Inactivation of intergenic enhancers by EBNA3A initiates and maintains polycomb signatures across a chromatin domain encoding CXCL10 and CXCL9.

Epstein-Barr virus (EBV) causes a persistent infection in human B cells by establishing specific transcription programs to control B cell activation and differentiation. Transcriptional reprogramming of EBV infected B cells is predominantly driven by the action of EBV nuclear antigens, among them the transcriptional repressor EBNA3A. By comparing gene expression profiles of wt and EBNA3A negative EBV infected B cells, we have previously identified a broad array of cellular genes controlled by EBNA3A. We now find that genes repressed by EBNA3A in these cells are significantly enriched for the repressive histone mark H3K27me3, which is installed by Polycomb group (PcG) proteins. This PcG-controlled subset of genes also carries H3K27me3 marks in a variety of other tissues, suggesting that the commitment to PcG silencing is an intrinsic feature of these gene loci that can be used by EBNA3A. In addition, EBNA3A targets frequently reside in co-regulated gene clusters. To study the mechanism of gene repression by EBNA3A and to evaluate the relative contribution of PcG proteins during this process, we have selected the genomic neighbors CXCL10 and CXCL9 as a model for co-repressed and PcG-controlled genes. We show that EBNA3A binds to CBF1 occupied intergenic enhancers located between CXCL10 and CXCL9 and displaces the transactivator EBNA2. This impairs enhancer activity, resulting in a rapid transcriptional shut-down of both genes in a CBF1-dependent manner and initiation of a delayed gain of H3K27me3 marks covering an extended chromatin domain. H3K27me3 marks increase gradually and are maintained by EBNA3A. Our study provides direct evidence that repression by EBNA3A requires CBF1 and that EBNA3A and EBNA2 compete for access to CBF1 at identical genomic sites. Most importantly, our results demonstrate that transcriptional silencing by EBNA3A precedes the appearance of repressive PcG marks and indicate that both events are triggered by loss of enhancer activity

Exploring group process

This study takes as a starting point the need for further observational research in identifying the phenomenon of cohesion in group psychotherapy. The purpose is to test the possibility that there are relevant observational technologies that, like the microscope, can reveal phenomena that cannot be perceived directly but that underpin events at the perceptual level. Two text analytical approaches will be applied to mutually support possible findings: conversation analysis as a qualitative tool and quantitative computer-assisted text analysis following the therapeutic cycles model (TCM). The text to be analyzed is a transcript of Session 9 of a psychodynamic psychotherapy group for seven women diagnosed with an eating disorder. Within the cycles there is higher category density, higher levels of coherence, and tying across turns. The TCM reliably can identify features of the therapeutic process that are of clinical interest

<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

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

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

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

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

EBF1 binds to EBNA2 and promotes the assembly of EBNA2 chromatin complexes in B cells.

Epstein-Barr virus (EBV) infection converts resting human B cells into permanently proliferating lymphoblastoid cell lines (LCLs). The Epstein-Barr virus nuclear antigen 2 (EBNA2) plays a key role in this process. It preferentially binds to B cell enhancers and establishes a specific viral and cellular gene expression program in LCLs. The cellular DNA binding factor CBF1/CSL serves as a sequence specific chromatin anchor for EBNA2. The ubiquitous expression of this highly conserved protein raises the question whether additional cellular factors might determine EBNA2 chromatin binding selectively in B cells. Here we used CBF1 deficient B cells to identify cellular genes up or downregulated by EBNA2 as well as CBF1 independent EBNA2 chromatin binding sites. Apparently, CBF1 independent EBNA2 target genes and chromatin binding sites can be identified but are less frequent than CBF1 dependent EBNA2 functions. CBF1 independent EBNA2 binding sites are highly enriched for EBF1 binding motifs. We show that EBNA2 binds to EBF1 via its N-terminal domain. CBF1 proficient and deficient B cells require EBF1 to bind to CBF1 independent binding sites. Our results identify EBF1 as a co-factor of EBNA2 which conveys B cell specificity to EBNA2