Mapping of motifs in active PNAS PLUS chromatin identifies IRF5 as key regulator in classical Hodgkin lymphoma

Stephan Krehera,b,1, M. Amine Bouhlelc,1, Pierre Cauchyd, Björn Lamprechta,b,e, Shuang Lia,b, Michael Grauf, Franziska Hummela,b, Karl Köcherta,b, Ioannis Anagnostopoulosg, Korinna Jöhrensg, Michael Hummele,g, John Hiscotth, Sören-Sebastian Wenzelb, Peter Lenzf, Markus Schneideri, Ralf Küppersi, Claus Scheidereita, Maciej Giefingj,k, Reiner Siebertj, Klaus Rajewskya, Georg Lenzb, Peter N. Cockerilld, Martin Janza,b, Bernd Dörkena,b,e, Constanze Boniferd,2, and Stephan Mathasa,b,e,2

aMax-Delbrück-Center for Molecular Medicine, 13125 Berlin, Germany; bHematology, Oncology, and Tumor Immunology, Charité–Universitätsmedizin Berlin, 13353 Berlin, Germany; cLeeds Institute of Molecular Medicine, University of Leeds, Leeds LS9 7TF, United Kingdom; dSchool of Cancer Sciences, College of Medical and Dental Sciences, University of Birmingham, Birmingham B15 2TT, United Kingdom; eGerman Cancer Consortium (DKTK), German Cancer Research Center (DKFZ), 69120 Heidelberg, Germany; fDepartment of Physics, Philipps University, 35052 Marburg, Germany; gInstitute of Pathology, Charité– Universitätsmedizin Berlin, 10117 Berlin, Germany; hVaccine and Therapy Institute of Florida, Port St. Lucie, FL 34987; iInstitute of Cell Biology (Cancer Research), University of Duisburg-Essen, 45122 Essen, Germany; jInstitute of Human Genetics, Christian-Albrechts University Kiel and University Hospital Schleswig-Holstein, Campus Kiel, 24105 Kiel, Germany; and kInstitute of Human Genetics, Polish Academy of Sciences, 60-479 Poznan, Poland

Edited by Louis M. Staudt, National Cancer Institute, National Institutes of Health, Bethesda, MD, and approved September 4, 2014 (received for review April 17, 2014) Deregulated transcription factor (TF) activities are commonly ob- lineages. However, the nature of the TFs initiating and maintaining served in hematopoietic malignancies. Understanding tumorigene- HRS-specific remains poorly understood. sis therefore requires determining the function and hierarchical role As an unbiased approach for the identification of deregulated of individual TFs. To identify TFs central to lymphomagenesis, we TF activities central to lymphoma biology, we identified HL-specific identified lymphoma type-specific accessible chromatin by global accessible chromatin regions by global mapping of DNaseI hy- mapping of DNaseI hypersensitive sites and analyzed enriched TF- persensitive sites (DHSs). DHSs mark cis-regulatory elements MEDICAL SCIENCES binding motifs in these regions. Applying this unbiased approach to bound by TF complexes (9) and differ between normal and classical Hodgkin lymphoma (HL), a common B-cell–derived lym- malignant cells (10, 11). We then performed an unbiased genome- phoma with a complex pattern of deregulated TFs, we discovered wide search for TF binding motifs enriched within HRS-specific interferon regulatory factor (IRF) sites among the top enriched motifs. High-level expression of the proinflammatory TF IRF5 was specific to HL cells and crucial for their survival. Furthermore, IRF5 Significance initiated a regulatory cascade in human non-Hodgkin B-cell lines and primary murine B cells by inducing the TF AP-1 and cooperating Human lymphomas and leukemias are characterized by mo- with NF-κB to activate essential characteristic features of HL. Our lecular and structural alterations of transcription factors (TFs). strategy efficiently identified a lymphoma type-specific key regula- The identification of such deregulated TFs is therefore central tor and uncovered a tumor promoting role of IRF5. to the understanding of lymphomagenesis. We addressed this question in classical Hodgkin lymphoma (HL), a common B-cell– ranscription factor (TF) activities have to be tightly con- derived malignancy that is one of the most prominent examples trolled because their aberrant regulation alters tissue-specific for complex patterns of deregulated TFs including the activation T κ gene expression programs and contributes to cancer pathogenesis. of NF- B or AP-1 and a profound deregulation of lineage- κ Therefore, the identification of altered TF activities in malig- specific TFs. We found that IRF5 together with NF- Binduces nancies is of crucial importance to understand malignant trans- a number of HL characteristic features in non-Hodgkin cells, formation and to develop new treatment strategies. Deregulated such as expression of cytokines and chemokines or AP-1 acti- TF activities are commonly observed in hematopoietic malignan- vation. Our work exemplifies how the global lymphoma type- cies including human lymphomas and leukemias, and the link specific characterization of TF activities can improve the between structural or functional alterations in TFs and malignant understanding of tumor biology. transformation has been documented in various in vitro and in – Author contributions: S.K., M.A.B., P.C., S.L., G.L., P.N.C., M.J., C.B., and S.M. designed vivo studies (1 3). Apart from the direct modulation of cellular research; S.K., M.A.B., P.C., B.L., S.L., F.H., S.-S.W., and M.S. performed research; S.K., processes like cellular growth or cell death, alterations of the ac- M.A.B., P.C., B.L., S.L., M. Grau, F.H., K.K., I.A., K.J., M.H., J.H., S.-S.W., P.L., M.S., R.K., C.S., tivity of even single TFs might enforce malignant transformation M. Giefing, R.S., K.R., G.L., P.N.C., M.J., B.D., C.B., and S.M. analyzed data; C.B. and S.M. by switching differentiation programs and consequently altering wrote the paper; S.K., M.A.B., P.C., R.K., C.S., M. Giefing, R.S., K.R., G.L., P.N.C., M.J., and B.D. contributed to writing of the manuscript; K.K. analyzed microarray data; I.A. and K.J. the cellular fate of the respective cells, as exemplarily demon- performed and interpreted IHC analyses; J.H. provided material; J.H., C.S., R.S., K.R., and strated for the B-lymphoid TF PAX5 (4, 5). B.D. interpreted data; and C.B. and S.M. supervised the project. Among lymphoid malignancies, one of the most prominent The authors declare no conflict of interest. examples for complex patterns of deregulated TFs is classical This article is a PNAS Direct Submission. Hodgkin lymphoma (HL), a common B cell-derived malignancy (6). Freely available online through the PNAS open access option. Pathogenic hallmarks of the malignant Hodgkin/Reed-Sternberg Data deposition: The datasets reported in the manuscript have been deposited in the (HRS) cells of HL include the constitutive activation of TFs that Gene Expression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession nos. are only transiently activated in normal B cells, such as nuclear GSE51726, GSE51813, GSE51717, and GSE51719). κ factor kappa B (NF- B) or activator -1 (AP-1), and a pro- 1S.K. and M.A.B. contributed equally to this work. – found deregulation of lineage-specific TFs such as E2A (6 8). 2To whom correspondence may be addressed. Email: [email protected] or stephan. Thus, although originating from B-lymphoid cells, HRS cells have [email protected]. lost their B cell-specific gene expression pattern and instead up- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. regulate expression of characteristic for other hematopoietic 1073/pnas.1406985111/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1406985111 PNAS | Published online October 6, 2014 | E4513–E4522 Downloaded by guest on October 1, 2021 accessible chromatin and functionally analyzed the role of the Identification of TF-Binding Motifs Enriched in HRS Cell-Specific corresponding factors. The identification of binding motifs for the Accessible Chromatin Regions. To identify TFs responsible for TFs AP-1, NF-κB, and STAT in HL-specific accessible chromatin the HRS-specific gene expression program, we searched for TF confirmed their essential role for HL biology (6). In addition, we DNA binding motifs specifically enriched in HRS-specific DHSs, revealed that IFN regulatory factor (IRF) binding motifs are using both L1236 and L428 cells as references (Fig. 2 A and B). among the top enriched motifs in HL. We detected in HRS cells an In the B (NH)-specific DHS signature (Fig. 1D; A), binding abundant expression and increased activity of IRF5, which is an motifs for TFs important for the B-cell program such as E-box IRF TF family member that plays a central role in Toll-like re- and OCT motifs (18) were enriched (Fig. 2 A and B), in accor- ceptor (TLR)-mediated immune responses and is a key regulator dance with their role in maintaining the B-cell phenotype. In of TLR-induced proinflammatory gene expression (12, 13). IRF5 contrast, HRS-specific DHSs (Fig. 1D; C) were enriched in directed HL-specific cytokine expression in cooperation with NF- binding motifs for members of the inducible TF families AP-1, κB and protected HRS cells from cell death. Alone or in combi- NF-κB, STAT, and IRF (Fig. 2 A and B), whereas, as previously nation with NF-κB, IRF5 was capable of inducing gene expression demonstrated (19), TF motifs important for the B-cell program alterations in non-Hodgkin and primary murine splenic B cells that were found with decreased frequency. Mapping the different were reminiscent of those found in HL. Furthermore, we identified binding motifs back to enriched sequences showed their associ- a transcriptional cross-talk between IRF5 and AP-1, as IRF5 di- ation with NH- or HRS-specific DHSs, respectively, and dem- rected the activation of the known abundant and HL-specific AP-1 onstrated that they clustered around the central position of the complex. These data describe a powerful method for the identifi- DHS (SI Appendix, Fig. S2 A–D). Motif distribution was not cation of deregulated TF activities in human lymphoma, which led random, as AP-1, STAT, and IRF motifs showed a preferred to the identification of IRF5 as a key regulator of HRS cell biology. distance with respect to the NF-κB binding motif (SI Appendix, Fig. S2E). A comparison of the DNaseI cutting frequency at the Results binding motifs for AP-1, NF-κB, and IRF across all HRS- and Definition of an HL-Specific Active Chromatin Landscape. To identify NH-specific, as well as shared DHSs, revealed that these motifs transcriptional key regulators of HL biology, we mapped DHSs were protected from DNaseI digestion in HRS cells only, sug- in both Hodgkin and non-Hodgkin cells, followed by the func- gesting that they were occupied by a protein complex (Fig. 2 C tional analysis of TFs binding to corresponding TF binding and D). In contrast, RUNX sites were not protected in any of the motifs enriched within lymphoma type-specific accessible chro- analyzed cell types, and CTCF sites were only protected in DHSs matin (Fig. 1A). Due to the low number of HRS cells in affected shared between HRS and NH cells, in accordance with an in- lymph nodes (6), we applied this strategy to three classical HL- variant binding of CTCF in different blood cell types (20). derived cell lines (L1236, L428, and L591; in the following re- ferred to as HRS cell lines) in comparison with two non-Hodgkin Abundant IRF5 Expression and Activation in HRS Cells. AP-1, NF-κB, B-cell lines (Reh and Namalwa; in the following referred to as and STAT TFs play an important role in HL pathogenesis (6), and non-Hodgkin or NH cell lines; Fig. 1 and SI Appendix, Fig. S1 our data confirmed their crucial role in shaping active chromatin and Table S1). These cell lines have been successfully used for in HRS cells. Because IRF binding motifs ranked among the top the identification of key molecular and genomic defects in HL (8, motifs within HRS-specific DHSs, we investigated IRF expression 14, 15). Permeabilized cells were treated with DNaseI, and small and function in HL in detail. First, we determined mRNA ex- genomic fragments were isolated and subjected to next-generation pression of all known human IRF genes, IRF1–IRF9,inHRSand sequencing (DNaseI-Seq) (16). DNaseI-Seq patterns for selected NH cell lines (Fig. 3A, Upper). IRF1–IRF3 and IRF6–IRF9 were genes are shown in Fig. 1B and SI Appendix, Fig. S1A. We next expressed at similar levels in all cell lines, whereas IRF8 expression determined the ratio of tag counts at DHSs between pairs of appeared to be lower in HRS cell lines (Fig. 3A, Upper). IRF4 was HRS and NH cell lines and ranked them according to their robustly expressed in HRS cells as described, but not exclusively change in DNaseI-seq signal (Fig. 1C). We concentrated on (21). Notably, IRF5 was highly expressed in all of the HRS cell lines distal (nonpromoter) sites, as they comprise the majority (about in accordance with previously published microarray data (22). The 75–80%) of all DHSs. Many of these elements are enhancers IRF5 expression levels exceeded that in non-Hodgkin cell lines that are more likely to bind tissue-specific factors than promoters including ABC-type diffuse large B-cell lymphoma (DLBCL) cell (17). The DHS rankings compared with the NH cell lines lines, in which IRF5 mRNA expression has been shown previously revealed highly similar DHS accessibility profiles for each of the (22) (Fig. 3A and SI Appendix,Fig.S3A).Weconfirmedanacti- three HRS cell lines (Fig. 1C). We next divided DHSs into three vation of the IRF5 locus in HRS cells at the chromatin level (SI groups, based on fold change (Fig. 1D and SI Appendix, Fig. Appendix,Fig.S3B–E), demonstrating an increase in DNaseI ac- S1B): 7,800 NH-specific peaks (marked in green as A); 12,027 cessibility across the entire IRF5 5′-regulatory region (SI Appendix, shared peaks (marked in black as B); and 6,959 HRS-specific Fig. S3B), an enrichment of histone H3 lysine 4 trimethylation peaks (marked in red as C). To validate the functional relevance (H3K4me3) (SI Appendix,Fig.S3C), and the presence of RNA of these data with respect to gene expression, we identified the polymerase II (RNA-Pol II) (SI Appendix,Fig.S3D), reflecting genes closest to the respective DHSs and determined their cor- ongoing transcription; in contrast, the repressive H3K9me3 mark responding mRNA expression level in HRS cell lines by mining was absent (SI Appendix,Fig.S3E). In NH cell lines, the IRF5 gene microarray expression data (Fig. 1 E and F and SI Appendix, Fig. displayed only limited accessibility with low-level enrichment of S1 C–E). In agreement with the requirement of accessible RNA-Pol II and H3K4me3 (SI Appendix,Fig.S3B–E), which is chromatin for ongoing transcription, genes associated with HRS- consistent with a primed state for an inducible gene. Northern specific DHSs displayed a higher expression level in HRS than in blotting analysis showed abundant and selective expression of two NH cell lines, irrespective of which HRS was compared with major IRF5 variants (23) in HRS cell lines (SI Appendix,Fig.S3F), which NH cell line. For both distal and promoter DHSs, the fold corresponding to high IRF5 protein expression (Fig. 3A, Lower). increase in HRS-specific nuclease accessibility correlated well IRF TFs are activated by phosphorylation in the cytoplasm with relative gene expression levels (Fig. 1G). Furthermore, and subsequent translocation into the nucleus (24, 25). In HRS promoter DNA methylation levels showed an expected inverse cor- cell lines, we found IRF5 activation as indicated by its nuclear relation with accessibility at promoter and nearby distal DHSs localization (Fig. 3A, Lower) and the presence of DNA-binding in L1236 and L428 HRS cell lines (Fig. 1H and SI Appendix, Fig. IRF5 complexes (Fig. 3B and SI Appendix,Fig.S3G–I). We S1 F and G). Taken together, these data defined a specific active confirmed HL-specific IRF5 expression by immunohistochemistry chromatin landscape in HRS cells. of various primary human lymphomas. IRF5 was abundantly and

E4514 | www.pnas.org/cgi/doi/10.1073/pnas.1406985111 Kreher et al. Downloaded by guest on October 1, 2021 CCL5 CD79B PNAS PLUS A B 75 75 L1236 0 L1236 0 DNaseI HS mapping in HL and non-HL 75 75 cell lines L428 L428 0 0 75 75 L591 L591 Identify HL-specific DH sites 0 0 75 75 Namalwa Namalwa Determine HL-specific enriched 0 0 75 75 TF binding motifs Reh Reh 0 0

Test biological relevance of Mammal Mammal corresponding TFs 1 1 conservation 0 conservation 0

L1236L428 L591 Reh Namalwa L1236L428 L591 Reh Namalwa Gene Gene expression expression -2.0 -0.4 0.4 2.0 -2.0 -0.4 0.4 2.0

C D E h e h h e e h h /R e 2/R y /R 2/R 8 540C L1236/RehL428/RehL591/RehL1236/GM12878 L1236/RehGroups L1236/RehL1236/RehL42 HDLM-KM-H L540/ReL Namalwa/Reh +4 +4 80 Reh +4 +1 0 0 0 L1236 0 A -1 -4 -4 40 *** -4 6 0 --2000 200 4 80 Reh g qFC n 2 B L1236 e

40 tag count FC I-s I easi 0 e

cr 0 --2000 200 Increasing Increasing In Nas NaseI-seq FC -2 D DN aseI-seq FC D DNase

DHS reads per bp 80 Reh C L1236 -4 40 up MEDICAL SCIENCES 0 down --2000 200 DNaseI-seq FC Distance to mRNA expression FC . DHS (bp)

F GHPromoters 2.0 . h r=0.707 /Re y/Reh 8 Reh Reh C 8/ 0/ 0 0.0 L1236/RehL1236/RehL42 Namalwa/Reh 2 4 4 +0.36 L1236/RehHDLM-2/RehKM-H2/RehL4 L5 L5 +4 0 0

*** *** *** *** *** *** FC (log2) −2.0 -4 -0.36 1.5 FC mRNA expression n 1.0 −4 −2 02 DNaseI-seq FC (log2)

0.5 qFC e s

0 I- e

Distal regions s a ncreasing

-0.5 I 1.5 r=0.732 DN

mRNA expressio -1.0

0.0 DNaseI-seq FC p p p up up u u up u own own own FC (log2) L1236/Reh down down d d d down −1.5 DNA methylation FC mRNA expression −15 015 (nearest TSS) DNaseI-seq FC (log2)

Fig. 1. Definition of an HL-specific active chromatin landscape. (A) Experimental strategy. (B) Screenshots of the CCL5 (Left) and CD79B (Right) loci depicting DNaseI-Seq profiles of HRS (red) and NH (green) cell lines. Underneath, microarray-based gene expression data for the respective genes. (C) HRS cell lines show similar patterns of distal DHSs. The L1236 over Reh DNaseI-Seq fold change (FC) sorted by increasing values and corresponding signals from comparisons L428 or L591 over Reh, respectively, are shown. Spearman rank correlation coefficients compared with L1236/Reh FCs were 0.82 for both L428/Reh and L591/ Reh, respectively. Far right, comparison using the ENCODE lymphoblastoid GM12878 DNaseI-Seq dataset showed a similar result (r = 0.87). (D) Classification of HRS- and NH-specific distal DHSs showing the L1236 over Reh DNaseI-Seq FC (left heat map). Bar next to the heat map: NH (green; group A) and HRS (red; group C) -specific DHSs, respectively. Nonvarying DHSs: black (group B). Panels next to the bar: average DNaseI-Seq profiles for each group. Outermost right, − box plot demonstrating the significance of differences in DNaseI-Seq FC for NH- and HRS- specific DHSs (***P < 2.2 × 10 16). (E) The L1236 over Reh increasing DNaseI FC correlates with the FC of mRNA expression of the nearest gene of multiple HRS cell lines compared with the NH cell line Reh (Spearman rank correlation coefficients: 0.73, 0.61, 0.51, 0.5, 0.49, and 0.45, respectively), but there is no such correlation with the NH cell line Namalwa (r = −0.09). (F) Box plots depicting the correlation in mRNA expression FC in HRS cell lines over Reh of nearest genes to sites with low (NH-specific sites, green) and high (HRS- specific sites, red) L1236 over Reh DNaseI-Seq FC. ***P < 2.2 × 10−16.(G) mRNA expression FC (L1236 vs. Reh) correlates positively with DNaseI-seq fold change in both promoter (Upper) and distal (Lower) regulatory regions. Linear fits are indicated by dashed red lines, Spearman correlation coefficients are shown in red. (H) Changes in chromatin accessibility inversely correlate with changes in DNA methylation levels. The heat maps show the L1236 over Reh DNaseI FC sorted by increasing L1236 over Reh DNaseI ratio and the methylation FC of the corresponding element, respectively. Spearman rank correlation coefficients compared with L1236/Reh DHS FC were −0.45, −0.303, and 0.041 for L1236/Reh, L428/Reh, and Namalwa/Reh m5C FC, respectively.

consistently detectable in the nucleus and cytoplasm of HRS Table S2). Although IRF5 expression was observed in several cells (37 of 38 HL cases) and was absent in the vast majority of DLBCL cases, nuclear IRF5 staining in DLBCL was only occa- non-Hodgkin lymphomas (Fig. 3C and SI Appendix, Fig. S3J and sionally detected (5 of 45 cases) without preference for ABC- or

Kreher et al. PNAS | Published online October 6, 2014 | E4515 Downloaded by guest on October 1, 2021 A DNaseI-seq FC L1236/Reh B DNaseI-seq FC L428/Reh

down up down up

ETS, p=10-103, 21.57% of seq. AP-1, p=10-458, 25.00% of seq. ETS, p=10-171, 16.68% of seq. AP-1, p=10-761, 48.58% of seq.

E-Box, p=10-103, 35.73% of seq. STAT, p=10-204, 29.93% of seq. OCT, p=10-123, 5.25% of seq. RUNX, p=10-206, 35.33% of seq.

OCT, p=10-97, 2.41% of seq. NF-κB, p=10-182, 4.92% of seq. E-Box, p=10-115, 26.38% of seq. NF-κB, p=10-139, 18.26% of seq.

PU.1 + IRF, p=10-48, 2.41% of seq. IRF, p=10-182, 4.31% of seq. PU.1 + IRF, p=10-78, 3.54% of seq. STAT, p=10-36, 15.97% of seq.

RUNX, p=10-46, 7.87% of seq. BLIMP1, p=10-91, 12.21% of seq. RUNX, p=10-27, 6.10% of seq. IRF, p=10-36, 3.28% of seq.

C DNaseI-seq FC L1236/Reh D DNaseI-seq FC L428/Namalwa down invariant up down invariant up 0.01 L1236 0.006 L428 Reh Namalwa IRF IRF

0 0 -100 0 100 -100 0 100 -100 0 100 -100 0 100 -100 0 100 -100 0 100 0.005 0.003

NF-κB NF-κB

0 0 -100 0 100 -100 0 100 -100 0 100 -100 0 100 -100 0 100 -100 0 100 0.01 0.01

AP-1 AP-1

0 0 DNaseI cut density -100 0 100 -100 0 100 -100 0 100 DNaseI cut density -100 0 100 -100 0 100 -100 0 100 0.01 0.01

RUNX RUNX

0 0 -100 0 100 -100 0 100 -100 0 100 -100 0 100 -100 0 100 -100 0 100 0.01 0.012 CTCF CTCF

0 0 -100 0 100 -100 0 100 -100 0 100 -100 0 100 -100 0 100 -100 0 100 Distance to motif centre (bp) Distance to motif centre (bp)

Fig. 2. Enriched TF binding motifs in HRS cell-specific accessible chromatin regions. (A and B) HRS-specific DHSs contain motifs for inducible TFs. Sequences within HRS (red) and NH (green) -specific DHSs (A, L1236 over Reh; B, L428 over Reh) as defined in Fig.1D were analyzed for the presence of enriched TF binding motifs. (C and D) Digital footprinting using average DNaseI cutting frequencies ± 100 bp around the HRS-enriched binding motifs IRF, NF-κB, and AP-1 for (C) L1236 vs. Reh and (D) L428 vs. Namalwa (red and green, respectively) in HRS-specific, shared (invariant), and NH-specific DHSs, corresponding to groups A, B, and C. Note the differences in average DNaseI cutting frequency in DHSs specific for HRS and NH cell lines and the reduction in cutting frequency around the motif centers. RUNX and CTCF motifs were examined as controls demonstrating (i) equal digestion around RUNX motifs in DHS of all cell types and (ii) increased digestion around CTCF motifs only within shared DHS.

GCB-type DLBCL. Furthermore, IRF5 expression in HRS cells analysis the TFs JUN, a known regulator of cytokine expression, was stronger than that observed in tonsils of patients with EBV- and LPS-induced TNF-α factor (LITAF), which mediates LPS- caused infectious mononucleosis (SI Appendix,Fig.S3J). induced cytokine and chemokine activation, and the LPS/IRF- inducible chemokine gene CXCL11, the latter two genes not yet IRF5 Controls Cytokine Activation in HRS Cells and Protects Them analyzed in HRS cells (Fig. 3D). All these genes were consis- from Cell Death. IRF5 attracted our attention because it orches- tently expressed in HRS but not in NH cell lines. trates the transcriptional activation of proinflammatory genes in IRFs and NF-κB can form a multiprotein complex that is re- response to various stimuli (12, 26), and a broad activation of quired for full target gene activation (13, 27). Given that per- proinflammatory response genes is a unique hallmark of HL (6). sistent activation of NF-κB is a key pathogenic feature of HRS To define the role of IRF5 in establishing the proinflammatory cells (6), we speculated that their unusual cytokine and chemo- phenotype of HRS cells, we first determined the mRNA ex- kine production resulted from IRF5 activation in combination pression levels of a panel of cytokine, chemokine, and chemo- with NF-κB. To explore this possibility, we analyzed in HRS kine genes in the various cell lines. We included in this cell lines the activities of wild-type (WT) or mutated reporter

E4516 | www.pnas.org/cgi/doi/10.1073/pnas.1406985111 Kreher et al. Downloaded by guest on October 1, 2021 PNAS PLUS -4 ABlwa a HL 428 540 Hodgkin cell lines L L1236 L Nam SU-D cell line wa 5 5 5 5 Cy l HL-4 F F 8 1 0 D ss IR IC IR IC IRF5IC IRF IC IRF IC 2 236 -H29 40 40 6 AB M 5 DLM-25 eh J cell line L4 L1 K L H L L5 R NamaBL- B SU- ss IRF1 IRF2 IRF3 IRF5 IRF4 n.s. IRF5 IRF6 free IRF7 RT-PCR probe IRF8 IRF9 GAPDH C HL IRF5 WC ß-actin IRF5 N PARP

Hodgkin cell lines D -4 E 2 L H M- Cy 0 B 1 L 0 -6 -D D eh U cell line L428L1236KM-H2L59 H L540L54R NamBLalwaBJAS IL6 60 L428 7 L1236 14 KM-H2 IL13 ) n TNFA o * 40 5 10 Mock LTA IκBαΔN tivati CSF2 c * * * * 3 * 6 *

RANTES da * l 20 * * * o * * * CXCL10 f * * * ( * * * * Luciferase activiy * * 1 2 * * CXCL11 * * * * 0 0 0 CCR7 3 t t t 3 t t t w L3 w ut w u MEDICAL SCIENCES mut G m m mu LITAF pGL E p Emu κB pGL E κB -κBmut R - R - ISR IS F IS JUN NF N NF GAPDH RANTES P RANTES P RANTES P

F G L428 L540Cy IL13 IL6 RANTES 80 80

1.2 s * 60 * * 60 L540Cy cell * * * * * * * + * * * siCTL mRNA 0.8 * *

P * ****** * * F * ** 40 40 * * * siIRF5_#1 * 0.4 *** siIRF5_#2 *** *** expression *** *** GAPDH

*** Percentage of 20 20 cytokine relative viable G to 0 Mock + + + 0 0 DNIRF5-4D + + + + + + hours after IκBαΔN + + + + + + 0 72 96 0 48 72 cell sorting

Fig. 3. Abundant IRF5 expression in HRS cells regulates cytokine expression and protects from cell death. (A, Upper) Analysis of IRF1 to IRF9 and, as a control, GAPDH mRNA expression by RT-PCR. (Lower) IRF5 protein expression in whole cell (WC) and nuclear (N) extracts. β-actin and PARP were analyzed as controls. One of four independent experiments is shown. (B) EMSA of complexes bound to the ISRE without (−) or with addition of IRF5 antibody or its isotype control (IC) [supershift (ss)]. n.s., nonspecific complex. Underneath, free probe of the gel. One of four independent experiments is shown. (C) Representative IHC analysis of IRF5 in classical HL. HRS cells showing nuclear and cytoplasmic IRF5 staining are marked by arrows. (D) RT-PCR analysis of various genes, as in- dicated. GAPDH was analyzed as control. One of four independent experiments is shown. (E) IRF and NF-κB binding sites are required for full RANTES promoter (P) activation. pGL3-basic, pGL3-RANTES P WT, or pGL3-RANTES P constructs with mutated ISRE or NF-κB site were transfected into HRS cell lines, together with Mock plasmid (filled bars) or IκBαΔN (open bars). Luciferase activity is shown as fold activation compared with pGL3-basic (set 1). Data are represented as mean ± SD. One of six independent experiments is shown. (F) IRF5 and NF-κB are required for endogenous IL13, IL6, and RANTES mRNA expression. L540Cy cells were transfected with control plasmid (Mock) or DNIRF5-4D– and/or IκBαΔN-expression plasmids. mRNA expression in enriched transfected cells was analyzed in comparison with the Mock control, set as 1, by real-time PCR. Error bars denote 95% CIs. One of six independent experiments is shown. (G) L428 and L540Cy cells were transfected with control (siCTL) or IRF5-specific siRNA (siIRF5_#1 and siIRF5_#2) constructs, and the percentage of viable cells was determined at the indicated times in enriched transfected cells. Data are represented as mean ± SD. One of four independent experiments is shown. P values are shown for the comparisons to the respective WT reporter activites or the Mock controls, respectively. *P < 0.05; **P < 0.01; ***P < 0.001.

constructs of the RANTES promoter (RANTES P), which contains HRS cells, we inhibited both factors by cotransfecting the domi- an IFN-stimulated response element (ISRE) and an NF-κB binding nant-negative variants DNIRF5-4D (24) and IκBαΔN, respectively site (27) (Fig. 3E). In accordance with the high RANTES mRNA (Fig. 3F and SI Appendix,Fig.S4A and B). Transfection of expression in these cells (Fig. 3D), the WT construct was highly DNIRF5-4D and IκBαΔN independently reduced IL13, IL6, and active (Fig. 3E). ISRE mutation reduced promoter activity, and RANTES mRNA expression, and both in combination resulted in mutation of the NF-κB site reduced activity to basal levels. Fur- a more pronounced reduction in mRNA expression (Fig. 3F and SI thermore, cotransfection with the NF-κB superrepressor IκBαΔN Appendix,Fig.S4A). These effects were confirmed by analyzing (28) reduced activities of the WT- and ISRE-mutated RANTES P IL-6 and RANTES protein expression in supernatants of trans- constructs (Fig. 3E), suggesting a cooperative activity of IRFs and fected HRS cell lines (SI Appendix,Fig.S4C). In addition to con- NF-κB in HRS cell lines. To determine the impact of IRF5 and trolling the expression of these proinflammatory genes, IRF5 was NF-κB on the endogenous expression of proinflammatory genes in essential for HRS cell viability. Following transient IRF5 knockdown

Kreher et al. PNAS | Published online October 6, 2014 | E4517 Downloaded by guest on October 1, 2021 using specific small-interfering (si)RNAs, we observed an in- a subset of proinflammatory genes in both cell lines, full activation duction of cell death of ∼35–50% of the transfected HRS cell of most genes to HRS-cell–like expression levels required their population (Fig. 3G and SI Appendix, Fig. S4 D and E). The combined activity. We demonstrated this synergistic induction ex- analysis of the cells by Annexin-V/propidium iodide (PI) and emplarily for RANTES and IL-6 also at the protein level (Fig. 4B active caspase-3 staining indicated that cell death occurred by and SI Appendix,Fig.S5B). Functionally, the production of cyto- induction of apoptosis (SI Appendix, Fig. S4 F and G). kines and chemokines following transfection of NH cell lines (Fig. 4A) led to an increased attraction of mononuclear cells (Fig. 4C), IRF5 Together with NF-κB Activates an HRS Cell-Like Inflammatory in accordance with the chemoattraction of various normal hema- Gene Expression Pattern in Non-Hodgkin Cells. Our results promp- topoietic cells by HRS cells (6). Finally, we performed microarray ted the question of whether IRF5 alone or in combination with profiling of IRF5-4D–,IKKβ(EE)-, and IRF5-4D– together with NF-κB was sufficient to induce the HL characteristic proin- IKKβ(EE)-transfected Reh cells and subsequent gene set enrich- flammatory gene expression program. In a subsequent series of ment analysis (GSEA). Evaluation of HRS signatures based on experiments, we therefore mimicked the HRS-cell–characteristic genes specifically up-regulated in HRS cell lines (Fig. 4D and SI activities of IRF5 and NF-κB by introducing the constitutively ac- Appendix, Table S3) and primary HRS cells (SI Appendix,Fig. tive variants IRF5-4D (29) and IKKβ(EE) (30), respectively, into S5C and Table S3) revealed a significant enrichment of the HRS non-Hodgkin cells. In HEK293 cells, either IRF5 or NF-κBacti- signature in IRF5-4D or IKKβ(EE) single transfectants, with the vation activated the RANTES P construct, but both together in- highest enrichment following transfection of both factors in duced a dramatic synergistic activation (SI Appendix, Fig. S5A). combination (Fig. 4D and SI Appendix,Fig.S5C). We confirmed the synergistic activation of endogenous proin- flammatory gene expression in the IRF5-4D- and/or IKKβ(EE)- IRF5 Induces Key Aspects of the HL-Specific Gene Expression Program transfected NH cell lines Reh and BJAB (Fig. 4 and SI Appendix, in Primary Murine Splenic B Cells. To assess the potential of IRF5 Fig. S5 B and C). Although IRF5-4D or IKKβ(EE) alone induced to initiate HRS-cell–characteristic gene expression in primary B

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Fig. 4. IRF5 together with NF-κB determines the inflammatory phenotype of HRS cells. (A) IRF5 alone or with NF-κB activates HL-specific inflammatory genes in NH cells. BJAB and Reh cells were transiently transfected with control plasmids (Mock) or IRF5-4D– and/or IKKβ(EE)-expression plasmids. Enriched trans- fected cells were analyzed for mRNA expression of the indicated genes by RT-PCR. L1236 cells and GAPDH expression served as controls. One of three in- dependent experiments is shown. (B) IL-6 and RANTES secretion by Mock and IRF5-4D and/or IKKβ(EE) transfected Reh cells analyzed by ELISA. One of four independent experiments is shown. (C) Chemotaxis assay of peripheral blood mononuclear cells toward supernatants (SN) of IRF5-4D and/or IKKβ(EE) transfected Reh and BJAB cells. Fold migration is shown as relating to spontaneous migration toward medium of Mock-transfectants, set 1.0. One of three in- dependent experiments is shown. (D) GSEA enrichment plots of the HRS cell line signature (SI Appendix,TableS3; signature based on the comparison of HRS vs. NH

cell lines, log2 FC cutoff 2) of up-regulated genes of IRF5-4D–,IKKβ(EE)-, or IRF5-4D–,andIKKβ(EE)-induced gene expression changes in transiently transfected Reh cells. Note, that the absolute value of NES is highest in IRF5-4D plus IKKβ(EE) cells, indicative of an additive or synergistic effect of the two factors with respect to a Hodgkin-like phenotype shift of transfected Reh cells. Error bars denote SDs. P values are shown for comparisons to the respective Mock controls. ns, not sig- nificant; *P < 0.05; **P < 0.01; ***P < 0.001.

E4518 | www.pnas.org/cgi/doi/10.1073/pnas.1406985111 Kreher et al. Downloaded by guest on October 1, 2021 cells, we infected purified murine splenic B cells with control affected B cell-associated genes and TFs, as demonstrated PNAS PLUS (Mock) or IRF5-4D encoding retroviruses (Fig. 5 and SI for down-regulation of AICDA and the TFs EBF1, PRDM1, Appendix,Fig.S5D and E). We characterized IRF5-4D– and XBP1, as well as their antagonists, as shown for the up- induced gene expression changes by microarray analyses regulation of ABF1 and ID2 (7) in a HRS-cell–characteristic and evaluated the HRS signature by GSEA (Fig. 5A and SI manner (Fig. 5B). Other typical features of HRS cells were Appendix,Fig.S5D). This analysis revealed a significant en- also recapitulated by IRF5 activity such as up-regulation of richment of the up- and down-regulated genes of the HRS CD30 (also called TNFRSF8)orthec-MET tyrosine-kinase signature in IRF5-infected B cells. Apart from the induction receptor and silencing of the epigenetic regulator CBFA2T3 of proinflammatory genes, IRF5-induced gene deregulation (31) (Fig. 5B).

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Fig. 5. IRF5 orchestrates HRS-specific gene expression in primary murine B cells and acts upstream of AP-1. (A) GSEA enrichment plots of IRF5-4D–induced gene expression changes in primary murine B cells of the HRS cell line signature for down (Left) and up (Right) -regulated genes are shown. Note, that IRF5-4D induces a highly significant and specific shift of murine splenic B cells to a Hodgkin-like phenotype. (B) IRF5-4D–induced mRNA expression changes of various HRS-characteristic genes were analyzed by RT-PCR. GAPDH was analyzed as control. Two (#1 and #2) of four independent experiments are shown. (C–E)IRF5 up-regulates AP-1 activity in murine splenic B cells. (C) Whole cell extracts of Mock- and IRF5-4D–transduced B cells were analyzed by EMSA for AP-1 DNA binding activity. L428, L1236, and KM-H2 HRS and Reh and Namalwa NH cells, and TF Sp1 DNA binding activity were included as controls. Note that the nonspecific complex is detectable in human but not murine cells and that AP-1 activation among the cell lines is restricted to HRS ones. One of three in- dependent experiments is shown. n.s., nonspecific complex. (D) IRF5-4D–transduced splenic B cells were analyzed by EMSA for AP-1 DNA binding activity without (−) or with addition of antibodies specific for c-JUN, JUNB, or c-JUN, JUNB, and JUND (pan-JUN). L428 and Reh cells were included as controls. One of three independent experiments is shown. ss, supershift. n.s., nonspecific complex. (E) IRF5-4D–transduced splenic B cells were analyzed for JUN, JUNB, and ATF3 mRNA expression by RT-PCR. GAPDH is shown in B. Two (#1 and #2) of three independent experiments are shown.

Kreher et al. PNAS | Published online October 6, 2014 | E4519 Downloaded by guest on October 1, 2021 We next focused on the analysis of the TF AP-1, whose de- NF-κBactivityinHRScells,whichisinpartmediatedbysecreted regulation is a molecular hallmark of HRS cells (28). In agree- factors (38), remains to be investigated. ment with the up-regulation of JUN in NH cell lines (Fig. 4A), The comparison of the DHSs data of HL and NH cells, as well IRF5-4D caused a strong induction of AP-1 DNA binding ac- as an Epstein Barr virus (EBV)-transformed lymphoblastoid cell tivity (Fig. 5C). The binding intensity and aberrant migration line dataset from the Encyclopedia of DNA Elements (ENCODE), pattern of the IRF5-induced AP-1 complex closely resembled highlighted an HRS-specific active chromatin landscape. This that observed in HRS cells (28), as demonstrated by supershift finding indicates that, although EBV infection is linked to HL analyses with JUN antibodies (Fig. 5D). In line with these pathogenesis (6, 39), it is per se not sufficient to reprogram the observations, IRF5-4D induced the AP-1/CREB factor genes epigenome of B-lymphoid cells toward a HRS cell-like conforma- JUN, JUNB, and ATF3, which encode three main components of tion. Furthermore, the HL-specific epigenetic landscape signifi- the HL-specific AP-1 complex (28, 32), at the transcriptional level cantly correlated with the gene expression pattern in the various (Fig. 5E), indicating that AP-1 is a downstream effector of IRF5. HRS cell lines, including those of T-cell origin (HDLM-2 and L540). This result suggests that a common transformation process Discussion with dominant transcriptional alterations leads to the HL pheno- The genome-wide mapping of DHSs has been used successfully type rather than that the HL gene expression pattern reflects that to characterize cell type-specific regulatory elements, as well as of a putative physiological counterpart. occupied TF binding motifs within these regions (33, 34). Only The important role of IRF5 in the orchestration of the recently, the comparison of DHSs in various normal and ma- HL-specific gene expression program points toward potential initi- lignant cell types revealed characteristic reprogramming patterns ating events in this disease. In innate immunity, IRF5 is positioned of the epigenome in distinct cancer entities including hemato- downstream of pattern recognition receptors (PRR) (13) and is poietic malignancies (10, 11). Here, we extended such analyses required for the induction of various inflammatory mediators. For − − by combining the definition of cell type-specific accessible chro- example, IRF5 / mice are resistant to LPS-induced lethal shock, matin regions with the functional characterization of TFs binding emphasizing the role of IRF5 in the coordination of innate immune to enriched binding motifs in such regions. responses (12, 24, 26, 40). In addition, IRF5 gene polymorphisms In the HRS-specific DHSs, we identified enriched binding and expression alterations have been linked to autoimmune and motifs particularly for inducible TFs including NF-κB, STAT, inflammatory diseases such as rheumatoid arthritis and inflamma- AP-1, and IRFs. For NF-κB, STAT, and AP-1 factors, an im- tory bowel diseases (12, 41, 42). With respect to IRF5 activation, an portant role not only for lymphomagenesis in general but also for involvement of MyD88-dependent signaling complexes following the pathogenesis of HL has been already established (6). For TLR activation and a role of the IKK-related kinases TBK1 and example, NF-κB and STATs are required for growth and survival IKKe have been described (12, 43). Thus, in HRS cells, high-level of the HRS cells, and various genomic defects in respective IRF5 expression might be a result of constitutive PRR activation, pathway components confirm their essential role for HL patho- a hypothesis that can now be experimentally tested. genesis (6). Our data confirm their important role for HRS-specific Full target gene induction by IRF5 requires the cooperation gene regulation and, vice versa, validated our experimental ap- with other TFs or coactivators (13, 44, 45). In line with these proach. Given the established importance of these factors for HL data, we observed a strong transcriptional synergism of IRF5 biology,wefocusedinthisworkonIRFfactors,whichhaveim- with NF-κB, which only in this combination resulted in full portant functions in the differentiation and transformation of he- activation of a whole set of proinflammatory genes in NH cells. matopoietic cells (13). Our data indicate that this synergism is the basis for the abun- HL represents a lymphoid malignancy with highly complex TF dant production of cytokines and chemokines in HRS cells, as alterations and dramatic changes of the lymphoid-specific gene NF-κB activation alone can be observed in a variety of other expression program. The most important result from this study is hematopoietic malignancies, which, however, lack the widespread therefore that the deregulated activity of only a few TFs might be and abundant activation of proinflammatory genes characteris- sufficient to initiate this program. The constitutive activity of tic for HL. It remains to be investigated how IRF5 and NF-κB IRF5 protects HRS cells from cell death and in combination with coordinate the protection of HRS cells from apoptotic cell κ – NF- B, IRF5 induces key aspects of the HRS-cell characteristic death, and whether other transcription factors aberrantly acti- gene expression program. Even in primary splenic B cells, IRF5 vated in HL, such as GATA3 (7, 46), AP-1, or the here de- activation results in the abundant induction of proinflammatory scribed LITAF, contribute to full induction of proinflammatory genes (6), the down-regulation of genes required for initiation genes in HL. and maintenance of the B-lineage differentiation program Together, our studies uncovered a key role of IRF5 in a lym- including terminal plasma cell differentiation, and the up- phoid malignancy with a unique inflammatory phenotype and regulation of their transcriptional antagonists. Moreover, IRF5 exemplifies how the global lymphoma type-specific identification promotes the HL-characteristic silencing of the epigenetic reg- of aberrant TF activities can improve the understanding of CBFA2T3, ulator which is involved in the aberrant activation of tumor biology. DNA long terminal repeat regions in HRS cells (8). We also identified AP-1 as a downstream effector of IRF5. AP-1 Methods deregulation has been recognized as a hallmark of HRS cells, being Cell Lines, Culture Conditions, and Transfections. HRS [L428, L1236, KM-H2, involved in the HRS cell-specific deregulation of CD30 (35), the L591 (EBV+), HDLM-2, L540, L540Cy], pro-B lymphoblastic leukemia (Reh), immunomodulatory gene LGALS1 (also called galectin-1) (36), and Burkitt´s lymphoma (Namalwa, BL-60, BJAB), diffuse large B-cell lymphoma their unique dedifferentiation process (7, 8). In HRS cells AP-1 is (OCI-Ly3, OCI-Ly10, HBL1, TMD8, HT, OCI-Ly1, OCI-Ly7, OCI-Ly19, SU-DHL-4), activated by an unusual mechanism, as its activation is partly and HEK293 cell lines were cultured as previously described (8). Cells were MAPK independent (28, 37), and it is primarily composed of electroporated (EP) in OPTI-MEM I using Gene-Pulser II (Bio-Rad) with 950 μF and 0.18 kV (L428, L1236, L540Cy, BJAB, HEK293), 50 μF and 0.5 kV (KM-H2), JUN and ATF subunits instead of JUN and FOS (28, 32). The μ IRF5-mediated transcriptional activation of the AP-1 complex de- and 500 F and 0.3 kV (Reh, L591). Transfection efficiency was determined by pEGFP-N3 (Clontech Laboratories) cotransfection and FACS analysis. scribed here positions IRF5 upstream of AP-1 in the transcriptional Reh and BJAB cells were transfected with 60–70 μg of a pcDNA3-FLAG- hierarchy in HRS cells and provides a molecular explanation for the IRF5-4D and/or 20–40 μg of a pRK5-IKKβ(EE) expression plasmid along with thus far poorly understood AP-1 activation in HL. Whether the 10 μg pEGFP-N3. L428, L591, and L540Cy cells were transfected with 60 μg IRF5-mediated gene expression alterations, including the induction of a pcDNA3-FLAG-DNIRF5-4D and/or 40 μg of a pcDNA3-IκBαΔN expres- of the various cytokines and chemokines, modify the constitutive sion plasmid or controls or with 50 μg of pSUPER plasmid (47)-based siIRF5

E4520 | www.pnas.org/cgi/doi/10.1073/pnas.1406985111 Kreher et al. Downloaded by guest on October 1, 2021 expression plasmids or respective scrambled siRNA controls along with Motif Discovery, Heatmaps, and Distributions. We used the findMotifsGenome PNAS PLUS 10 μg pEGFP-N3 for enrichment of transfected cells by FACS sorting, or function in Homer for primary motif detection, where de novo discovery additionally along with 10 μg of an expression plasmid encoding truncated was performed in regions ranging [−200bp; +200bp] around the maximum mouse MHC class I molecule H-2Kk (Miltenyi Biotec) for enrichment by + DHS. Motif reinjection was carried out in RSA Tools Matrix-Scan (49). MACS sorting. Forty-eight to 72 h after transfection, GFP cells were Resulting outputs were converted to gff format and subsequently computed k enriched by FACS sorting or MACS sorting using of MACSelect K as frequencies every 10 bp for all regions and ordered according to fold MicroBeads according to the manufacturer´s recommendations (Miltenyi changes. Heatmaps were generated via Java Treeview. Motif densities were Biotech). For analysis of luciferase activity, L428, L1236, KM-H2, and computed relative to each DHS maximum, where distances used were that HEK293 cells were transfected by EP with 10 μg of reporter constructs, together with 100 ng pRL-TKLuc as an internal control. Where indicated, between the start of each motif (regardless of the strand) and the DHS κ cells were additionally transfected with pcDNA3-IκBαΔN, pRK5-IKKβ(EE), maximum. Motif distances to NF- B were obtained using BedTools closest and/or pcDNA3-FLAG-IRF5-4D expression constructs or the respective and plotted in R. controls. Twenty-four to 48 h after transfection, the ratio of the two luciferases was determined (Dual luciferase kit; Promega). Murine splenic DNaseI Digestion. To perform DNaseI accessibility assays with HRS and NH B cells were isolated from C57BL6/J mice, purified by CD43 depletion (Miltenyi cell lines, the optimal cell number and DNaseI (DPFF DNaseI; Worthington Biotec), and cultured in the presence of 20 μg/mL LPS (Sigma), all according Biochemical Corporation) concentration were titrated for each cell line. The to standard protocols. After 24 h, cells were retrovirally transduced with DNA digestion extent was comparable in all of the generated samples as MSCV-IRF5-4D-IRES-CFP (IRF5-4D) expression plasmid or MSCV-IRES-CFP measured by RT-PCR (10). Briefly, 1 × 106 to 5 × 106 cells were permeabilized (Mock) as a control. Forty-eight hours after retroviral transduction, CFP- with detergent and immediately digested at 3 × 107 cells per mL for 3 min positive cells were FACS sorted, and mRNA or protein extracts were pre- at 22 °C with DNaseI at 1–12 U/mL in digestion buffer supplemented with pared. The use of human material was approved by the Ethikkommision of 1 mM CaCl (10). The nuclei were lysed, the nuclear were digested the Charité - Universitätsmedizin Berlin and was performed in accordance 2 with 1 mg/mL Proteinase K overnight at 37 °C, and the DNA was isolated with the Declaration of Helsinki. Animal care and experiments were per- by phenol/chloroform extraction. Levels of DNaseI digestion were assessed formed in accordance with the institutional guidelines. using real-time PCR, measuring the ratio of presence of known DNaseI hy- DNA Constructs. Expression constructs pcDNA3-IκBαΔN and pRK5-IKKβ(EE) persensitive regions to more resistant gene-free regions. Sequences of real- ′ and luciferase reporter constructs pGL3-RANTES P WT, pGL3-RANTES P-ISRE time PCR primers used were, for the active region, TBP promoter 5 - ′ mut, and -NF-κB mut have been previously described (8, 27, 28). To generate CTGGCGGAAGTGACATTATCAA and 5 -GCCAGCGGAAGCGAAGTTA, and for the pcDNA3-FLAG-IRF5-4D expression construct, full-length human IRF5 the inactive region, a gene free control region of 18: 5′- cDNA (GenBank accession no. U51127) was cloned with N-terminal FLAG ACTCCCCTTTCATGCTTCTG and 5′-AGGTCCCAGGACATATCCATT. DNaseI-Seq epitope into pcDNA3 (Invitrogen). Activating mutations according to Lin et al. samples were generated from a size selection of DNaseI-digested DNA frag- (29) were introduced by site-directed mutagenesis using the QuikChange ments comprised within a range of 100–600 bp and subjected to library prep- MEDICAL SCIENCES Multi Site-Directed Mutagenesis Kit (Stratagene). pcDNA3-FLAG-DNIRF5- aration as per the manufacturer´s instructions (Illumina). Libraries were run on – 4D lacks AA 1 137, which comprise the N-terminally located nuclear lo- an Illumina GAIIx sequencer. calization signal and the DNA binding domain and was cloned through More detailed information is provided in SI Appendix, SI Methods. BamHI and EcoRI restriction sites into pcDNA3-FLAG from pcDNA3-FLAG- IRF5-4D. For cloning of the retroviral expression construct MSCV-IRF5- ACKNOWLEDGMENTS. We thank S. Kressmann and B. Wollert-Wulf for 4D-IRES-CFP, the plasmid pcDNA3.1/Zeo-IRF5-4D (29) was digested with outstanding technical assistance, P. Rahn for cell sorting, F. Rosenbauer for pro- BamHI followed by a Klenow fill-in reaction and EcoRI cleavage. The re- viding the MSCV-IRES-CFP construct, and S. A. Assi and D. Westhead for hosting sulting IRF5-4D fragment was cloned into the vector MSCV-IRES-CFP (a gift the DNaseI data and providing data links. This work was supported in part by of F. Rosenbauer, Institute of Molecular Tumor Biology, University of Deutsche Forschungsgemeinschaft Grants SFB/TRR54 and JA1847/1-1 (to S.M. Münster, Germany) via EcoRI and XhoI (the latter converted to a blunt end and M.J.) and KU1315/7-1 and GRK1431 (to R.K.), the Berliner Krebsgesellschaft, by Klenow fill-in). Sequences for IRF5 siRNAs were selected based on pre- the Experimental and Clinical Research Center, a joint cooperation between viously described design rules (48). siRNA constructs were generated by the Charité-Universitätsmedizin Berlin and the Max Delbrück Center for Molec- ular Medicine, and the German Cancer Consortium. Research in Leeds and cloning target sequences siRNA_2113 5′-GAGTGAGCACTTAGGTATCAT-3′ ′ ′ Birmingham was supported by grants from Cancer Research UK (to C.B.), as (siIRF5 #1) and siRNA_1025 5 -GTGGAACTCTTCGGCCCCATA-3 (siIRF5 #2) well as Leukemia Lymphoma Research (to C.B. and P.N.C.). M. Giefing was through BglII and HindIII restriction sites into pSUPER (47). The scrambled supported by an Federation of European Biochemical Societies Long-Term Fel- siRNA construct has been previously described (8). All constructs were ver- lowship and Support for International Mobility of Scientists fellowship of the ified by sequencing. Polish Ministry of Sciences and Higher Education.

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