMP1, or infected with GFP-EBV recombinant virus were treated with Bay11 or the equivalent volume of DMSO (Mock). (<b>A</b>) mRNA levels of <i>LMP1</i> and <i>DOK1</i> were measured by real time PCR, and normalized to <i>GAPDH</i> expression. (<b>B</b>) The indicated proteins were detected using western blotting. RPMI cells were transfected with pcDNA3 empty plasmid (Vector), expression vector pcDNA3-LMP1 and/or expressing the super-repressor IκBα (ΔIκBα), while RPMI cells infected with GFP-EBV recombinant virus were transfected only with pcDNA3 empty (Vector) or expression vector of the super-repressor IκBα (ΔIκBα). After 48 hours, cells were collected for analysis. (<b>C</b>) mRNA levels of <i>LMP1</i> and <i>DOK1</i> were measured by real time PCR, and normalized to <i>GAPDH</i> expression. (<b>D</b>) The indicated proteins were detected using western blotting. RPMI cells were transfected with empty pLXSN (Vector), or expression vector pLXSN-LMP1 wild type (WT), LMP1 mutant for the CTAR1 domain (AxAxA), and CTAR2 domain (378 stop), or both CRAT1 and 2 domains (AxAxA/378 stop). After 48 hours, cells were harvested for expression analysis. (<b>E</b>) mRNA levels of <i>LMP1</i>, <i>GAPDH</i> and <i>DOK1</i> were measured using real time PCR. (<b>F</b>) The indicated proteins were detected using western blotting. DOK1 protein levels were quantified from two independent immunoblots and normalized to the corresponding β-actin level (bottom of B, D and F). Stable RPMI cells with empty pLXSN (Vector), or expression vector pLXSN-LMP1, were treated with Bay11 or the equivalent volume of DMSO (Mock). (<b>G</b>) Cells were subjected to quantitative ChIP assay using the indicated antibody or IgG. The <i>DOK1</i> promoter was amplified by real-time PCR using specific primers flanking the E2F-response element located at −498/−486. Data were calculated as percentages of enrichment of total input. Error bars indicate the standard deviation from two independent experiments performed in triplicate. (<b>H</b>) <i>In vitro</i> DNA pull-down assay. The <i>DOK1</i> promoter region containing ERE1 was incubated with total lysate from RPMI cells expressing LMP1 treated with Mock or Bay11, and then pulled down using streptavidin-agarose beads. Immunoblotting was used to check the recruitment of E2F1, pRB, DNMT1, EZH2. β-Actin was used as a negative control of binding to DNA.</p

5-Aza treatment rescue <i>DOK1</i> expression in EBV infected cells.

<p>Cells were treated with 1 µM methyl-transferase inhibitor 5-Aza-2′deoxycytidine (5-Aza) for 4 days or equivalent volume of DMSO (Mock), then collected for analysis. (<b>A</b>) DNA methylation levels of the <i>DOK1</i> promoter were measured using pyrosequencing. Each bar represents the percentage of methylation for individual CpG sites. (<b>B</b>) Quantitative ChIP assay using anti-E2F1 (KH 95) antibody or IgG. The <i>DOK1</i> promoter was amplified by real-time PCR using specific primers flanking the E2F-response element located at (−498/−486). Data were calculated as percentages of enrichment of input. Error bars indicate the standard deviation (SD) from two independent experiments performed in triplicate. (<b>C</b>) The mRNA expression levels of <i>LMP1</i>, <i>GAPDH</i> and <i>DOK1</i> were determined using real time PCR. (<b>D</b>) The indicated proteins were analyzed using western blotting. DOK1 protein levels were quantified from two independent immunoblots and normalized to the corresponding β-actin level (bottom). (<b>E</b>) ChIP assays were carried out using anti-H3K27 trimethylation antibody, anti-H3K4 trimethylation antibody or IgG. The <i>DOK1</i> promoter and <i>GAPDH</i> promoter were amplified by real-time PCR. Data were calculated as percentages of enrichment of input.</p