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Supplementary Material and Methods
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Electrophoretic Mobility Shift Assays (EMSA)
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PRE
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ACAAGGGACTTTCCGCTGGGGACTTTCCAGG-3’
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ACAACTCACTTTCCGCTGCTCACTTTCCAGG-3’. The NF-B binding B sites are in
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bold type and mutated bases have been underlined. Probe containing AP-1 site (Activating
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Protein-1
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CGCTTGATGAGTCAGCCGGAA-3’. The oligonucleotides were end-labelled with [-
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32
and
PREmut
consensus
(PRE
site)
with
was
a
B
used
mutated
as
an
site
(27))
probes
were:
and
irrelevant
oligonucleotide:
5’5’-
5’-
PATP (Amersham) using T4 polynucleotide kinase (Roche, Neuilly sur Seine, France).
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Five µg of nuclear extract were preincubated in binding buffer (50 mM Tris pH7.5, 100 mM
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NaCl, 2 mM EDTA, 1 mM dTT, 1 µg poly[d(I-C)]) for 1 hour on ice. For supershift assays, 3
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µg isotypic irrelevant or anti-NF-B rabbit polyclonal antibodies [Oct-2 (H-120), p65 (H-
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286), p50 (NLS), c-Rel (N), p52 (447), and RelB (C-19); Santa Cruz Biothechnology,
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Heidelberg, Germany] were added. For competition experiments, a 50-fold molar excess of
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unlabeled competitor oligonucleotide was used. Binding reactions were performed using 2 ng
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[-32P-labeled probe at room temperature for 30 minutes. Samples were separated onto a
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0.25x Tris-borate-EDTA-4% polyacrylamide gel at 150 V, dried and exposed to storage
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Phosphor Screen (Perkin Elmer, Massachusetts, USA). Image was digitalized using the
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CycloneTM scanner (Perkin Elmer).
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Luciferase analysis
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Analysis of Luciferase expression was done on 20 µg proteins extracted in Passive Lysis
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Buffer (Promega, Paris, France) the using Luciferase Assay System (Promega) and TD20/20
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luminometer (Turner Designs, CA, USA).
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Immunoprecipitation
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Proteins were extracted in RIPA lysis buffer (150 mM NaCl, 1.0% IGEPAL® CA-630, 0.5%
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sodium deoxycholate, 0.1% SDS, and 50 mM Tris, pH 8.0) supplemented with protease and
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phosphatase inhibitors: 200mM PMSF (Phenylmethylsulfonyl Fluoride), 100mM NA VO and a
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protease inhibitor cocktail (Santa Cruz Biotechnology, CA, USA). P50 was immunoprecipitated
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from 250 µg proteins using: 4 µg rabbit polyclonal antibody anti-p50 (sc-114, Santa Cruz
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Biotechnology) and 100 µL μMACS™ Protein A MicroBeads (Myltenyl Biotech, Cologne,
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Germany). Then, immunoprecipitated proteins were separated through μcolumn according to the
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manufacturer’s protocol (Miltenyl Biotech).
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4
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Western-blot
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The antibodies used were anti-LMP1 (Hybridoma S12) at 1/100, anti-IB (sc-847, Santa
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Cruz Biotechnology, CA, USA) at 1/200, anti-p65 (sc-7151, Santa Cruz Biotechnology) at
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1/2000, anti-p100/p52 (Cell Signaling Technology, Saint Quentin Yvelines, France) at 1/700,
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anti-RelB (sc-226, Santa Cruz Biotechnology) at 1/2000, anti-SAM68 (sc-333, Santa Cruz
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Biotechnology) at 1/1000, and anti-αTubulin (B-5-1-2, Sigma-Aldrich, Saint Louis, USA) at
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1/10 000.
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EREB2.5 cell culture conditions for Gene expression profiling (GEP)
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E0hEBV-72h, E6hEBV, and E24hEBV: 72 hours estradiol deprived EREB2.5 cells (E0hEBV-72h)
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were estradiol induced for EBV-latency III program for 6 (E6hEBV), and 24 hours
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(E24hEBV).
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EcontEBV: EREB2.5 cells growing in continuous (cont) presence of estradiol.
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E0hEBV-120h+Dox, and E0hEBV-120h+Dox-Luc: 120 hours estradiol deprived pRT-1-
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Luciferase vector transfected EREB2.5 cells were NGFRt (truncated Nerve Growth Factor)
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sorted, as previously described [1], after addition of doxycycline (+Dox) for 48 hours.
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Positive (E0hEBV-120h+Dox-Luc) and negative (E0hEBV-120h+Dox) fractions were used for
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GEP.
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E0hEBV-120h+Dox-LMP1: 120 hours estradiol deprived pRT-1-LMP1 vector transfected
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EREB2.5 cells were NGFRt sorted after addition of doxycycline (+Dox) for 48 hours.
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Positive fraction was used for GEP.
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EcontEBV+Dox, and EcontEBV+Dox-Luc: pRT-1-Luciferase (Luc) vector transfected EREB2.5
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cells cultured with continuous (cont) estradiol (EBV-latency III) were NGFRt sorted after
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addition of doxycycline (+Dox) for 48 hours. Positive (EcontEBV+Dox-Luc) and negative
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(EcontEBV +Dox) fractions were used for GEP.
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EcontEBV+Dox-RelA, EcontEBV+Dox-IBS32,36A, EcontEBV+Dox-RelB, and EcontEBV+Dox-
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p100: EREB2.5 cells cultured in continuous (cont) estradiol (EBV-latency III) were stably
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transfected with pRT-1-Luciferase (Luc), pRT-1-RelA, pRT-1-IBS32,36A, pRT-1-RelB or
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pRT-1-p100 and then NGFRt sorted after addition of doxycycline (+Dox) for 48 hours.
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Gene expression profiling (GEP)
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Amplification of RNAs and hybridization onto microarrays were performed by the Affimetrix
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platform. Briefly, 100 ng total RNAs were reverse-transcribed to double-stranded cDNAs that
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were used as templates for in vitro transcription to produce amplified biotin-labelled RNAs
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using the GeneAtlas™ 3’ IVT Express kit (Affimetrix, CA, USA). Biotin-labelled RNAs
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were fragmented at 95oC in the presence of high magnesium concentration to prepare samples
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for hybridization onto an Affymetrix Human genome U219 Array Strip. Hybridized RNAs
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were stained in the Fluidics Station 400 (Affymetrix) as followed: 1.streptavidin-
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phycoerythrin (Molecular Probes), 2.biotinylated anti-streptavidin antibody for signal
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amplification (Vector Labs), and 3.streptavidin-phycoerythrin (Molecular Probes). The
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distribution of fluorescent signal on the processed microarray was determined using the
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Agilent GeneArray laser scanner (Affymetrix).
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Before analysis, expression values were log2-transformed using RMA (Robust Microarray
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Analysis) and gene-filtered. Briefly, probes showing minimal variation across all samples
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were filtered out, i.e. probes with a coefficient of variation greater than 0.1 were retained. For
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statistical analyses, the R and Bioconductor software was used. The empirical Bayesian
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method for differential expression calculation [2] implemented in the Linear Models for
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Microarray Analysis (LIMMA) package was used to identify genes specifically differentially-
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expressed between two groups. For each pairwise comparison, the p-values were adjusted
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after multiple testing corrections using the Benjamini and Hochberg method [3]. The
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significant differentially-expressed probes were selected based on an adjusted p-value of at
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least 0.05. We also used Significant Analysis of Microarrays (SAM) with a false discovery
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rate of at least 0.05 [4]. The selected genes were those detected as differentially expressed by
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SAM and/or LIMMA methods.
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Method for selection of the 726 EBV up-regulated genes and the 46 RelA and/or RelB
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regulated genes
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We first selected EBV up-regulated genes in B-cell lines. To perform supervised analysis, the
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different B-cell lines were divided in two groups, those in which the EBV-latency III program
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was on or off, i.e. EBV-on or EBV-off respectively. Selected genes yielded a Cell Line-list of
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1 550 genes up-regulated by the EBV-latency III program (Supplementary Table S1). Most of
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these genes (1 142, 74%) were up-regulated with a fold change of at least 1.5 in estradiol
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deprived EREB2.5 cells (E0hEBV) induced for LMP1 expression only (Supplementary Figure
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S2 and Table S1). Secondly, comparison of EBV-DLBCL tumors (cases 1 to 8) with non-
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tumoral reactive lymph nodes (LN n° 1 to 3) led to a Tumor-list of 3 415 genes
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(Supplementary Table S2). Seven hundred twenty six genes were in common between both
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Cell Line and Tumour lists (Supplementary Table S3). Thus, these 726 genes were bona fide
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EBV-induced genes associated with transformation of EBV-related DLBCLs in humans.
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These 726 EBV up-regulated genes were K-mean clustered [5] and analysis of biological
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functions was performed using the GSEA molecular signatures database (Supplementary
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Figure S3). The seven K-mean clusters correlated with the main functional categories such as
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mitochondrion, DNA replication, cell cycle and proliferation or cytokines as well as
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transcription factor pathways such as c-Myc, E2F1, Jun or NF-B. For the latter, most known
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NF-B target genes such as CD80/B7.1, ICAM1/CD54 (Intercellular adhesion molecule-1),
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EBI3/IL27B (EBV-induced gene 3), NFKBIA/IKBA (inhibitor of Rel/NF-B), IL1R2
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(interleukin 1 receptor, type II), and TRAF1 (TNF-receptor associated factor 1) were indeed
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K-mean clustered together. Interestingly, genes from the c-Myc, c-Jun and NF-B K-mean
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clusters were induced early at 6 hours after estradiol exposure of EREB2.5 cells whereas
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E2F1-regulated genes were induced later at 24 hours, which suggests that the EBV-latency III
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program was directly responsible for activation of c-Myc, c-Jun and NF-B whereas E2F1
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was secondarily activated.
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RelA and RelB regulated genes were a priori segregated as follows: RelA or RelB regulated
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genes were those that were over-expressed in E.RelA or E.RelB when compared to
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E.IBS32,36A or E.p100 cells respectively and that were down-regulated in E.IBSS32,36A or
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E.p100 cells respectively when compared to E.Luc cells. Intersection between the RelA and
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RelB lists gave the genes co-dominantly regulated by RelA and RelB (Figure 5a and
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Supplementary Table S4). Genes that were dominantly regulated by RelA (Figure 5b and
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Supplementary Table S4) or RelB were those belonging to the RelA but no to the RelB list
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and reciprocally. Since RelA and RelB over-expression induced the reciprocal loss of RelB
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and RelA DNA binding activity respectively, genes very likely to be regulated by RelA or
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RelB only were those whose expression was repressed by RelB or RelA respectively (Figure
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5c and Supplementary Table S4).
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Gene quantification with TaqMan Low Density array (TLDA)
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Relative quantities (RQ) of gene expressions were calculated according to 2-DDCT method,
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where DDCT is the delta delta cycle threshold, using the ExpressionSuite software version
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1.0.1 (Life Technologies). References are annotated on Supplementary Figure S5 and S6. The
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18S and ACTB genes were used as endogenous control genes (TaqMan Assay references:
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18S-Hs99999901_s1 and ACTB-Hs01060665_g1). All amplification steps were performed in
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duplicate. RQ of gene expressions were calculated as the ratio of the test biological group to
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its reference control group according to experiments. RQ minimum and maximum values
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were calculated with 95% confidence levels and used to calculate the standard error and p-
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value which is the probability that RQ ≠1 is not due to chance.
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References
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