SUPPLEMENTAL INFORMATION Titels and legends to 5 Supplemental Figures and 4 Supplemental Tables 5 Supplementary Figures 4 Supplementary Tables SI METHODS Titles and Legends to Supplemental Figures and Tables Supplemental Figure 1. Definition of Hodgkin- and non-Hodgkin-specific accessible chromatin. (A) Examples of genes showing NH-specific (IGH, left) and HRS-specific (LTA, TNF, right) increased chromatin accessibility. For details see legend to Fig. 1. (B) Definition of HRS- and NH-specific distal DHS-sets. The heat map on the left shows the L428 over Reh DNaseI-Seq FC. HRS-(red) and NH-(green) specific DHSs, respectively, are represented on the bar next to the heat map and are defined by statistical thresholds corresponding to 2! significance. Non-varying genes are represented in black. The panels next to the bar show the average DNaseI-Seq profiles for each group indicating an average peak width of about 200 – 400 bp. Outermost right, box plot demonstrating that the differences in DNaseI-Seq FC for NH-(green) and HRS-(red) specific DHSs are significant (***, p < 2.2*10-16). (C) Changes in chromatin accessibility correspond to changes in mRNA expression levels in HRS as compared to the NH cell lines. The L428 over Reh increasing DNAseI FC in distal elements depicted on the heat map on the left is correlated with the FC of mRNA expression of the nearest gene of multiple HRS cell lines compared to the NH cell line Reh. Spearman rank correlation coefficients are 0.71, 0.57, 0.52, 0.49, 0.53, 0.5, respectively. (D) Changes in chromatin accessibility at distal elements correspond to changes in mRNA expression levels compared to different NH cell lines. Comparison of FC in mRNA expression in multiple HRS cell lines compared to the NH cell lines Namalwa (left panel; Spearman rank correlation coefficients are 0.68, 0.62, 0.62, 0.63, 0.76, 0.61, respectively) or SU-DHL-4 (right panel; Spearman rank correlation coefficients are 0.58, 0.48, 0.58, 0.49, 0.71, 0.47, respectively) with DNaseI-Seq FC L1236 versus Reh as base line. (E) Box plots depicting the overall correlation in mRNA expression FC in different HRS cell lines over Reh of genes nearest to sites with low (NH-specific sites, green) and high (HRS-specific sites, red) L428 over Reh DNaseI-Seq FC, revealing consistently up- and down-regulated genes common to all HRS cell lines. (F,G) Changes in chromatin accessibility inversely correlate to changes in methylation levels between NH and HRS cell lines. Direct comparison of the fold increase in DNaseI accessibility for the L1236 and L428 HRS cell lines relative to the NH cell line Reh with the DNA-methylation pattern in the corresponding promoter regions as described in Fig. 1. The heat maps show the (F) L1236 over Reh and (G) L428 over Reh DNaseI FC sorted by increasing DNaseI ratio and corresponding methylation FC of the associated TSSs for promoter (left) and distal (right) DHSs in the indicated cell lines. Spearman rank correlation coefficients are: (F) L1236/Reh DHS vs. expr./m5C 0.707/-0.603 (promoters), and 0.723/- 0.45 (distal elements); (G) L428/Reh DHS vs expr./m5C 0.833/-0.671 (promoters), and 0.717/-0.346 (distal elements). Supplemental Figure 2. Transcription factor binding motifs enriched in HRS- or NH- specific DHSs are centered around the DHS center. (A,B) Alignment of motifs in the different classes of distal DHSs (A, B, or C) with the DHS center for NH and HRS motifs in total L428 and Reh genes. Each heatmap represents the position, in yellow, of NH (left panels) and HRS (right panels) enriched motifs with respect to the maximum DNaseI-Seq signal within +/-200 bp, sorted by increasing (A) L1236 or (B) L428 over Reh DNaseI FC. (C,D) Plots depicting average motif densities +/-200 bp around each DHS maximum for (C) L1236 and (D) L428 versus Reh. Enriched motifs in HRS-specific DHSs are shown in red, shared DHSs in black, and motifs in NH-specific DHSs are depicted in green. (E) Binding motifs enriched in HRS-specific DHSs co-localize with NF-!B. Distribution of distances between AP-1, STAT and IRF motifs with regard to the position of the NF-!B motif in the union of L1236 over Reh distal regions. In each case, motifs exhibit preferentially proximal positioning in relation to the NF-!B motif. For STAT motifs, a preferential position was found at +7 bases from the start of the NF-!B site, thus forming a GGGGAWTTCCNNAGAA composite consensus. Supplemental Figure 3. Quantification of IRF5 mRNA levels by real-time PCR; increased chromatin accessibility and transcriptional activity at the IRF5 locus; detection of IRF5 protein-DNA complexes in HRS cells; IRF5 IHC analyses. (A) mRNA expression of IRF5 in various Hodgkin (L428, L1236), non-Hodgkin (Reh, Namalwa) as well as ABC-type- (OCI-Ly3, OCI-Ly10, HBL1, TMD8) and GCB-type- (HT, OCI-Ly1, OCI- Ly7, OCI-Ly19) DLBCL cell lines was analyzed by quantitative real-time PCR relative to GAPDH mRNA expression. Error bars denote 95% confidence intervals. (B) DNaseI-Seq profiles in the IRF5 gene locus reveal elevated DNaseI-accessibility as well as the usage of an alternative promoter in HRS cell lines. IGV screenshot of DNaseI-Seq profiles of the IRF5 gene locus in HRS and NH cell lines, as indicated on the left. HRS (L1236, L428 and L591; red) and NH (Namalwa and Reh; green) DNaseI-seq profiles are shown above the IRF5 gene annotation (black), where both TSSs (TSS1 and 2) are represented by red arrows, and correspond to phylogenetically conserved regions (blue, bottom). RefSeq annotations are shown underneath all profiles, where thin lines represent introns, thick blocks exons and thickest blocks coding sequences. Gene transcription direction is shown by arrows. Note, that the DNaseI-Seq signal upstream of TSS1 as well as in the corresponding 1st intron is higher in HRS compared to NH cell lines, while TSS2 exhibits equivalent levels. (C-E) Epigenetic and transcriptional signatures of the IRF5 locus in the HRS cell lines L428 and L591 and the NH cell lines Reh and Namalwa, as indicated. One of three independent experiments is shown, respectively. Chromatin immunoprecipitation (ChIP) assay measuring (C) H3K4me3, (D) RNA PolII P-Ser5 (upper panel) and RNA PolII P-Ser2 (bottom panel) and (E) H3K9me3 at the indicated positions of the IRF5 locus, revealing higher enrichment for H3K4me3 as well as paused and elongating RNA PolII in the HRS cell lines at both TSSs. Histone modification marks and RNA PolII enrichment levels are shown as percentages of those of input or H3, respectively. (F) Northern blot analysis of IRF5 mRNA expression in HRS and NH cell lines, as indicated. GAPDH expression was analyzed as a control. (G-I) EMSA analyses of whole cell extracts using wt and mutated ISRE sites from the ISG15 promoter as a probe. (G) EMSA analysis of whole cell extracts of various HRS and NH cell lines, as indicated, using the wild- type ISRE site (ISRE wt) or an ISRE site with mutated IRF binding site (ISRE mut) as probe. Positions of specific protein-DNA complexes are indicated. n.s., non-specific complex. The free probe of the gel using ISRE wt as probe is shown underneath. Note, that IRF5-containing complexes are detectable only in HRS cell lines, that mutation of the ISRE site results in the loss of the IRF-containing complexes but not the non-specific complexes, that IRF5- containing complexes are selectively detectable in HRS cell lines and that the complex detectable in Reh cells (marked by an open circle) does not react with the IRF5 antibody. (H,I) EMSA analysis of whole cell extracts of the indicated cell lines using the (H) ISRE wt or (I) mutated site from the ISG15 promoter as a probe. Positions of specific protein-DNA complexes without (-) or with addition of IRF5 antibody (IRF5) or its isotype control (IC) are indicated (supershift, ss). n.s., non-specific complex. The free probe of the gel is shown underneath. Note, that in (I) mutation of the IRF5 site results in the loss of the IRF-containing complexes but not the non-specific complexes. (J) IRF5 immunohistochemistry of reactive tonsillar tissue (top panel), a tonsil from an infectious mononucleosis patient (center) and an IRF5-negative DLBCL case (bottom panel). Note, that in reactive tonsillar tissue IRF5 was detectable in some dendritic cells and macrophages. Supplemental Figure 4. IRF5 is required for the HRS cell-characteristic inflammatory gene expression and for HRS cell survival. (A) TFs IRF5 and NF-!B are required for endogenous IL13, IL6 and RANTES mRNA expression in HRS cell lines. The HRS cell line L591 was transfected with control plasmid (Mock) or a dominant-negative variant of IRF5 (DNIRF5-4D) and/or the NF-!B super-repressor I!B"#N, respectively. Enriched transfected cells were analyzed for mRNA expression of IL13, IL6 and RANTES by real-time PCR. Error bars denote 95% confidence intervals. One of six experiments is shown. P-values are shown for the comparisons to the respective Mock control. (B) The HRS cell lines L540Cy and L591 were transfected with control plasmid (Mock), or plasmids encoding a dominant-negative variant of IRF5 (DNIRF5-4D) and/or the NF-!B super-repressor I!B"#N, respectively. After enrichment of transfected cells, whole cell extracts were prepared and protein expression of DNIRF5-4D and I!B"#N was analyzed by use of antibodies (Ab) specific for IRF5 and I!B", respectively, as indicated. Extracts of L428 and Namalwa cells and the analysis of $- actin were included as controls.