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

<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

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

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

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

Subcellular localization of read-through transcripts in HSV-1 infection.

<p>(a) Boxplots indicating the distribution of the percentage of transcription downstream of genes identified before infection in total, cytoplasmic, nucleoplasmic and chromatin-associated RNA. Numbers in boxes indicate median values. (b) Boxplots indicating the distribution of read-through at 8h p.i. HSV-1 infection in all RNA fractions. Numbers in boxes indicate median values. (c) Boxplots indicating the distribution of log2 ratios of nucleoplasm enrichment (= gene FPKM in nucleoplasmic RNA/ gene FPKM in cytoplasmic RNA) at 8h p.i. compared to uninfected cells. Ratios are shown separately for groups of genes with different amounts of read-through in 7-8h p.i. 4sU-RNA. The value for IRF1 is highlighted in red. (d) Heatmap of read counts (sum of 2 replicates) in total, cytoplasmic, nucleoplasmic and chromatin-associated RNA for the intergenic splicing events shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006954#ppat.1006954.g003" target="_blank">Fig 3C</a>. Results for the intergenic splicing junction connecting SRSF2 and JMJD6 are highlighted by a blue box. (e) Spearman correlation between read-through (calculated from 4sU-seq data) in all conditions and the percentage of transcription downstream of genes identified in chromatin-associated RNA of uninfected/untreated cells. Correlation to mock read-through values is shown below. Mock read-through values were calculated as described in methods and correlations were averaged for each condition. (f) Mapped sequencing reads (negative strand) for total (light/dark pink), cytoplasmic (light/dark green), nucleoplasmic (light/dark red) and chromatin-associated RNA (light/dark cyan) in uninfected cells (light colors) and at 8h p.i. (dark colors) for the IRF1 gene. Read coverage ranges and RefSeq gene annotation are indicated as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006954#ppat.1006954.g001" target="_blank">Fig 1C</a>. (g) Scatterplot of read-through at 2-3h p.i. against the percentage of transcription downstream of genes identified in chromatin-associated RNA of uninfected/untreated cells. Colors indicate density of points (red = highest density, blue = lowest density).</p

Global characteristics of DoTT/DoG transcription in salt and heat stress and HSV-1 infection.

<p>(a) Boxplots showing the distribution of read-through in salt (orange) and heat stress (red) and HSV-1 infection (cyan). Results for individual replicates are shown in Fig D in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006954#ppat.1006954.s003" target="_blank">S3 File</a>. (b) Median read-through values for each condition and time-point are plotted against the standard deviation in gene expression (= gene FPKM) fold-changes (log2). The gray curve indicates the result of a locally weighted polynomial regression (LOWESS) on all HSV-1 infection time-points. Spearman correlation (Cor) between median read-through and standard deviation in log2 expression fold-changes across all samples is also indicated. (c) 6-mers whose frequency in the 100nt up- or downstream of gene 3’ ends is significantly correlated to read-through in at least one sample (FDR adjusted p<0.0001). FDR adjusted p-values for all samples are color-coded (red for negative correlations, blue for positive correlations). (d) Boxplots showing the distribution of read-through in 2h salt and heat stress and 4-5h and 7-8h p.i. for genes without (w/o) or with (w/) at least one occurrence of the AAUAAA motif in the 100nt upstream of gene 3’ends. P-values of Wilcoxon rank sum tests comparing read-through in each sample between the two groups are indicated above the x-axis.</p

Comparison of DoTT/DoG transcription and association with aberrant splicing.

<p>(a) Spearman correlation for read-through values between all samples. (b) Heatmap of read-though for all 3,682 analyzed genes in salt and heat stress and HSV-1 infection (excluding the first two time-points with very low levels of read-through). Colors indicate read-though >5%. Hierarchical clustering was performed using average linkage clustering based on Euclidean distances. (c) Heatmap of read counts (sum of 2 replicates) for intergenic splicing events connecting exons of neighboring genes on the same strand. Junctions are annotated with the upstream and downstream gene symbol. Results for the intergenic splicing junction connecting SRSF2 and JMJD6 are highlighted by a blue box. Only junctions are shown with >2 reads covering at least 5bp of both exons in either 2h salt stress, 2h heat stress or 7-8h p.i. HSV-1 infection. Hierarchical clustering was performed as for (b). (d) Percentage of splicing junctions that are part of protein-coding transcripts, novel (using either 2 or 1 known exon boundary), nonsense-mediated-decay (NMD)-associated or only observed in a processed transcript. Results are shown separately for non-regulated, up-regulated and down-regulated junctions (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006954#sec012" target="_blank">methods</a> for definition) for 2h salt and heat stress and 4–5hs and 7-8h p.i. HSV-1 infection.</p