H3K27me3 marks are elevated throughout the <i>CXCL10/9</i> domain in EBNA3A positive LCLs.

<p>(A) Schematic representation of the <i>CXCL10</i> and <i>9</i> encompassing domain indicating the positions of primer pairs A-T used for qPCR quantification of ChIPed DNA relative to the TSS of the analyzed genes. (B–G) ChIP analysis of established wt and EBNA3A negative LCLs (D2 wt 1 and D2 E3AmtB 3) showing the abundance of (B) Pol II, (C) H3ac, (D) H3K4me3, (E) H3K27me3, (F) SUZ12, and (G) EZH2. Bars indicate the enrichment of Pol II, of histone modifications and of PRC2 subunits at the individual loci as assessed by qPCR with primer pairs A-T. Primer pairs for the TSS of <i>GAPDH</i> (ctrl^ac) and a pericentromeric region on chromosome 1 (ctrl^si) were included as a control for active and silenced chromatin, respectively. Bar height was calculated as percentage of ChIPed DNA recovered from input DNA, after subtraction of values from negative control IgG precipitation. Data are representative of three independent experiments. Error bars indicate SD of triplicate qPCR reactions (with exception of data in panel G, which are given as mean ± range of two independent experiments).</p

A 2-step model for EBNA3A's mode of action.

<p>(A) EBNA3A displaces the transactivator EBNA2 from CBF1 occupied intergenic enhancers. Reduction of EBNA2 triggered enhancer activity by EBNA3A binding causes a rapid transcriptional shut-down of adjacent <i>CXCL10</i> and <i>9</i> genes. In the absence of EBNA2, however, EBNA3A acts by its intrinsic repressor activity, rendering <i>CXCL10</i> and <i>9</i> refractory to IFNγ-mediated induction. (B) The transcriptionally repressed state of <i>CXCL10</i> and <i>9</i> is subsequently fixed on the chromatin level by PcG proteins. PRC2-catalyzed H3K27me3 marks spread in a domain-wide fashion, potentially starting from remote enhancers. The gain of H3K27me3 levels to full range is a slow process that requires a time period of at least 14 days. When EBNA3A expression is discontinued, PcG repression is reversed and re-expression of distal genes is permitted (blue stars: H3K27ac; red hexagons: H3K27me3).</p

<i>CXCL10</i> and <i>CXCL9</i> reside within a PcG-controlled chromatin domain of 118 kb and are rapidly repressed upon EBNA3A expression.

<p>(A) Schematic representation of a genomic region on human chromosome 4 showing the location of the EBNA3A repressed genes <i>CXCL10</i> and <i>CXCL9</i> as well as flanking genes and the H3K27me3 coverage in wt LCLs (GM12878) according to ENCODE data. <i>CXCL10</i> and <i>9</i> comprise a region of 22 kb, which is embedded in an H3K27me3 positive domain of 118 kb. <i>CXCL11</i> and <i>ART3</i> also reside within this domain but are neither expressed in wt nor EBNA3A negative LCLs, while <i>SDAD1</i> and <i>NUP54</i> show similar expression levels irrespective of the EBNA3A status. Dotted lines demarcate an alternative TSS of <i>ART3</i>, which is not used in LCLs. (B) Validation of differential <i>CXCL10</i> and <i>9</i> expression in wt and EBNA3A negative LCLs derived from 5 unrelated B cell donors. Transcripts of <i>CXCL10</i> and <i>9</i> were quantified by qPCR in triplicate cDNA preparations from LCLs established by infection of B cells with EBVwt or either EBV-E3AmtA (D1, D4, D5) or EBV-E3AmtB (D2, D3). Data were normalized to 18S rRNA levels and are given as mean ± standard deviation (SD). (C) Western blot analysis of EBNA3A expression in ΔE3A-LCL^doxE3A cells prior to and 24 or 48 h post treatment with 100 ng/ml Dox. Protein extracts of the parental EBNA3A negative LCL (D2 E3AmtB 3) and a corresponding wt LCL (D2 wt 1) served as a negative and positive control, respectively. GAPDH immunodetection was used as loading control. Protein band intensities were quantified by densitometry. EBNA3A protein levels were normalized to GAPDH and are given as x-fold expression relative to the expression level in the corresponding wt LCL. (D) Flow cytometric analysis of NGFR expression in ΔE3A-LCL^doxE3A cells prior to and 24 h post treatment with 100 ng/ml Dox. Staining of cells with isotype-matched nonspecific antibodies served as a negative control. (E) EBNA3A induction in conditional LCLs rapidly down-regulates <i>CXCL10</i> and <i>9</i> expression. ΔE3A-LCL^doxE3A cells were induced for EBNA3A (E3A) expression by treatment with 100 ng/ml Dox for 24 or 48 h or left untreated. For metabolic labeling of nascent RNA, cells were cultured in the presence of 4sU for 1 h prior to harvesting. <i>CXCL10</i> and <i>9</i> transcripts in total RNA were quantified by qPCR, normalized to total 18S rRNA levels, and are given as mean ± SD of two biological replicates analyzed in triplicates. (F) <i>CXCL10</i> and <i>9</i> repression by EBNA3A is achieved by reduction of <i>de novo</i> transcription. Nascent RNA was isolated from total RNA prepared in (E). Nascent <i>CXCL10</i> and <i>9</i> transcripts were quantified by qPCR, normalized to nascent 18S rRNA levels, and are given as mean ± SD of two biological replicates analyzed in triplicates.</p

Transcriptional down-regulation precedes the gain of repressive H3K27me3 chromatin marks.

<p>ChIP analysis of ΔE3A-LCL^doxE3A cells showing the abundance of (A) Pol II, (B) H3ac, (C) H3K4me3, and (D) H3K27me3 across the <i>CXCL10</i> locus (primer pairs H-J, see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003638#ppat-1003638-g003" target="_blank">Figure 3A</a>) prior to and 24 h post EBNA3A induction with 100 ng/ml Dox. Primer pair S was used as a control. Bars were calculated and displayed as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003638#ppat-1003638-g003" target="_blank">Figure 3</a>. ChIP analyses of a wt LCL were included for comparison.</p

EBNA3A directly targets intergenic enhancers between <i>CXCL10</i> and <i>9</i> that are also bound by CBF1 and EBNA2.

<p>(A) Close-up of enhancer regions R1–R3 which are clustered within an intergenic 6 kb region located between <i>CXCL10</i> and <i>9</i>. R1–R3 are bound by CBF1 and EBNA2 in LCLs according to published ChIP-seq results <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003638#ppat.1003638-Zhao1" target="_blank">[19]</a>, which are displayed as raw read data for EBNA2, CBF1, and input DNA duplicates. The depicted region was additionally analyzed for CBF1 consensus binding sites <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003638#ppat.1003638-Kovall1" target="_blank">[100]</a> and aligned with ENCODE DNase-seq data, ChIP-seq data for H3K4me1, H3K27ac, p300, and Pol II, as well as strong enhancer annotations revealed by chromatin state segmentation. All displayed ENCODE data were generated with wt LCLs (GM12878). Black lines demarcate region R1, R2, and R3. (B) ChIP analysis with α-HA antibody showing the binding of HA-tagged EBNA3A to regions R1–R3 24 h post HA-EBNA3A induction with 100 ng/ml Dox in ΔE3A-LCL^doxHA-E3A cells. Results were either calculated as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003638#ppat-1003638-g003" target="_blank">Figure 3</a> (left panel) or displayed as fold enrichment of α-HA precipitated DNA over negative control IgG precipitation (right panel). Primer pair Q (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003638#ppat-1003638-g003" target="_blank">Figure 3A</a>) shows neither CBF1 nor EBNA2 binding and was used as a negative control. (C) ChIP analysis of EBNA2 occupancy at regions R1–R3 prior to and 24 h post HA-EBNA3A induction with 100 ng/ml Dox in ΔE3A-LCL^doxHA-E3A cells. Results were calculated and displayed as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003638#ppat-1003638-g003" target="_blank">Figure 3</a>. Primer pair Q was used as a negative control. (D) ChIP analysis of EBNA2 occupancy at regions R1–R3 in established wt and EBNA3A negative LCLs. Results are shown as mean ± SD of two independent experiments analyzed in duplicates. Primer pair Q was used as a negative control.</p

EBNA3A binding to intergenic enhancers reduces enhancer activity.

<p>ChIP analysis of (A) histone H3, (B) H3K4me1, (C) H3K27ac, and (D) Pol II occupancy at regions R1–R3 prior to and 24 h post HA-EBNA3A induction with 100 ng/ml Dox in ΔE3A-LCL^doxHA-E3A cells. Data are shown as mean ± SD of three independent experiments analyzed in duplicates. Results for H3K4me1 and H3K27ac were normalized to total histone H3 levels to account for the low nucleosomal occupancy at regions R1–R3. Asterisks indicate the <i>p</i>-value as determined by Student's <i>t</i>-test. Primer pair Q was used as a negative control.</p

EBNA3A impairs <i>CXCL10</i> and <i>9</i> induction by IFNγ via a CBF1-dependent mechanism.

<p>(A) Analysis of <i>CXCL10</i> and <i>9</i> repression by EBNA3A in DG75 wt^doxE3A and DG75 ko^doxE3A cell lines. EBNA3A expression was induced with 100 ng/ml Dox for 24 h either post (upper panels) or prior to (lower panels) IFNγ treatment for 6 h. <i>CXCL10</i> and <i>9</i> transcripts were quantified by qPCR prior to and post IFNγ or Dox treatment and normalized to 18S rRNA levels. X-fold repression and induction values are given as mean ± SD of two biological replicates analyzed in triplicates. (B) qPCR quantification of <i>CXCL10</i> and <i>9</i> transcripts prior to and post IFNγ treatment of a wt and an EBNA3A negative LCL established from the same B cell donor. Transcript levels were normalized to 18S rRNA levels and are shown as mean ± SD of three independent experiments.</p

The influence of EBNA 3C on chromosome looping at the <i>ADAM28/ADAMDEC1</i> locus.

<p>(A) Diagram (not to scale) showing the <i>Hind</i>III restriction fragments around the <i>ADAM28</i> locus that encompass the promoter (P), the ADAM enhancer (E, located downstream of <i>ADAM28</i>) and two intervening control regions (con1 and con2). The arrow indicates the direction of transcription. (B) Chromosome conformation analysis of the <i>ADAM28</i> locus in the pz1 control BJAB cell line (−) and the E3C-3 stable EBNA 3C expressing cell line (+) using primer pairs that amplify across promoter-enhancer or promoter-control ligation junctions. Positive controls show PCR amplification from control digestion and ligation reactions carried out using PCR-amplified DNA fragments encompassing the promoter, enhancer and control regions. (C) Diagram (not to scale) showing the <i>Aci</i>I restriction fragments around the <i>ADAMDEC1</i> locus that encompass the promoter (P), the ADAM enhancer (E, located upstream of <i>ADAMDEC1</i>) and an intervening control region (con). The arrow indicates the direction of transcription. (D) Chromosome conformation analysis of the <i>ADAMDEC1</i> locus in the pz1 control BJAB cell line (−) and the E3C-3 stable EBNA 3C expressing cell line (+) using primer pairs that amplify across promoter-enhancer or promoter-control ligation junctions. Positive controls show PCR amplification from control digestion and ligation reactions carried out using PCR-amplified DNA fragments encompassing the promoter, enhancer and control region.</p

The influence of EBNA 2 and 3A on chromosome looping at the <i>CTBP2</i> locus.

<p>(A) Diagram (not to scale) showing the <i>Eco</i>R1 restriction fragments at the <i>CTBP2</i> locus that encompass the promoter (P), enhancer (E) and an intervening control region (con). The arrow indicates the direction of transcription. (B) Chromosome conformation analysis in LCLs infected with wild-type or EBNA 3A knock-out EBV using primer pairs that amplify across promoter-enhancer or promoter-control ligation junctions. Positive controls show PCR amplification from control digestion and ligation reactions carried out using PCR-amplified DNA fragments encompassing the promoter, enhancer and control regions. (C). Chromosome conformation capture analysis in the ER-EB 2.5 LCL expressing EBNA 2 that is active in the presence of β-estradiol (+ est) and inactive in the absence of β-estradiol (−est). (D) Model for the control of chromatin looping by EBNA 2 and 3 proteins at <i>CTBP2</i>. (E) Re-ChIP analysis using anti-EBNA 2 antibodies in the first round of ChIP followed by a second round of ChIP in absence of antibody or using anti-EBNA 2, EBNA 3A, EBNA 3B or EBNA 3C antibodies. Results show mean percentage primary input −/+ range of two independent Q-PCR reactions from a representative experiment. (F) Control re-ChIP analysis using anti-EBNA 3A antibodies in the first round followed by re-precipitation in the absence of antibody or using anti-EBNA 3A antibodies. (G) Control re-ChIP analysis using anti-EBNA 3B antibodies in the first round followed by re-precipitation in the absence of antibody or using anti-EBNA 3B antibodies. (H) Control re-ChIP analysis using anti-EBNA 3C antibodies in the first round followed by re-precipitation in the absence of antibody or using anti-EBNA 3C antibodies.</p

The Effect of EBNA 2 and 3 proteins on <i>ITGAL</i> expression.

<p>(A) Q-PCR analysis of <i>ITGAL</i> transcript levels using cDNA from wild-type LCLs infected with B95.8 virus (wt) and EBNA 3B knock-out LCLs (KO) in two different donor backgrounds (PER142 and PER253). Transcript levels were normalised to GAPDH levels and expressed relative to the level in wt cells for each donor. Results show the mean −/+ range of two independent QPCR reactions each performed in duplicate. (B) Luciferase reporter assays carried out in DG75 cells transiently transfected with 2 µg of the control vector pGL3 basic, an <i>ITGAL</i> promoter-luciferase reporter (pGL3 <i>ITGAL</i>p) or the EBV C promoter reporter (pCp1425GL2) (right panel) in the absence or presence of 10 or 20 µg of EBNA 2, 3A, 3B or 3C expressing constructs. Firefly luciferase signals were normalised to Renilla luciferase signals from the co-transfected control plasmid pRL-TK (1 µg). Results show the mean −/+ standard deviation of 3 independent experiments and are expressed relative to the pGL3 basic signal (left panel) or the Cp1425GL2 signal (right panel) in the absence of EBNA 2. Western blot analysis of EBNA 2, 3A, 3B and 3C expression levels in transfected cells. Each set of blots was also probed for actin as a loading control. (C) Re-ChIP analysis in Mutu III cells using anti-EBNA 2 antibodies in the first round of ChIP followed by a second round of ChIP in absence of antibody or using anti-EBNA 2, EBNA 3B or EBNA 3C antibodies. Primers at peak 3 were used for analysis. Results show mean percentage primary input −/+ range of two independent Q-PCR reactions from a representative experiment. (D) Control re-ChIP analysis using anti-EBNA 3B antibodies in the first round followed by re-precipitation in the absence of antibody or using anti-EBNA 3B antibodies. (E) Control re-ChIP analysis using anti-EBNA 3C antibodies in the first round followed by re-precipitation in the absence of antibody or using anti-EBNA 3C antibodies.</p